CN1933854A - Therapeutic agent for auris interna disorder containing il-6 antagonist as active ingredient - Google Patents

Abstract

A therapeutic and/or preventive agent for auris interna disorder, comprising an IL-6 antagonist, preferably anti-IL-6R antibody as an active ingredient.

Description

Contain of the internal ear treating dysfunction agent of interleukin-6 antagonist as effective ingredient

Technical field

The present invention relates to internal ear treating dysfunction and/or preventive.Of the present inventionly treat and/or prevent agent and contain interleukin-6 (IL-6) antagonist.

Background technology

In the deafness, the deafness that causes because of interior remebrance (cochlea) or fan's property walked (the 8th cranial nerve) is called sensorineural hearing loss.Deaf reason is varied, for example can enumerate Meniere syndrome (Meniere ' s disease), drug-induced property internal ear obstacle (because anticancer preparation, the internal ear obstacle that causes as aminoglycoside and cisplatin etc.), viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor.Sudden deafness, old man's induced deafness, noise deafness (noise deafness) are also included within the sensorineural hearing loss in addition.Noise deafness is that chain saw, internal combustion engine, heavy device, rifle, aircraft etc. send that big noise causes the internal ear obstacle and the result that takes place is relevant with mobile on shooting, the snow, airline traveling, Springsteen etc.

Think that the reason of sensorineural hearing loss is varied, the acute immunoreation of known internal ear can cause permanent auditory dysesthesia (hearing loss) (Satoh H et al., Laryngoscope.2002 Sep; 112 (9): 1627-34).The document reports that also the internal ear of KLH (keyhole limpet hemocyanin) or subarachnoid injection cause the immunoreation at cochlea, express TNF-α and IL-1 β, TNF-α causes the disease progression of cochlea, and because the TNF-alpha inhibitor causes audition partly to reduce., so far, still fully not about with interleukin-6 to the contributive report of sensorineural hearing loss.

Non-patent literature 1 Satoh H et al., laryngoscope.2002 Sep; 112 (9) 1627-34

Summary of the invention

The object of the present invention is to provide the newtype drug that is used for the treatment of and/or prevents the internal ear obstacle.

Lucubrates such as the inventor are illustrated interleukin-6 and the disease of sensorineural hearing loss and are formed relevantly, and the interleukin-6 antagonist has sensorineural hearing loss treatment of diseases effect.

Therefore, the invention provides internal ear treating dysfunction and/or the preventive that contains the interleukin-6 antagonist as effective ingredient.

Above-mentioned internal ear obstacle, sensorineural hearing loss for example, this sensorineural hearing loss is the sensorineural hearing loss that causes of Meniere syndrome, drug-induced property internal ear obstacle, viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor for example, perhaps sudden deafness, old man's induced deafness or noise deafness.

Perhaps, above-mentioned internal ear obstacle, vestibular disorder for example, this vestibular disorder, for example vestibular disorder that causes of Meniere syndrome, vestibular neuritis or medicine internal ear obstacle.

Above-mentioned interleukin-6 antagonist, for example anti-interleukin-6 receptor antibody, the monoclonal antibody that this anti-interleukin-6 receptor antibody for example is anti-interleukin-6 receptor, the monoclonal antibody of preferred antihuman interleukin-6 receptor, or the monoclonal antibody of anti-mice interleukin-6 receptor, the monoclonal antibody of above-mentioned antihuman interleukin-6 receptor is for example: PM-1 antibody, the monoclonal antibody of above-mentioned anti-mice interleukin-6 receptor for example: MR16-1 antibody.

The preferred recombinant antibodies of the antibody of above-mentioned anti-interleukin-6 receptor, this recombinant antibodies for example, is chimeric antibody, humanized antibody or people's antibody.This humanized antibody for example is a humanization PM-1 antibody.

The present invention can take following mode.

(1) application of interleukin-6 antagonist in making internal ear treating dysfunction and/or preventive.

(2) above-mentioned (1) described application, wherein, described internal ear obstacle is a sensorineural hearing loss.

(3) above-mentioned (2) described application, wherein, described sensorineural hearing loss is the sensorineural hearing loss that Meniere syndrome, drug-induced property internal ear obstacle, viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor cause.

(4) above-mentioned (2) described application, wherein, described sensorineural hearing loss is sudden deafness, old man's induced deafness or noise deafness.

(5) above-mentioned (1) described application, wherein, described internal ear obstacle is a vestibular disorder.

(6) above-mentioned (5) described application, wherein, described vestibular disorder is the vestibular disorder that Meniere syndrome, vestibular neuritis or drug-induced property internal ear obstacle cause.

(7) each described application of above-mentioned (1)～(6), wherein, described interleukin-6 antagonist is the antibody of anti-interleukin-6 receptor.

(8) above-mentioned (7) described application, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-interleukin-6 receptor.

(9) above-mentioned (8) described application, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of antihuman interleukin-6 receptor.

(10) above-mentioned (8) described application, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-mice interleukin-6 receptor.

(11) each described application of above-mentioned (7)～(10), wherein, the antibody of described anti-interleukin-6 receptor is recombinant antibodies.

(12) above-mentioned (9) described application, wherein, the monoclonal antibody of described antihuman interleukin-6 receptor is a PM-1 antibody.

(13) above-mentioned (10) described application, wherein, the monoclonal antibody of described anti-mice interleukin-6 receptor is a MR16-1 antibody.

(14) each described application of above-mentioned (7)～(13), wherein, the antibody of described anti-interleukin-6 receptor is chimeric antibody, humanized antibody or people's antibody of anti-interleukin-6 receptor.

(15) above-mentioned (14) described application, wherein, the humanized antibody of described anti-interleukin-6 receptor is a humanization PM-1 antibody.

The present invention can also take following mode:

(1) treats and/or prevents the method for internal ear obstacle, comprise giving the interleukin-6 antagonist.

(2) above-mentioned (1) described method, wherein, described internal ear obstacle is a sensorineural hearing loss.

(3) above-mentioned (2) described method, wherein, described sensorineural hearing loss is the sensorineural hearing loss that Meniere syndrome, drug-induced property internal ear obstacle, viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor cause.

(4) above-mentioned (2) described method, wherein, described sensorineural hearing loss is sudden deafness, old man's induced deafness or noise deafness.

(5) above-mentioned (1) described method, wherein, described internal ear obstacle is a vestibular disorder.

(6) above-mentioned (5) described method, wherein, described vestibular disorder is the vestibular disorder that Meniere syndrome, vestibular neuritis or drug-induced property internal ear obstacle cause.

(7) each described method in above-mentioned (1)～(6), wherein, described interleukin-6 antagonist is the antibody of anti-interleukin-6 receptor.

(8) above-mentioned (7) described method, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-interleukin-6 receptor.

(9) above-mentioned (8) described method, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of antihuman interleukin-6 receptor.

(10) above-mentioned (8) described method, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-mice interleukin-6 receptor.

(11) each described method in above-mentioned (7)～(10), wherein, the antibody of described anti-interleukin-6 receptor is recombinant antibodies.

(12) above-mentioned (9) described method, wherein, the monoclonal antibody of described antihuman interleukin-6 receptor is a PM-1 antibody.

(13) above-mentioned (10) described method, wherein, the monoclonal antibody of described anti-mice interleukin-6 receptor is a MR16-1 antibody.

(14) each described method in above-mentioned (7)～(13), wherein, the antibody of described anti-interleukin-6 receptor is chimeric antibody, humanized antibody or people's antibody of anti-interleukin-6 receptor.

(15) above-mentioned (14) described method, wherein, the humanized antibody of described anti-interleukin-6 receptor is a humanization PM-1 antibody.

Description of drawings

Fig. 1 is the result of expression embodiment 1, be illustrated in the deaf model of the mice that makes by 3 kinds of frequencies, give using group and giving the figure of audition threshold value reduction degree (dB) of the matched group of mouse immuning ball protein G of interleukin-6 R humanized antibody of the present invention (MR16-1).

Fig. 2 is the electrophoresis result of the RT-PCR of expression among the embodiment 2, this result be illustrated in the cochlea that carries out the SD rat behind the sound load various cytokines through the time change.

Fig. 3 is the result of the quantitative RT-PCR of expression among the embodiment 2, this result be illustrated in the cochlea that carries out the SD rat behind the sound load TNF α through the time change.

Fig. 4 is the result of the quantitative RT-PCR of expression among the embodiment 2, this result be illustrated in the cochlea that carries out the SD rat behind the sound load interleukin-6 through the time change.

Fig. 5 is the result's of the quantitative RT-PCR of expression among the embodiment 2 figure, this result be illustrated in the cochlea that carries out the SD rat behind the sound load IL-1 β through the time change.

Fig. 6 is that the pass through Vectastein ABC test kit of expression among the embodiment 2 uses the antibody test of anti-mice interleukin-6 to carry out result's the alternative photo of figure of the tissue of the SD rat of sound load after 6 hours.

Fig. 7 is that expression is passed through Vectastein ABC test kit uses the result of anti-mice interleukin-6 antibody test interleukin-6 to the tissue of the SD rat of sound load after 6 hours the alternative photo of figure among the embodiment 2.

After Fig. 8 is the sound of amplification of 2 hours 124dB of C57BL/6J mice load, be determined in the internal ear spiral organ of Corti of using behind MR16-1 of the present invention (at the anti-interleukin-6 humanized antibody of reference example 4 preparations) or the rat IgG (contrast) result's the electrophoretogram of the expression of STAT3, the Erk of always STAT3, Erk and Akt and phosphorylation and Akt.

The specific embodiment

Therapeutic agent of the present invention have suppress the cochlear tissue obstacle, be the effect of the development of the sexy auditory dysesthesia of feeling nerve deafness (sensorineural hearing loss) of internal ear.Therapeutic agent particularly of the present invention has the effect of the high pitch zone auditory dysesthesia that suppresses in the sensorineural hearing loss.As and obstacle diseases associated hair cell identical, can enumerate vestibular disorder with sensorineural hearing loss.Cochlea generation obstacle in sensorineural hearing loss, although cochlea is administered different sensations with vestibule, structurally, any one all links to each other with lymph fluid with hair cell, sustenticular cell, is bearing physiological function by their similar tissues and physical property thereof.That is, the hair cell perception becomes the tissue that carries out perception with same structure owing to the difference sensation that the difference of the motion of shaking of causing of sound, lymph that acceleration causes produces.Any one of cochlea and vestibule is associated with the function reduction with the obstacle that hair cell is arranged, and its susceptibility to medicine is similar.So therapeutic agent of the present invention can be used for the treatment of internal ear obstacles such as sensorineural hearing loss, vestibular disorder.

Interleukin-6 is the cytokine that is called as B-cell stimulating factor 2 (BSF2) or interferon beta2.Interleukin-6 is found (Hirano as participating in the activatory differentiation factor of bone-marrow-derived lymphocyte, T.et al., Nature (1986) 324,73-76), being illustrated afterwards is the multifunctional cytokine (Akira of the various cell functions of influence, S.et al., Adv.inImmunology (1993) 54,1-78).(J.Exp.Med. (1988) 167,1253-1258) for Lotz, M.et al. to it is reported the maturation of interleukin-6 inducer T lymphocyte.

Interleukin-6 transmits its biologic activity by two kinds of protein on the cell.A kind of be in conjunction with the about 80kD of molecular weight of interleukin-6 part binding proteins matter interleukin-6 receptor (Taga, T.et al., J.Exp.Med. (1987) 166,967-981, Yamasaki, K.et al., Science (1987) 241,825-828).The interleukin-6 receptor exists mainly as the SIL-6R that is made of this zone, extracellular except the perforation cell membrane, the film conjunction type of expressing on the cell membrane.

Another kind is the memebrane protein gp130 of the molecular weight about 130kD relevant with signal transduction of non-part associativity.Interleukin-6 and interleukin-6 receptor form interleukin-6/interleukin-6 receptor complex, and then by combining with gp130, the biologic activity of interleukin-6 is passed to that (Cell (1989) 58,573-581) for Taga, T.et al. in the cell.

The interleukin-6 antagonist is to suppress the material that the interleukin-6 biologic activity is transmitted.Up to the present, known antibody (anti-interleukin-6 antibody) at interleukin-6 is arranged, at the antibody (anti-interleukin-6 receptor antibody) of interleukin-6 receptor, at partial peptide of antibody (anti-gp130 antibody), interleukin-6 variant, interleukin-6 or the interleukin-6 receptor of gp130 etc.

The existing relevant report (Novick of several and anti-interleukin-6 receptor antibody, D.et al., Hybridoma (1991) 10,137-146, Huang, Y.W.et al., Hybridoma (1993) 12,621-630, International Patent Application Publication No. WO 95-09873, french patent application publication number FR 2694767, U.S. Patent number US 521628).(J.Immunol. (1989) 143 for Hirata, Y.et al., complementary determining region (CDR 2900-2906) by the mouse antibodies PM-1 with one of them; Complementarity determiningregion) is transplanted to the humanization PM-1 antibody known (International Patent Application Publication No. WO 92-19759) that people's antibody obtains.

The antibody of the preferably anti-interleukin-6 receptor of above-mentioned interleukin-6 antagonist, the preferably monoclonal antibody of the monoclonal antibody of antihuman interleukin-6 receptor or anti-mice interleukin-6 receptor.As the monoclonal antibody such as the PM-1 antibody of above-mentioned antihuman interleukin-6 receptor, in addition as the monoclonal antibody such as the MR16-1 antibody of anti-mice interleukin-6 receptor.Above-mentioned antibody is chimeric antibody, humanized antibody or people's antibody preferably, for example: humanization PM-1 antibody.Humanized antibody is to be called the humanized antibodies (altered antibody) or the reshaping antibody of structure (reshaped) people antibody again.

The interleukin-6 antagonist that uses among the present invention can be used as the active component of internal ear treating dysfunction and/or preventive, no matter its source, kind and shape can be used so long as suppress internal ear obstacle cell proliferation.

The interleukin-6 antagonist is the signal transduction that depends on interleukin-6 capable of blocking, suppresses the material of interleukin-6 biologic activity.The interleukin-6 antagonist preferably among interleukin-6, interleukin-6 receptor and the gp130 any in conjunction with inhibited material.As the interleukin-6 antagonist, for example: the partial peptide of anti-interleukin-6 antibody, anti-interleukin-6 receptor antibody, anti-gp130 antibody, interleukin-6 variant, SIL-6R variant or interleukin-6 or interleukin-6 receptor and showing and they same active lower-molecular substances.

The anti-interleukin-6 antibody that uses among the present invention can use well-known means to obtain with polyclone or monoclonal antibody form.As the anti-interleukin-6 antibody that uses among the present invention, especially preferably from mammiferous monoclonal antibody.As from mammiferous monoclonal antibody, antibody that is produced by hybridoma and the antibody that is contained the expression vector host transformed generation of antibody gene by genetic engineering means by usefulness are arranged.This antibody is by combining with interleukin-6, and the inhibition interleukin-6 combines with the interleukin-6 receptor, and blocking-up interleukin-6 biologic activity is transmitted in cell.

As such antibody, (Eur.J.Immunol. (1988) 18 for Matsuda, T.et al., 951-956) and SK2 antibody (the 21st Japanese immunology can be always for Sato, K.et al., academic record (1991) 21,166) etc. as MH166.

Can use well-known technology basically, be prepared as follows the hybridoma that produces anti-interleukin-6 antibody.Promptly, use interleukin-6 as sensitization antigen, carry out immunity according to common immunization method, immunocyte that uses common cell fusion method to make to obtain and well-known parental cell merge, and by the screening technique of routine, the screening preparation produces the cell of monoclonal antibody.

Specifically, can resemble and prepare anti-interleukin-6 antibody following.For example: can be by using Eur.J.Biochem (1987) 168,543-550, J.Immunol. (1988) 140, the disclosed interleukin-6 gene of 1534-1541 or Agr.Biol.Chem. (1990) 54,2685-2688/aminoacid sequence obtains as the sensitization antigenic human interleukin-6 that obtains antibody.

The gene order of interleukin-6 is inserted into well-known expression vector system, after transforming appropriate host cell, with well-known method purification purpose interleukin-6 protein from this host cell or in the culture supernatant, the interleukin-6 protein that can use this purification is as sensitization antigen.In addition, also can use interleukin-6 protein and other proteinic fused protein as sensitization antigen.

Can use well-known means, obtain the antibody of the anti-interleukin-6 receptor that uses among the present invention with polyclone or monoclonal antibody form.As the antibody of the anti-interleukin-6 receptor that uses among the present invention especially preferably from mammiferous monoclonal antibody.As from mammiferous monoclonal antibody, have by the antibody of hybridoma generation and by the antibody that produces in order to the expression vector host transformed that contains antibody gene by genetic engineering means.This antibody by with the interleukin-6 receptors bind, suppress combining of interleukin-6 and interleukin-6 receptor, the biologic activity of blocking interleukin-6 is to intracellular transmission.

As such antibody, as: MR16-1 antibody (Tamura, T.et al.Proc.Natl.Acad.Sci.USA (1993) 90,11924-11928), PM-1 antibody (Hirata, Y.et al., J.Immunol. (1989) 143,2900-2906), AUK12-20 antibody, AUK64-7 antibody or AUK146-15 antibody (International Patent Application Publication No. WO 92-19759) etc.Wherein particularly preferred antibody is PM-1 antibody.

To produce the strain of PM-1 antibody hybridoma cell as PM-1, in putting down into the first year in (1989) July 12, with FERM BP-2998, be preserved in the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst's patent center (the Ibaraki county builds a kind of ground of 1 fourth order, east, ripple city, 1 central authorities the 6th) according to budapest treaty.In addition, to produce the strain of MR16-1 antibody hybridoma cell as Rat-mousehybridoma MR16-1, in putting down into 9 years (1997) March 13, with FERM BP-5875, be preserved in the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst's patent center (the Ibaraki county builds a kind of ground of 1 fourth order, east, ripple city, 1 central authorities the 6th) according to budapest treaty.

Can use well-known technology basically, be prepared as follows the hybridoma that produces anti-interleukin-6 acceptor monoclonal antibody.Promptly, use the interleukin-6 receptor as sensitization antigen, carry out immunity according to common immunization method, cell fusion method by routine merges the immunocyte and the well-known parental cell that obtain, uses common screening technique, can produce the cell preparation of monoclonal antibody by screening.

Specifically, can be prepared as follows the antibody of anti-interleukin-6 receptor.For example: human interleukin-6 receptor that uses as the sensitization antigen that is used to obtain antibody can obtain by the gene/aminoacid sequence that uses the Japanese Unexamined Patent Publication No spy open the disclosed interleukin-6 receptor of flat 3-155795 by using Europe patent application publication number EP 325474, mice interleukin-6 receptor.

The interleukin-6 receptor protein has (SIL-6R) expressed and break away from from cell membrane on cell membrane (J.Biochem. (1990) 108 for Yasukawa, K.et al., 673-676) two kinds.The antibody of SIL-6R is made of the zone, substantial extracellular that is combined in the interleukin-6 receptor on the cell membrane, cell membrane connects the zone or cell membrane connects zone and intracellular region territory because of lacking, and SIL-6R is different with film conjunction type interleukin-6 receptor.The interleukin-6 receptor protein can use any interleukin-6 receptor so long as can be used as the sensitization antigen of the antibody of the anti-interleukin-6 receptor that preparation uses among the present invention and use.

The gene order of interleukin-6 receptor can be inserted in the well-known expression vector system, after transforming appropriate host cell, with well-known method purification purpose interleukin-6 receptor protein from this host cell or in the culture supernatant, this purification interleukin-6 receptor protein is used as sensitization antigen.In addition, also can use the cell of expressing the interleukin-6 receptor or interleukin-6 receptor protein and other proteinic fusion rotein as sensitization antigen.

The escherichia coli (E.coli) of plasmid pIBIBSF2R of cDNA that contain coding human interleukin-6 receptor are preserved in the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst's patent center (the Ibaraki county builds a kind of ground of 1 fourth order, east, ripple city, 1 central authorities the 6th) according to budapest treaty in putting down into the first year with HB101-pIBIBSF2R, preserving number FERM BP-2232 in (1989) January 9.

The anti-gp130 antibody that uses among the present invention can use well-known means to obtain with polyclone or monoclonal antibody form.As the anti-gp130 antibody that uses among the present invention especially preferably from mammiferous monoclonal antibody.As from mammiferous monoclonal antibody, have by the antibody of hybridoma generation and by the antibody that produces with the expression vector host transformed that contains antibody gene by genetic engineering means.This antibody suppresses interleukin-6/interleukin-6 receptor complex and combines with gp130's by combining with gp130, and the biologic activity of blocking-up interleukin-6 is to intracellular transmission.

As such antibody, as AM64 antibody (spy opens flat 3-219894 communique), 4B11 antibody and 2H4 antibody (US 5571513), B-S12 antibody and B-P8 antibody (spy opens flat 8-291199 communique) etc.

Can use well-known technology to be prepared as follows the hybridoma that produces anti-gp130 monoclonal antibody basically.Promptly, use gp130 as sensitization antigen, carry out immunity according to common immunization method, cell fusion method by routine merges the immunocyte and the well-known parental cell that obtain, uses conventional screening technique, produces cell by the screening monoclonal antibody and is prepared.

Specifically, can be prepared as follows monoclonal antibody.For example: can obtain gp130 by the gene/aminoacid sequence that uses disclosed gp130 among the patent application publication number EP 411946 of Europe as the sensitization antigen use that obtains antibody.

The gene order of gp130 can be inserted in the well-known expression vector system, after being converted into appropriate host cell, use well-known method purification purpose gp130 protein from this host cell or in the culture supernatant, the gp130 receptor protein of this purification is used as sensitization antigen.In addition, also can use as sensitization antigen expressing the cell of gp130 or gp130 protein and other proteinic fused protein.

Mammal as with the sensitization antigen immune is not particularly limited, preferably consider with cell fusion in select after the fitness of the parental cell that uses, generally use rodent, for example: mice, rat, hamster etc.

According to well-known method with the sensitization antigen-immunized animal.For example, as general method, by sensitization antigen is expelled to mammiferous intraperitoneal or subcutaneous.Specifically, sensitization antigen is diluted to appropriate amount with PBS (Phosphate-Buffered Saline) or normal saline etc., as requested can be with suspension solution and common adjuvant, for example: Freund's complete adjuvant mixes in right amount, and preferably every 4-21 day is to the mammal injection for several times after the emulsifying.In addition, when the sensitization antigen immune, can use appropriate carriers.

Resemble and carry out immunity above-mentioned, confirm that the antibody horizontal of expecting in the serum rises after, take out immunocyte from mammal, be used for cell fusion.Enumerate splenocyte especially as the ideal immunocyte that is used for cell fusion.

The well-known various cell strain of suitable use is as the mammal myeloma cell of the opposing party's parental cell that can merge with above-mentioned immunocyte, for example: P3X63Ag8.653 (Kearney, J.F.et al.J.Immnol. (1979) 123,1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology andImmunology (1978) 81,1-7), NS-1 (Kohler.G.and Milstein, C.Eur.J.Immunol. (1976) 6,511-519), MPC-11 (Margulies.D.H.etal., Cell (1976) 8,405-415), SP2/0 (Shulman, M.et al., Nature (1978) 276,269-270), FO (de St.Groth, S.F.et al., J.Immunol.Methods (1980) 35,1-21), S194 (Trowbridge, I.S.J.Exp.Med. (1978) 148,313-323), R210 (Galfre, G.et al., Nature (1979) 277,131-133) etc.

Basically (Kohler.G.and Milstein, C., MethodsEnzymol. (1981) 73 3-46) wait and carry out above-mentioned immunocyte and myeloma cell's cell fusion according to people's such as well-known method, for example Milstein method.

More particularly, above-mentioned cell fusion can be implemented in the nutrient medium of routine under the situation that for example has cell fusion promoter to exist.As merging promoter, for example, can use Polyethylene Glycol (PEG), Sendai virus (HVJ) etc., in addition as requested,, also can add adjuvant such as using dimethyl sulfoxide in order further to improve fusion efficiencies.

Immunocyte and myeloma cell's usage ratio, for example, preferably with respect to 1～10 times of myeloma cell's immunocyte.Culture fluid as above-mentioned cell fusion use, for example, can use the RPMI1640 culture fluid that is fit to above-mentioned myeloma cell strain propagation, MEM culture fluid, other the conventional culture fluid that in this cell culture, uses, in addition also can and with hyclone serum annex solutions such as (FCS).

Cell fusion can be by fully mixing quantitative above-mentioned immunocyte of institute and myeloma cell in above-mentioned culture fluid, concentration according to 30～60% (w/v) is added the PEG solution that is heated in advance about 37 ℃, for example: the PEG solution of mean molecule quantity about 1000～6000 forms purpose fused cell (hybridoma) by mixing.Then, add suitable culture fluid one by one,, can remove the cell fusion agent that influences hybridoma fertility etc. by the operation that repeated centrifugation is removed supernatant.

This hybridoma is by cultivating, select in common selection culture fluid, for example HAT culture fluid (culture fluid that contains hypoxanthine, aminopterin and thymidine).For the thorough death for the cell (non-fused cell) beyond the purpose hybridoma provides adequate time, make the cultivation in this HAT culture fluid continue a few days～several weeks usually.Then, implement conventional Method of Limited Dilution method, produce the screening and the clone of the hybridoma of purpose antibody.

Obtain outside the above-mentioned hybridoma in addition, except the animal the people being carried out antigen immune, also can be at the antigen protein or the antigen presentation cell sensitization human lymphocyte of external use expectation, sensitization bone-marrow-derived lymphocyte and human myeloma cell, for example U266 are merged, also can obtain having the people's antibody (with reference to special fair 1-59878) that combines active expectation with antigen of expecting or antigen presentation cell.In addition, antigen or antigen presentation cell had the transgenic animal of human immunoglobulin gene storehouse (repertoire), the people's antibody (with reference to International Patent Application Publication No. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735) that uses said method also can obtain to expect.

The hybridoma of Zhi Bei the generation monoclonal antibody cultivation of can going down to posterity in the culture fluid of routine like this can be preserved in that liquid nitrogen is medium-term and long-term in addition.

In order to obtain monoclonal antibody, can adopt following method: according to conventional method this hybridoma is cultivated, obtained its culture supernatant, or following method: hybridoma is fit to its mammal, makes its propagation, obtain its ascites etc. from this hybridoma.The method of front is suitable for obtaining highly purified antibody, and the latter's method is suitable for the mass production of antibody.

For example, can open the hybridoma that flat 3-139293 disclosed method preparation produces anti-interleukin-6 receptor antibody according to the spy.Will be in putting down into the first year in (1989) July 12, with FERMBP-2998, the intraperitoneal that the hybridoma that is preserved in the generation PM-1 antibody at the biological preservation of Independent Administrative Leged Industrial Technology Complex Inst patent center (the Ibaraki county builds a kind of ground of 1 fourth order, east, ripple city, 1 central authorities the 6th) according to the budapest treaty world injects BALB/c mouse obtains ascites, from this ascites purification PM-1 antibody, or with this hybridoma in suitable culture medium, for example, contain 10% hyclone, the RPMI1640 culture medium of 5%BM-Condimed H1 (Boehringer Mannheim manufacturing), hybridoma SFM culture medium (GIBCO-BRL manufacturing), PFHM-II culture medium (GIBCO-BRL manufacturing) etc. is cultivated, from this culture supernatant purification PM-1 antibody.

In the present invention, as monoclonal antibody, can use from hybridoma clonal antibody gene, be incorporated into appropriate carriers, this carrier is imported the host, use the recombinant antibodies that gene recombination technology produces (for example, with reference to Borrebaeck C.A.K.and Larrick J.W.THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the UnitedKingdom by MACMILLAN PUBLISHERS LTD, 1990).

Specifically, from the cell that produces purpose antibody, the mRNA that for example hybridoma separates encoding antibody variable (V) district.The separation of mRNA is according to well-known method, for example, guanidine ultracentrifugation (Chirgwin, J.M.et al., Biochemistry (1979) 18,5294-5299), (Anal.Biochem. (1987) 162 for Chomczynski, P.et al. for the AGPC method, 156-159) wait the total RNA of preparation, use preparation mRNA such as mRNA purification kit (Pharmacia system).In addition by using QuickPrep mRNA purification kit (Pharmacia manufacturing) can directly prepare mRNA.

Use the reverse transcriptase can be from the cDNA in the mRNA synthetic antibody V district that obtains.The 1st chain cDNA synthetic agent of use AMV reverse transcriptase box etc. carries out the synthetic of cDNA.In addition in order to carry out the synthetic and amplification of cDNA, can use 5 '-Ampli FINDER RACE test kit (Clontech manufacturing) and use 5 of PCR '-RACE method (Frohman, M.A.et al., Proc.Natl.Acad.Sci.USA (1988) 85,8998-9002; Belyavsky, A.et al., Nucleic Acids Res. (1989) 17,2919-2932).PCR product purification target DNA fragment by obtaining is connected with carrier DNA.Make recombinant vector more thus, import escherichia coli etc., select bacterium colony, the recombinant vector of preparation expectation.By well-known method, for example: the deoxidation method is confirmed the base sequence of target DNA.

The DNA in purpose antibody V district if obtain encoding is connected its DNA with the encoding antibody constant region (C district) of expectation, is incorporated into expression vector.Or also the DNA in encoding antibody V district can be incorporated in the expression vector that contains Antibody C region DNA.

In order to make the antibody that uses among the present invention,, antibody gene is incorporated into the expression vector that under the regulation and control of expression regulation district, for example enhancer, promoter, to express as what narrate below.Then, with this expression vector transformed host cell, make antibody expression.

In the present invention, in order to make the purpose of heteroantigen reduction to the people etc., can end user's gene recombinaton type antibody for a change, for example: chimeric (Chimeric) antibody, humanization (Humanized) antibody, people (human) antibody.Can use known method to make these and change antibody.

Chimeric antibody can be connected with the DNA of coding people Antibody C region by the DNA in the encoding antibody V district that will obtain as mentioned above, be incorporated into expression vector then, import the host, make its generation obtain (with reference to Europe patent application publication number EP 125023, International Patent Application Publication No. WO92-19759).Use this known method, can obtain being used for chimeric antibody of the present invention.

For example, the plasmid that contains the DNA in coding chimeric PM-1 light chain of antibody and H chain V district is named as pPM-k3 and pPM-h1 respectively, the escherichia coli that contain this plasmid on February 12nd, 1991 respectively with NCIMB 40366 and NCIMB 40362, be preserved in National Collections of Industrial and Marine BacteriaLimited (Ferguson Building according to budapest treaty, Craibst one Estate, Bucksburn, Aberdeen AB 21 9YA, United Kindom).

Humanized antibody is also referred to as structure (reshaped) people antibody again, be that the complementary determining region (CDR) of the mammal beyond the people, for example mouse antibodies is transplanted to product after people's complementary antibody determining area, its general gene recombination method known (with reference to Europe patent application publication number EP125023, International Patent Application Publication No. WO 92-19759).

It is synthetic according to the CDR of mouse antibodies and the framework region (FR of people's antibody by the PCR method specifically, to have several oligonucleotide of lap by the portion endways of being made into; Frameworkregion) connect the DNA sequence that designs like that.The DNA that obtains is connected with the DNA of coding people Antibody C region, is incorporated into then in the expression vector, this carrier is imported the host, make its generation obtain (with reference to Europe patent application publication number EP 239400, International Patent Application Publication No. WO92-19759).

The FR of the people's antibody that connects by CDR selects to make complementary determining region form the zone of good antigen-binding site.As required, (Cancer Res. (1993) 53,851-856) for Sato, K.et al. for the aminoacid that makes again the complementary determining region of structure people antibody form the framework region that suitable antigen-binding site also can the antagonist variable region is replaced.

For chimeric antibody, humanized antibody, can end user's Antibody C region.As people's Antibody C region, as C γ, for example: can use C γ 1, C γ 2, C γ 3 or C γ 4.And in order to improve the stability of antibody or its generation, also can modify people's Antibody C region.

Chimeric antibody is by constituting from the variable region of the mammiferous antibody beyond the people with from the C district of people's antibody, humanized antibody is by constituting with framework region and C district from people's antibody from the complementary determining region of the mammiferous antibody beyond the people, since low in the intravital antigenicity of people, so can be used as the antibody that uses among the present invention.

As the ideal object lesson of the humanized antibody that uses among the present invention, as humanization PM-1 antibody (with reference to International Patent Application Publication No. WO 92-19759).

And as the method that obtains people's antibody, except the method for narrating previously, known end user's antibody library in addition by elutriation, obtains the technology of people's antibody.For example, make the variable region of people's antibody be illustrated in phage surface, also can select and the bonded phage of antigen as single-chain antibody (scFv) by the phage display method.If the gene of the phage of resolve selecting, the DNA sequence with the variable region of the bonded people's antibody of antigen of can determining to encode.If illustrated with the DNA sequence of the bonded scFv of antigen, can utilize this sequence to prepare suitable expression, obtain people's antibody.These methods are well-known, can be with reference to WO 92/01047, and WO 92/20791, WO93/06213, WO 93/11236, and WO 93/19172, and WO 95/01438, and WO 95/15388.

Resembling the antibody gene that makes up above-mentioned can express it by well-known method to obtain.Under the situation that is cells of mamma animals, by make useful promoter commonly used, the functionally bonded DNA of a-signal peptide or contain the vector expression of this DNA is got together for fun under the antibody gene of being expressed, its 3 ' side.For example:, be early promoter/enhancer (human cytomegalovirus immediate earlypromoter/enhancer) as human cytomegalic inclusion disease virus as promoter/enhancer.

As the promoter/enhancer that uses in being used in of other antibody expression of the present invention, can use the promoter/enhancer from cells of mamma animals such as retroviral, polyoma virus, adenovirus, simian virus 40 viral promotors/enhancers such as (SV 40) or people's EF-1 a (HEF1a) in addition.

When for example, using SV 40 promoteres/enhancer, method (Mulligan according to people such as Mu11igan, R.C.et al., Nature (1979) 277,108-114), and when using HEF1 α promoter/enhancer, method (Mizushima according to people such as Mizushima, S.andNagata, S.Nucleic Acids Res. (1990) 18,5322) implement easily.

The antibody gene of when using escherichia coli, can make useful promoter commonly used, be used for antibody secreted signal peptide sequence, being expressed combination functionally, expression.For example as promoter, as lacZ promoter, araB promoter.When using the lacZ promoter, can be according to people's such as Ward method (Ward, E.S.et al., Nature (1989) 341,544-546; Ward, E.S.et al.FASEB J. (1992) 6 2422-2427) implements, when using the araB promoter, can (Better, M.et al.Science (1988) 240,1041-1043) implement according to people's such as Better method.

As being used for antibody secreted signal peptide sequence, make antibody when colibacillary pericentral siphon produces, can use the pelB signal peptide sequence (Lei, S.P.et al J.Bacteriol. (1987) 169,4379-4383).After being separated in the antibody of pericentral siphon generation, the structure of antagonist is carried out suitable refolding (refold) back and is used (for example, with reference to WO96/30394).

As origin of replication, can use origin of replication from SV 40, polyoma virus, adenovirus, bovine papilloma virus (BPV) etc., in addition, in order to carry out the gene copy number amplification in host cell system, expression vector can contain aminoglycoside phosphotransferase (APH) gene as selection marker, thymidine kinases (TK) gene, escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene etc.

In order to make the antibody that uses among the present invention, can use production system arbitrarily.The production system that is used for the antibody manufacturing has external and intravital production system.As external production system, as use eukaryotic production system and the production system of using prokaryotic cell.

When using eukaryotic cell, the production system of using zooblast, plant cell or fungal cell is arranged.As zooblast, known (1) cells of mamma animals, for example: CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero etc.; (2) amphibian cell, for example: Africa xenopus oocyte or (3) insect cell, for example, sf9, sf21, Tn5 etc.As plant cell, known cell from Nicotiana tabacum L. (Nicotianat abacum) can carry out callus culture to them.As the fungal cell, known have a yeast, for example: yeast (Saccharomyces) genus, for example saccharomyces cerevisiae (Saccharomyces cerevisiae), filamentous bacteria, for example: aspergillus (Aspergillus), for example: aspergillus niger (Aspergillusniger) etc.

When using prokaryotic cell, the production system of using bacterial cell is arranged.As bacterial cell is known escherichia coli (E.coli), bacillus subtilis arranged.

By transforming the purpose antibody gene is imported in these cells,, can obtain antibody by being cultivated external by cell transformed.Cultivate according to well-known method.For example, as culture fluid, can use DMEM, MEM, RPMI 1640, IMDM, also can and with hyclone serum annex solutions such as (FCS).Also can arrive the abdominal cavity of animal etc. in addition by the cell transfer that will import antibody gene, produce antibody in vivo.

In addition, as intravital production system, as the production system of using animal and the production system of using plant.The production system of use mammal, insecticide etc. is arranged when using animal.

As mammal, can use (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993) such as goat, pig, sheep, mice, cattle.And can use silkworm as insecticide.When using plant, for example can use Nicotiana tabacum L..

Antibody gene is imported in these animal or plants, make antibody in the body of animal or plant, produce, reclaim.For example: after antibody gene being inserted into the proteinic gene centre of the such intrinsic generation in milk of coding goat β casein, be prepared into fusion gene.The dna fragmentation that will contain the fusion gene that has inserted antibody gene injects the embryo of goat, and this embryo is imported female goat.The milk that the transgenic goat that can give birth to from the goat of having accepted embryo or its descendants produce obtains the antibody of expectation.For the milk amount of expecting antibody that contains that produces from transgenic goat is increased, also can use suitable hormone to transgenic goat.(Ebert，K.M.et al.，Bio/Technology(1994)12，699-702)。

In addition, when using silkworm, with the baculovirus infection silkworm that has inserted the purpose antibody gene, (Nature (1985) 315,592-594) for Maeda, S.et al. to obtain the antibody of expectation from the body fluid of this silkworm.In addition, when using Nicotiana tabacum L., the purpose antibody gene is inserted expression of plants with carrier, for example pMON 530, this carrier is imported in the such antibacterial of Agrobacterium tumefaciens.Make this bacterial infection Nicotiana tabacum L., for example Nicotiana tabacum, (Eur.J.Immunol. (1994) 24,131-138) for Julian, K.-C.Ma et al. for the antibody that can obtain expecting from the leaf of this Nicotiana tabacum L..

Resemble when producing antibody by external or intravital production system above-mentioned, after also the DNA of encoding antibody heavy chain (H chain) or light chain (L chain) can being incorporated into expression vector respectively, transform the host simultaneously, also the DNA of coding H chain and L chain can be incorporated in one the expression vector, transform host (referring to International Patent Application Publication No. WO 94-11523).

Spendable antibody among the present invention is so long as be fit to that the present invention uses, even the fragment of antibody or its trim also can.For example: as the fragment of antibody, as Fab, F (ab ') ₂, Fv or connect the H chain of Fv and the strand Fv (scFv) of L chain with suitable joint.

Specifically, with antibody with enzyme, for example: papain, pepsin are handled, make it to generate antibody fragment, or the gene of these antibody fragments of structure coding, behind this gene importing expression vector, express (for example, with reference to Co with appropriate host cell, M.S.et al., J.Immunol. (1994) 152,2968-2976, Better, M.﹠amp; Horwitz, A.H.Methods in Enzymology (1989) 178,476-496, Plueckthun, A.﹠amp; 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, R.E.et al., TIBTECH (1991) 9,132-137).

Be connected with the V district of L chain by V district and can obtain scFv the H chain of antibody.In this scFv, the V district of H chain and the V district of L chain by joint, preferably peptide linker be connected (Huston, J.S.et al., Proc.Natl.Acad.Sci.U.S.A. (1988) 85,5879-5883).The V district of the H chain among the ScFv and the V district of L chain also can be from above-mentioned any antibody as the antibody record.Peptide linker as connecting the V district for example can use the peptide of strand arbitrarily that is made of 12-19 amino acid residue.

Can by with the DNA in the V district of the DNA in the V district of the H chain of the above-mentioned antibody of encoding or H chain and coding L chain or L chain as template, the DNA of the aminoacid sequence expected in these sequences of encoding partly used the primer of stipulating its two ends to increasing by the PCR method, and then with the DNA of encoded peptide blank area and stipulate to increase after primer that its two ends can be connected with each H chain, L chain is to combination and obtain the DNA of coding scFv.

And in case prepare the DNA of coding scFv, can obtain to contain the expression vector of these DNA and with this expression vector host transformed, can use this host to obtain scFv according to conventional methods in addition by conventional method.

The fragment of these antibody can with above-mentioned its gene that similarly obtains, produce by host expresses." antibody " mentioned among the present invention also comprises the fragment of these antibody.

As the trim of antibody, also can use and the Polyethylene Glycol bonded antibody of various molecules such as (PEG)." antibody " mentioned among the present invention also comprises these antibody modification things.In order to obtain such antibody modification thing,, the antibody that obtains to obtain by being implemented chemical modification.These methods are established in this field.

Can be inside and outside cell, separate from the host and to resemble the antibody that produces, expresses above-mentioned, be purified to homogeneous.Can carry out separation, the purification of the antibody that uses among the present invention by affinity chromatograph.As the pillar that is used for affinity chromatograph, for example protein A post, Protein G post.The carrier that uses in the protein A post, for example, Hyper D, POROS, Sepharose F.F. etc.In addition, can use the separation, the purification process that use in the common protein, without any qualification.

For example: can select suitably, make up that chromatography, filtration, exclusion beyond the above-mentioned affinity chromatograph filters, saltouts, dialysis etc., can the antibody that use among the present invention be separated, purification.As chromatography, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration etc.These chromatographies are applicable to HPLC (High performance liquid chromatography).In addition, also can use reversed-phase HPLC (reverse phase HPLC).

Can carry out the concentration determination of the above-mentioned antibody that obtains by absorbance measurement or ELISA etc.Promptly, when utilizing absorbance measurement, suitably after the dilution, measure the absorbance of 280nm, calculate as 1.35OD with 1mg/ml with PBS (-).When measuring, can followingly measure by ELISA.Promptly, 100 μ l are added to 96 orifice plates (Nunc manufacturing) with the anti-human IgG of goat (TAG manufacturing) that 0.1M bicarbonate acid buffer (pH9.6) is diluted to 1 μ g/ml, be incubated overnight sessile antibody in 4 ℃.Sealing back, add the antibody that uses among the present invention of suitably dilution or the sample that contains antibody or as human IgG (CAPPEL system) the 100 μ l of standard substance, incubation is 1 hour under room temperature.

After the washing, add anti-human IgG (BIOSOURCE system) the 100 μ l of the alkali phosphatase enzyme mark that has diluted 5000 times, incubation is 1 hour under room temperature.After the washing, add substrate solution, behind the incubation, use MICROPLATE READER Model 3550 (Bio-Rad systems) to measure the absorbance of 405nm, calculate the concentration of purpose antibody.

The interleukin-6 variant that uses among the present invention is to have actively with combining of interleukin-6 receptor, and does not transmit the material of interleukin-6 biologic activity.That is, the interleukin-6 variant combines the interleukin-6 receptor competitively with interleukin-6, owing to do not transmit the biologic activity of interleukin-6, so the signal transduction that depends on interleukin-6 capable of blocking.

The interleukin-6 variant is to import the sudden change preparation by the amino acid residue of the aminoacid sequence of interleukin-6 is replaced.As the interleukin-6 of the body of interleukin-6 variant no matter its source, if consider antigenicity etc., preferably human interleukin-6.

Specifically, use well-known molecule model program, for example WHATIF (Vriend etal., J.Mol.Graphics (1990) 8,52-56) secondary structure of the aminoacid sequence of interleukin-6 is predicted, again by to the influence of integral body being estimated by metathetical amino acid residue.After determining suitable replacement amino acid residue, as template, import the sudden change that can make amino acid replacement, the gene of the interleukin-6 variant that can obtain encoding by using the PCR method of carrying out usually with the carrier of the base sequence that contains coding human interleukin-6 gene.As required can be with this gene integration to suitable expression, expression, generation and purification process by above-mentioned recombinant antibodies can obtain the interleukin-6 variant.

As the object lesson of interleukin-6 variant, as Brakenhoff et al., J.Biol.Chem. (1994) 269,86-93 and Savino et al., EMBO J. (1994) 13,1357-1367, WO 96-18648, the disclosed example of WO 96-17869.

Interleukin-6 partial peptide that uses among the present invention or interleukin-6 acceptor portion peptide are to have actively with combining of interleukin-6 receptor or interleukin-6 respectively, and do not transmit the material of interleukin-6 biologic activity.That is, interleukin-6 partial peptide or interleukin-6 acceptor portion peptide combine with interleukin-6 receptor or interleukin-6, by catching them, suppress combining of interleukin-6 and interleukin-6 receptor specifically.Its result is not owing to transmitting the biologic activity of interleukin-6, so blocked the signal transduction that depends on interleukin-6.

Interleukin-6 partial peptide or interleukin-6 acceptor portion peptide are the peptides that part or all aminoacid sequence by zone relevant with the interleukin-6 receptors bind with interleukin-6 in the aminoacid sequence of interleukin-6 or interleukin-6 receptor constitutes.Such peptide usually by 10～80, preferred 20～50, more preferably 20～40 amino acid residues constitute.

Interleukin-6 partial peptide or interleukin-6 acceptor portion peptide can be prepared as follows.District relevant with the combination of interleukin-6 and interleukin-6 receptor in the aminoacid sequence of interleukin-6 or interleukin-6 receptor is specified, and known method, for example genetic engineering means or the method for peptide synthesis by routine prepares its part or all aminoacid sequence.

In order to prepare interleukin-6 partial peptide or interleukin-6 acceptor portion peptide by genetic engineering means, the DNA sequence of the peptide of expecting encoding is incorporated in the expression vector, can obtain described partial peptide according to above-mentioned recombinant antibodies expression, generation and purification process.

In order to prepare interleukin-6 partial peptide or interleukin-6 acceptor portion peptide by method of peptide synthesis, can use normally used method, for example solid-phase synthesis or liquid phase synthesizing method in peptide is synthetic.

Specifically, can be according to the exploitations of pharmaceuticals " continuous " the 14th volume, " peptide is synthetic ", prison repair vow the island control bright, wide river bookstore, 1991, described method was carried out.As solid-phase synthesis; for example can use the C-terminal aminoacid combination that makes on the carrier of organic solvent corresponding to wanting synthetic peptide being insoluble to; the aminoacid that uses the due care base that alpha-amido and side chain functional group have been carried out protection by alternate repetition carries out the reaction of amino acid condensation one by one according to the order from C-terminal to the N-terminal direction and makes the aminoacid that is combined on the resin or reaction that alpha-amino this protecting group of peptide breaks away from, makes the method for peptide chain extension.The solid phase method of peptide synthesis roughly is divided into Boc method and Fmoc method because of the kind difference of the protecting group of use.

Resemble synthesized the purpose peptide above-mentioned after, the reaction of carrying out deprotection reaction and peptide chain being downcut from carrier.In the cut-out reaction of downcutting peptide chain, if use the Boc method to use fluohydric acid gas or trifluoromethayl sulfonic acid usually, and if use the Fmoc method to use TFA usually.If use the Boc method, for example in fluohydric acid gas, in the presence of methoxybenzene, above-mentioned protection peptide resin is handled.Deprotection base and downcut recovering peptide then from carrier.By this peptide is carried out lyophilization, obtain thick peptide.In addition, if use the Fmoc method, for example in TFA with above-mentioned same operation, the reaction of carrying out deprotection reaction and peptide chain being downcut from carrier.

HPLC can separate the thick peptide that obtains, purification by using.When carrying out eluting, use normally used water-acetonitrile series solvent in the protein purification, under optimal condition, carry out.The peak fraction that is equivalent to whole tomographic map that obtains is collected, this fraction is carried out lyophilization.For the peptide fraction of such purification, the molecular weight parsing of being undertaken by mass spectral analysis, amino acid composition analysis or identify by aminoacid sequence parsing etc.

The object lesson of interleukin-6 partial peptide and interleukin-6 acceptor portion peptide opens as the spy that flat 2-188600, spy open flat 7-324097, the spy opens flat 8-311098 and the disclosed example of U.S. Patent bulletin US5210075.

The interleukin-6 signal transduction of the interleukin-6 antagonist that uses among the present invention suppresses active and can estimate by the method for common usefulness.Specifically, by to interleukin-6 dependency human myeloma strain (S6B45, KPMM2), people Lennert T lymphoma cell strain KT3 or interleukin-6 dependent cell MH60.BSF2 cultivate, add interleukin-6, make interleukin-6 antagonist coexistence simultaneously, measure the interleukin-6 dependent cell in view of the above ³The H-thymidine mixes.

In addition, by the U266 as interleukin-6 expression of receptor cell is cultivated, add ¹²⁵The interleukin-6 of I labelling adds the interleukin-6 antagonist simultaneously, measures with interleukin-6 expression of receptor cell bonded ¹²⁵The interleukin-6 of I labelling.In above-mentioned analysis system, except the group that makes the existence of interleukin-6 antagonist, the negative control group that does not contain the interleukin-6 antagonist is set, if the result that both are obtained compares, the interleukin-6 that can estimate the interleukin-6 antagonist suppresses active.

Provide as following embodiment,, show that the interleukin-6 antagonisies such as antibody of anti-interleukin-6 receptor can be used as the therapeutic agent of internal ear obstacle owing to confirmed the proliferation inhibiting effect of the antibody of anti-interleukin-6 receptor to internal ear obstacle cell.

Treatment target of the present invention is a mammal.The preferred people of treatment target mammal.

Internal ear treating dysfunction of the present invention and/or preventive or internal ear obstacle inhibition of cell proliferation can be by oral or non-oral way whole body or local applications.For example, can select intravenous injection, intramuscular injection, intrathoracic injection, intraperitoneal injection, subcutaneous injection, suppository, coloclysis, oral property enteric agents etc. such as drop, can select the suitable method that gives according to patient's age, symptom.Effectively administered dose can be from each, every 1kg body weight, in the scope of 0.01mg to 100mg, select.Or can select the administered dose of 1～1000mg, preferred 5～50mg for each patient.

Ideal amount of application, application process, when for example being anti-interleukin-6 receptor antibody, there to be free antibodies to have the amount of degree in the blood is effective amount of application, as concrete example, divide 1 time in 1 month (4 week) of every 1kg body weight to for several times, for example use method afford 0.5mg to 40mg such as intravenous injection, subcutaneous injection such as drop, preferred 1mg to 20mg etc. according to the plan of using in 2 times/week, 1 time/week, 1 time/2 weeks, 1 time/4 weeks etc.Plan also can observe the trend of the state of an illness and blood test value on one side, carry out with administration interval from 2 times/week or extended to for 1 time/2 weeks 1 time/week, adjustment such as 1 time/3 weeks, 1 time/4 weeks.

Internal ear treating dysfunction of the present invention and/or preventive or internal ear obstacle inhibition of cell proliferation can contain at carrier of pharmaceutically allowing or additive simultaneously according to route of administration.As the such carrier and the example of additive, as water, the organic solvent of pharmaceutically allowing, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxy vinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble glucan, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, Radix Acaciae senegalis, casein, gelatin, agar, diglycerol, third (support) glycol, Polyethylene Glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, Sorbitol, lactose, surfactant of allowing as medical additive etc.The additive that uses can be according to dosage form, suitable or combination selection from above-claimed cpd, but be not limited to these chemical compounds.

Embodiment

Below, by embodiment and reference example the present invention is specifically described, but the present invention is not limited to these embodiment.

Embodiment 1.

Connect experimental technique

(1) audiometrist of mice before the sound load

For the C57BL/6J male mice in 4 ages in week, before the sound load, carry out audiometrist by auditory brainstem response (ABR).Before mensuration, by to intraperitoneal injection Xyladine and ketamine (Ketamine), implement sufficient deep anaesthesia, in mensuration, as required, implement to append anesthesia with ketamine.

Under (auditory brainstem response) above-mentioned noise conditions, in order to know that noise brings out deaf degree, threshold value shift to auditory brainstem response (ABR) experimentizes, use PowerLabsystem (Model:PowerLob2/20, the waveform storing of Scope software ADInstruments CastleHillAustralia) and stimuluscontrol carry out the mensuration of ABR, use extracellular amplifier Digital Biomap System (Model:BAL-1, Tucker-Davis Technologies FL/USA) record EEG.Sonic stimulation is to produce by insert coupled mode speaker (Model:ESlspc, BioResearch Center, Nagoya/Japan) in the external auditory meatus of mice.Make it to produce the plosive stimulation of 0.1ms lifting/lowering time (cosine gate) and the smooth fragment (flatsegment) of 1ms, specify amplitude with sound generator, Attention Real-time Processor and Programable Attenater (Model:RP2.1 and PA5, Tucker-DavisTechnologies FL/USA).Use Sound Level Meter (Model:LA-5111,0no Sokki Yokohama/Japan) to measure sound levels.For record, the needle electrode of stainless steel is arranged at the top and the empty sidepiece of left ear and auris dextra.Usually, use the filter apparatus of 50-5000Hz, with the sample rate of 40000Hz, be the ABR waveform of 12.8ms writing time, the average waveforms from 256 stimulations in the 9Hz frequency.With the interval of 5-dB SPL, from the crest amplitude opening entry till waveform is invisible to vision.In order to measure threshold value, the frequency of the sound that gives is decided to be three kinds of 4kHz, 12kHz and 20kHz, measure each audition threshold value.

(2) making of deaf model

(this device is to make an uproar device as sound generator with the system of RION Audiometyr AA67N at the sound load device, use SONY SRP-P150, FOSTEXD-1405 is as amplifier, after carrying out amplification, the device of in confined space, loading) in the microphone of the about 12cm of diameter, the mice of in the wire netting system cage of the high 3cm that 1cm under microphone leaves standstill at the place, packing into.With the wire netting of same character with cage with radial separation, simultaneously 4 mices are installed to respectively in each chamber.Then, in the sound load device, preestablish sound pressure (CASELA company, repeatedly measures by CEL-231) in 2 hours load process, with this understanding, carry out 2 hours high pitch load according to 124 ± 1db.Use the sound of the Octaveband noise of 4kHzSPL.In addition, laying temperature meter in audio unit, the temperature before and after the mensuration sound load.

(3) drug administration

After above-mentioned (2), animal is divided into A group and B group, at once with rat IgG according to 2mg/ only or MR16-1 (the anti-interleukin-6 R humanized antibody of preparation in reference example 4) only be applied to each group according to 2mg/.When using, as blind experiment, when the audiometrist of (4) finished, the experimenter was unclear to the individual group of content of using.

(4) mensuration of audition threshold value

Identical with the mode of above-mentioned (1), measure audition from sound load and drug administration after one week.After mice after confirming mensuration reaches sufficient depth of anesthesia, broken end, extraction side skull after fixing, is preserved to be ready for use on histology's investigation.

(5) interpretation of result

Get the poor of audition that each individuality obtains in above-mentioned (4) and audition before the disposal that above-mentioned (1) obtains, calculate audition threshold value reduction degree (dB).Calculate MR16-1 respectively and use group and the average audition threshold value reduction (dB) of matched group at each frequency place.

The result

When carrying out the anesthesia of above-mentioned (4), dead 1, owing to can not measure audition, so deduction.The number of elements that obtains the audition threshold degrees is 4 in the MR16-1 group, uses in the group to being 2 at IgG.In addition, the temperature in the sound load device is 25 ℃ before load, is 27 ℃ behind the load.Result such as following table 1 and shown in Figure 1.

Table 1

Handle frequency	IgG uses matched group	MR16-1 uses group	Changes of threshold is poor
				4kHz	22.5	37.5	-15
12kHz	35	27.5	+7.5
				20kHz	55	28.8	+26.3

Discuss

Think that the sound wound is a this physical property external force of sound pressure, the model of Shi Yonging is physical organization's damage model of cochlea in this experiment.In this experiment, be the center with the frequency of 4kHz, the sound that load amplifies.At the cochlea place, the sensor of experiencing sound carries out spatiality distribution (to notropic) corresponding to frequency, audition in matched group reduces up to 4kHz, set out thus, diminish gradually up to the high pitch zone, the sound that amplifies because of load in the zone of 4kHz can be described, so caused the closer to this zone, the damage of tissue is big more.

On the other hand, use in the group at the interleukin-6 receptor antibody, the tissue injury of the sound that load amplifies is far away more from direct acceptor site, compares with matched group, can suppress audition more and reduce.From the above mentioned, think that the interleukin-6 receptor antibody has the tissue disorder of the gradual inhibition cochlea in space, that is, the effect that the audition of interior remebrance nerve deafness reduces perhaps has the effect that the audition in the high pitch zone of the nerve deafness of being suppressed at reduces.

Embodiment 2. struvite cytokines are in the expression of the post-traumatic cochlea of sound

The male SD rat that used for 3～4 ages in week is with the identical method of embodiment 1, make deaf model, and then the sound load extracts the side skull respectively from each rat after 3 hours, after 6 hours, after 12 hours, after 24 hours, after 3 days, after 5 days, after 7 days, after 28 days.Keep not spill i-coch lymph liquid condition, only extract cochlea, measure the expression of various inflammatory cytokine by the RT-PCR method.The result as shown in Figure 2.As shown in Figure 2, the cochlea place of wound model outside sound, the expression of TNF α, IL-1 α, IL-1 β, IL-1 receptor antagonist (IL-1RA), interleukin-6 is risen.On the other hand, do not find the expression of IL-12p40 and GM-CSF.

Then, by quantitative RT-PCR, measure that TNF α, interleukin-6, IL-1 β, interleukin-6 express through the time change.In the quantitative RT-PCR, use 18SrRNA as interior mark (reference gene), by Δ Δ Ct method analysis result.Result such as Fig. 3～shown in Figure 5.From Fig. 3～Fig. 5 as can be known, 24 hours commitment of less than after the sound wound confirms to have TNF α, the IL-1 β at the peak that shows expression, the expression of interleukin-6.

In addition, use 6 hours tissue of sound load, on frozen section, carry out immuning tissue's dyeing.Animal is used the SD rat, makes deaf model, broken end after noise was loaded back 6 hours with the method identical with embodiment 1.Extract the side skull immediately, use liquid nitrogen to fix 6 hours with 4% paraformaldehyde, the detection sample that obtains with 0.5mol/1EDTA loss of thick fluid mineral is freezing rapidly, uses low-temperature preservation device (CM3000, Leica company), and it is thick to be cut into 7 μ m, obtains frozen section.In this section, implement immunohistochemical staining.In dyeing, use Vectastein ABC test kit, develop the color with DAB.As painted contrast, use not add the dyeing that first antibody carries out.Result such as Fig. 6～shown in Figure 7, by Fig. 6～Fig. 7 as can be known, at the cochlea of sound load after 6 hours, the basement membrane that demonstrates under cochlea outer wall and spiral organ of Corti (Corti ' s organ) is expressed interleukin-6.

The affirmation of the drug effect due to the systemic administration of embodiment 3. interleukin-6 antagonisies

The same with embodiment 1, to the excessive sound of 2 hours 124dB of C57BL/6J male mice load, and then, with MR16-1 only, rat IgG is only used to intraperitoneal according to 2mg/ according to 2mg/.After the administration of antibodies, extraction side skull after 8 hours, the internal ear of extraction both sides is separated and is cut spiral organ of Corti, is divided into first spiral (tip), second spiral (substrate), respectively from about reclaim, merge.By Western trace method, measure STAT3, the Erk of total STAT3, Erk, Akt and phosphorylation, the expression of Akt.The result as shown in Figure 8.As shown in Figure 8, confirm very clearly in the matched group that STAT3, the phosphorylation Erk of phosphorylation, the signal of phosphorylation Akt are suppressed in MR16-1 use group.According to this result, can confirm that the MR16-1 of systemic administration brings into play drug effect in cochlea.

According to the result of this result and embodiment 2, think that the mechanism of action that inhibition audition due to using by the intraperitoneal of the MR16-1 shown in the embodiment 1 reduces is: since after the sound wound interleukin-6 signal of the i-coch expression when early stage be subjected to the effect of the inhibition of MR16-1.

The preparation of reference example 1. human soluble interleukin-6 receptors

(Science (1988) 241 for Yamasaki, K.et al., and the plasmid pBSF2R.236 of the cDNA that contains coding interleukin-6 receptor that 825-828) obtains is prepared into SIL-6R by the PCR method according to people's such as Yamasaki method in use.Plasmid pBSF2R.236 with restricted enzyme Sph I digestion, is obtained the interleukin-6 receptor cdna, this cDNA is inserted among the mp18 (Amersham manufacturing).In order to import termination codon to the interleukin-6 receptor cdna, use the synthetic oligonucleotide primer thing of design, import sudden change by in vitro mutagenesis system (Amersham manufacturing) with PCR normal direction interleukin-6 receptor cdna.By this operation, termination codon is directed to the position of aminoacid 345, the cDNA of the SIL-6R that obtains encoding.

For at expressing cho cell SIL-6R cDNA, it is connected with plasmid pSV (Pharmacia manufacturing), obtain plasmid pSVL344.Insert the SIL-6R cDNA that cuts off with Hind III-Sal I to the plasmid pECEdhfr of the cDNA that contains dhfr, obtain expressing cho cell plasmid pECEdhfr 344.

(Mol.Cell.Biol. (1987) 7 for Chen, C.et al. with the calcium phosphate precipitation method, 2745-2751) with the plasmid pECEdhfr344 transfection dhfr-CHO cell strain DXB-11 (Urlaub of 10 μ g, G.et al., Proc.Natl.Acad.Sci.USA (1980) 77,4216-4220).The Chinese hamster ovary celI of transfection is selected to cultivate for 3 weeks in the culture fluid at the α MEM that does not contain nucleotide of the streptomycin of penicillin that contains 1mM glutamine, 10% dialysis FCS, 100U/ml and 100 μ g/ml.

With the Method of Limited Dilution method Chinese hamster ovary celI of selecting is screened, obtain single Chinese hamster ovary celI clone.This Chinese hamster ovary celI is cloned in the methotrexate of 20nM～200nM concentration and increases, obtain to produce the Chinese hamster ovary celI strain of human soluble interleukin-6 receptor.Chinese hamster ovary celI strain 5E27 is cultivated in the improved Dulbecco culture fluid of the Iscove that contains 5%FBS (IMDM, Gibco make).Reclaim culture supernatant, the SIL-6R concentration in the culture supernatant is measured by ELISA.There is SIL-6R in this results verification culture supernatant.

The preparation of reference example 2. antihuman interleukins-6 antibody

The recombinant type interleukin-6 (Immunol.Lett. (1988) 17 for Hirano, T.et al., 41) of 10 μ g with the immune BALB/c mouse of Freund's complete adjuvant, is continued immunity week about and can detect in serum till the anti-interleukin-6 antibody.From regional nodes's joint extraction immunocyte, use polyethylene glycol 1500, merge with myeloma cell strain P3U1.Method (Selective Methodsin CellularImmunology according to people's such as Oi use HAT culture fluid, W.H.Freeman and Co., San Francisco, 351,1980) select hybridoma, set up the hybridoma that produces antihuman interleukin-6 antibody.

The hybridoma that produces antihuman interleukin-6 antibody can resemble and carry out the interleukin-6 binding analysis following.Promptly, will be with 96 hole micro plate (DynatechLaboratories of softish polyethylene system, Inc. make, Alexandria, VA) in the bicarbonate buffer (pH9.6) of 0.1M with goat anti-mouse Ig (the 10 μ l/ml of 100 μ l, Cooper Biomedical, Inc makes Malvern, PA) wraps down in 4 ℃ and is spent the night.Then plate was handled under room temperature 2 hours with the PBS that contains 100 μ l1% bovine serum albumins (BSA).

Then with PBS with after the plate washing, the hybridoma culture supernatant of 100 μ l is added to each hole, under 4 ℃, be incubated overnight.Wash plate is added to each hole like that according to 2000cpm/0.5ng/well ¹²⁵The recombinant type interleukin-6 of I labelling, after the washing, (Fullerton CA) measures the radioactivity in each hole for Beckman Gamma9000, Beckman Instruments with gamma counter.By the interleukin-6 binding analysis, there are 32 hybridoma clones positive among 216 hybridoma clones.In these clones, finally obtain stable MH166.BSF2.The anti-interleukin-6 antibody MH166 that this hybridoma produces has the IgG1K hypotype.

Use the dependent mice hybridoma clone of interleukin-6 MH60.BSF2 then, research MH166 antibody and the relevant neutralization activity of hybridoma propagation.With the MH60.BSF2 cell according to 1 * 10 ⁴Dispensing is carried out in/200 μ l/ holes, adds the sample that contains MH166 antibody then, cultivated 48 hours, adds 0.5 μ Ci/ hole ³The H thymidine (New England Nuclear, Boston, MA) after, continue to cultivate again 6 hours.Cell is placed on the glass filter paper, and (Labo Mash Science Co., Tokyo Japan) handle with automatic collector.Use the anti-interleukin-6 antibody of rabbit in contrast.

This result shows what MH166 antibody capacity dependency ground suppressed by the inductive MH60.BSF2 cell of interleukin-6 ³The H thymidine mixes.Show thus MH166 antibody can in and the activity of interleukin-6.

The preparation of reference example 3. antihuman interleukins-6 receptor antibody

Make method (Hirata according to incidental prescription by people such as Hirata, Y.et al.J.Immunol. (1989) 143, anti-interleukin-6 receptor antibody MT18 that 2900-2906) is prepared into and the activatory sepharose 4B of usefulness CNBr (Pharmacia Fine Chemicals system, Piscataway, NJ) combination, purification interleukin-6 receptor (Yamasaki, K.et al., Science (1988) 241,825-828).With human myeloma cell strain U266 with containing 1% digitonin (Wako Chemicals system), the dissolving of the 1mM p-aminophenyl sulfonyl methane villiaumite hydrochlorate (Wako Chemicals system) (digitonin buffer) of 10mM triethanolamine (pH7.8) and 0.15MNaCl and is combined in the particulate MT18 antibody of sepharose 4B and mixes.Then, granule is washed 6 times with the digitonin buffer, as the partially purified interleukin-6 receptor that is used to carry out immunity.

With from 3 * 10 ⁹The above-mentioned partial purification interleukin-6 receptor that individual U266 cell obtains is every BALB/c mouse of immunity on the 10th, and immunity 4 times is made hybridoma by conventional method then.Active with combining of interleukin-6 receptor by following method research from the hybridoma culture supernatant in the positive hole of growing up.With 5 * 10 ⁷Individual U266 cell is used ³⁵S-methionine (2.5mCi) carries out labelling, dissolves with above-mentioned digitonin buffer.Dissolved U266 cell is mixed with the particulate MT18 antibody of sepharose 4B that is combined in of 0.04ml capacity, then, wash 6 times with the digitonin buffer, the digitonin buffer (pH3.4) of usefulness 0.25ml will ³⁵S-methionine mark interleukin-6 receptor eluting goes out, and neutralizes with the 1MTris (pH7.4) of 0.025ml.

The hybridoma culture supernatant of 0.05ml is mixed with the Protein G agarose (Phramacia system) of 0.01ml.After the washing, with the 0.005ml's of agarose and above-mentioned preparation ³⁵The interleukin-6 receptor solution of S labelling is incubation together.The immunoprecipitation material is analyzed the hybridoma culture supernatant of research and interleukin-6 receptor response by SDS-PAGE.This result has set up reacting positive hybridoma clone PM-1 (FERM BP-2998).The antibody that is produced by hybridoma PM-1 has IgG1 κ hypotype.

The antibody that end user's myeloma cell strain U266 research hybridoma PM-1 produces is to the inhibition activity of interleukin-6 and human interleukin-6 receptors bind.(Immunol.Lett. (1988) 17 for Hirano, T.et al., 41-45), (New England Nuclear, Boston MA) carries out by Bolton-Hunter reagent to prepare people's recombinant type interleukin-6 from escherichia coli ¹²⁵(J.Exp.Med. (1987) 166,967-981) for Taga, T.et al. for the I labelling.

With 4 * 10 ⁵The culture supernatant of the hybridoma PM-1 of individual U266 cell and 70% (v/v) and 14000cpm's ¹²⁵The interleukin-6 of I labelling was cultivated 1 hour together.The sample of 70 μ l is layered on above the FCS of 300 μ l of little fusion polyethylene tube of 400 μ l, centrifugal after, measure the radioactivity on the cell.

This result shows the antibody inhibition interleukin-6 of hybridoma PM-1 generation and combining of interleukin-6 receptor.

The preparation of reference example 4. anti-mice interleukin-6 receptor antibodies

Utilize Saito, T.et al., the described method of J.Immunol. (1991) 147,168-173 prepares the monoclonal antibody of anti-mice interleukin-6 receptor.

The Chinese hamster ovary celI that produces the mice SIL-6R is cultivated in containing the IMDM culture fluid of 10%FCS, affinity column (with reference to above-mentioned Saito, T.et al) the purification mice SIL-6R from the anti-mice interleukin-6 receptor antibody RS12 of this culture supernatant has been fixed in use on Affigel 10 gels (Biorad manufacturing).

The mice SIL-6R 50 μ g that obtain are mixed with Freund's complete adjuvant, be expelled to the abdominal part of Wistar rat.Carry out supplementary immunization with incomplete Freund after 2 weeks.In the time of the 45th day, obtain the Rats Spleen cell, with 2 * 10 ⁸Individual cell and 1 * 10 ⁷After the PEG1500 (Boehringer Mannheim system) of individual murine myeloma cell P3U1 use 50% carries out cell fusion by conventional method, by HAT culture medium screening hybridoma.

The hybridoma culture supernatant is added to bag by behind the plate of the Mus IgG of rabbit Chinese People's Anti-Japanese Military and Political College antibody (Cappel manufacturing), makes it to react with the mice SIL-6R.Then, produce the hybridoma of the anti-interleukin-6 receptor antibody of little mouse soluble by the ELISA method screening of using rabbit anti-mice interleukin-6 receptor antibody and alkali phosphatase enzyme mark goat anti-rabbit igg.Confirm that the hybridoma clone who produces antibody carries out 2 subsieve choosings, obtains single hybridoma clone.Should clone called after MR16-1.

(J.Immunol. (1988) 18 for Matsuda, T.et al., 951-956) by using the MH60.BSF2 cell ³The neutralization activity of the antibody that mixes this hybridoma generation of research in the information transduction of mice interleukin-6 of H thymidine.According to 1 * 10 ⁴Individual/200 μ l/ holes are preparation MH60.BSF2 cell in 96 orifice plates like that.Add mice interleukin-6 and MR16-1 antibody or the RS12 antibody 12.3～1000ng/ml of 10pg/ml to this plate, in 37 ℃, 5%CO ₂Under cultivate 44 hours after, add 1 μ Ci/ hole ³The H thymidine.After 4 hours, measure ³Mixing of H thymidine.This result shows MR16-1 antibody inhibition MH60.BSF2 cell ³The H thymidine mixes.

Therefore show the antibody inhibition interleukin-6 of hybridoma MR16-1 (FERM BP-5875) generation and combining of interleukin-6 receptor.

Claims (45)

1. contain internal ear treating dysfunction and/or the preventive of interleukin-6 antagonist as effective ingredient.

2. claim 1 is described treats and/or prevents agent, and wherein, described internal ear obstacle is a sensorineural hearing loss.

3. claim 2 is described treats and/or prevents agent, wherein, described sensorineural hearing loss is the sensorineural hearing loss that Meniere syndrome, drug-induced property internal ear obstacle, viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor cause.

4. claim 2 is described treats and/or prevents agent, and wherein, described sensorineural hearing loss is sudden deafness, old man's induced deafness or noise deafness.

5. claim 1 is described treats and/or prevents agent, and wherein, described internal ear obstacle is a vestibular disorder.

6. claim 5 is described treats and/or prevents agent, and wherein, described vestibular disorder is the vestibular disorder that Meniere syndrome, vestibular neuritis or drug-induced property internal ear obstacle cause.

Claim 1～6 each describedly treat and/or prevent agent, wherein, described interleukin-6 antagonist is the antibody of anti-interleukin-6 receptor.

8. claim 7 is described treats and/or prevents agent, and wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-interleukin-6 receptor.

9. claim 8 is described treats and/or prevents agent, and wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of antihuman interleukin-6 receptor.

10. claim 8 is described treats and/or prevents agent, and wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-mice interleukin-6 receptor.

11. each describedly treats and/or prevents agent claim 7～10, wherein, the antibody of described anti-interleukin-6 receptor is recombinant antibodies.

Treat and/or prevent agent 12. claim 9 is described, wherein, the monoclonal antibody of described antihuman interleukin-6 receptor is a PM-1 antibody.

Treat and/or prevent agent 13. claim 10 is described, wherein, the monoclonal antibody of described anti-mice interleukin-6 receptor is a MR16-1 antibody.

14. each describedly treats and/or prevents agent claim 7～13, wherein, the antibody of described anti-interleukin-6 receptor is chimeric antibody, humanized antibody or people's antibody of anti-interleukin-6 receptor.

Treat and/or prevent agent 15. claim 14 is described, wherein, the humanized antibody of described anti-interleukin-6 receptor is a humanization PM-1 antibody.

16. the application of interleukin-6 antagonist in making internal ear treating dysfunction and/or preventive.

17. the described application of claim 16, wherein, described internal ear obstacle is a sensorineural hearing loss.

18. the described application of claim 17, wherein, described sensorineural hearing loss is the sensorineural hearing loss that Meniere syndrome, drug-induced property internal ear obstacle, viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor cause.

19. the described application of claim 17, wherein, described sensorineural hearing loss is sudden deafness, old man's induced deafness or noise deafness.

20. the described application of claim 16, wherein, described internal ear obstacle is a vestibular disorder.

21. the described application of claim 20, wherein, described vestibular disorder is the vestibular disorder that Meniere syndrome, vestibular neuritis or drug-induced property internal ear obstacle cause.

22. each described application of claim 16～21, wherein, described interleukin-6 antagonist is the antibody of anti-interleukin-6 receptor.

23. the described application of claim 22, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-interleukin-6 receptor.

24. the described application of claim 23, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of antihuman interleukin-6 receptor.

25. the described application of claim 23, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-mice interleukin-6 receptor.

26. each described application of claim 22～25, wherein, the antibody of described anti-interleukin-6 receptor is recombinant antibodies.

27. the described application of claim 24, wherein, the monoclonal antibody of described antihuman interleukin-6 receptor is a PM-1 antibody.

28. the described application of claim 25, wherein, the monoclonal antibody of described anti-mice interleukin-6 receptor is a MR16-1 antibody.

29. each described application of claim 22～28, wherein, the antibody of described anti-interleukin-6 receptor is chimeric antibody, humanized antibody or people's antibody of anti-interleukin-6 receptor.

30. the described application of claim 29, wherein, the humanized antibody of described anti-interleukin-6 receptor is a humanization PM-1 antibody.

31. treat and/or prevent the method for internal ear obstacle, comprise to object giving the interleukin-6 antagonist.

32. the described method that treats and/or prevents of claim 31, wherein, described internal ear obstacle is a sensorineural hearing loss.

33. the described method that treats and/or prevents of claim 32, wherein, described sensorineural hearing loss is the sensorineural hearing loss that Meniere syndrome, drug-induced property internal ear obstacle, viral otitis interna, otitis interna purulenta, side skull fracture, auditory nerve tumor cause.

34. the described method that treats and/or prevents of claim 32, wherein, described sensorineural hearing loss is sudden deafness, old man's induced deafness or noise deafness.

35. the described method that treats and/or prevents of claim 31, wherein, described internal ear obstacle is a vestibular disorder.

36. the described method that treats and/or prevents of claim 35, wherein, described vestibular disorder is the vestibular disorder that Meniere syndrome, vestibular neuritis or drug-induced property internal ear obstacle cause.

37. each described method that treats and/or prevents of claim 31～36, wherein, described interleukin-6 antagonist is the antibody of anti-interleukin-6 receptor.

38. the described method that treats and/or prevents of claim 37, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-interleukin-6 receptor.

39. the described method that treats and/or prevents of claim 38, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of antihuman interleukin-6 receptor.

40. the described method that treats and/or prevents of claim 38, wherein, the antibody of described anti-interleukin-6 receptor is the monoclonal antibody of anti-mice interleukin-6 receptor.

41. each described method that treats and/or prevents of claim 37～40, wherein, the antibody of described anti-interleukin-6 receptor is recombinant antibodies.

42. the described method that treats and/or prevents of claim 39, wherein, the monoclonal antibody of described antihuman interleukin-6 receptor is a PM-1 antibody.

43. the described method that treats and/or prevents of claim 40, wherein, the monoclonal antibody of described anti-mice interleukin-6 receptor is a MR16-1 antibody.

44. each described method that treats and/or prevents of claim 37～43, wherein, the antibody of described anti-interleukin-6 receptor is chimeric antibody, humanized antibody or people's antibody of anti-interleukin-6 receptor.

45. the described method that treats and/or prevents of claim 44, wherein, the humanized antibody of described anti-interleukin-6 receptor is a humanization PM-1 antibody.

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