HK1185887B

HK1185887B - Improved antibody molecules

Info

Publication number: HK1185887B
Application number: HK13113190.6A
Authority: HK
Inventors: 井川智之; 石井慎也; 前田敦彦; 樱井实香; 小岛哲郎; 橘达彦; 白岩宙丈; 角田浩行; 樋口义信
Original assignee: 中外制药株式会社
Priority date: 2008-09-26
Filing date: 2013-11-26
Publication date: 2016-01-29

Description

Improved antibody molecules

The present application is a divisional application of an invention patent application having an application date of 2009, 9/25, application No. 200980100709.6, and an invention name of "improved antibody molecule".

Technical Field

The present invention relates to a pharmaceutical composition containing an anti-IL-6 receptor antibody as an active ingredient, a process for producing the same, and the like.

Background

Antibodies have attracted attention as pharmaceutical products because of their high stability in plasma and few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs are currently under development (non-patent documents 1 and 2). IL-6 is a cytokine involved in various autoimmune diseases, inflammatory diseases, malignant tumors, and the like (non-patent document 3), and TOCILIZUMAB (トシリズマブ), which is a humanized anti-IL-6 receptor IgG1 antibody, specifically binds to an IL-6 receptor. TOCILIZUMAB is considered to be useful as a therapeutic agent for IL-6-related diseases such as rheumatoid arthritis (patent documents 1, 2 and 3 and non-patent document 4), because TOCILIZUMAB neutralizes the biological action of IL-6, and is recognized as a therapeutic agent for Karlmann's disease and rheumatoid arthritis (non-patent document 5) in Japan.

Humanized antibodies such as TOCILIZUMAB are generation 1 antibody drugs, and generation 2 antibody drugs improved in potency, convenience and cost by improving generation 1 antibody drugs are currently under development. As technologies applicable to 2 nd generation antibody drugs, various technologies have been developed, and there have been reported technologies for improving effector functions, antigen binding ability, pharmacokinetics, and stability, and technologies for reducing the risk of immunogenicity. As a method for enhancing the drug efficacy or reducing the dose, a technique for enhancing antibody-dependent cellular cytotoxicity (ADCC activity) or complement-dependent cytotoxicity (CDC activity) by substituting an amino acid in the Fc region of an IgG antibody has been reported (non-patent document 6). In addition, as a technique for enhancing the antigen binding ability and the antigen neutralizing ability, an affinity maturation technique has been reported (non-patent document 7), in which the binding activity to an antigen can be enhanced by introducing a mutation into an amino acid such as a Complementarity Determining Region (CDR) of a variable region. By enhancing the antigen-binding ability, the biological activity in vitro can be improved, or the dose can be reduced, and the drug efficacy in vivo can be further improved (non-patent document 8). Currently, clinical trials of Motavizumab (Motavizumab) (manufactured by affinity maturation technology) that exerts an effect superior to that of Palivizumab (Palivizumab), which is the first-generation drug of an anti-RSV antibody, have been ongoing (non-patent document 9). Among anti-IL-6 receptor antibodies, an antibody having an affinity of about 0.05nM, which is stronger than TOCILIZUMAB, has been reported (patent document 4), but a human antibody, a humanized antibody or a chimeric antibody having an affinity of more than 0.05nM has not been reported.

The problems faced by current antibody drugs are: high manufacturing costs due to the very large amount of protein administered. The humanized anti-IL-6 receptor IgG1 antibody, TOCILIZUMAB, was also administered by intravenous injection at a dose of about 8 mg/kg/month (non-patent document 4). With regard to the mode of administration, when chronic autoimmune diseases are concerned, subcutaneous administration preparations are preferred. Generally, a high concentration preparation is required for subcutaneous administration, and in the case of an IgG-type antibody preparation, a preparation of about 100mg/mL is considered to be the limit in terms of stability and the like (non-patent document 10). In order to exert a sustained therapeutic effect, the amount of protein to be administered is reduced by increasing the half-life of the antibody in plasma, and high stability is imparted, so that subcutaneous administration can be performed at long administration intervals, and a 2 nd generation antibody drug which is low in cost and highly convenient can be provided.

In terms of pharmacokinetics of antibodies, FcRn is involved in many cases, and regarding differences in half-life in plasma among antibody isotypes, it is known that IgG1 and IgG2 have the most excellent half-life in plasma, and IgG3 and IgG4 are inferior to them (non-patent document 11). As a method for further improving the plasma half-life of IgG1 and IgG2 antibodies having excellent plasma half-life, amino acid substitutions in the constant region that enhance the binding to FcRn have been reported (non-patent documents 12 and 13). Further, from the viewpoint of immunogenicity, it is more desirable to further increase the half-life in plasma by amino acid substitution of the variable region than the constant region (patent document 5), but no report has been made so far on of increasing the half-life in plasma of an IL-6 receptor antibody by modifying the variable region.

One of the most important issues in the development of biopharmaceuticals is immunogenicity. Generally, mouse antibodies are humanized to reduce immunogenicity. The risk of immunogenicity can be further reduced by using germline (ジヤ - ムライン, germline) sequences in the template framework during humanization (non-patent document 14). However, Adalimumab (Adalimumab), which is a fully human anti-TNF antibody, exhibits high-frequency (13 to 17%) immunogenicity, and a decrease in therapeutic effect is observed in patients exhibiting immunogenicity (non-patent documents 15 and 16). Even in the case of human antibodies, T cell epitopes may be present in CDRs, and the T cell epitopes on the CDRs may cause immunogenicity. Methods for predicting T cell epitopes in silico or in vitro have been reported (non-patent documents 17 and 18), and it is considered that by removing T cell epitopes predicted by the above methods, the risk of immunogenicity can be reduced (non-patent document 19).

TOCILIZUMAB as a humanized anti-IL-6 receptor IgG1 antibody was an IgG1 antibody humanized from a mouse PM1 antibody. The H chain and the L chain were CDR-grafted using sequences of human NEW and human REI as template frameworks, respectively, but 5 amino acids, which are important for maintaining activity, were left in the frameworks as mouse sequences (non-patent document 20). There has been no report of complete humanization without reducing the activity of a mouse sequence remaining in the framework of TOCILIZUMAB as a humanized antibody. In addition, the CDR sequence of TOCILIZUMAB is a mouse sequence, and like adalimumab, there is a possibility that a T cell epitope is present in the CDR, and the risk of immunogenicity cannot be denied. In clinical trials of TOCILIZUMAB, the appearance of an anti-TOCILIZUMAB antibody was not confirmed at a drug effect dose of 8mg/kg, but the antibody was confirmed at 4mg/kg and 2mg/kg (patent document 6). It is thus assumed that: there is also room for improvement in the immunogenicity of TOCILIZUMAB. However, there has been no report of TOCILIZUMAB with an improved risk of immunogenicity through amino acid substitution.

The isotype of TOCILIZUMAB is IgG1, but the difference in isotype is a difference in constant region sequence, and the sequence of the constant region is considered to have a great influence on effector function, pharmacokinetics, physicochemical properties, and the like, so that the selection of the constant region sequence is very important for the development of antibody drugs (non-patent document 11). In recent years, great importance has been placed on the safety of antibody drugs, and it is considered that one of the causes of major side effects found in phase I clinical trials of TGN1412 is the interaction between the Fc part of the antibody and Fc γ receptor (effector function) (non-patent document 21). In an antibody drug for the purpose of neutralizing the biological effect of an antigen, it is not necessary to bind to an Fc γ receptor important for effector functions such as ADCC, and it is considered that binding to an Fc γ receptor may not be preferable from the viewpoint of side effects. As a method for reducing binding to Fc γ receptor, there is a method of changing the isotype of IgG antibody from IgG1 to IgG2 or IgG4 (non-patent document 22), but IgG2 is more preferable than IgG4 in terms of binding to Fc γ receptor I and pharmacokinetics (non-patent document 11). Since the isotype of TOCILIZUMAB is IgG1, i.e., an IL-6 receptor neutralizing antibody, IgG2 may be preferable in the case where effector functions such as ADCC are not required and the possibility of side effects is considered.

On the other hand, in the development of antibodies as pharmaceuticals, the physicochemical properties of proteins, among them, uniformity and stability are extremely important, and there are reports that: the IgG2 isotype has a very large heterogeneity in disulfide bonds (heterogeneity) in the hinge region (non-patent document 23). It is not easy to manufacture a large amount of the target substance/related substance as a medicine while maintaining the difference in the heterogeneity between the substances, and it is preferable to use a single substance as much as possible in view of the increase in cost. And as heterogeneity of the antibody H chain C-terminal sequence, there have been reported: in the case of developing an antibody of IgG2 isotype as a pharmaceutical product, it is desired to reduce the heterogeneity while maintaining high stability, due to deletion of lysine residue at the C-terminal amino acid and amidation of the C-terminal carboxyl group caused by deletion of glycine and lysine at the 2C-terminal amino acids (non-patent document 24). In order to produce a stable high-concentration subcutaneous preparation excellent in convenience, it is desirable that the stability is high and the half-life in plasma is more excellent than IgG1 which is an isotype of TOCILIZUMAB. However, there has been no report of a constant region sequence having reduced heterogeneity associated with an antibody having a constant region of IgG2 isotype, high stability, and a half-life in plasma superior to that of an antibody having a constant region of IgG1 isotype.

The prior art documents of the present invention are as follows.

Patent document 1: WO92/19759

Patent document 2: WO96/11020

Patent document 3: WO96/12503

Patent document 4: WO2007/143168

Patent document 5: WO2007/114319

Patent document 6: WO2004/096273

Non-patent document 1: janic M Reichert, Clark J Rosenssweig, Laura BFaden & Matthew C Dewitz, Monoclonal antibody vaccines in the clinic, Nature Biotechnology 23, 1073-.

Non-patent document 2: pavlou AK, Belsey MJ., The therapeutic anthropod to 2008, Eur J Pharm biopharmm.2005 Apr; 59(3): 389-96.

Non-patent document 3: nishimoto N, kisimoto t, Interleukin 6: from bench marks, Nat Clin practice Rheumato1.2006 Nov; 2(11): 619-26.

Non-patent document 4: maini RN, Taylor PC, Szechinski J, Pavelka K, BrollJ, Balint G, Emery P, Raemen F, Petersen J, Smolen J, Thomson D, Kishimoto T; charisma Study group, Double-blind randomised controlled clinical trial of the interface-6 receptor antagnostist, Tocilizumab, in European Patients with rhematoid reaction with lead and complex reaction to method, Arthritis Rheum.2006 Sep; 54(9): 2817-29.

Non-patent document 5: nishimoto N, Kanakura Y, Aozasa K, johnkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, Nishioka K, HaraM, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S, Kodama F, Nakahara H, Hagihara K, Yoshizaki K, Kishimoto T. 106(8): 2627-32.

Non-patent document 6: kim SJ, Park Y, Hong HJ., Antibody engineering for the degradation of therapeutic antibodies, Mol cells.2005aug 31; 20(1): review.

Non-patent document 7: rothe A, Hosse RJ, Power B E.Ribosome display for improved biological samples, expert Opin Biol. The.2006 Feb; 6(2): 177-87.

Non-patent document 8: rajpal A, Beyaz N, Haber L, Cappuccil G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A general method for grease simulation of the affinity of antibiotics by using composite libraries, ProcNal Acad Sci U.S. 2005 Jun 14; 102(24): 8466-71.Epub 2005 Jun 6.

Non-patent document 9: wu H, Pfar DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA. development of Motavizumab, an Ultra-potential Antibody for the prediction of Respiratory synthetic Virus Infection in the Upper and Lower Respiratory tract transfer. Jmol biol.2007, 368, 652-665.

Non-patent document 10: shire SJ, Shahrokh z, Liu j. variations in the concentration of high protein concentrations formulations.j Pharm sci.2004 Jun; 93(6): 1390-402.

Non-patent document 11: salfeld JG, Isotype selection in antibody engineering, Nat Biotechnol.2007 Dec; 25(12): 1369-72.

Non-patent document 12: hinton PR, Xiong JM, Johnfs MG, Tang MT, KellerS, Tsoushita N., An engineered human IgG1 antibody with ringer serum half life, J Immunol.2006 Jan 1; 176(1): 346-56.

Non-patent document 13: ghetie V, Popov S, Borvak J, Radu C, matheoi D, Medesan C, Ober RJ, Ward es, incorporated the serum persistence of igg fragment by random mutagenesis, Nat biotechnol.1997 Jul; 15(7): 637-40.

Non-patent document 14: hwang WY, Almagro JC, Buss TN, Tan P, Foote J.use of human germline genes in a CDR homology-based aproach reactivity method.2005 May; 36(1): 35-42.

Non-patent document 15: bartelds GM, Wijbrandts CA, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA, Tak P, Wollink GJ. clinical response to adalimumab: the relationship with anti-aldimum antibiotics and serum adalimumab conjugates in rheumoid arthritis, Ann Rheum Dis.2007 Mar 9; [ Epub ahead of print ].

Non-patent document 16: bender NK, Heilig CE, Droll B, Wohlgemuth J, Armbruster FP, Heilig B. immunogenicity, efficcy and overture events of adalimumab in RA Patients Rheumatotol int.2007 Jan; 27(3): 269-74.

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Non-patent document 18: jones TD, Phillips WJ, Smith BJ, BamHord CA, Nayee PD, Baglin TP, Gaston JS, Baker MP.identification and removalof a promiscuous CD4+ T cell epitope from the C1 domain of factor VIII.J Thromb Haemost.2005 May; 3(5): 991-1000.

Non-patent document 19: chirino AJ, Ary ML, Marshall SA. minimizing the informatics of protein therapeutics. drug discovery Today.2004 Jan 15; 9(2): 82-90.

Non-patent document 20: sato K, Tsuchiya M, Saldanha J, Koishihara Y, Ohsugi Y, Kishimoto T, Bendig MM. Reshaping a human anti body to inhibit the interleukin 6-dependent tumor cell growth. cancer Res.1993Feb 15; 53(4): 851-6.

Non-patent document 21: strand V, Kimberly R, Isaacs JD. biological therapisin rhematology: messons left future directions. nat Rev Drug Discov.2007 Jan; 6(1): 75-92.

Non-patent document 22: gessner JE, Heiken H, Tamm A, Schmidt RE, the IgG Fc receiver family, Ann Hematol.1998 Jun; 76(6): 231-48.

Non-patent document 23: dillon TM, Ricci MS, Vezina C, Flynn GC, Liu YD, Rehder DS, Plant M, Henkle B, Li Y, Deechingkit S, Varnum B, WypychJ, Balland A, Bondarenko PV, structural and functional charateristicionof diagnostic aspects of the human IgG2 sublamps.J Biol chem.2008 Jun 6; 283(23): 16206-15.

Non-patent document 24: johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC.Caption exchange-HPLC and mass spectrometrotropic C-terminal amino of an IgG1 heavy chain.anal biochem.2007Jan 1; 360(1): 75-83.

Disclosure of Invention

Invention of the inventionProblems to be solved

The present invention has been made in view of the above circumstances, and an object thereof is to provide: a pharmaceutical composition comprising a 2 nd generation molecule superior to TOCILIZUMAB (hereinafter, sometimes referred to as "agent" or "preparation" in the present specification), which has an enhanced neutralizing activity against an antigen and improved pharmacokinetics by modifying the amino acid sequences of the variable and constant regions of a humanized anti-IL-6 receptor IgG1 antibody, TOCILIZUMAB, thereby reducing the administration frequency, continuously exerting a therapeutic effect, and having improved immunogenicity, safety, physicochemical properties (stability and uniformity), and a method for producing the pharmaceutical composition.

Means for solving the problems

The present inventors have conducted intensive studies to develop a 2 nd generation molecule superior to TOCILIZUMAB, and have enhanced the drug efficacy and simultaneously improved the pharmacokinetics by modifying the amino acid sequences of the variable and constant regions of the 1 st generation humanized anti-IL-6 receptor IgG1 antibody, TOCILIZUMAB, thereby reducing the administration frequency, continuously exerting the therapeutic effect, and improving the immunogenicity, safety, physicochemical properties (stability and uniformity). As a result, the present inventors found a plurality of CDR mutations in the variable region of TOCILIZUMAB, which improve the binding ability (affinity) to an antigen, and succeeded in greatly improving the affinity by using these combinations. In addition, the present inventors succeeded in improving pharmacokinetics by introducing a modification that lowers the isoelectric point of the variable region sequence. The present inventors have succeeded in improving pharmacokinetics by neutralizing an antigen multiple times with 1 molecule of antibody by imparting pH dependence to the binding to an IL-6 receptor as an antigen. The inventors have succeeded in reducing the risk of immunogenicity by reducing the number of T-cell epitope peptides in the variable region predicted on-computer chips by fully humanizing the mouse-derived sequence remaining in the framework of TOCILIZUMAB. The present inventors have succeeded in finding a novel constant region sequence in the constant region of TOCILIZUMAB, which has improved pharmacokinetics compared to IgG1 by reducing binding to Fc γ receptor to less than IgG1, and which has reduced heterogeneity of disulfide bond derived from the hinge region of IgG2 and heterogeneity derived from the C-terminal of H chain without reducing stability, in order to improve safety. By properly combining the modification of the amino acid sequence of the CDR region, the modification of the amino acid sequence of the variable region and the modification of the amino acid sequence of the constant region, the generation 2 molecule superior to TOCILIZUMAB is successfully developed.

The present invention relates to a pharmaceutical composition comprising a humanized anti-IL-6 receptor IgG antibody having more excellent ability to bind to an antigen (IL-6 receptor), more excellent pharmacokinetics, more excellent safety, immunogenicity risk, physicochemical properties (stability, uniformity), by modifying the amino acid sequences of the variable and constant regions of a humanized anti-IL-6 receptor IgG1 antibody, tocilizab, and a method for producing the pharmaceutical composition. More specifically, the present invention provides the following [1] to [11 ].

[1] A polypeptide according to any one of the following (a) to (f):

(a) a polypeptide comprising a polypeptide having the sequence of SEQ ID NO: 1 (CDR 1 of VH4-M73), a CDR1 having the sequence of SEQ ID NO: 2(VH 4-CDR 2 of M73) and a CDR2 having the sequence of SEQ ID NO: 3 (CDR 3 of VH4-M73) CDR 3;

(b) a polypeptide comprising a polypeptide having the sequence of SEQ ID NO: 4 (CDR 1 of VH3-M73), a CDR1 having the sequence of SEQ ID NO: 5 (CDR 2 of VH3-M73) and a CDR2 having the sequence of SEQ ID NO: 6 (CDR 3 of VH3-M73) CDR 3;

(c) a polypeptide comprising a polypeptide having the sequence of SEQ ID NO: 7 (CDR 1 of VH5-M83), a CDR1 having the sequence of SEQ ID NO: 8 (CDR 2 of VH5-M83) and a CDR2 having the sequence of SEQ ID NO: CDR3 of the sequence of 9 (CDR 3 of VH 5-M83);

(d) a polypeptide comprising a polypeptide having the sequence of SEQ ID NO: 10 (CDR 1 of VL1), a CDR1 having the sequence of SEQ ID NO: 11 (CDR 2 of VL1) and a CDR2 having the sequence of SEQ ID NO: 12 (CDR 3 of VL1) CDR 3;

(e) a polypeptide comprising a polypeptide having the sequence of SEQ ID NO: 13 (CDR 1 of VL3), a CDR1 having the sequence of SEQ ID NO: 14 (CDR 2 of VL3) and a CDR2 having the sequence of SEQ ID NO: 15 (CDR 3 of VL3) CDR 3;

(f) a polypeptide comprising a polypeptide having the sequence of SEQ ID NO: 16 (CDR 1 of VL5), a CDR1 having the sequence of SEQ ID NO: 17 (CDR 2 of VL5) and a CDR2 having the sequence of SEQ ID NO: 18 (CDR 3 of VL5) of CDR 3.

[2] An antibody according to any one of the following (a) to (c):

(a) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 (CDR 1 of VH4-M73), a CDR1 having the sequence of SEQ ID NO: 2(VH 4-CDR 2 of M73) and a CDR2 having the sequence of SEQ ID NO: 3 (CDR 3 of VH4-M73) CDR 3;

the light chain variable region comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 10 (CDR 1 of VL1), a CDR1 having the sequence of SEQ ID NO: 11 (CDR 2 of VL1) and a CDR2 having the sequence of SEQ ID NO: 12 (CDR 3 of VL1) CDR 3;

(b) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 (CDR 1 of VH3-M73), a CDR1 having the sequence of SEQ ID NO: 5 (CDR 2 of VH3-M73) and a CDR2 having the sequence of SEQ ID NO: 6 (CDR 3 of VH3-M73) CDR 3;

the light chain variable region comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 13 (CDR 1 of VL3), a CDR1 having the sequence of SEQ ID NO: 14 (CDR 2 of VL3) and a CDR2 having the sequence of SEQ ID NO: 15 (CDR 3 of VL3) CDR 3;

(c) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 (CDR 1 of VH5-M83), a CDR1 having the sequence of SEQ ID NO: 8 (CDR 2 of VH5-M83) and a CDR2 having the sequence of SEQ ID NO: CDR3 of the sequence of 9 (CDR 3 of VH 5-M83);

the light chain variable region comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 16 (CDR 1 of VL5), a CDR1 having the sequence of SEQ ID NO: 17 (CDR 2 of VL5) and a CDR2 having the sequence of SEQ ID NO: 18 (CDR 3 of VL5) of CDR 3.

[3] A variable region according to any one of the following (a) to (f):

(a) a heavy chain variable region having SEQ ID NO: 19 (variable region of VH 4-M73);

(b) a heavy chain variable region having SEQ ID NO: 20 (variable region of VH 3-M73);

(c) a heavy chain variable region having SEQ ID NO: 21 (variable region of VH 5-M83);

(d) a light chain variable region having SEQ ID NO: 22 (variable region of VL 1);

(e) a light chain variable region having SEQ ID NO: 23 (variable region of VL 3);

(f) a light chain variable region having SEQ ID NO: 24 (variable region of VL 5).

[4] An antibody according to any one of the following (a) to (c):

(a) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19(VH 4-M73) and the light chain variable region has the sequence of SEQ ID NO: 22 (variable region of VL 1);

(b) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20(VH 3-M73), the light chain variable region having the sequence of SEQ ID NO: 23 (variable region of VL 3);

(c) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 21(VH 5-M83) and the light chain variable region has the sequence of SEQ ID NO: 24 (variable region of VL 5).

[5] A heavy or light chain of any one of (a) to (f) below:

(a) a heavy chain having the sequence of SEQ ID NO: 25(VH 4-M73);

(b) a heavy chain having the sequence of SEQ ID NO: 26(VH 3-M73);

(c) a heavy chain having the sequence of SEQ ID NO: 27(VH 5-M83);

(d) a light chain having the sequence of SEQ ID NO: 28(VL 1);

(e) a light chain having the sequence of SEQ ID NO: 29(VL 3);

(f) a light chain having the sequence of SEQ ID NO: 30(VL 5).

[6] An antibody according to any one of the following (a) to (c):

(a) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 25(VH4-M73), the light chain having the sequence of SEQ ID NO: 28(VL 1);

(b) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 26(VH3-M73), the light chain having the sequence of SEQ ID NO: 29(VL 3);

(c) an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 27(VH5-M83), the light chain having the sequence of SEQ ID NO: 30(VL 5).

[7] A gene encoding the polypeptide according to any one of [1] to [6 ].

[8] A vector comprising the gene according to [7 ].

[9] A host cell comprising the vector of [8 ].

[10] A method for producing the polypeptide according to any one of [1] to [6] by culturing the host cell according to [9 ].

[11] A pharmaceutical composition comprising the polypeptide according to any one of [1] to [6] or the polypeptide produced by the method according to [10 ].

Effects of the invention

The humanized anti-IL-6 receptor IgG antibody obtained according to the present invention can reduce the frequency of administration by enhancing the drug effect and improving the pharmacokinetics, and can continuously exert the therapeutic effect.

Drawings

FIG. 1 is a diagram showing a list of mutation sites that collectively increase the affinity of TOCILIZUMAB for the IL-6 receptor. The TOCILIZUMAB sequence of HCDR2 is shown in SEQ ID NO: 81, post-mutation sequence of HCDR2 (upper panel) is shown in SEQ ID NO: 82, post-mutation sequence of HCDR2 (lower panel) see SEQ ID NO: 83, the TOCILIZUMAB sequence of HCDR3 is shown in SEQ ID NO: 84, the post-mutation sequence of HCDR3 (upper) is shown in SEQ ID NO: 85, and the post-mutation sequence of HCDR3 (lower) is shown in SEQ ID NO: 86, LCDR1 is shown in SEQ ID NO: 87, the mutated sequence of LCDR1 (upper panel) is shown in SEQ ID NO: 88, LCDR1 (lower) see SEQ ID NO: 89, the TOCILIZUMAB sequence of LCDR3 is shown in SEQ ID NO: 90, post-mutation sequence of LCDR3 (upper panel) shown in SEQ ID NO: 91, post-mutation sequence of LCDR3 (lower panel) see SEQ ID NO: 92.

FIG. 2 is a graph showing the neutralizing activity of TOCILIZUMAB and RDC-23 in BaF/gp 130.

FIG. 3 is a graph showing a list of mutation sites that can decrease the isoelectric point of the variable region without significantly decreasing the binding of TOCILIZUMAB to the IL-6 receptor. The asterisks in the figure are intended to form sites where the human sequence has been mutated, although it does not affect the isoelectric point. The TOCILIZUMAB sequence of HFR1 is shown in SEQ ID NO: 93, post-mutation sequence of HFR1 is shown in SEQ ID NO: 94, the TOCILIZUMAB sequence of HCDR1 is shown in SEQ ID NO: 95, the sequence of HCDR1 after mutation is shown in SEQ ID NO: 96, the TOCILIZUMAB sequence of HFR2 is shown in SEQ ID NO: 97, post-mutation sequence of HFR2 is shown in SEQ ID NO: 98, the TOCILIZUMAB sequence of HCDR2 is shown in SEQ ID NO: 81, the post-mutation sequence of HCDR2 is shown in SEQ ID NO: 99, the TOCILIZUMAB sequence of HFR4 is shown in SEQ ID NO: 100, post-mutation sequence of HFR4 is shown in SEQ ID NO: 101, TOCILIZUMAB sequence of LFR1 is shown in SEQ ID NO: 102, the post-mutation sequence of LFR1 is set forth in SEQ ID NO: 103, the TOCILIZUMAB sequence of LCDR1 is shown in SEQ ID NO: 87, the mutated sequence of LCDR1 is shown in SEQ ID NO: 104, the TOCILIZUMAB sequence of LFR2 is shown in SEQ ID NO: 105, post-mutation sequence of LFR2 is shown in SEQ ID NO: 106, LCDR2 is shown in SEQ ID NO: 107, LCDR2 is shown in SEQ ID NO: 108, 109, TOCILIZUMAB sequence of LFR3 is shown in SEQ ID NO: 110, post-mutation sequence of LFR3 is shown in SEQ ID NO: 111, TOCILIZUMAB sequence of LFR4 is shown in SEQ id no: 112, the post-mutation sequence of LFR4 is shown in SEQ ID NO: 113.

FIG. 4 is a graph showing the neutralizing activity of TOCILIZUMAB and H53/L28 in BaF/gp 130.

FIG. 5 is a graph showing the change in plasma concentration after intravenous administration of TOCILIZUMAB and H53/L28 to mice.

FIG. 6 is a graph showing the change in plasma concentration following subcutaneous administration of TOCILIZUMAB and H53/L28 to mice.

FIG. 7 is a schematic diagram showing that IgG molecules are bound to a new antigen again by dissociation from a membrane-type antigen in the nucleus.

FIG. 8 is a graph showing a list of mutation sites that can impart pH dependence (binding at pH7.4, dissociation at pH 5.8) to the binding of TOCILIZUMAB to IL-6 receptor. The TOCILIZUMAB sequence of HFR1 is shown in SEQ ID NO: 93, post-mutation sequence of HFR1 is shown in SEQ ID NO: 114, the TOCILIZUMAB sequence of HCDR1 is shown in SEQ ID NO: 95, the sequence of HCDR1 after mutation is shown in SEQ ID NO: 115, LCDR1 is shown in SEQ ID NO: 87, the mutated sequence of LCDR1 is shown in SEQ ID NO: 116, the TOCILIZUMAB sequence of LCDR2 is shown in SEQ ID NO: 107, LCDR2 is shown in SEQ ID NO: 117.

FIG. 9 is a graph showing the neutralizing activity of TOCILIZUMAB and H3pI/L73 in BaF/gp 130.

FIG. 10 is a graph showing the change in plasma concentration following intravenous administration of TOCILIZUMAB and H3pI/L73 to cynomolgus monkeys (カニクイザル).

FIG. 11 is a graph showing the change in plasma concentration after intravenous administration of TOCILIZUMAB and H3pI/L73 to human IL-6 receptor transgenic mice.

FIG. 12 is a graph showing the results of evaluation of heterogeneity from the C-terminus of TOCILIZUMAB, TOCILIZUMAB Δ K and TOCILIZUMAB Δ GK by cation exchange chromatography.

FIG. 13 is a graph showing the results of evaluation of heterogeneity derived from disulfide bonds of TOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2 and TOCILIZUMAB-SKSC by cation exchange chromatography.

FIG. 14 is a graph showing the denaturation curves of TOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2, and TOCILIZUMAB-SKSC, and the Tm values of the respective Fab domains, as determined by Differential Scanning Calorimetry (DSC).

FIG. 15 is a graph showing the change in plasma concentration following intravenous administration of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73 to human FcRn transgenic mice.

FIG. 16 is a graph showing the neutralizing activity of TOCILIZUMAB, control and Fv5-M83 in BaF/gp 130.

FIG. 17 is a graph showing the neutralizing activity of TOCILIZUMAB, Fv3-M73, and Fv4-M73 in BaF/gp 130.

FIG. 18 is a graph showing the change in plasma concentration following intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys.

FIG. 19 is a graph showing the change in CRP concentration after intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys.

FIG. 20 is a graph showing the change in the rate of non-binding soluble IL-6 receptor after intravenous administration of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, and Fv5-M83 to cynomolgus monkeys.

FIG. 21 is a graph showing the inhibitory effect of TOCILIZUMAB and Fv4-M73 on MCP-1 production by synovial cells from human RA patients.

FIG. 22 is a graph showing the inhibitory effect of TOCILIZUMAB and Fv4-M73 on VEGF production by synovial cells from human RA patients.

Best Mode for Carrying Out The Invention

The present invention provides a polypeptide according to any one of the following (a) to (f).

In the present invention, the polypeptide is not particularly limited, but is preferably an antigen-binding substance having a binding activity to a human IL-6 receptor. Preferred examples of antigen-binding substances are: a heavy chain variable region (VH) of an antibody, a light chain variable region (VL) of an antibody, a heavy chain of an antibody, a light chain of an antibody, and the like.

Among the polypeptides (a) to (f), preferred examples of the polypeptides (a) to (c) include: preferred examples of the antibody heavy chain variable region and the polypeptides (d) to (f) include: the variable region of the light chain of the antibody.

The variable regions described above may be used as part of an anti-human IL-6 receptor antibody. An anti-human IL-6 receptor antibody using the variable region has excellent binding activity, excellent pharmacokinetics, excellent safety and immunogenicity, and/or excellent physicochemical properties. In the present invention, the excellent pharmacokinetics or the improvement of pharmacokinetics means a decrease in "Clearance (CL)", an increase in "Area Under the concentration Curve (AUC)", an increase in "Mean Residence Time", an increase in "half-life in plasma (t) which is one of pharmacokinetic parameters calculated from the change in plasma concentration when an antibody is administered in vivo_1/2) "any one of the increases. In the present invention, the excellent physicochemical properties or the improvement of physicochemical properties are not particularly limited, and means improvement of stability, reduction of heterogeneity, and the like.

Framework Regions (FRs) of a human antibody to which the CDRs are linked are selected as framework regions in which the CDRs form a good antigen binding site. The FR used in the variable region of the present invention is not particularly limited, and any FR can be used, but it is preferable to use FR derived from human. The human-derived FR may be one having a natural sequence, or one having 1 or more amino acids of a framework region having a natural sequence may be substituted, deleted, added and/or inserted as necessary to form a suitable antigen-binding site in the CDR. For example, a mutant FR sequence having desired properties can be selected by measuring and evaluating the binding activity of an antibody using an FR with an amino acid substitution to an antigen (Sato, K. et al, Cancer Res. (1993)53, 851 856).

In the above CDR sequences, 1 or more amino acids and the like may be substituted, deleted, added and/or inserted. It is preferable that the CDR sequence after substitution, deletion, addition and/or insertion of 1 or more amino acids has the same activity as the CDR sequence before modification in binding activity, neutralizing activity, stability, immunogenicity and/or pharmacokinetics. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, but is preferably within 3 amino acids, more preferably within 2 amino acids, and still more preferably 1 amino acid per CDR.

As a method for substituting 1 or more amino acid residues into other target amino Acids, there are, for example, site-specific mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotides-directed double amber method for site-directed mutagenesis (oligo-deoxyribotide-directed double amber site directed mutagenesis) Gene 152, 271-induced 275, Zoller, MJ, and Smith, M. (1983) Olincolotide-directed mutagenesis of DNA fragments of linked O M13vectors (oligo-directed mutagenesis of DNA fragments cloned into vector of HM 13), method.100, Enzym 500, Jaameter 500, Jatam-induced DNA fragments of linked O M13vectors (oligo-directed mutagenesis of DNA fragments of DNA sequences of DNA fragments cloned into vector), method.100, Oligonucleotide-induced DNA, DNA of DNA sequences, DNA fragments of DNA sequences of linked DNA sequences, DNA sequences of interest, DNA sequences of The like, DNA sequences of The genus of interest (oligo-directed mutagenesis, DNA sequences of The genus DNA of The genus of The family of, 9441-9456, Kramer W, and Fritz HJ (1987) Oligonucleotide-directed restriction of mutated duplex DNA Methods (Oligonucleotide directed structure mutated by the gapped duplex DNA method), enzymol.154, 350-367, Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis with phenotyping selection (Rapid and efficient site-specific mutagenesis without phenotypic selection), Proc Natl Acad Sci U S A.82, 488-492). This method allows substitution of the desired amino acid of the antibody with another target amino acid. In addition, amino acids in the framework and CDRs can also be substituted with other appropriate amino acids by using library techniques such as framework shuffling (Mol Immunol.2007 Apr; 44 (11): 3049-60) and CDR repair (US 2006/0122377).

The present invention also provides an antibody according to any one of the following (a) to (c).

(a) An antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 (CDR 1 of VH4-M73), a CDR1 having the sequence of SEQ ID NO: 2(VH 4-CDR 2 of M73) and a CDR2 having the sequence of SEQ ID NO: 3 (CDR 3 of VH4-M73), the light chain variable region comprising a CDR3 having the sequence of seq id NO: 10 (CDR 1 of VL1), a CDR1 having the sequence of SEQ ID NO: 11 (CDR 2 of VL1) and a CDR2 having the sequence of SEQ ID NO: 12 (CDR 3 of VL1) CDR 3;

(b) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4 (CDR 1 of VH3-M73), a CDR1 having the sequence of SEQ ID NO: 5 (CDR 2 of VH3-M73) and a CDR2 having the sequence of SEQ ID NO: 6 (CDR 3 of VH3-M73), and the light chain variable region comprises a CDR3 having the sequence of SEQ ID NO: 13 (CDR 1 of VL3), a CDR1 having the sequence of SEQ ID NO: 14 (CDR 2 of VL3) and a CDR2 having the sequence of SEQ ID NO: 15 (CDR 3 of VL3) CDR 3;

(c) an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 (CDR 1 of VH5-M83), a CDR1 having the sequence of SEQ ID NO: 8 (CDR 2 of VH5-M83) and a CDR2 having the sequence of SEQ ID NO: 9 (CDR 3 of VH5-M83), the light chain variable region comprising a CDR3 having the sequence of seq id NO: 16 (CDR 1 of VL5), a CDR1 having the sequence of SEQ ID NO: 17 (CDR 2 of VL5) and a CDR2 having the sequence of SEQ ID NO: 18 (CDR 3 of VL5) of CDR 3.

The above antibody can be used as an anti-human IL-6 receptor antibody having excellent binding activity, excellent pharmacokinetics, excellent safety and immunogenicity, and/or excellent physicochemical properties.

Framework regions of the human antibody of the present invention to which CDRs are linked are selected as framework regions in which CDRs form a good antigen-binding site. The FR used in the variable region of the present invention is not particularly limited, and any FR can be used, but it is preferable to use FR derived from human. As the human-derived FR, those having a natural sequence can be used, and if necessary, 1 or more amino acids of the framework region having a natural sequence can be substituted, deleted, added and/or inserted so that CDRs form an appropriate antigen-binding site. For example, by measuring and evaluating the binding activity of an antibody using amino acid-substituted FRs to an antigen, a mutant FR sequence having desired properties can be selected (Sato, K. et al, Cancer Res. (1993)53, 851 856).

The constant region used in the antibody of the present invention is not particularly limited, and any constant region may be used. Preferred examples of constant regions for use in the antibodies of the invention are: constant regions from human (constant regions from IgG1, IgG2, IgG3, IgG4, ck, C λ, etc.). 1 or more amino acids may be substituted, deleted, added and/or inserted in the constant region derived from human. For example, as a preferred example of a constant region derived from human, in the case of a heavy chain constant region, there are, for example: has the sequence shown in SEQ ID NO: 31 (constant region of VH4-M73), a constant region having the amino acid sequence of SEQ ID NO: 32 (constant region of VH3-M73), a constant region having the amino acid sequence of SEQ ID NO: 33 (constant region of VH 5-M83); in the case of the light chain constant region, for example, there are: has the sequence shown in SEQ ID NO: 34(VL1), a constant region having the amino acid sequence of SEQ ID NO: 35(VL3), a constant region having the amino acid sequence of SEQ id no: 36(VL 5).

1 or more amino acids may be substituted, deleted, added and/or inserted in the above CDR sequences, etc. It is preferable that the CDR sequence after substitution, deletion, addition and/or insertion of 1 or more amino acids has equivalent activity in binding activity, neutralizing activity, stability, immunogenicity and/or pharmacokinetics to the CDR sequence before modification. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, and each CDR is preferably within 3 amino acids, more preferably within 2 amino acids, and still more preferably 1 amino acid.

Amino acid substitutions, deletions, additions, and/or insertions can also be made by the above-described methods.

The present invention also provides a variable region described in any one of (a) to (f) below.

The variable regions described above may be used as part of an anti-human IL-6 receptor antibody. An anti-human IL-6 receptor antibody using the variable region has excellent binding activity, excellent pharmacokinetics, excellent safety and immunogenicity, and/or excellent physicochemical properties.

The variable region may be substituted, deleted, added, and/or inserted with 1 or more (for example, up to 5 amino acids, preferably up to 3 amino acids) amino acids. Examples of the method for substituting 1 or more amino acid residues with other target amino acids include the above-mentioned methods.

The present invention also provides polypeptides comprising the above-described variable regions.

The constant region used in the antibody of the present invention is not particularly limited, and any constant region may be used. Preferred examples of constant regions for use in the antibodies of the invention are: constant regions from human (constant regions from IgG1, IgG2, IgG3, IgG4, kappa chain, lambda chain, etc.). 1 or more amino acids may be substituted, deleted, added and/or inserted in the constant region derived from human. For example, as a preferred example of a constant region derived from human, in the case of a heavy chain constant region, there are, for example: has the sequence shown in SEQ ID NO: 31 (constant region of VH4-M73), a constant region having the amino acid sequence of SEQ ID NO: 32 (constant region of VH3-M73), a constant region having the amino acid sequence of SEQ ID NO: 33 (constant region of VH 5-M83); in the case of the light chain constant region, for example, there are: has the sequence shown in SEQ ID NO: 34(VL1), a constant region having the amino acid sequence of SEQ ID NO: 35(VL3), a constant region having the amino acid sequence of SEQ id no: 36(VL 5).

The present invention also provides a heavy chain or a light chain as described in any one of (a) to (f) below.

(a) A heavy chain having the amino acid sequence of SEQ ID NO: 25(VH 4-M73);

(b) a heavy chain having the amino acid sequence of SEQ ID NO: 26(VH 3-M73);

(c) a heavy chain having the amino acid sequence of SEQ ID NO: 27(VH 5-M83);

(d) a light chain having the sequence of SEQ ID NO: 28(VL 1);

(e) a light chain having the sequence of SEQ ID NO: 29(VL 3);

(f) a light chain having the sequence of SEQ ID NO: 30(VL 5).

The heavy or light chain can be used as part of an anti-human IL-6 receptor antibody. The anti-human IL-6 receptor antibody using the heavy chain or the light chain has excellent binding activity, excellent pharmacokinetics, excellent safety and immunogenicity, and/or excellent physicochemical properties.

The heavy chain or the light chain may be substituted, deleted, added, and/or inserted with 1 or more (for example, within 10 amino acids, preferably within 5 amino acids, and more preferably within 3 amino acids) amino acids. As a method for substituting 1 or more amino acid residues with other target amino acids, for example, the above-mentioned method can be used.

The substitution, deletion, addition and/or insertion of 1 or more amino acids may be made in the variable region, also in the constant region, or in both the variable region and the constant region.

The above antibody is an anti-human IL-6 receptor antibody having excellent binding activity, excellent pharmacokinetics, excellent safety and immunogenicity, and/or excellent physicochemical properties.

The above antibody may be substituted, deleted, added, and/or inserted with 1 or more (for example, within 20 amino acids, preferably within 10 amino acids, and more preferably within 5 amino acids) amino acids. Examples of the method for substituting 1 or more amino acid residues with other target amino acids include the above-mentioned methods.

The antibody of the invention is preferably a humanized antibody.

A humanized antibody is also called a reshaped (reshaped) human antibody, which is obtained by grafting CDRs from a non-human mammal to CDRs of a human antibody, and a general genetic recombination technique thereof is also known (see european patent application publication nos. EP125023 and WO 96/02576).

Specifically, for example, a DNA sequence designed to link a target CDR and a target Framework Region (FR) is synthesized by a PCR method using a plurality of oligonucleotides prepared to have overlapping portions in the terminal regions of the CDR and FR as primers (see the method described in WO 98/13388). The obtained DNA is ligated with a DNA encoding a human antibody constant region or a human antibody constant region variant, and then inserted into an expression vector, which is then introduced into a host to produce a humanized antibody (see european patent application publication No. EP239400 and international patent application publication No. WO 96/02576).

Framework regions of human antibodies to which CDRs are linked framework regions in which CDRs form good antigen binding sites are selected. Amino acids in the framework regions of the antibody variable regions may be substituted, deleted, added, and/or inserted, as necessary.

In the constant region of the humanized antibody, a human antibody constant region or a human antibody constant region variant in which 1 or more amino acids are substituted, deleted, added and/or inserted in the human antibody constant region may be used.

For example, C γ 1, C γ 2, C γ 3, C γ 4, C μ, C, C α 1, C α 2, C may be used in the H chain, and C κ and C λ may be used in the L chain. The amino acid sequence of ck is shown in SEQ ID NO: 38, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 37. the amino acid sequence of C gamma 1 is shown in SEQ ID NO: 40, the nucleotide sequence for coding the amino acid sequence is shown in SEQ ID NO: 39. the amino acid sequence of C γ 2 is set forth in SEQ ID NO: 42, and the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 41. the amino acid sequence of C γ 4 is shown in SEQ ID NO: 44, the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 43.

in addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production. The human antibody used for humanization may be a human antibody of any isotype such as IgG, IgM, IgA, IgE, IgD, etc., but IgG is preferably used in the present invention. As IgG, IgG1, IgG2, IgG3, IgG4, and the like can be used.

After the humanized antibody is prepared, amino acids in the variable region (for example, CDR and FR) or the constant region may be substituted with other amino acids, deleted, added, and/or inserted, and the humanized antibody of the present invention further includes the above-mentioned humanized antibody having amino acid substitution.

The antibody of the present invention may contain not only a bivalent antibody represented by IgG but also a monovalent antibody or a multivalent antibody represented by IgM as long as it has a binding activity and/or a neutralizing activity to an IL-6 receptor. The multivalent antibody of the present invention includes a multivalent antibody having an identical antigen binding site or a multivalent antibody having a portion of different or completely different antigen binding sites. The antibody of the present invention is not limited to the full length molecule of the antibody, and may be a small molecule antibody or a modified product thereof as long as it binds to the IL-6 receptor protein.

The small molecule antibody is not particularly limited as long as it has a binding activity to an IL-6 receptor and/or a neutralizing activity, and is an antibody containing an antibody fragment in which a part of a full length antibody (e.g., a whole IgG) is deleted. In the present invention, the small molecule antibody is not particularly limited as long as it contains a part of the full length antibody, and is preferably a small molecule antibody containing VH or VL, and particularly preferably containing both VH and VL. In addition, other preferable examples of the small molecule antibody of the present invention are: a small molecule antibody comprising the CDRs of the antibody. The CDRs contained in the small molecule antibody may comprise all 6 CDRs of the antibody, or may comprise a portion of the CDRs.

The small molecule antibody of the present invention is preferably smaller in molecular weight than the full length antibody, and may form a polymer such as a dimer, trimer or tetramer, and the molecular weight may be larger than the full length antibody.

Specific examples of antibody fragments are: for example, Fab ', F (ab') 2, Fv, etc. Specific examples of small molecule antibodies are: for example, Fab ', F (ab') 2, Fv, scFv (single chain Fv), diabody, sc (Fv)2 (single chain (Fv)2), and the like. Multimers (e.g., dimers, trimers, tetramers, polymers) of these antibodies are also encompassed within the small molecule antibodies of the invention.

The antibody fragment can be obtained by treating an antibody with an enzyme to produce an antibody fragment. Enzymes for producing antibody fragments, such as papain, pepsin, and plasmin, are known. Alternatively, genes encoding the above antibody fragments can be constructed, introduced into expression vectors, and then expressed in appropriate host cells (see, e.g., Co, M.S. et al, J.Immunol. (1994)152, 2968-2976; Better, M. & Horwitz, A.H.Methodsin Enzymology (1989)178, 476-496; Pluckthun, A. & Skerra, A.Methodsin Enzymology (1989)178, 476-496; Lamoyi, E., Methods in Enzymology (1989)121, 652-663; Rousseaux, J. et al, Methods in Enzymology (1989)121, 663-669; rd, R.E. et al, TIH (1991)9, ECH 132).

The digestion enzyme cleaves a specific site of the antibody fragment, producing an antibody fragment of the specific structure described below. In the case of antibody fragments obtained by using the above enzymes, any part of the antibody may be deleted when a genetic engineering method is applied.

The antibody fragments obtained when the above-mentioned digestive enzymes were used were as follows.

And (3) papain digestion: f (ab)2 or Fab

And (3) pepsin digestion: f (ab ') 2 or Fab'

Plasmin digestion: facb

The small molecule antibody of the present invention may contain an antibody fragment in which any region is deleted, as long as it has a binding activity and/or a neutralizing activity to an IL-6 receptor.

Diabodies refer to bivalent (bivalent) antibody fragments constructed by gene fusion (Holliger P et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993), EP404,097, WO93/11161, etc.). Diabodies are dimers consisting of two polypeptide chains. Typically, the polypeptide chains that make up the dimer are each bound via a linker in the same chain by VL and VH. The linker in diabodies is typically so short that VL and VH cannot bind to each other. Specifically, the amino acid residues constituting the linker are, for example, about 5 residues. Thus, VL and VH encoded on the same polypeptide chain cannot form single chain variable fragments, but rather form dimers with other single chain variable fragments. As a result, the diabody has 2 antigen binding sites.

scFv Antibodies are Antibodies which bind VH and VL via linkers or The like to form single chain polypeptides (Huston, J.S. et al, Proc. Natl.Acad.Sci.U.S.A. (1988)85, 5879-5883; Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol.113, edited by Resenburg and Moore, Springer Verlag, New York, p. 269-315, (1994)). The H chain V region and the L chain V region in the scFv may be derived from any of the antibodies described in the present specification. The peptide linker connecting the V regions is not particularly limited. For example, any single-chain peptide containing about 3 to 25 residues can be used as a linker. Specifically, for example, a peptide linker described later can be used.

The V regions of both strands can be ligated by, for example, the PCR method described above. In order to connect V regions by PCR, a DNA encoding all or a desired partial amino acid sequence is used as a template in the following DNA.

A DNA sequence encoding H chain or H chain V region of an antibody, and

DNA sequence encoding L chain or L chain V region of antibody

DNAs encoding the V regions of the H chain and the L chain are amplified by a PCR method using a pair of primers having sequences corresponding to the sequences at both ends of the DNA to be amplified. Thereafter, a DNA encoding the peptide linker moiety is prepared. DNA encoding a peptide linker can also be synthesized using PCR. In this case, a nucleotide sequence capable of being ligated to an amplification product of each V region separately synthesized may be added to the 5' -side of the primer used. Then, PCR reaction was carried out using each of the DNAs [ H chain V region DNA ] - [ peptide linker DNA ] - [ L chain V region DNA ] and a primer for assembly PCR.

The assembly PCR primer includes a combination of a primer that anneals to the 5 '-side of [ H chain V region DNA ] and a primer that anneals to the 3' -side of [ L chain V region DNA ]. That is, the assembly of PCR primers refers to a primer set capable of amplifying DNA encoding the full-length sequence of scFv to be synthesized. On the other hand, a nucleotide sequence capable of linking to each V region DNA is added to [ peptide linker DNA ]. As a result, the DNAs are ligated together, and PCR primers are assembled to finally produce a full-length scFv as an amplification product. When DNA encoding scFv is prepared in the first place, an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. As a result, the recombinant cell obtained is cultured to express DNA encoding the scFv, whereby the scFv can be obtained.

The order of the VH and VL to be combined is not particularly limited, and they may be arranged in any order, for example, the following arrangement.

[ VH ] linker [ VL ]

[ VL ] linker [ VH ]

sc (fv)2 is a small molecule antibody in which 2 VH and 2 VL are combined by a linker or the like to form a single chain (Hudson et al, J Immunol. methods 1999; 231: 177-189). sc (fv)2 can be prepared by linking scFv to a linker, for example.

Preferably, the antibody is characterized in that 2 VH and 2 VL are arranged in the order of VH, VL, VH, VL ([ VH ] linker [ VL ] linker [ VH ] linker [ VL ]) with the N-terminal side of the single-chain polypeptide as a base point, and the order of 2 VH and 2 VL is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangement is provided.

[ VL ] linker [ VH ] linker [ VL ]

[ VH ] linker [ VL ] linker [ VH ]

[ VH ] linker [ VL ]

[ VL ] linker [ VH ]

[ VL ] linker [ VH ] linker [ VL ] linker [ VH ]

The amino acid sequence of VH or VL in a small molecule antibody may be substituted, deleted, added, and/or inserted. In addition, when VH and VL are associated, a part may be deleted or another polypeptide may be added as long as they have antigen binding activity. In addition, the variable region may be chimeric or humanized.

In the present invention, any peptide linker or synthetic compound linker that can be introduced by genetic Engineering, such as those disclosed in Protein Engineering, 9(3), 299-305, 1996, can be used as a linker for linking the antibody variable region.

In the present invention, a preferred linker is a peptide linker. The length of the peptide linker is not particularly limited, and may be appropriately selected by those skilled in the art according to the purpose, but is usually 1 to 100 amino acids, preferably 3 to 50 amino acids, more preferably 5 to 30 amino acids, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

Examples of the amino acid sequence of the peptide linker include the following sequences.

Ser

Gly·Ser

Gly·Gly·Ser

Ser·Gly·Gly

Gly·Gly·Gly·Ser(SEQ ID NO：45)

Ser·Gly·Gly·Gly(SEQ ID NO：46)

Gly·Gly·Gly·Gly·Ser(SEQ ID NO：47)

Ser·Gly·Gly·Gly·Gly(SEQ ID NO：48)

Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO：49)

Ser·Gly·Gly·Gly·Gly·Gly(SEQ ID NO：50)

Gly·Gly·Gly·Gly·Gly·Gly·Ser(SEQ ID NO：51)

Ser·Gly·Gly·Gly·Gly·Gly·Gly(SEQ ID NO：52)

(Gly·Gly·Gly·Gly·Ser(SEQ ID NO：47))n

(Ser·Gly·Gly·Gly·Gly(SEQ ID NO：48))n

[ n is an integer of 1 or more ], and the like.

Regarding the amino acid sequence of the peptide linker, those skilled in the art can appropriately select it according to the purpose. For example, n, which determines the length of the peptide linker, is usually 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

Synthetic compound linkers (chemical crosslinkers) are crosslinkers commonly used for peptide crosslinking, such as: n-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl suberate) (BS)³) Dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene glycol bis (sulfosuccinimidyl succinate) (Sulfo-EGS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (Sulfo-DST), bis [2- (succinimidyloxycarbonyloxy) ethyl ] ethyl]Sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl]Sulfone (Sulfo-BSOCOES), and the like, and the above-mentioned crosslinking agents are commercially available.

In linking 4 antibody variable regions, usually 3 linkers are required. These joints may be the same or different.

The antibodies of the invention also include: an antibody having 1 or more amino acid residues added to the amino acid sequence of the antibody of the present invention. Fusion proteins of the above antibodies with other peptides or proteins are also included. The fusion protein can be prepared by a method known to those skilled in the art, for example, by ligating a polynucleotide encoding the antibody of the present invention with a polynucleotide encoding another peptide or polypeptide so that the frameworks are identical, and then introducing the resulting construct into an expression vector to express the resulting construct in a host. As other peptides or polypeptides to be fused with the antibody of the present invention, there are known peptides such as FLAG (Hopp, T.P. et al, Bio Technology (1988)6, 1204-1210), 6 XHis comprising 6 His (histidine) residues, 10 XHis, influenza Hemagglutinin (HA), human C-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lcktag, alpha-tubulin fragment, B-tag, protein C fragment, and the like. In addition, as other polypeptides for fusion with the antibody of the present invention, for example, there are: GST (glutathione-S-transferase), HA (influenza hemagglutinin), immunoglobulin constant region, beta-galactosidase, MBP (maltose binding protein), etc. A fusion polypeptide can be prepared by fusing a polynucleotide encoding the above-mentioned commercially available peptide or polypeptide with a polynucleotide encoding the antibody of the present invention and expressing the thus-prepared fusion polynucleotide.

The antibody of the present invention may be a conjugated antibody that binds to various molecules such as a polymer substance such as polyethylene glycol (PEG) or hyaluronic acid, a radioactive substance, a fluorescent substance, a luminescent substance, an enzyme, and a toxin. The above-mentioned conjugated antibody can be obtained by chemically modifying the obtained antibody. It is to be noted that methods for modifying antibodies have been established in this field (e.g., US5057313, US 5156840). The "antibody" in the present invention also encompasses the above-mentioned conjugated antibody.

The antibody of the present invention further comprises an antibody having a modified sugar chain.

The antibody used in the present invention may be a bispecific antibody. A dual specificity antibody is one that has variable regions that recognize different epitopes within the same antibody molecule. In the present invention, the dual specific antibody may be a dual specific antibody recognizing different epitopes on the IL-6 receptor molecule, or may be a dual specific antibody in which one antigen binding site recognizes the IL-6 receptor and the other antigen binding site recognizes other substances. Examples of the antigen to be bound by the other antigen-binding site of the dual-specific antibody comprising the antibody of the present invention recognizing the IL-6 receptor include: IL-6, TNF α, TNFR1, TNFR2, CD80, CD86, CD28, CD20, CD19, IL-1 α, IL- β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxin α, lymphotoxin β, LIGHT ligand, LIGHT, VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21, IL-21R, GM-CSF, GM-CSFR, M-CSFR, IFN- α, VEGF, VEGFR, EGF, EGFR, 5, APRIL, APRILR, and the like.

Methods for making dual specificity antibodies are well known. For example, a dual specificity antibody can be produced by binding 2 kinds of antibodies recognizing different antigens. The antibody to be bound may be 1/2 molecules having an H chain and an L chain, respectively, or 1/4 molecules containing only an H chain. Alternatively, a hybridoma producing a different monoclonal antibody may be fused to produce a double-specific antibody-producing fused cell. The dual specificity antibody can also be prepared by using a genetic engineering method.

As described later, the antibody of the present invention differs in amino acid sequence, molecular weight, isoelectric point, presence or absence of a sugar chain, conformation, and the like depending on the cell or host producing the antibody or the purification method. However, the antibody obtained is included in the present invention as long as it has the same function as the antibody of the present invention. For example, when the antibody of the present invention is expressed in prokaryotic cells, for example, Escherichia coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody (original antibody). The antibodies of the invention also include such antibodies.

The polypeptide such as an anti-IL-6 receptor antibody of the present invention can be prepared by a method known to those skilled in the art.

For example, an anti-IL-6 receptor antibody can be prepared by using a gene recombination technique known to those skilled in the art based on the sequence of the obtained anti-IL-6 receptor antibody. Specifically, polynucleotides encoding antibodies are constructed based on the sequence of antibodies that recognize the IL-6 receptor, introduced into an expression vector, and then expressed in an appropriate host cell (see, for example, Co, M.S. et al, J.Immunol. (1994)152, 2968-2976; Better, M.and Horwitz, A.H., Methods Enzyl. (1989)178, 476-496; Pluckthun, A.and Skerra, A., Methods Enzyl. (1989)178, 497-515; Lamoyi, E., Methods Enzyl. (1986)121, 652-663; Rousseaux, J.et al, Methods Enzyl. (1986)121, 663 9; Bind, R.66137, Trenchol., 132).

Accordingly, the present invention provides a method for producing a polypeptide of the present invention, or a polypeptide encoded by a gene encoding a polypeptide of the present invention, comprising the step of culturing a host cell comprising a vector into which a polynucleotide encoding a polypeptide of the present invention has been introduced.

More specifically, a method for producing the polypeptide of the present invention is provided, which comprises the following steps.

(a) A step of culturing a host cell comprising a vector into which a gene encoding a polypeptide of the present invention is introduced;

(b) a step of obtaining a polypeptide encoded by the gene.

Examples of carriers are: m13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like. For subcloning and excision of cDNA, there are, for example, pGEM-T, pDIRECT, pT7 and the like in addition to the above-mentioned vector. Expression vectors are particularly useful when vectors are used for the production of the antibodies of the invention. As an expression vector, for example, in order to express in Escherichia coli, it is necessary to have a promoter which can be expressed with high efficiency in Escherichia coli hosts such as JM109, DH 5. alpha., HB101, XL1-Blue, for example, lacZ promoter (Ward et al, Nature (1989)341, 544-546; FASEB J. (1992)6, 2422-2427), araB promoter (Better et al, Science (1988)240, 1041-1043) or T7 promoter, in addition to the characteristic of amplification in Escherichia coli. As such vectors, there are pGEX-5X-1 (Pharmacia), "QIAexpress system" (Quiagen), pEGFP and pET (in this case, BL21 expressing T7RNA polymerase is preferable as the host), and the like, in addition to the above vectors.

The signal sequence for antibody secretion may be contained in the vector of the expression plasmid. As a signal sequence for antibody secretion, the pelB signal sequence can be used when produced into the periplasm of e.coli (Lei, s.p. et al, j.bacteriol. (1987)169, 4379). For example, the vector can be introduced into a host cell by the calcium chloride method or the electroporation method.

As a vector for producing the antibody of the present invention, in addition to Escherichia coli, there are, for example: expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen corporation) or pEF-BOS (Nucleic acids. Res.1990, 18(17), p. 5322), pEF, pCDM 8); expression vectors derived from insect cells (e.g., "Bac-to-BAC baculovirus expression System" (manufactured by Gibco-BRL), pBacPAK 8); plant-derived expression vectors (e.g., pMH1, pMH 2); expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw); an expression vector derived from a retrovirus (e.g., pZIPneo); yeast-derived Expression vectors (e.g., "Pichia Expression kit" (manufactured by Invitrogen), pNV11, SP-Q01); expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH 50).

In order to express in animal cells such as CHO cells, COS cells, and NIH3T3 cells, the vector for the expression plasmid must have a promoter necessary for intracellular expression, for example, SV40 promoter (Mullingan et al, Nature (1979)277, 108), MMLV-LTR promoter, EF1 a promoter (Mizushima et al, Nucleic Acids Res. (1990)18, 5322), CMV promoter, etc., and more preferably has a gene for selecting transformed cells (for example, a drug-resistant gene distinguishable by drugs (neomycin, G418, etc.)). Examples of the carrier having the above-mentioned properties include: pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13 and the like.

In order to stably express a gene and amplify the copy number of the gene in a cell, a method of introducing a vector having a DHFR gene (for example, pSV2-DHFR ("Molecular Cloning second edition," Cold Spring Harbor Laboratory Press, (1989)) which fills up the pathway into a CHO cell lacking a nucleic acid synthesis pathway and then amplifying the vector using Methotrexate (MTX) can be mentioned. In order to express a gene transiently, a method of transformation using a vector having a replication origin of SV40 (pcD or the like) using COS cells having a gene expressing SV40T antigen on their chromosomes can be exemplified. As the replication origin, those derived from polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), etc. can be used. In order to amplify the gene copy number in the host cell line, the expression vector may further contain an aminoglycoside transferase (APH) gene, a Thymidine Kinase (TK) gene, E.coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, etc., as a selection marker.

The antibody of the present invention thus obtained can be isolated from the inside or outside of the host cell (such as a culture medium) and purified to a substantially pure and homogeneous antibody. The antibody can be isolated and purified by a method of isolation and purification generally used for purification of an antibody, but is not limited thereto. For example, the antibody can be separated and purified by appropriately selecting and combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, recrystallization, and the like.

Examples of chromatography include: affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and characterization guidelines for Protein Purification: A Laboratory CourseMicroanal. Ed Daniel R. Marshark et al, Cold Spring Harbor Laboratory Press, 1996). The above chromatography can be performed using liquid chromatography such as HPLC, FPLC, and the like. The columns used for affinity chromatography are: protein A column and protein G column. Examples of protein a columns are: HyperD, POROS, Sepharose FF (GE Amersham Biosciences), and the like. The present invention also encompasses an antibody which is highly purified by the above-described purification method.

The binding activity of the resulting antibody to the IL-6 receptor can be determined by methods known to those skilled in the art. For example, as a method for measuring the antigen binding activity of an antibody, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay) or a fluorescent antibody method can be used. For example, in the case of an enzyme immunoassay, a sample containing an antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added to a plate coated with an antigen. The antigen binding activity can be evaluated by adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating the plate, washing, adding an enzyme substrate such as p-nitrophenyl phosphate, and measuring the absorbance.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition containing the above polypeptide as an active ingredient. The pharmaceutical composition can be used for diseases such as IL-6 related rheumatoid arthritis and the like. That is, the present invention also provides a therapeutic agent for diseases such as rheumatoid arthritis, which comprises the above antibody as an active ingredient. Preferred examples of the diseases to be treated by the present invention include: rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile idiopathic arthritis, castleman's disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, Crohn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, Still disease, amyloidosis, multiple sclerosis, transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, Chronic Obstructive Pulmonary Disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetic nephropathy, GVHD, endometrium, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin's lymphoma, pancreatic cancer, lung cancer, esophageal cancer, colorectal cancer, cancer cachexia, cancer neuro-infiltration, myocardial infarction, multiple myeloma, and bone marrow cancer, Myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, panuveitis, anterior uveitis, intermediate uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry eye, post-operative inflammation, and the like, but is not limited thereto.

"containing an anti-IL-6 receptor antibody as an active ingredient" means that an anti-IL-6 receptor antibody is contained as at least one active ingredient, and the content ratio thereof is not limited. The pharmaceutical composition of the present invention may contain other active ingredients in addition to the above-mentioned polypeptide.

It is to be noted that the pharmaceutical composition of the present invention can be used not only for therapeutic purposes but also for prophylactic purposes.

The polypeptides of the invention can be formulated according to conventional methods (e.g., Remington's pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.). And, if necessary, may contain a pharmaceutically acceptable carrier and/or additive together. For example, it may contain: surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, fragrances, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, glidants, flavoring agents, etc. However, the prophylactic or therapeutic agent for inflammatory diseases of the present invention is not limited to these, and other conventional carriers may be appropriately contained. The method specifically comprises the following steps: light silicic anhydride, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic base, and the like. In addition, other small molecules of polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, and amino acids may be contained. When the antibody is prepared as an aqueous solution for injection, for example, the anti-IL-6 receptor antibody may be dissolved in an isotonic solution containing physiological saline, glucose or other adjuvants. As auxiliaries, for example: d-sorbitol, D-B-Shibata, D-mannitol, and sodium chloride may be used in combination with an appropriate cosolvent, for example, alcohol (ethanol, etc.), polyol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), and the like.

Further, the polypeptide may be encapsulated in a microcapsule (a microcapsule of hydroxymethylcellulose, gelatin, polymethylmethacrylate, or the like) or may be formulated into a colloidal administration system (liposome, albumin microsphere, microemulsion, nanoparticle, nanocapsule, or the like) as required (refer to Remington's pharmaceutical science, 16 th edition, Oslo Ed. (1980), or the like). Also, methods for formulating drugs into sustained release formulations are known and can be applied to polypeptides (Langer et al, J.biomed.Mater.Res. (1981) 15: 167-277; Langer, chem.Tech. (1982) 12: 98-105; U.S. Pat. No. 3,773,919; European patent application publication (EP) No. 58,481; Sidman et al, Biopolymers (1983) 22: 547-56; EP No. 133,988). Further, the amount of subcutaneous liquid can be increased by adding or mixing hyaluronidase to the present agent (e.g., WO 2004/078140).

The pharmaceutical composition of the present invention may be administered orally or parenterally, but parenteral administration is preferred. Specifically, the injection and transdermal administration are performed to the patient. Examples of injection formulations are: for example, systemic or local administration can be carried out by intravenous injection, intramuscular injection, subcutaneous injection, or the like. Local injection, particularly intramuscular injection, may be performed at or around the treatment site. Examples of transdermal administration forms are: such as ointments, gels, creams, wet cloths, patches and the like, may be administered systemically or topically. In addition, the appropriate administration method can be selected according to the age and symptoms of the patient. The dosage may be, for example: the active ingredient is selected in the range of 0.0001 mg-100 mg per 1kg body weight per time. Alternatively, for example, when the antibody is administered to a patient, the active ingredient is selected in the range of 0.001 to 1000mg/kg body weight per patient, and the amount of the antibody of the present invention is preferably about 0.01 to 50mg/kg body weight per administration. However, the pharmaceutical composition of the present invention is not limited to the above-mentioned dosage.

It is to be noted that amino acids contained in the amino acid sequence described in the present invention may be subjected to post-translational modification (for example, N-terminal glutamine is modified to pyroglutamic acid by pyroglutamylation (known to those skilled in the art)), and even if the amino acids are modified post-translationally in this manner, they are naturally included in the amino acid sequence described in the present invention.

The structure of the sugar chain to be bound may be any structure. The sugar chain at position 297 in the EU numbering system may have any sugar chain structure (preferably, a fucosylated sugar chain), or may not be bound (for example, the sugar chain may be produced in Escherichia coli, or may be modified so as not to be bound at position 297 in the EU numbering system).

It is to be noted that all the prior art documents cited in the present specification are incorporated herein by reference.

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 identification of variable region mutation sites that improve the affinity of TOCILIZUMAB for IL-6 receptor

In order to improve the affinity of TOCILIZUMAB (H chain WT-IgG1/SEQ ID NO: 53, L chain WT-. kappa./SEQ ID NO: 54) to the IL-6 receptor, a library having mutations introduced into the CDR sequences was prepared and studied. As a result of screening the library having mutations introduced into CDRs, mutations that improve the affinity for IL-6 receptor were found, and these are summarized in FIG. 1. Examples of high affinity TOCILIZUMAB combined with these mutations are: RDC-23(H chain RDC23H-IgG1/SEQ ID NO: 55, L chain RDC-23L-. kappa./SEQ ID NO: 56). The affinity of RDC-23 for the soluble IL-6 receptor and the biological activity of BaF/gp130 production were compared with TOCILIZUMAB (see reference examples for methods).

The results of the affinity measurements are shown in Table 1. The results of measurement of the biological activity produced by BaF/gp130 (final IL-6 concentration of 30ng/mL) are shown in FIG. 2. As a result, it was found that: compared with TOCILIZUMAB, the affinity of RDC-23 is improved by about 60 times, and the activity is improved by about 100 times as 100% inhibition concentration of BaF/gp 130.

[ Table 1]

EXAMPLE 2 identification of mutations that increase pharmacokinetics by lowering of isoelectric Point of TOCILIZUMAB

To improve the pharmacokinetics of TOCILIZUMAB, a mutation site that can lower the isoelectric point of the variable region without greatly reducing the binding to the IL-6 receptor was studied. The mutant sites of the variable region predicted from the three-dimensional structure model of TOCILIZUMAB were screened, and as a result, it was found that the isoelectric point of the variable region could be lowered without significantly reducing the binding to the IL-6 receptor, and these are summarized in FIG. 3. Examples of isoelectric point lowering tocilizab combined with these mutations are: H53/L28(H chain H53-IgG1/SEQ ID NO: 57, L chain L28- κ/SEQ ID NO: 58). The affinity of H53/L28 for the soluble IL-6 receptor, the biological activity of BaF/gp130, the isoelectric point and the pharmacokinetics in mice were compared with TOCILIZUMAB (method reference).

The results of the affinity measurements are shown in Table 2. The results of measurement of the biological activity produced by BaF/gp130 (final IL-6 concentration of 30ng/mL) are shown in FIG. 4. Compared with TOCILIZUMAB, the affinity of H53/L28 is improved by about 6 times, and the activity is improved by about several times as 100% inhibition concentration of BaF/gp 130.

[ Table 2]

The isoelectric point is determined by isoelectric point electrophoresis which is well known by the technicians in the field, and the result shows that the isoelectric point of TOCILIZUMAB is about 9.3, the isoelectric point of H53/L28 is about 6.5-6.7, and the isoelectric point of H53/L28 is reduced by about 2.7 compared with that of TOCILIZUMAB. When the theoretical isoelectric point of the variable region VH/VL was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of TOCILIZUMAB was 9.20, the theoretical isoelectric point of H53/L28 was 4.52, and the isoelectric point of H53/L28 was decreased by about 4.7 as compared with TOCILIZUMAB.

To evaluate the decrease of isoelectric pointThe pharmacokinetics of the modified antibody H53/L28 were compared with those of TOCILIZUMAB and H53/L28 in normal mice. TOCILIZUMAB and H53/L28 were administered to mice (C57BL/6J, Charles river, Japan) at 1mg/kg Intravenously (IV) and Subcutaneously (SC) in a single dose, and concentration changes in plasma were evaluated. The changes in plasma concentration after intravenous administration of TOCILIZUMAB and H53/L28 are shown in FIG. 5, the changes in plasma concentration after subcutaneous administration are shown in FIG. 6, and the pharmacokinetic parameters (clearance (CL), half-life (T) obtained by WinNonlin (manufactured by Pharsight) are shown in FIG. 6_1/2) See table 3). Half-life in plasma (T) following intravenous administration of H53/L28_1/2) The increase was about 1.3 times that of TOCILIZUMAB and the decrease in clearance was about 1.7 times. T after subcutaneous administration of H53/L28_1/2The increase was about 2 times that of TOCILIZUMAB and the decrease in clearance was about 2.1 times. As a result, it was found that: by thus lowering the isoelectric point of TOCILIZUMAB using amino acid substitution, pharmacokinetics can be greatly improved.

[ Table 3]

EXAMPLE 3 identification of mutation sites that reduce the immunogenicity of TOCILIZUMAB

Of mutant sites which reduce the risk of immunogenicity by T-cell epitopes present in the variable region Identification

T cell epitopes present in the variable region sequence of TOCILIZUMAB were analyzed using TEPITOPE (methods.2004 Dec; 34 (4): 468-75). As a result, it was predicted that a plurality of HLA-binding T-cell epitopes (sequences with high risk of immunogenicity) were present in L chain CDR 2. Thus, in TEPITOPE analysis, amino acid substitutions that reduce the immunogenic risk of L chain CDR2 but do not reduce stability, binding activity, neutralizing activity were investigated.

The screening result shows that: the risk of immunogenicity can be reduced without loss of stability, binding activity, neutralizing activity by substituting threonine to glycine and arginine to glutamate (SEQ ID NO: 60) of L51(Kabat numbering, Kabat EA et al, 1991.Sequences of Proteins of Immunological interest. NIH) of L chain CDR2(SEQ ID NO: 59) of TOCILIZUMAB as follows.

TOCILIZUMAB L chain CDR2(SEQ ID NO: 59)

TOCILIZUMAB L chain CDR2(SEQ ID NO: 60) with T cell epitopes removed

EXAMPLE 4 reduction of the immunogenicity Risk caused by complete humanization of the variable region framework sequences of TOCILIZUMAB

Among the variable region sequences of TOCILIZUMAB, H27, H28 of H chain FR1, H29, H30 and H71 of H chain FR3 (Kabat numbering, Kabat EA et al, 1991.Sequencesof Proteins of Immunological interest. NIH) in order to maintain the binding activity during humanization, a mouse sequence was left in the framework sequence (Cancer Res.1993Feb 15; 53 (4): 851-6). Since the residual mouse sequence is responsible for increasing the risk of immunogenicity, to further reduce the risk of immunogenicity of TOCILIZUMAB, the framework sequences were fully humanized.

As a result, it was found that: the entire framework of TOCILIZUMAB can be completely humanized without decreasing stability, binding activity, and neutralizing activity by substituting H chain FR1(SEQ ID NO: 61) of TOCILIZUMAB with humanized H chain FR1-A (SEQ ID NO: 62) described below and H chain FR3(SEQ ID NO: 63) of TOCILIZUMAB with humanized H chain FR3(SEQ ID NO: 64) described below.

TOCILIZUMAB H chain FR1(SEQ ID NO: 61)

Humanized H chain FR1-A (SEQ ID NO: 62) (from germline IMGT hVH _4)

TOCILIZUMAB H chain FR3(SEQ ID NO: 63)

Humanized H chain FR3(SEQ ID NO: 64) (from mol. Immunol.2007, 44 (4): 412-

EXAMPLE 5 identification of the site of mutation for improving the pharmacokinetics of pH-dependent binding of TOCILIZUMAB to the IL-6 receptor

One of the methods for improving the pharmacokinetics of TOCILIZUMAB is to modify a molecule such that 1 molecule of TOCILIZUMAB repeatedly binds to and neutralizes a plurality of IL-6 receptors. Consider that: after binding to the membrane-type IL-6 receptor, TOCILIZUMAB is incorporated into intracellular endosomes by internalization while remaining in a state of binding to the membrane-type IL-6 receptor, and then, while being bound to the membrane-type IL-6 receptor, TOCILIZUMAB is directly transferred to lysosomes and is decomposed by lysosomes. That is, it is considered that 1 molecule of TOCILIZUMAB binds to1 to 2 molecules of membrane-type IL-6 receptor (monovalent to divalent), and is degraded by lysosome after internalization, so that 1 molecule of tocilizab can bind to and neutralize only 1 to 2 molecules of membrane-type IL-6 receptor.

Therefore, as shown in fig. 7, it is considered that, if a pH-dependent binding tocizumab in which binding of tocizumab is maintained under neutral conditions and binding of tocizumab is significantly reduced under acidic conditions can be produced, the pH-dependent binding tocizumab dissociates from a membrane-type IL-6 receptor as an antigen in vivo in the nucleus, binds to FcRn present in vivo in the nucleus, and returns to plasma, and the pH-dependent binding tocizumab returned to plasma can bind to the membrane-type IL-6 receptor again. It is considered that 1 molecule of TOCILIZUMAB can repeatedly bind to and neutralize a plurality of molecules of IL-6 receptor by repeating the above-described binding in plasma and dissociation in vivo, and thus it is considered that: the pharmacokinetics of the pH-dependent bound tocilizab are improved compared to TOCILIZUMAB.

In order for TOCILIZUMAB to dissociate from IL-6 receptors under acidic conditions in the nucleus, it is necessary to significantly reduce binding under acidic conditions compared to neutral conditions. Since TOCILIZUMAB must bind strongly to the IL-6 receptor on the cell surface to neutralize it, the antibody must bind to the IL-6 receptor to an extent greater than the same extent at pH7.4 on the cell surface as TOCILIZUMAB. The pH in vivo in the nucleus is reported to be usually pH5.5 to pH6.0(Nat Rev Mol Cell biol.2004 Feb; 5 (2): 121-32), and thus: any TOCILIZUMAB modified to have a pH-dependent binding property that weakly binds to IL-6 receptor at pH5.5 to pH6.0 can be dissociated from IL-6 receptor under acidic conditions in the nuclear body. That is, it is considered that 1 molecule of TOCILIZUMAB modified to strongly bind to IL-6 receptor at pH7.4 on the cell surface and to weakly bind to IL-6 receptor at pH5.5 to pH6.0 in the nuclear body can bind to and neutralize a plurality of IL-6 receptors, thereby improving pharmacokinetics.

To confer pH-dependence on the binding of TOCILIZUMAB to the IL-6 receptor, the following methods are considered: a histidine residue having a pKa of about 6.0 to 6.5 and a proton dissociation state that changes between neutral conditions (pH7.4) and acidic conditions (pH5.5 to pH6.0) is introduced into the variable region of TOCILIZUMAB. Therefore, the histidine-introducing site of the variable region predicted from the three-dimensional structure model of TOCILIZUMAB was selected. In addition, a library in which selected variable region sequences designed as TOCILIZUMAB were randomly substituted with histidine was prepared and screened. The screening was carried out using as an index the binding to the IL-6 receptor at pH7.4, the dissociation from the IL-6 receptor at pH5.5 to pH5.8, or the decrease in affinity.

As a result, it was found that TOCILIZUMAB can be imparted with a mutation site showing pH-dependent binding (binding at pH7.4, dissociation at pH 5.8) to IL-6 receptor, and they are summarized in FIG. 8. The tyrosine of H27 of fig. 8 was substituted with histidine, which is not a mutation of the CDR, but of FR1 of the H chain, but as in eur.j.immunol.1992.22: 1719-1728 since the sequence in which H27 is histidine is present as the human sequence (SEQ ID NO: 65), it can be fully humanized together with example 4 by using the following framework.

Humanized H chain FR1-B (SEQ ID NO: 65)

Examples of pH-dependent binding TOCILIZUMAB in combination with the above mutations are: h3pI/L73(H chain H3pI-IgG1/SEQ ID NO: 66, L chain L73- κ/SEQ ID NO: 67). The affinity of H3pI/L73 for soluble IL-6 receptor at pH7.4, the dissociation rate from membrane-type IL-6 receptor at pH7.4 and pH5.8, the biological activity of BaF/gp130 production, and the pharmacokinetics in cynomolgus and human IL-6 receptor transgenic mice were compared to TOCILIZUMAB (method reference).

The results of the determination of the affinity for the soluble form of the IL-6 receptor at pH7.4 are shown in Table 4. The results of measurement of the biological activity produced by BaF/gp130 (final IL-6 concentration of 30ng/mL) are shown in FIG. 9. H3pI/L73 was shown to have approximately the same affinity for the soluble IL-6 receptor and BaF/gp130 activity at pH7.4 as compared to TOCILIZUMAB.

[ Table 4]

The results of the determination of the dissociation rates of TOCILIZUMAB and H3pI/L73 from membrane-type IL-6 receptors at pH7.4 and pH5.8 are shown in Table 5. Shows that: compared with TOCILIZUMAB, the dissociation rate of H3pI/L73 at pH5.8 was faster, and the pH dependence of the dissociation rate from the membrane-type IL-6 receptor was increased by about 2.6 times.

[ Table 5]

Tocilizumab and H3pI/L73 were administered to cynomolgus monkeys at 1mg/kg in a single intravenous dose to assess changes in plasma concentrations. The changes in plasma concentrations following intravenous administration of TOCILIZUMAB and H3pI/L73 are shown in FIG. 10. As a result, the pharmacokinetics of H3pI/L73 in cynomolgus monkeys was significantly improved as compared with TOCILIZUMAB.

TOCILIZUMAB and H3pI/L73 were administered at 25mg/kg to human IL-6 receptor transgenic mice (hIL-6R tg mice, Proc Natl Acad Sci U S A.1995 May 23; 92 (11): 4862-6) in a single intravenous dose to assess changes in plasma concentrations. The changes in plasma concentrations following intravenous administration of TOCILIZUMAB and H3pI/L73 are shown in FIG. 11. As a result, the pharmacokinetics of H3pI/L73 in human IL-6 receptor transgenic mice was greatly improved compared to TOCILIZUMAB.

The pharmacokinetics of the pH-dependent binding TOCILIZUMAB, H3pI/L73, in cynomolgus and human IL-6 receptor transgenic mice was greatly improved compared to TOCILIZUMAB, thus suggesting that: by imparting the property of binding to an antigen at pH7.4 and dissociating from the antigen at pH5.8, 1 molecule of H3pI/L73 can bind to and neutralize a plurality of IL-6 receptors. In addition, it is thought that pharmacokinetics can be further improved by imparting a stronger pH dependence on binding to the IL-6 receptor than H3 pI/L73.

EXAMPLE 6 optimization of constant region of TOCILIZUMAB

Reduction of heterogeneity of the C-terminal of TOCILIZUMAB H chain

As for the heterogeneity of the H chain C-terminal sequence of IgG antibodies, amidation of the C-terminal carboxyl group caused by deletion of the lysine residue at the C-terminal amino acid and deletion of 2 amino acids, i.e., glycine and lysine at the C-terminal, has been reported (Anal biochem.2007Jan 1; 360 (1): 75-83). In TOCILIZUMAB, although the main component is a sequence in which lysine, which is a C-terminal amino acid present in a nucleotide sequence, is deleted by post-translational modification, a side component in which lysine remains and an amidated side component of a C-terminal carboxyl group caused by deletion of both glycine and lysine exist as heterogeneity. Since it is not easy and cost-intensive to produce a large amount of the antibody as a pharmaceutical product while maintaining the difference between the target substance and the related substance in production, it is desirable to use a single substance as much as possible, and to reduce the heterogeneity in the case of developing an antibody as a pharmaceutical product. Therefore, when the pharmaceutical product is developed, it is desired that the heterogeneity of the C-terminal of the H chain is not present.

The C-terminal amino acid is modified in order to reduce the heterogeneity of the C-terminal amino acid. As a result, it was found that heterogeneity derived from the C-terminus can be avoided by deleting lysine and glycine at the C-terminus of the H chain constant region of TOCILIZUMAB in advance in the nucleotide sequence. The heterogeneity of TOCILIZUMAB, TOCILIZUMAB with a C-terminal deletion of lysine (TOCILIZUMAB. DELTA. K, H chain WT-IgG 1. DELTA.K/SEQ ID NO: 68, L chain WT-. kappa./SEQ ID NO: 54), and TOCILIZUMAB with a C-terminal deletion of lysine and glycine (TOCILIZUMAB. DELTA. GK, H chain WT-IgG 1. DELTA. GK/SEQ ID NO: 69, L chain WT-. kappa./SEQ ID NO: 54) was evaluated by cation exchange chromatography. The column used ProPac WCX-10, 4X 250mm (Dionex), mobile phase A used 25mmol/L MES/NaOH, pH 6.1; the mobile phase B was carried out using 25mmol/L MES/NaOH, 250mmol/L NaCl, pH6.1, with appropriate flow rates and gradients. The results of the evaluation by cation exchange chromatography are shown in FIG. 12. As a result, it was found for the first time that heterogeneity of C-terminal amino acids can be reduced by deleting not only C-terminal lysine in the H chain constant region but also C-terminal lysine and glycine in the H chain constant region in advance in the nucleotide sequence. In human antibody constant regions IgG1, IgG2, and IgG4, the C-terminal sequences are all: the EU numbering system (refer to Sequences of proteins of immunologicalinterest, NIH Publication No.91-3242) changed from the 447 th to the 447 th and from the 446 th to the glycine, and thus considered that: the methods found in this study to reduce heterogeneity of C terminal amino acids may also be applied to IgG2 constant regions and IgG4 constant regions or variants thereof.

Reduction of disulfide bond-derived heterogeneity in IgG2 isotype of TOCILIZUMAB

The isotype of TOCILIZUMAB is IgG1, but since tocilizab is a neutralizing antibody, binding to Fc γ receptor may not be considered preferable in consideration of immunogenicity and side effects. As a method for reducing the binding to the Fc γ receptor, a method of changing the isotype of an IgG antibody from IgG1 to IgG2 or IgG4 (Ann Hematol.1998 Jun; 76 (6): 231-48) is considered, and IgG2(Nat Biotechnol.2007 Dec; 25 (12): 1369-72) is more preferable than IgG4 from the viewpoint of binding to the Fc γ receptor I and pharmacokinetics. On the other hand, when an antibody is developed as a pharmaceutical product, the physicochemical properties of the protein, and the uniformity and stability thereof are extremely important, and it has been reported that the heterogeneity of disulfide bonds derived from the hinge region is very large in the IgG2 isotype (J Biol chem.2008 Jun 6; 283 (23): 16206-15). It is not easy and cost-intensive to manufacture a large amount of a drug while maintaining the production difference of heterogeneity of a target substance/related substance derived therefrom, and therefore it is desirable to use a single substance as much as possible. Therefore, in the development of antibodies of the IgG2 isotype as pharmaceuticals, it is desirable to reduce disulfide-derived heterogeneity without reducing stability.

In order to reduce the heterogeneity of the IgG2 isotype, various variants were studied and found to: heterogeneity can be reduced without reducing stability in the IgG2 constant region sequence by WT-SKSC (SEQ ID NO: 70) which refers to a constant region in which a cysteine at position 131 and an arginine at position 133 in the EU numbering system of the CH1 domain of the H chain is substituted with a serine and a lysine, respectively, and a cysteine at position 219 in the EU numbering system of the upper hinge (upper hinge) of the H chain is substituted with a serine. TOCILIZUMAB-IgG1(H chain WT-IgG1/SEQ ID NO: 53, L chain WT- κ/SEQ ID NO: 54), TOCILIZUMAB-IgG2(H chain WT-IgG2/SEQ ID NO: 71, L chain WT- κ/SEQ ID NO: 54) and TOCILIZUMAB-SKSC (H chain WT-SKSC/SEQ ID NO: 70, L chain WT- κ/SEQ ID NO: 54) were prepared and evaluated for heterogeneity and stability. The heterogeneity was evaluated by cation exchange chromatography. The column was used ProPacWCX-10(Dionex), mobile phase A was used 20mM sodium acetate, pH 5.0; mobile phase B used 20mM sodium acetate, 1M NaCl, ph 5.0; with appropriate flow rates and gradients. The results of the evaluation by cation exchange chromatography are shown in FIG. 13. The stability was evaluated by evaluating the thermal denaturation intermediate temperature (Tm value) by Differential Scanning Calorimetry (DSC) (VP-DSC, manufactured by Microcal). The DSC measurement and Tm value of Fab domain under the conditions of 20mM sodium acetate, 150mM NaCl, pH6.0 are shown in FIG. 14.

As a result, it was found that: although TOCILIZUMAB-IgG2 shows a significant increase in heterogeneity as compared with TOCILIZUMAB-IgG1, the heterogeneity can be greatly reduced by forming TOCILIZUMAB-SKSC. In comparison with TOCILIZUMAB-IgG1, TOCILIZUMAB-IgG2 confirmed a shoulder (Fab) that is considered to be generated by a hetero component and has low stability, i.e., low Tm, in the heat-denatured peak of the Fab domain in DSC (Fab)^*) Component (b), but by forming TOCILIZUMAB-SKSC, the shoulder which is considered to be low in Tm value due to the hetero component disappears, showing that TOCILIZUMAB-The Tm values of about 94 ℃ equivalent to those of the Fab domains of IgG1 and TOCILIZUMAB-IgG2 were found to be: TOCILIZUMAB-SKSC has high stability.

Identification of constant region mutation sites that improve the pharmacokinetics of TOCILIZUMAB

As described above, it was found that heterogeneity of the C-terminal and antibody that can reduce the constant region of IgG2 isoform in a state of reducing binding to Fc γ receptor and maintaining high stability can be reduced by IgG1 that is an isotype of TOCILIZUMAB, but regarding pharmacokinetics, it is also desirable to have an excellent constant region than IgG1 that is an isotype of TOCILIZUMAB.

In order to find a constant region having an excellent half-life in plasma as compared with an antibody having a constant region of IgG1 isotype, for TOCILIZUMAB-SKSC having high stability and reduced heterogeneity as described above with respect to an antibody having a constant region of IgG2 isotype, mutation sites were screened in order to improve pharmacokinetics thereof, and as a result, it was found that: WT-M58(SEQ ID NO: 72 (amino acid sequence)) in which glutamic acid at position 137, serine at position 138, histidine at position 268, arginine at position 355, and glutamine at position 419 were substituted with glycine at position 446 and lysine at position 447, respectively, in the WT-SKSC in accordance with the EU numbering system, respectively, to form glycine, glycine at position 268, glutamine at position 355, and glutamine at position 419, respectively, and in addition thereto, glycine at position 446 and lysine at position 447 were deleted in order to reduce heterogeneity at the C-terminus of the H chain. On the other hand, WT-M44(SEQ ID NO: 73 (amino acid sequence)) was prepared in which asparagine at position 434 of IgG1 was substituted with alanine. Furthermore, WT-M83(SEQ ID NO: 74 (amino acid sequence)) in which the 446 th glycine and 447 th lysine of M44 were deleted was prepared in order to reduce the heterogeneity of the H chain C-terminus of M44. WT-M73(SEQ ID NO: 75 (amino acid sequence)) was prepared in which asparagine at position 434 of WT-M58 was substituted with alanine.

TOCILIZUMAB-M44(H chain WT-M44/SEQ ID NO: 73, L chain WT- κ/SEQ ID NO: 54), TOCILIZUMAB-M58(H chain WT-M58/SEQ ID NO: 72, L chain WT- κ/SEQ ID NO: 54) and TOCILIZUMAB-M73(H chain WT-M73/SEQ ID NO: 75, L chain WT- κ/SEQ ID NO: 54) were prepared and evaluated for affinity to human FcRn and pharmacokinetics in human FcRn transgenic mice (see reference examples for methods).

The binding of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 to human FcRn was evaluated by Biacore and shown in Table 6, with TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 binding about 2.7 times, about 1.4 times and about 3.8 times stronger than TOCILIZUMAB-IgG1, respectively.

[ Table 6]

The pharmacokinetics of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZU-MAB-M58 and TOCILIZUMAB-M73 in human FcRn transgenic mice were evaluated and the results are shown in FIG. 15. As shown in FIG. 15, it was found that TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZU-MAB-M73 all had improved pharmacokinetics compared to TOCILIZUMAB-IgG 1. This pharmacokinetic enhancing effect is associated with the binding capacity to human FcRn. Among them, TOCILIZUMAB-M73 showed an approximately 16-fold improvement in plasma concentration after 28 days, compared to TOCILIZUMAB-IgG1, and thus it was considered that: even in humans, the pharmacokinetics of antibodies with the constant region of M73 was greatly improved compared to antibodies with the constant region of IgG 1.

EXAMPLE 7 preparation of fully humanized IL-6 receptor antibody having improved PK/PD

When a variant of tocizumab in which a plurality of mutations of the variable region and the constant region of tocizumab found in the above examples are combined was prepared and subjected to various screens, it was found that: fv3-M73(H chain VH4-M73/SEQ ID NO: 25, L chain VL1- κ/SEQ ID NO: 28), Fv4-M73(H chain VH3-M73/SEQ ID NO: 26, L chain VL3- κ/SEQ ID NO: 29), Fv5-M83(H chain VH5-M83/SEQ ID NO: 27, L chain VL5- κ/SEQ ID NO: 30) as fully humanized IL-6 receptor antibodies.

The affinities of Fv3-M73, Fv4-M73 and Fv5-M83 to IL-6 receptor were compared with TOCILIZUMAB (see reference examples for methods). The affinity of the above antibody to the soluble IL-6 receptor was determined at pH7.4, and the results are shown in Table 7. In addition, the neutralizing activity of BaF/gp130 was compared with TOCILIZUMAB and a control (a known high affinity high IL-6 receptor antibody of reference example, VQ8F11-21hIgG1 of US 2007/0280945) (method reference example). The biological activities of BaF/gp130 produced by the above antibodies were measured, and the results are shown in FIG. 16 (final concentration of IL-6: 300 ng/mL: TOCILIZUMAB, control, Fv5-M83) and FIG. 17 (final concentration of IL-6: 30 ng/mL: TOCILIZUMAB, Fv3-M73, Fv 4-M73). As shown in Table 7, Fv3-M73 and Fv4-M73 have a strong affinity of about 2 to 3 times that of TOCILIZUMAB, and Fv5-M83 has a strong affinity of about 100 times that of TOCILIZUMAB (since the affinity is difficult to measure in Fv5-M83, Fv5-IgG1(H chain VH5-IgG1/SEQ ID NO: 76 and L chain VL5- κ/SEQ ID NO: 30) in which a constant region is formed into IgG1 were used to measure the affinity, and it is considered that the constant region does not generally affect the affinity). As shown in FIG. 17, Fv3-M73 and Fv4-M73 showed slightly stronger activity than TOCILIZUMAB, and as shown in FIG. 16, Fv5-M83 showed 100-fold or more strong activity at 50% inhibitory concentration than TOCILIZUMAB, and also showed about 10-fold higher neutralizing activity at 50% inhibitory concentration than the control (known high-affinity high-IL-6 receptor antibody).

[ Table 7]

The dissociation rates of TOCILIZUMAB, Fv3-M73 and Fv4-M73 from the membrane-type IL-6 receptor were determined at pH7.4 and pH5.8, and the results are shown in Table 8 (methods refer to reference examples). The results show that: the pH dependence of dissociation rates from the membrane-type IL-6 receptor was increased by approximately 11-fold and 10-fold for Fv3-M73 and Fv4-M73, respectively, compared to TOCILIZUMAB. The pH dependence of the dissociation rate was greatly improved compared to H3pI/L73 in example 5, and it is therefore considered that: compared with H3pI/L73, the pharmacokinetics of Fv3-M73 and Fv4-M73 are greatly improved.

[ Table 8]

The isoelectric points of TOCILIZUMAB, a control, Fv3-M73, Fv4-M73 and Fv5-M83 are measured by isoelectric point electrophoresis by a method known by a person skilled in the art, and as a result, the isoelectric point of TOCILIZUMAB is about 9.3, the isoelectric point of the control is about 8.4-8.5, the isoelectric point of Fv3-M73 is about 5.7-5.8, the isoelectric point of Fv4-M73 is about 5.6-5.7, and the isoelectric point of Fv5-M83 is 5.4-5.5, and the isoelectric points of all antibodies are greatly reduced compared with TOCILIZUMAB and the control. When the theoretical isoelectric point of the variable region VH/VL is calculated by genetyx (genetyxcoraperture), the theoretical isoelectric point of tocizumab is 9.20, the theoretical isoelectric point of the control is 7.79, the theoretical isoelectric point of Fv3-M73 is 5.49, the theoretical isoelectric point of Fv4-M73 is 5.01, and the theoretical isoelectric point of Fv5-M83 is 4.27, and the isoelectric points of all antibodies are greatly reduced compared with tocizumab and the control. Example 2 shows that: by lowering the isoelectric point, pharmacokinetics is improved, and it is thus believed that: compared with TOCILIZUMAB and the control, the pharmacokinetics of Fv3-M73, Fv4-M73 and Fv5-M83 are improved.

T-cell epitopes present in the variable region sequences of TOCILIZUMAB, Fv3-M73, Fv4-M73 and Fv5-M83 were analyzed using TEPITOPE (methods.2004 Dec; 34 (4): 468-75). As a result, as shown in example 3, although T-cell epitopes that bind to HLA were predicted to exist in the plurality of sequences of TOCILIZUMAB, the sequences of Fv3-M73, Fv4-M73 and Fv5-M83 that are predicted to bind to T-cell epitopes were greatly reduced. Furthermore, Fv3-M73, Fv4-M73 and Fv5-M83 were fully humanized, leaving no mouse sequences in the framework. This indicates that: compared with TOCILIZUMAB, the immunogenicity risks of Fv3-M73, Fv4-M73 and Fv5-M83 are likely to be greatly reduced.

EXAMPLE 8 PK/PD assay of fully humanized IL-6 receptor antibody in monkeys

TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 were administered to cynomolgus monkeys at 1mg/kg in a single intravenous administration to evaluate the change in plasma concentration (method reference example). The changes in plasma concentrations following intravenous administration of TOCILIZUMAB, Fv3-M73, Fv4-M73 and Fv5-M83 are shown in FIG. 18. As a result, Fv3-M73, Fv4-M73 and Fv5-M83 showed significantly improved pharmacokinetics in cynomolgus monkeys compared to TOCILIZUMAB and the control. In which the pharmacokinetics of Fv3-M73 and Fv4-M73 were greatly improved compared to TOCILIZUMAB.

To evaluate the efficacy of neutralizing the cynomolgus monkey membrane type IL-6 receptor to some extent, cynomolgus monkey IL-6 was subcutaneously administered in an amount of 5. mu.g/kg continuously on the lumbar and back on days 6 to 18 (days 3 to 10 in TOCILIZUMAB) after antibody administration, and CRP concentration was measured 24 hours later for each individual (see reference example for the method). The change in CRP concentration at each antibody administration is shown in figure 19. To evaluate the efficacy of neutralizing cynomolgus monkey soluble IL-6 receptor to some extent, the concentration of non-binding cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured and the rate of non-binding soluble IL-6 receptor was calculated (see reference example for the method). The change in the rate of non-conjugated soluble IL-6 receptor upon administration of each antibody is shown in FIG. 20.

Fv3-M73, Fv4-M73, and Fv5-M83 all neutralized the cynomolgus monkey membrane type IL-6 receptor more consistently and inhibited the increase in CRP for a longer period of time than TOCILIZUMAB and controls (well known high affinity anti-IL-6 receptor antibodies). Fv3-M73, Fv4-M73, and Fv5-M83 all more consistently neutralized cynomolgus soluble IL-6 receptors and inhibited the increase in non-binding cynomolgus soluble IL-6 receptors for a longer period of time than TOCILIZUMAB and controls. It was thus found that: with respect to the persistence of neutralization of the membrane type IL-6 receptor and the soluble type IL-6 receptor, Fv3-M73, Fv4-M73 and Fv5-M83 were all superior to TOCILIZUMAB and the control. Among them, Fv3-M73 and Fv4-M73 were extremely excellent in the persistence of neutralization. On the other hand, compared with Fv3-M73 and Fv4-M73, Fv5-M83 inhibits CRP and a non-conjugated cynomolgus monkey soluble type IL-6 receptor at a low level, and thus it is considered that: fv5-M83 neutralized both membrane-type IL-6 receptor and soluble IL-6 receptor more strongly than Fv3-M73, Fv4-M73, and the control (well-known high affinity anti-IL-6 receptor antibodies). This is considered to be: compared with the control, Fv5-M83 has strong affinity with IL-6 receptor and strong biological activity in BaF/gp130, and is reflected in cynomolgus monkey.

It is thus assumed that: compared with TOCILIZUMAB and the control, Fv3-M73 and Fv4-M73 have extremely excellent persistence of action as anti-IL-6 receptor neutralizing antibodies, and can greatly reduce the administration frequency and the administration amount; fv5-M83 was found to have extremely excellent action intensity and excellent persistence of action as an anti-IL-6 receptor neutralizing antibody. Therefore, Fv3-M73, Fv4-M73 and Fv5-M83 are believed to be useful as IL-6 antagonists for pharmaceutical products.

[ example 9]

Monocyte Chemotactic Protein (MCP) -1 is known to be involved in cell invasion of monocytes, T cells, NK cells, basophils. It has been reported that: MCP-1 is highly expressed in synovial tissue and fluid of RA patients (J Clin invest.1992 Sep; 90 (3): 772-9), and is considered to be involved in the pathogenesis of RA (Inflamm early Drug targets.2008 Mar; 7 (1): 53-66).

VEGF is known to be a potent angiogenic factor produced by macrophages, fibroblasts, synovial cells, etc. in the synovium of RA patients (J Rheumatotol.1995 Sep; 22 (9): 1624-30). In addition, VEGF levels in the serum of RA patients are associated with disease activity or radiation progression (radiologic progression) (Arthritis Rheum.2003 Jun; 48 (6): 1521-9., Arthritis Rheum.2001 Sep; 44 (9): 2055-64), and by treating RA patients with anti-IL-6R antibody TOCILIZUMAB, VEGF levels in the serum are reduced, thereby suggesting that: VEGF also plays an important role in the pathogenesis of RA (Mod Rheumatotol. 2009; 19 (1): 12-9., Mediators Inflamm. 2008; 2008: 129873).

Thus, the following method was used to investigate whether TOCILIZUMAB and Fv4-M73 could inhibit MCP-1 and VEGF production from synovial cells of human RA patients, which were stimulated by sIL-6R and IL-6.

Synoviocytes (TOYOBO) from human RA patients were added to IMDM medium with 5% FCS at 2X 10⁴0.05 mL/well was inoculated into a 96-well plate and allowed to stand in an incubator at 37 ℃ with 5% CO2 for 90 minutes. Adding 0.05mL of appropriately diluted TOCILIZUMAB and Fv4-M73, and standingAfter 15 minutes, 0.05mL of soluble IL-6 receptor (SR 344: prepared by the method described in reference example) was added, and the mixture was allowed to stand for 30 minutes, followed by further addition of 0.05mL of IL-6(TORAY) (both soluble IL-6 receptor and IL-6 were added at a final concentration of 50 ng/mL). Culture supernatants were recovered after 2 days of culture, and the concentrations of MCP-1 and VEGF in the culture supernatants were determined using ELISA kits (Biosource and Pierce Biotechnology). The results are shown in FIGS. 21 and 22. TOCILIZUMAB and Fv4-M73 concentration-dependently inhibited MCP-1 and VEGF production from synovial cells of human RA patients stimulated by soluble IL-6 receptors and IL-6.

Thus, it can be seen that: fv4-M73, which is an anti-IL-6 receptor neutralizing antibody, is extremely superior in persistence of its action (binding to IL-6 receptor, blocking signals of membrane type IL-6 receptor and soluble type IL-6 receptor) as compared with TOCILIZUMAB, can greatly reduce the frequency and dosage as compared with TOCILIZUMAB, and Fv4-M73 inhibits MCP-1 and VEGF production from synovial cells of human RA patients, thereby showing that: fv4-M73 is a very useful therapeutic agent for RA.

[ reference example ]

Preparation of recombinant soluble human IL-6 receptor

As an antigen of the human IL-6 receptor recombinant soluble type of IL-6 receptor preparation as follows. A CHO cell constant expression strain (a known strain) was prepared for the soluble human IL-6 receptor comprising the amino acid sequences from position 1 to position 344 on the N-terminal side as reported in J.biochem.108, 673-once 676(1990) (Yamasaki et al, Science 1988; 241: 825-once 828(GenBank # X12830)). Soluble human IL-6 receptor was purified from the culture supernatant obtained from SR 344-expressing CHO cells by using 3 kinds of column chromatography, namely Blue Sepharose 6FF column chromatography, affinity chromatography using a column to which an SR 344-specific antibody is immobilized, and gel filtration column chromatography. The component eluted as the main peak was used as the final pure product.

Preparation of recombinant soluble cynomolgus IL-6 receptor (cIL-6R)

An oligo DNA primer was prepared based on the disclosed rhesus IL-6 receptor gene sequence (Birney et al, Ensembl2006, Nucleic Acids Res.2006 Jan 1: 34(Database issue): D556-61), and a DNA fragment encoding the full length of the cynomolgus IL-6 receptor gene was prepared by PCR using the cDNA prepared from the cynomolgus monkey pancreas as a template. The obtained DNA fragment was inserted into a mammalian cell expression vector, and a CHO constant expression strain (cyno. sIL-6R-producing CHO cell) was prepared using the vector. The culture broth of sIL-6R-producing CHO cells was purified by HisTrap column (GE Healthcare Bioscience), concentrated by Amiconultra-15 Ultracel-10k (Millipore), and further purified by Superdex200pg16/60 gel filtration column (GE Healthcare Bioscience) to obtain a final pure soluble cynomolgus IL-6 receptor (hereinafter referred to as "cIL-6R").

Preparation of recombinant cynomolgus monkey IL-6(cIL-6)

Cynomolgus IL-6 was prepared as follows. A nucleotide sequence encoding 212 amino acids registered in SWISSPROT access No. p79341 was prepared, cloned into a mammalian cell expression vector, and introduced into CHO cells to prepare a constantly expressing cell line (cyno. il-6-producing CHO cells). IL-6 CHO cell-producing culture broth was purified by SP-Sepharose/FF column (GE Healthcare Bioscience), concentrated by Amicon Ultra-15 Ultracel-5k (Millipore), further purified by Superdex75pg26/60 gel filtration column (GE Healthcare Bioscience), and concentrated by Amicon Ultra-15 Ultracel-5k (Millipore) to give a pure cynomolgus IL-6 (hereinafter referred to as "cIL-6") as a final product.

Preparation of high affinity anti-IL-6 receptor antibody

As a known high affinity anti-IL-6 receptor antibody, a vector for mammalian cell expression was constructed in order to express VQ8F11-21hIgG1(US 2007/0280945A1, H chain amino acid sequence: SEQ ID NO: 77, L chain amino acid sequence: SEQ ID NO: 78), which is a high affinity anti-IL-6 receptor antibody described in US 2007/0280945A 1. Antibody variable regions were prepared by PCR (assembly PCR) combined with synthetic oligo-DNA, and IgG1 was used as the constant region. The antibody variable region and the constant region were bound by the assembly PCR method and inserted into a vector for mammalian expression to prepare the desired H chain expression vector and L chain expression vector. The nucleotide sequence of the resulting expression vector is determined by methods well known to those skilled in the art. The expression vector thus prepared was used for expression and purification. Expression and purification were carried out by the method described in example 1 to obtain a high-affinity anti-IL-6 receptor antibody (hereinafter referred to as "control").

Preparation, expression and purification of TOCILIZUMAB mutant

The mutants of TOCILIZUMAB were prepared by the method described in the attached manual using QuikChange site-directed mutagenesis kit (Stratagene), and the obtained plasmid fragments were inserted into mammalian cell expression vectors to prepare the objective H chain expression vector and L chain expression vector. The nucleotide sequence of the resulting expression vector is determined according to methods well known to those skilled in the art. The expression of the antibody was carried out by the following method. HEK293H strain (Invitrogen) derived from human embryonic kidney cancer cells was suspended in a DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen) at 5-6 × 10⁵Cell Density/mL 10mL of each adherent cell culture dish (10 cm diameter, CORNING) was seeded with 5% CO at 37 ℃%₂After culturing in the incubator of (1) for a whole day and night, the medium was aspirated, and 6.9mL of CHO-S-SFM-II (Invitrogen) medium was added. The prepared plasmid was introduced into cells by lipofection. The resulting culture supernatant was recovered, then centrifuged at room temperature at a centrifugal force of about 2000g for 5 minutes to remove the cells, and sterilized by passing through a 0.22 μm filter MILLEX (R) -GV (Millipore) to obtain a culture supernatant. Using rProtein A Sepharose^TMFast Flow (Amersham Biosciences) and the antibody was purified from the resulting culture supernatant according to a method known to those skilled in the art. As for the purified antibody concentration, the absorbance at 280nm was measured using a spectrophotometer. The absorbance was calculated from the obtained values by the PACE method, and the antibody concentration was calculated using the absorbance (Protein Science 1995; 4: 2411-2423).

Establishment of human gp130 expression BaF3 cell line

To obtain a cell line showing IL-6-dependent proliferation, a BaF3 cell line expressing human gp130 was established as follows.

The DHFR gene expression site of pCHOI (Hirata et al, FEBSLetter 1994; 356: 244-. The full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1 (+).

Mu.g of pCOS2Zeo/gp130 were mixed with BaF3 cells (0.8X 10) suspended in PBS⁷Cells), a pulse was applied at a capacity of 950. mu. FD and 0.33kV using Gene Pulser (Bio-Rad). BaF3 cells into which a gene had been introduced by electroporation were cultured in RPMI1640 medium (Invitrogen) containing 0.2ng/mL of mouse interleukin-3 (Peprotech) and 10% fetal bovine serum (hereinafter referred to as "FBS" and HyClone) for a whole day and night, and human interleukin-6 (R) containing 100ng/mL was added&D systems), 100ng/mL human interleukin-6 soluble receptor (R)&D systems) and 10% FBS in RPMI1640 medium, and establishing a human gp 130-expressing BaF3 cell line (hereinafter referred to as "BaF 3/gp 130"). Because the BaF/gp130 is in human interleukin-6 (R)&D systems) and soluble human IL-6 receptor, and can be used to evaluate the proliferation inhibitory activity of anti-IL-6 receptor antibodies (i.e., IL-6 receptor neutralizing activity).

Evaluation of the biological Activity of human gp130 expressing BaF3 cells (BaF/gp130)

IL-6 receptor neutralizing activity was evaluated using BaF3/gp130, which showed IL-6/IL-6 receptor dependent proliferation. The BaF3/gp130 was washed 3 times with 10% FBS-containing RPMI1640 medium, and suspended in 600 ng/mL-60 ng/mL human interleukin-6 (TORAY) (final concentration 300 ng/mL-30 ng/mL), appropriate amount of soluble human IL-6 receptor and 10% FBS-containing RPMI1640 medium to 5X 10⁴At a concentration of cells/mL, 50. mu.L of each well of a 96-well plate (CORNING) was injected. Next, the purified antibody is subjected toDiluted with 10% FBS in RPMI1640, 50. mu.L of each well was mixed. At 37 deg.C, 5% CO₂Was cultured for 3 days under the conditions of (1), WST-8 reagent (Cell Counting Kit-8, Kyowa Kagaku Co., Ltd.) diluted 2-fold with PBS was added at 20. mu.L/well, and immediately thereafter absorbance at 450nm (reference wavelength: 620nm) was measured using SUNRISE CLASSIC (TECAN). After 2 hours of incubation, the absorbance at 450nm (reference wavelength: 620nm) was measured again, and IL-6 receptor neutralizing activity was evaluated using the change in absorbance for 2 hours as an index.

Evaluation of binding to soluble human IL-6 receptor by Biacore

Analysis of velocity theory of antigen-antibody reaction was performed using Biacore T100(GE Healthcare). An appropriate amount of protein A or protein A/G or anti-IgG (gamma-chain specific) F (ab') 2 was immobilized on a sensor chip by an amine coupling method, followed by binding of a target antibody at pH7.4, flowing soluble IL-6 receptor prepared at pH7.4 at various concentrations in the form of an analyte, and determining the interaction of the antibody with soluble human IL-6 receptor. All measurements were performed at 37 ℃. Calculating a kinetic parameter, i.e., a binding rate constant k, from the sensor map obtained by the measurement_a(1/Ms) and dissociation rate constant k_d(1/s) and calculating K from the value_D(M). Each parameter was calculated using biacore t100 evaluation software (GE Healthcare).

Evaluation of pH-dependent dissociation from membrane-type IL-6 receptor using Biacore

Biacore T100(GE Healthcare) was used to observe antigen-antibody reactions with membrane type IL-6 receptors at pH5.8, pH 7.4. Binding to the membrane-type IL-6 receptor was evaluated by evaluating binding to the soluble-type human IL-6 receptor immobilized on the sensor chip. SR344 is biotinylated according to methods well known to those skilled in the art, and the biotinylated soluble form of human IL-6 receptor is immobilized on the sensor chip via streptavidin using the affinity of streptavidin for biotin. All assays were performed at 37 ℃ with the mobile phase buffer 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20, and injected at pH7.4After pH-dependent binding clones were allowed to bind to the soluble human IL-6 receptor (sample buffer 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 was injected), pH-dependent dissociation of each clone was observed at pH5.8 in the mobile phase. Using Biacore T100 evaluation software (GEHealthcare), only the dissociation phase at pH5.8 was fitted when the sample concentration was 0.25. mu.g/mL, bound in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20, and dissociated in 10mM MES pH5.8, 150mM NaCl, 0.05% Tween20, to calculate the dissociation rate constant (k.k.sub.5.8) at pH5.8_d(1/s)). Similarly, the dissociation rate constant (k.k.sub.k.sub.7.4) at pH7.4 was calculated by fitting only the dissociation phases at pH7.4 when the sample concentration was 0.5. mu.g/mL, bound in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20, and dissociated in 10mM MES pH7.4, 150mM NaCl, 0.05% Tween20 using Biacore T100 evaluation software (GE Healthcare)_d(1/s))。

Evaluation of binding to human FcRn

FcRn is a complex of FcRn and β 2-microglobulin. Oligo DNA primers were prepared based on the published sequence of the human FcRn gene (J.Exp.Med.180(6), 2377-2381 (1994)). Using the Human cDNA (Human centa Marathon-Ready cDNA, Clontech) as a template, a DNA fragment encoding the full length of the gene was adjusted by PCR using the prepared primers. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular domain (Met1-Leu290) containing a signal region was amplified by PCR and inserted into a mammalian cell expression vector (human FcRn amino acid sequence/SEQ ID NO: 79). Similarly, oligo DNA primers were prepared based on the disclosed human β 2-microglobulin gene sequence (Proc. Natl. Acad. Sci. U.S.A.99(26), 16899-169903 (2002)). Using the prepared primers, a DNA fragment encoding the full length of the gene was prepared by PCR using human cDNA (Hu-planta Marathon-Ready cDNA, CLONTECH) as a template. The resulting DNA fragment was used as a template to amplify a DNA fragment encoding the full length of β 2-microglobulin (Met1-Met119) comprising a signal region by PCR, and inserted into a mammalian cell expression vector (human β 2-microglobulin amino acid sequence/SEQ ID NO: 80).

Expression of soluble human FcRn was performed according to the following procedure. The prepared plasmids of human FcRn and human β 2-microglobulin were introduced into cells of HEK293H strain (Invitrogen) derived from human embryonic kidney cancer cells by lipofection using 10% fetal bovine serum (Invitrogen). The resulting culture supernatant was collected and then purified by the method of (J Immunol.2002 Nov 1; 169 (9): 5171-80) using IgG Sepharose 6 Fast Flow (Amersham biosciences). Thereafter, purification was performed by HiTrap Q HP (GE Healthcare).

Determination of antibody concentration in mouse plasma

The antibody concentration in the mouse plasma was measured by ELISA using a method known to those skilled in the art.

Antibody concentration, CRP concentration, non-conjugated soluble IL-6 in plasma of monkey PK/PD assay Assays for receptors

The concentration in cynomolgus monkey plasma was measured by ELISA method using a method known to those skilled in the art.

CRP concentration was measured using an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.) using Cias R CRP (Kanto chemical Co., Ltd.).

The concentration of non-binding soluble cynomolgus IL-6 receptor in cynomolgus plasma was determined as follows. mu.L of cynomolgus plasma was placed in a 0.22 μm filter (Millipore), and an appropriate amount of dried rProtein A Sepharose Fast Flow (GE Healthcare) resin was added to adsorb all IgG type antibodies (cynomolgus IgG, anti-human IL-6 receptor antibody, and anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex) present in the plasma on protein A. Thereafter, the solution passed through the centrifuge was spun down (spin-down) to recover the solution. Since the solution passed through does not contain the anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex bound to protein A, the concentration of non-bound soluble cynomolgus IL-6 receptor can be measured by measuring the concentration of soluble cynomolgus IL-6 receptor in the solution passed through protein A. The concentration of soluble cynomolgus monkey IL-6 receptor was measured by a method known to those skilled in the art for measuring the concentration of human IL-6 receptor, using the soluble cynomolgus monkey IL-6 receptor (cIL-6R) prepared as described above as a standard. The rate of the non-binding soluble IL-6 receptor was calculated by the following formula.

(concentration of non-binding soluble IL-6 receptor after antibody administration ÷ concentration of soluble IL-6 receptor before antibody administration). times.100.

Claims

1. An antibody comprising a heavy chain variable region comprising CDR1 consisting of the sequence of SEQ ID NO: 1, CDR2 consisting of the sequence of SEQ ID NO: 2 and CDR3 consisting of the sequence of SEQ ID NO: 3, wherein SEQ ID NO: 1 is CDR1 of VH4-M73, SEQ ID NO: 2 is CDR2 of VH4-M73, and SEQ ID NO: 3 is CDR3 of VH 4-M73;

the light chain variable region comprises CDR1 consisting of the sequence of SEQ ID NO. 10, CDR2 consisting of the sequence of SEQ ID NO. 11 and CDR3 consisting of the sequence of SEQ ID NO. 12, wherein SEQ ID NO. 10 is CDR1 of VL1, SEQ ID NO. 11 is CDR2 of VL1, and SEQ ID NO. 12 is CDR3 of VL 1.

2. An antibody comprising a heavy chain variable region consisting of the sequence of SEQ ID NO 19 and a light chain variable region consisting of the sequence of SEQ ID NO 22, wherein SEQ ID NO 19 is the variable region of VH4-M73 and SEQ ID NO 22 is the variable region of VL 1.

3. An antibody comprising a heavy chain consisting of the sequence of SEQ ID NO. 25 and a light chain consisting of the sequence of SEQ ID NO. 28, wherein SEQ ID NO. 25 is VH4-M73 and SEQ ID NO. 28 is VL 1.

4. A gene encoding the antibody according to any one of claims 1 to 3.

5. A vector comprising the gene of claim 4.

6. A host cell comprising the vector of claim 5.

7. A method for producing the antibody according to any one of claims 1 to 3 by culturing the host cell according to claim 6.

8. A pharmaceutical composition for treating IL-6 related diseases comprising an antibody according to any one of claims 1 to 3 or an antibody prepared according to the method of claim 7.