TWI440470B - Pharmaceutical formulation containing improved antibody molecules - Google Patents

Description

Pharmaceutical formula containing modified antibody molecules

The present invention relates to a formulation containing a multi-peptide and/or antibody, and more particularly to a stable liquid formulation containing a high concentration of a modified anti-IL6 receptor antibody.

In recent years, various antibody formulations have been developed and put into practical use. A variety of formulations are used for intravenous injection. Since the amount of antibody administered per time is large (about 100 mg to 200 mg), and the volume of subcutaneous injection is usually limited, the antibody-containing formulation designed for subcutaneous injection needs to increase the concentration of the antibody in the liquid.

Undesirable disintegration occurs in solutions containing high concentrations of antibodies, which include the formation of insoluble and/or soluble agglomerates. Such insoluble and soluble agglomerates may form in a liquid state due to binding to antibody molecules. When the formulation liquid is stored for a long period of time, the biological activity of the antibody molecule may be lost or reduced due to deamidation of the asparagine residue. The freezing and thawing cycles also form disintegrating and agglutinating antibody molecules.

A number of proposals have been made for providing a stabilizing formulation in which the loss of active ingredient is reduced even when the formulation is stored for a long period of time. Such a formulation is obtained by dissolving an active ingredient and various additives in a buffer solution. There is still a need for a formulation containing a high concentration of antibodies that inhibits dimerization and deuteration during prolonged storage, and is stable and suitable for subcutaneous administration.

Since antibodies are highly stable in plasma and have few side effects, they are attracting attention as pharmaceuticals. Among them, a variety of IgG-type antibody drugs are available on the market, and many antibody drugs are currently under development (Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Monoclonal antibody successes in the clinic, Nature Biotechnology 23, 1073-1078 (2005) and Pavlou AK, Belsey MJ., The therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59(3): 389-96). IL-6 is a cytokine involved in a variety of autoimmune diseases, inflammatory diseases, malignant tumors, etc. (Nishimoto N, Kishimoto T., Interleukin 6: from bench to bedside., Nat Clin Pract Rheumatol. 2006 Nov; 2 (11 ): 619-26). TOCILIZUMAB is a humanized anti-IL-6 receptor IgG1 antibody that is specifically linked to the IL-6 receptor. TOCILIZUMAB neutralizes the biological activity of IL-6 and is therefore considered to be a therapeutic agent for IL-6-related diseases, such as rheumatoid arthritis (WO 92/19759, WO 96/11020, WO 96/12503, and Maini). RN, Taylor PC, Szechinski J, Pavelka K, Broll J, Balint G, Emery P, Raemen F, Petersen J, Smolen J, Thomson, D, Kishimoto T; CHARISMA Study Group., Double-blind randomized controlled clinical trial of the Interleukin-6 receptor antagonist, Tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate., Arthritis Rheum, 2006 Sep; 54(9): 2817-29). In Japan, TOCILIZUMAB has been proven to be a therapeutic agent for Castleman disease and rheumatoid arthritis (Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y ,Sasaki T,Nishioka K,Hara M,Taguchi H,Kimura Y,Kato Y,Asaoku H,Kumagai S,Kodama F,Nakahara H,Hagihara K,Yoshizaki K,Kishimoto T. Humanized anti-interleukin-6 receptor antibody treatment of Multicentric Castleman disease. Blood. 2005 Oct 15;106(8):2627-32).

Humanized antibodies, such as TOCILIZUMAB, are first generation antibody drugs. In order to improve the efficacy, convenience and cost of first-generation antibody drugs, second-generation antibody drugs are currently being developed. A variety of techniques are available for the development of second-generation antibody drugs. Techniques for enhancing effector function, antigen binding ability, pharmacokinetics, and stability, as well as techniques for reducing the risk of immunogenicity have been reported. As a method for enhancing drug potency or reducing dose, it has been reported to enhance antibody-dependent cellular cytotoxic activity (ADCC activity) or complement-dependent cytotoxic activity (CDC activity) by amino acid substitution of the Fc region of an IgG antibody. (Kim SJ, Park Y, Hong HJ., Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1): 17-29. Review). Furthermore, Affinity maturation has been reported as a technique for enhancing antigen binding ability or antigen neutralization ability (Rothe A, Hosse RJ, Power BE. Ribosome display for improved biotherapeutic molecules. Expert Opin Biol Ther. 2006 Feb;6(2):177-87). This technique enhances antigen binding activity by introducing an amino acid mutation to the complementarity determining (CDR) region of the variant region or the like. This enhanced antigen binding capacity improves in vitro biological activity or reduces dosage, and thus improves efficacy in vivo (Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R. A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci USA. 2005 Jun 14; 102(24): 8466-71. Epub 2005 Jun 6). Now, Motavizumab (produced by affinity maturation) is undergoing clinical trials, and it is expected to have superior effects compared to the first generation of anti-RSV antibody drugs, Palivizumab (Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM , Patel NK, White WI, Young JF, Kiener PA. Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract. J Mol Biol. 2007, 368, 652-665). An anti-IL-6 receptor antibody having an affinity of about 0.05 nM has been reported (i.e., has greater affinity than TOCILIZUMAB) (WO 2007/143168). However, human antibodies, humanized antibodies or chimeric antibodies having an affinity higher than 0.05 nM have not been reported.

One problem faced by current antibody pharmaceuticals is the high production cost associated with the administration of extremely large amounts of protein. For example, TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody, is administered intravenously at an estimated dose of about 8 mg/kg/month (Maini RN, Taylor PC, Szechinski J, Pavelka K, Broll J, Balint G, Emery P, Raemen F, Petersen J, Smolen J, Thomson D, Kishimoto T; CHARISMA Study Group., Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist. Tocilizumab, In European patients with rheumatoid arthritis who had an incomplete response To methotrexate., Arthritis Rheum. 2006 Sep;54(9):2817-29). In chronic autoimmune diseases, the preferred form of administration is a subcutaneous formulation. In general, subcutaneous formulations require a high concentration formulation. From the standpoint of stability and the like, the limit of the IgG type antibody formulation is generally about 100 mg/ml (Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004 Jun; 93 (6 ): 1390-402). By increasing the half-life of the antibody in plasma to prolong its efficacy and impart high stability to the antibody, it provides a low-cost, convenient second-generation antibody drug that can be administered subcutaneously and at long intervals, thereby reducing The amount of protein administered.

FcRn is closely related to antibody pharmacokinetics. Regarding the difference in plasma half-life of isotype antibodies, it is known that IgG1 and IgG2 have superior plasma half-life compared to IgG3 and IgG4 (Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25(12): 1369-72) . As a method for further improving IgG1 and IgG2 antibodies having superior plasma half-lives, a method of replacing the amino acid in a constant region to enhance binding to FcRn is known (Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurshita N, m An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1;176(1):346-56 and Ghetie V, Popov S, Borvak J, RaduC, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul; 15(7): 637-40). From the standpoint of immunogenicity, the substitution of the amino acid of the variable region compared to the constant region further improves the plasma half-life (WO 2007/114319). However, to date, no method for improving the plasma half-life of IL-6 receptor antibodies by altering the variable region has been reported.

Another important issue encountered in the development of biopharmaceuticals is immunogenicity. In general, the immunogenicity of mouse antibodies is reduced by antibody humanization. It is speculated that the risk of immunogenicity can be further reduced by using the germline framework sequence as a template for antibody humanization (Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of vital germline) Genes in a CDR homology-based approach to antibody humanization. Methods. 2005 May; 36(1): 35-42). However, even human anti-TNF antibodies, Adalimumab, showed high frequency (13% to 17%) of immunogenicity, and the therapeutic effect was found to be reduced in patients with immunogenicity (Bartelds GM, Wijbrandts CA, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA, Tak P, Wolbink GJ. Clinical response to adalimumab: The relationship with anti-adalimumab antibodies and serum adalimumab concentration in rheumatoid arthritis. Ann Rheum Dis. 2007 Mar 9; Epub ahead of print]and Bender NK, Heilig CE, Droll B, Wohlgemuth J, Armbruster FP, Heilig B. Immunogenecity, efficacy and adverse events of adalimumab in RA patients. Rheumatol Int. 2007 Jan;27(3):269-74 ). Even the CDRs of human antibodies may have T cell epitopes, and the T cell epitopes of the CDRs may be immunogenic. In silico and in vivo methods for predicting T cell epitopes (Van Walle I, Gansemans Y, Parren PW, Stas P, Lasters I. Immunogenicity screening in protein drug development. Expert Opin Biol Ther 2007 Mar;7(3):405-18 and Jones TD,Philips WJ,Smith BJ,Bamford CA,Nayee PD,Baglin TP,Gaston JS,Baker MP. Identification and removal of a promiscuous CD4+ T cell epitope from the C1 Domain of factor VIII. J Thromb Haemost. 2005 May;3(5):991-1000). It is speculated that the risk of immunogenicity can be reduced by using these methods to remove T cell epitopes (Chirino AJ, Ary ML, Marshall SA. Minimizing the immunogenicity of protein therapeutics. Drug Discov Today. 2004 Jan 15; 9 ( 2): 82-90).

The humanized anti-IL-6 receptor IgG1 antibody, TOCILIZUMAB, is an IgG1 antibody obtained by humanizing mouse antibody PM1. The CDR grafting was carried out using the human NEW and REI sequences as template frameworks for the H and L chains, respectively. However, in order to maintain activity, there are 5 mouse sequence amino acids remaining in the framework as essential amino acids (Sato K, Tsuchiya M, Saldanha J, Koishihara Y, Ohsugi Y, Kishimoto T, Bendig MM. Reshaping a human antibody To inhibit the interleukin 6-dependent tumor cell growth. Cancer Res. 1993 Feb 15; 53(4): 851-6). None of the remaining mouse sequences in the humanized antibody TOCILIZUMAB framework have been previously reported to be humanized without reducing the activity. Furthermore, the CDR sequences of TOCILIZUMAB are mouse sequences and, therefore, like Adalimumab, there may be T cell epitopes in the CDRs, and there may be a potential risk of immunogenicity. In the clinical trial of TOCILIZUMAB, no anti-TOCILIZUMAB antibody was detected in the effective dose of 8 mg/kg, but was observed at doses of 2 mg/kg and 4 mg/kg (WO 2004/096273). This shows that there is still room for improvement regarding the immunogenicity of TOCILIZUMAB. However, there is no report of reducing the risk of TOCILIZUMAB immunogenicity by amino acid substitution.

The TOCILIZUMAB isotype is IgG1. The difference in this isoform refers to the difference in the sequence of the constant region. Since the constant region sequence is presumed to have a strong influence on effector function, drug kinetics, physical properties, etc., it is important to select a constant region sequence when developing antibody pharmaceuticals (Salfeld JG. Isotype selection in antibody engineering. Nat Biotechnol. 2007 Dec;25(12):1369-72). In recent years, the safety of antibody pharmaceuticals has become very important. The interaction of the Fc portion of the antibody with the Fc gamma receptor (effector function) may cause severe negative effects in the Phase I clinical trial of TGN1412 (Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned future directions Nat Rev Drug Discov. 2007 Jan;6(1):75-92). For antibody pharmaceuticals designed to neutralize the biological activity of antigens, binding to Fc gamma receptors is important for effector functions such as ADCC. From a negative effect point of view, binding to an Fc gamma receptor is even unfavorable. A method for reducing binding to an Fc gamma receptor is to change an IgG antibody isoform from IgG1 to IgG2 or IgG4 (Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann Hematol. 1998 Jun; 76 ( 6): 231-248). From the standpoint of pharmacokinetics and Fcγ receptor binding, IgG2 is more suitable than IgG4 (Salfeld JG. Isotyper selection in antibody engineering. Nat Biotechnol. 2007 Dec; 25(12): 1369-72). TOCILIZUMAB is a neutralizing antibody of IL-6 receptor, and its isoform is IgG1. Therefore, IgG2 may be a preferred isoform from the standpoint of potential adverse effects, since effector functions such as ADCC are not required.

At the same time, when developing antibody pharmaceuticals, the physicochemical properties of proteins, especially homogeneity and stability, are critical. IgG2 isoforms are known to have significant heterogeneity derived from the disulfide bond of the hinge region (DillonTM, Ricci MS, Vezina C, Flynn GC, Liu YD, Rehder DS, Plant M, Henkle B, Li Y, Deechongkit S, Varnum B, Wypych J, Balland A, Bondarenko PV. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008 Jun 6; 283(23): 16206-15). In order to maintain the heterogeneity associated with a target substance/related substance derived from a disulfide bond between productions, mass production into a pharmaceutical product is not easy and may be more costly. Therefore, it is desirable to be as single substance as possible. Furthermore, for the heterogeneity of the C-terminal sequence of the H chain of the antibody, the C-terminal amino acid lysine residue has been deleted, and the guanamine which removes the amino acid of the two C-terminal glycine and the lysine has been removed. Report on the C-terminal carboxyl group (Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2007 Jan 1;360 (1): 75-83). When developing an IgG2 isotype antibody as a pharmaceutical, it is preferred to reduce such heterogeneity and maintain high stability. In order to produce a convenient, stable, high-concentration, subcutaneous administration formulation, it is preferred to have not only high stability, but also a plasma half-life superior to that of TOCILIZUMAB isoform IgG1. However, no one has reported a cross-sectional region sequence of an antibody having an IgG2 isoform constant region, which has reduced heterogeneity, high stability, and superior plasma half-life compared to an antibody having an IgG1 isoform constant region.

It is an object of the present invention to provide a pharmaceutical formulation (hereinafter also referred to as "agent" or "pharmaceutical composition" comprising a second generation molecule superior to the humanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB by modification The amino acid sequence of the variable and constant regions of TOCILIZUMAB enhances antigen neutralization and improves drug motility, enabling prolonged therapeutic effects at lower frequency of administration, and improving immunogenicity, safety, and physics. Chemical properties (stability and homogeneity).

Furthermore, the formulation of the present invention provides a formulation containing a high concentration of multi-peptide and/or antibody, which is dimerized and deproteinized during prolonged storage or multiple freeze/thaw cycles. The amide amination is inhibited and the formulation is stable and suitable for subcutaneous administration.

Intensive research has been conducted to solve the above problems, and it has been found that a stable, high concentration multipeptide/antibody-containing formulation can be obtained by adding an amino acid squalic acid or a salt thereof as a stabilizer in a solution.

The invention will be described in detail below. The study focused on obtaining a pharmaceutical formula that is stable from a second-generation molecular perspective, and the second-generation molecule is superior to the first-generation humanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB. Regarding the results of the antibodies of interest, the inventors of the present invention found that the multiplex mutation of the variable region of TOCILIZUMAB improves the binding ability (affinity) to the antigen. The inventors of the present invention thus succeeded in improving the affinity by using a combination of such mutations. The inventors of the present invention have also succeeded in improving pharmacokinetics by introducing modifications that reduce the isoelectric point of the variable region sequence. The inventors of the present invention have also succeeded in improving pharmacokinetics by making the binding to the IL-6 receptor antigen pH-dependent, so that a single antibody molecule can neutralize multiple antigens. Furthermore, they succeeded by completely humanizing the sequence of mouse-derived residues remaining in the TOCILIZUMAB framework and reducing the number of T cell epitopes in the variable region predicted by computer simulations. Reduce the risk of immunogenicity. Furthermore, the inventors of the present invention have succeeded in finding that the novel constant region sequence directed against the constant region of TOCILIZUMAB has improved binding to the Fc gamma receptor compared to IgG1, which improves safety and improves pharmacokinetics compared to IgG1. And reduce the heterogeneity caused by the disulfide bond in the hinge region of IgG2, and the heterogeneity caused by the C-terminus of the H chain without detracting from stability. The inventors of the present invention succeeded in producing a second generation molecule superior to TOCILIZUMAB by appropriately combining these amino acid sequence changes in the CDR, variable region and constant region.

The invention relates to a pharmaceutical formulation comprising a humanized anti-IL-6 receptor IgG antibody with superior antigen (IL-6 receptor) binding ability, superior pharmacokinetics, superior safety and physical properties (stable And homogeneity), and further, can reduce immunogenicity by altering the amino acid sequence of the variable region and the constant region of the humanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB; A method of treating a pharmaceutical composition. More specifically, the present invention provides the following:

(1) A pharmaceutical composition comprising at least one multi-peptide selected from the group consisting of:

(a) a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 comprising SEQ ID NO: 2 (CDR2 of VH4-M73), and CDR3 comprising the sequence Identification number 3 (CDR3 of VH4-M73);

(b) a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising SEQ ID NO: 5 (CDR2 of VH3-M73), and CDR3 comprising the sequence Identification number 6 (CDR3 of VH3-M73);

(c) a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising SEQ ID NO: 8 (CDR2 of VH5-M83), and CDR3 comprising the sequence Identification number 9 (CDR3 of VH5-M83);

(d) a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 10 (CDR1 of VL1), CDR2 comprising SEQ ID NO: 11 (CDR2 of VL1), and CDR3 comprising SEQ ID NO: 12 CDR3 of VL1);

(e) a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 13 (CDR1 of VL3), CDR2 comprising SEQ ID NO: 14 (CDR2 of VL3), and CDR3 comprising SEQ ID NO: 15 CDR3 of VL3);

(f) a multi-peptide comprising CDR1, CDR2, CDR3 comprising SEQ ID NO: 16 (CDR1 of VL5), CDR2 comprising SEQ ID NO: 17 (CDR2 of VL5), and CDR3 comprising SEQ ID NO: 18 ( CDR3 of VL5).

(2) A pharmaceutical formulation comprising at least one antibody selected from the group consisting of:

(a) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 1 (CDR1 of VH4-M73) comprising SEQ ID NO: 2 (VH4) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1), comprising SEQ ID NO: 11 CDR2 of CDR2 of VL1, and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1);

(b) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 4 (CDR1 of VH3-M73) comprising SEQ ID NO: 5 (VH3) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 13 (CDR1 of VL3) comprising SEQ ID NO: 14 CDR2 of CDR2 of VL3, and CDR3 comprising SEQ ID NO: 15 (CDR3 of VL3);

(c) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83) comprising SEQ ID NO: 8 (VH5) CDR2 of CDR2) of M83, and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5) comprising SEQ ID NO: 17 CDR2 of CDR2 of VL5, and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5);

(d) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprising: Sequence identification number 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73); the light chain variable region comprising: Sequence identification number 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 21 (variable region of VH5-M83); the light chain variable region comprising: Sequence identification number 24 (variable region of VL5);

(g) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 25 (VH4-M73); the light chain comprising: SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprising: SEQ ID NO: 29 (VL3);

(i) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 27 (VH5-M83); the light chain comprising: SEQ ID NO: 30 (VL5).

(3) A stable pharmaceutical formulation according to (1) or (2) comprising a group of amine acid and/or citrate buffers.

(4) A stable pharmaceutical formulation according to any one of (1) to (3) comprising at least one cationic amino acid.

(5) A stable pharmaceutical formulation according to any one of (1) to (4) comprising 1 to 500 mM histidine and/or citrate buffer, 1 to 1500 mM of at least one cationic amino acid, 1 ~200mg/mL of antibody, and 1~400mM carbohydrates.

(6) The formulation of (4) or (5), wherein the cationic amino acid is arginine.

(7) The formulation of (5) or (6), wherein the carbohydrate is sucrose or trehalose.

(8) The formulation according to any one of (1) to (7), further comprising a surfactant.

(9) The formulation according to any one of (1) to (8), wherein the amount of the multi-peptide and/or antibody is at least 10 mg/ml.

(10) The formulation according to any one of (1) to (9), wherein the amount of the multi-peptide and/or antibody is at least 50 mg/ml.

(11) The formulation according to any one of (1) to (10), wherein the amount of the multi-peptide and/or antibody is at least 80 mg/ml.

(12) The formulation according to any one of (1) to (11), wherein the amount of the multi-peptide and/or antibody is less than or equal to 240 mg/ml.

(13) The formulation according to any one of (1) to (12), wherein the pH is between 4.5 and 7.0.

(14) The formulation of (13), wherein the pH is between 5.5 and 6.6.

(15) The formulation of any one of (1) to (14) wherein the formulation is a liquid.

(16) The formulation of (15), wherein the formulation is not freeze-dried during the formulation.

(17) The formulation according to any one of (1) to (16) wherein dimerization of the multi-peptide and/or antibody molecule is reduced.

(18) The formulation according to any one of (1) to (17) wherein the dimerization of the multi-peptide and/or antibody molecule is inhibited.

(19) The formulation according to any one of (1) to (18) for subcutaneous administration.

(20) A method for stabilizing a solution containing the antibody, comprising adding at least one cationic amino acid, wherein the antibody is at least one antibody selected from the group consisting of:

(a) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 1 (CDR1 of VH4-M73) comprising SEQ ID NO: 2 (VH4) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1), comprising SEQ ID NO: 11 CDR2 of CDR2 of VL1, and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1);

(b) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 4 (CDR1 of VH3-M73) comprising SEQ ID NO: 5 (VH3) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 13 (CDR1 of VL3) comprising SEQ ID NO: 14 CDR2 of CDR2 of VL3, and CDR3 comprising SEQ ID NO: 15 (CDR3 of VL3);

(c) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83) comprising SEQ ID NO: 8 (VH5) CDR2 of CDR2) of M83, and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5) comprising SEQ ID NO: 17 CDR2 of CDR2 of VL5, and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5);

(d) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprising: Sequence identification number 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73); the light chain variable region comprising: Sequence identification number 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 21 (variable region of VH5-M83); the light chain variable region comprising: Sequence identification number 24 (variable region of VL5);

(g) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 25 (VH4-M73); the light chain comprising: SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprising: SEQ ID NO: 29 (VL3);

(i) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 27 (VH5-M83); the light chain comprising: SEQ ID NO: 30 (VL5).

(21) A method for stabilizing an antibody, comprising: adding at least one cationic amino acid to a solution in a freeze/thaw cycle, wherein the antibody is selected from at least one of the following antibodies:

(a) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 1 (CDR1 of VH4-M73) comprising SEQ ID NO: 2 (VH4) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1), comprising SEQ ID NO: 11 CDR2 of CDR2 of VL1, and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1);

(b) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 4 (CDR1 of VH3-M73) comprising SEQ ID NO: 5 (VH3) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 13 (CDR1 of VL3) comprising SEQ ID NO: 14 CDR2 of CDR2 of VL3, and CDR3 comprising SEQ ID NO: 15 (CDR3 of VL3);

(c) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83) comprising SEQ ID NO: 8 (VH5) CDR2 of CDR2) of M83, and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5) comprising SEQ ID NO: 17 CDR2 of CDR2 of VL5, and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5);

(d) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprising: Sequence identification number 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73); the light chain variable region comprising: Sequence identification number 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 21 (variable region of VH5-M83); the light chain variable region comprising: Sequence identification number 24 (variable region of VL5);

(g) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 25 (VH4-M73); the light chain comprising: SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprising: SEQ ID NO: 29 (VL3);

(i) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 27 (VH5-M83); the light chain comprising: SEQ ID NO: 30 (VL5).

The above humanized anti-IL-6 receptor IgG antibody has enhanced potency and improved pharmacokinetics; therefore, it can exert an extended therapeutic effect with a small frequency of administration.

The invention provides a pharmaceutical formulation comprising at least one multi-peptide selected from the group consisting of:

(a) a multi-peptide comprising CDR1, CDR2 and CDR3 comprising SEQ ID NO: 1 (CDR1 of VH4-M73), the CDR2 comprising SEQ ID NO: 2 (CDR2 of VH4-M73) comprising sequence recognition No. 3 (CDR3 of VH4-M73);

(b) a multi-peptide comprising CDR1, CDR2 and CDR3 comprising SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising SEQ ID NO: 5 (CDR2 of VH3-M73) comprising sequence recognition No. 6 (CDR3 of VH3-M73);

(c) a multi-peptide comprising CDR1, CDR2 and CDR3 comprising SEQ ID NO: 7 (CDR1 of VH5-M83), the CDR2 comprising SEQ ID NO: 8 (CDR2 of VH5-M83), the CDR3 comprising sequence recognition No. 9 (CDR3 of VH5-M83);

(d) a multi-peptide comprising CDR1, CDR2 and CDR3 comprising SEQ ID NO: 10 (CDR1 of VL1) comprising SEQ ID NO: 11 (CDR2 of VL1) comprising SEQ ID NO: 12 (VL1) CDR3);

(e) a multi-peptide comprising CDR1, CDR2 and CDR3 comprising SEQ ID NO: 13 (CDR1 of VL3) comprising SEQ ID NO: 14 (CDR2 of VL3) comprising SEQ ID NO: 15 (VL3) CDR3);

(f) a multi-peptide comprising CDR1, CDR2 and CDR3 comprising SEQ ID NO: 16 (CDR1 of VL5) comprising SEQ ID NO: 17 (CDR2 of VL5) comprising SEQ ID NO: 18 (VL5) CDR3).

The multi-peptides and antibodies of the present invention can be formulated according to conventional methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA).

The "pharmaceutical formula", "pharmaceutical" and "pharmaceutical composition" as used in the present invention means a liquid or solid formulation containing a multi-peptide and/or an antibody as an active ingredient, which is prepared for direct or reconstitution. Post-administered to animals, such as humans. The formulation may contain a pharmaceutically acceptable carrier and/or additive if desired. For example, detergents (eg, PEG, Tween, Pluronic), excipients, antioxidants (eg, ascorbic acid, methionine), colorants, flavors, preservatives, stabilizers, buffers, chelating agents (eg, EDTA), suspending agents, isotonic agents, binders, disintegrating agents, lubricants, flow promoters, and flavoring agents.

The formulation containing the multi-peptide and/or antibody of the present invention preferably contains no HAS (human serum albumin), gelatin, and the like as a stabilizer.

The multi-peptide and/or antibody-containing formulation of the present invention is preferably a liquid pharmaceutical formulation comprising a high concentration of multi-peptide and/or antibody at a concentration of not less than 10 mg/mL, preferably not less than 50 mg. /mL, more preferably not less than 80 mg/mL. The concentration is between 10 and 240 mg/mL, and may be 10 mg/mL, preferably 50 mg/mL, more preferably 100 to 200 mg/mL.

The liquid pharmaceutical formulation of the present invention is preferably manufactured without a freeze drying step.

Buffering agents useful in the present invention are those which are pH adjustable to the desired range and which are pharmaceutically acceptable. The formulation of the present invention containing a high concentration of multi-peptide and/or antibody preferably has a pH of 4.5 to 7, more preferably 5.5 to 6.6. Such buffering agents are known to those skilled in the art and include, for example, inorganic salts such as phosphates (sodium or potassium salts), and sodium bicarbonate; organic acid salts such as citrate (sodium or potassium), Sodium acetate and sodium succinate; and acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid, and gluconic acid.

Further, Tris buffer, Good's buffer such as MES and MOPS, histidine (for example, histidine hydrochloride), and glycine may also be used. According to the formulation of the present invention containing a high concentration of multi-peptide and/or antibody, the buffer is preferably a group of amine acid or citrate buffer, more preferably a histidine buffer. The concentration of the buffer solution is generally from 1 to 500 mM, preferably from 5 to 100 mM, more preferably from 10 to 20 mM. When the histidine buffer is used, the buffer solution preferably has a histidine concentration of 5 to 25 mM, more preferably 10 to 20 mM.

The formulation of the present invention may further comprise a surfactant. Examples of typical surfactants include: nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprate, sorbitan monolaurate, and sorbitan Monopalmitate; glycerol fatty acid esters such as glycerol monocaprate, glycerol monomyristate, and glyceryl monostearate; polyglycerol fatty acid esters such as decaglyceryl monostearate, decaglycerin Fatty acid esters, and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, poly Oxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate; Polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan tetrastearate, and polyoxyethylene sorbitan tetraoleate; polyoxyethylene glyceryl esters, such as polyoxyethylene glycerol Stearate; polyethylene glycol fatty acid ester, such as polyethylene glycol Acid ester; polyoxyethylene alkyl ether, such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl ether, such as polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylene ether, and polyoxyethylene polymerization Oxypropylene propylene cetyl ether; polyoxyethylene alkyl phenyl ether, such as polyoxyethylene decyl phenyl ether; polyoxyethylene hardened castor oil, for example, polyoxyethylene castor oil, and polyoxyethylene hardened castor oil (poly Oxyethylene hydrogenated castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; HLB 6 to 18 surfactants, For example, a polyoxyethylene fatty acid decylamine such as polyoxyethylene octadecylamine; an anionic surfactant such as an alkyl sulfate having a C ₁₀ -C ₁₈ alkyl group such as sodium cetyl sulfate, sodium lauryl sulfate, and Sodium oleate; polyoxyethylene alkyl ether sulfate, wherein the added oxyethylene unit has an average number of moles of 2 to 4, and the alkyl group has 10 to 18 carbon atoms, such as polyoxyethylene lauryl sodium sulfate; having C ₈ -C ₁₈ alkyl group of a sulfo group Perkin salts, such as sodium lauryl sulfosuccinate; natural surfactant such as lecithin, and glycerol phosphate; entangled phospholipid, e.g. sphingomyelin; and C ₁₂ -C ₁₈ fatty acid esters of sucrose. These surfactants may be separately added to the formulation of the present invention, or two or more such surfactants may be added in combination.

Preferred surfactants are polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl ethers, more preferably polysorbate 20, 21, 40, 60, 65, 80, 81 and 85. A surfactant in the form of Pluronic, and most preferably polysorbate 20 and 80, and Pluronic F-68 (Poloxamer 188).

The surfactant dose to be added to the antibody formulation of the present invention is generally 0.0001 to 10% (w/v), preferably 0.001 to 5%, more preferably 0.005 to 3%.

The formulation of the present invention may further comprise an acidic amino acid such as arginine, and methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, aspartate Amine, sitamine, and such amino acids act as stabilizers.

In the antibody-containing formulation or antibody-containing solution formulation according to the present invention, the formation of a dimer during the freeze/thaw cycle can be inhibited by the addition of a carbohydrate such as a saccharide. The saccharide which can be used includes non-reducing oligosaccharides such as non-reducing disaccharides such as sucrose and trehalose, or non-reducing trisaccharides such as raffinose, more preferably non-reducing oligosaccharides. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

In the antibody-containing formulation or the antibody-containing solution formulation according to the present invention, the formation of a multimer and a decomposition product during long-term storage can be inhibited by the addition of a carbohydrate such as a saccharide. Sugars which may be used include sugar alcohols such as mannitol and sorbitol; and non-reducing oligosaccharides such as non-reducing disaccharides such as sucrose, and trehalose, or non-reducing trisaccharides such as raffinose, wherein Preferred are non-reducing oligosaccharides. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

The sugar may be added in an amount of 0.1 to 500 mg/mL, more preferably 10 to 300 mg/mL, still more preferably 25 to 100 mg/mL.

In another aspect, the formulation according to the invention preferably consists essentially of the following components:

A) a modified anti-IL-6 receptor antibody;

B) a cationic amino acid (such as arginine, histidine, and/or lysine);

C) a buffer (for example, histidine or citrate).

Carbohydrates (eg, sugars) and/or surfactants may be included in the formulation depending on the application.

As the stabilizer, it may include: methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, aspartame, situramine, and the like.

As used herein, "substantially composed" means not containing ingredients other than those normally added to the formulation, and the ingredients usually added to the formulation are selected from the following optional additive ingredients such as suspending agents, dissolving agents, Isotonic agents, preservatives, adsorption inhibitors, diluents, carriers, pH adjusters, sedatives, sulfur-containing reducing agents, and antioxidants.

Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum rabic, powdered tragacanth, sodium carboxymethylcellulose, and polyoxyethylene sorbitol. Anhydride monolaurate.

Examples of the dissolving agent include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil. Fatty acid ethyl ester.

Examples of isotonic agents include sodium chloride, potassium chloride, and calcium chloride.

Examples of the preservative include methylparaben, ethylparaben, sorbic acid, phenol, cresol, and chlorocresol.

Examples of adsorption inhibitors include: human serum albumin, lecithin, dextran, oxyethylene-oxypropylene copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene Glycol.

Examples of sulfur-containing reducing agents include compounds having a thiol group, such as N-ethyl decyl cysteine, N-ethinyl cystein, thioctic acid, thiodiethylene Alcohol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and C ₁ -C ₇ sulfane.

Examples of antioxidants include: erythorbic acid, dibutylhydroxytoluene, anisole, a-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L - ascorbic acid-based palmitate, L-ascorbic acid stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as ethylene Disodium edetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

However, the formulation (agent, pharmaceutical composition) according to the present invention can be used for the prevention or treatment of an IL-6-related disease including an inflammatory disease, and is not limited to the above, and other conventional carriers can be appropriately included. In particular, for example, slightly dehydrated citric acid, lactose, crystalline cellulose, mannitol, starch, calcium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose , polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid glyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, And inorganic salts.

It may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; and amino acids. When preparing an aqueous solution for injection, these anti-IL-6 receptor antibodies are dissolved in, for example, an isotonic solution containing physiological saline, glucose or other adjuvant. An adjuvant comprising, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Further, a suitable dissolving agent such as an alcohol (ethanol or the like), a polyhydric alcohol (propylene glycol, PEG, etc.), and a nonionic surfactant (polysorbate 80 and HCO-50) may be combined.

If necessary, these multi-peptides can be encapsulated in microcapsules (microcapsules made of hydroxycellulose, gelatin, poly(methyl methacrylate), etc.) or into a colloidal drug delivery system (lilipid, albumin microspheres, microemulsions, nano-particles, nano-capsules, etc.) (see e.g. ^{"Remington's Pharmaceutical Science 16 th edition} ", Oslo Ed. (1980)). Further, a method of preparing a pharmaceutical agent as a sustained release pharmaceutical agent is known and can be applied to this multi-peptide (Langer et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12: 98-105; US Patent No. 3,773,919; European Patent Application (EP) No. 58,481; Sidman et al., Biopolymers (1983) 22:547-56; EP No. 133,988). Also, the volume of liquid for subcutaneous administration can be increased by adding or mixing hyaluronidase into a medicament (see, for example, WO2004/078140).

The pharmaceutical composition of the present invention can be administered by both oral and parenteral administration, but is preferably administered parenterally. In particular, such compositions are administered to a patient by injection or transdermally. Injections include, for example, systemic and topical administration by intravenous, intramuscular or subcutaneous injection. Such compositions can be injected locally at the site of treatment or at the periphery of the site by, inter alia, intramuscular injection. Transdermal formulations include, for example, ointments, gels, creams, poultices, and patches, which may be administered topically or systemically. Furthermore, the method of administration can be appropriately selected depending on the age and symptoms of the patient. The dose to be administered can be selected from the range of administration of each active ingredient per kg body weight, for example, 0.0001 mg to 100 mg. Alternatively, when such compositions are administered to a human patient, for example, a dose may be administered from each patient, for example, from 0.001 mg to 1000 mg of active ingredient per kilogram of body weight per administration. The single administration dose preferably includes, for example, about 0.01 to 50 mg/kg body weight of the antibody of the present invention. However, the dose of the antibody of the present invention is not limited to such an equivalent dose.

As can be seen from the results of the following examples, according to the present invention, a stable formulation can be obtained in which, by adding one or more cationic amino acids (arginine, histidine, and/or lysine, more preferably In the case of arginine, or a combination of a cationic amino acid and other amino acids, in this formulation, dimerization and deamidation of the antibody are less during prolonged storage or freezing/thawing.

In order to evaluate the stability during storage of the formulation containing the high concentration of the antibody, the inventors of the present invention studied the effects of various additives by performing size exclusion chromatography and anion exchange chromatography test. As a result, it was found that a solution in which a high concentration of antibody was dissolved in a buffer solution containing amino acid arginine was used, and the amount of the dimer was lower than that in the case where no arginine solution was added. These results show that arginine as a stabilizer can effectively inhibit antibody dimerization.

Thus, by the addition of arginine as a stabilizer, a stable antibody formulation can be provided in which dimerization of the antibody is reduced.

One embodiment of the invention is a stable antibody-containing formulation characterized by comprising an antibody and arginine in a buffered solution.

The arginine acid used in the present invention may be any arginine compound itself, a derivative thereof and a salt thereof, preferably L-arginine and a salt thereof.

When the formulation according to the present invention contains arginine, the concentration of arginine is preferably from 1 to 1,500 mM, more preferably from 50 to 1,000 mM, still more preferably from 50 to 200 mM.

The multipeptide of the present invention is not particularly limited, however, an antigen-binding substance having an activity of binding to a human IL-6 receptor is preferred. Such antigen-binding substances preferably include, for example, an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), an antibody heavy chain, an antibody light chain, and an antibody.

Among the above multi-peptides (a) to (f), the multi-peptides of (a) to (c) are examples of preferred antibody heavy chain variable regions, and the multi-peptides of (d) to (f) are An example of an antibody light chain variable region.

These variable regions can be used as part of a primary antibody against human IL-6 receptor. The anti-human IL-6 receptor antibody using such a variable region has superior binding activity, excellent pharmacokinetics, excellent safety, reduced immunogenicity, and/or superior physicochemical properties. In the present invention, the improvement of excellent pharmacokinetics or pharmacokinetics means any of the following: "clearance rate (CL)" decreases, "area under the curve (AUC)" increases, and "average residual time" increases. And "plasma half-life (t1/2)" is an increase in the pharmacokinetic parameters calculated from the time of plasma concentration when the antibody is administered to the body. Here, superior physical and chemical properties or improved physicochemical properties refer to improved stability, reduced heterogeneity, and the like, but are not limited thereto.

The human antibody framework regions linked to the CDRs of the invention are selected such that the CDRs form a favorable antigen binding site. In the present invention, the FR used for these variable regions is not particularly limited, and any FR may be used; however, it is preferred to use a human-derived FR. Human derived FRs with native sequences can be used. Alternatively, more than one amino acid substitution, deletion, addition and/or insertion, etc., may be introduced into the framework region having the native sequence such that the CDR forms a sufficient antigen binding site. A mutant FR sequence having a desired property can be selected by, for example, measuring and evaluating the binding activity of an antibody having an amino acid-substituted FR to an antigen (Sato, K. et al., Cancer Res. (1993) 53, 851-856. ).

Further, one or more amino acids may be substituted, deleted, added, and/or inserted in the CDR sequence. Preferably, after substitution, deletion, addition and/or insertion of one or more amino acids, a CDR sequence binds to activity, neutralizing activity, stability, immunogenicity, and/or pharmacokinetics, and changes The former CDR sequences are equivalent in activity. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, however, it is preferably 3 or less of amino acids, more preferably 2 or less of amino acids, and more preferably each. CDR, 1 amino acid or less.

A method for substituting one or more amino acid residues for other amino acids of interest, for example: point mutation (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide -directed dual amber method for site-directed mutagenesis. Gene 152. 271-275: Zoller. MJ. and Smith. M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100,468-500; Kramer , W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, HJ (1984) The gapped duplex DAN approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12,9441-9456; Kramer W And Fritz HJ (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154,350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82,488-492 ). This method can be used to replace the desired amino acid in the antibody with other amino acids of interest. Furthermore, as a method of amino acid substitution, gene library technology such as framework shuffling (Mol. Immunol. 2007 Apr; 44(11): 3049-60) and CDR repair (US2006/0122377) can be utilized. Amino acid substitutions are made to obtain the appropriate framework and CDRs.

The invention also provides a pharmaceutical formulation comprising at least one antibody selected from the group consisting of:

(a) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 1 (CDR1 of VH4-M73) comprising SEQ ID NO: 2 (VH4) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 3 (CDR3 of VH4-M73); the light chain variable region comprises: CDR1 comprising SEQ ID NO: 10 (CDR1 of VL1), comprising SEQ ID NO: 11 CDR2 of CDR2 of VL1, and CDR3 comprising SEQ ID NO: 12 (CDR3 of VL1);

(b) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 4 (CDR1 of VH3-M73) comprising SEQ ID NO: 5 (VH3) CDR2 of CDR2) of M73, and CDR3 comprising SEQ ID NO: 6 (CDR3 of VH3-M73); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 13 (CDR1 of VL3) comprising SEQ ID NO: 14 CDR2 of CDR2 of VL3, and CDR3 comprising SEQ ID NO: 15 (CDR3 of VL3);

(c) an antibody comprising a heavy chain variable region and a light chain variable region comprising: CDR1 comprising SEQ ID NO: 7 (CDR1 of VH5-M83) comprising SEQ ID NO: 8 (VH5) CDR2 of CDR2) of M83, and CDR3 comprising SEQ ID NO: 9 (CDR3 of VH5-M83); the light chain variable region comprising: CDR1 comprising SEQ ID NO: 16 (CDR1 of VL5) comprising SEQ ID NO: 17 CDR2 of CDR2 of VL5, and CDR3 comprising SEQ ID NO: 18 (CDR3 of VL5);

(d) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 19 (variable region of VH4-M73); the light chain variable region comprising: Sequence identification number 22 (variable region of VL1);

(e) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 20 (variable region of VH3-M73); the light chain variable region comprising: Sequence identification number 23 (variable region of VL3);

(f) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: SEQ ID NO: 21 (variable region of VH5-M83); the light chain variable region comprising: Sequence identification number 24 (variable region of VL5);

(g) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 25 (VH4-M73); the light chain comprising: SEQ ID NO: 28 (VL1);

(h) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 26 (VH3-M73); the light chain comprising: SEQ ID NO: 29 (VL3);

(i) an antibody comprising a heavy chain and a light chain, the heavy chain comprising: SEQ ID NO: 27 (VH5-M83); the light chain comprising: SEQ ID NO: 30 (VL5).

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

The human antibody framework sequences linked to the CDRs of the invention are selected such that the CDRs form a favorable antigen binding site. In the present invention, the FR used for these variable regions is not particularly limited, and any FR may be used; however, it is preferred to use a human-derived FR. Human derived FRs with native sequences can be used. Alternatively, more than one amino acid substitution, deletion, addition and/or insertion, etc., may be introduced into the framework region having the native sequence such that the CDR forms a sufficient antigen binding site. A mutant FR sequence having a desired property can be selected by, for example, measuring and evaluating the binding activity of an antibody having an amino acid-substituted FR to an antigen (Sato, K. et al., Cancer Res. (1993) 53, 851-856. ).

Meanwhile, the constant region used for the antibody of the present invention is not particularly limited, and any constant region can be used. Preferred for use in the constant regions of the antibodies of the invention include, for example, human derived constant regions (constant regions derived from IgGl, IgG2, IgG3, IgG4, CK, Cλ, etc.). Substitution, deletion, addition and/or insertion of one or more amino acids can be carried out in this human-derived constant region. Preferred human-derived heavy chain constant regions include, for example, a constant region comprising amino acid sequence number 31 (variable region of VH4-M73), a constant region comprising amino acid sequence identifier 32 (constant VH3-M73) a region comprising a constant region of amino acid sequence number 33 (a constant region of VH5-M83), and preferably a human-derived light chain constant region comprising, for example, a constant region comprising amino acid sequence identifier 34 (VL1) ), a constant region (VL3) comprising amino acid sequence identifier 35, and a constant region (VL5) comprising amino acid sequence identifier 36.

Further, one or more amino acids may be substituted, deleted, added, and/or inserted in the CDR sequence. Preferably, after substitution, deletion, addition and/or insertion of one or more amino acids, a CDR sequence binds to activity, neutralizing activity, stability, immunogenicity, and/or pharmacokinetics, and changes The former CDR sequences are equivalent in activity. The number of amino acids to be substituted, deleted, added, and/or inserted is not particularly limited, however, it is preferably 3 or less of amino acids, more preferably 2 or less of amino acids, and more preferably each. CDR, 1 amino acid or less.

Each variable region of such antibodies can be part of a molecule that reacts with an anti-human IL-6 receptor. Such constant regions may also comprise substitutions, deletions, additions, and/or insertions of more than one amino acid (e.g., 5 or less amino acids, preferably 3 or less amino acids). A method for substituting one or more amino acid residues with other amino acids of interest, including, for example, the above methods.

The present invention also encompasses a multi-peptide comprising the above variable regions.

Each of the heavy and light chains of such antibodies can be part of a molecule that reacts with an anti-human IL-6 receptor. The anti-human IL-6 receptor antibodies using these heavy and light chains have superior binding activity, excellent pharmacokinetics, excellent safety, reduced immunogenicity, and/or superior physicochemical properties.

These heavy and light chains may further comprise one or more amino acids (for example, 10 or less amino acids, preferably 5 or less amino acids, more preferably 5 or less amino acids) Substitution, deletion, addition, and/or insertion. A method for substituting one or more amino acid residues with other amino acids of interest, including, for example, the above methods.

Substitution, deletion, addition, and/or insertion of one or more amino acids can be carried out for the variable region, the constant region, or both.

The present invention further encompasses a multi-peptide comprising the above heavy and light chains.

The antibody of the present invention is preferably a humanized antibody.

Humanized antibodies are also known as reshaped antibodies. This humanized anti-system is obtained by grafting a complementarity determining region (CDR) from a non-human mammal to the CDRs of a human antibody. Known genetic recombination techniques for the preparation of such antibodies are known (see European Patent Application No. EP 125023; WO 96/02576).

In particular, for example, a designed DNA sequence in which a CDR of interest and a framework region of interest (FR) are linked is obtained by using a plurality of oligonucleotides prepared by using a terminal having a portion overlapping with a CDR and a FR as an primer. PCR synthesis (see the method described in WO 98/13388). The humanized anti-system is obtained by ligating the resulting DNA to DNA encoding a human antibody constant region or a modified human antibody constant region; inserting the above into a expression vector; and introducing the vector into a host This antibody was produced (see European Patent Application No. EP 239400, and Internationa1 Patent Application Publication No. WO 96/02576).

The human antibody framework regions linked to the CDRs are selected such that the CDRs form a favorable antigen binding site. The amino acid may be substituted, deleted, added, and/or inserted into the framework region of the variable region of the antibody, as needed.

A human antibody constant region, or an altered human antibody constant region in which one or more amino acids have been substituted, deleted, added, and/or inserted, can serve as a constant region for a humanized antibody.

For example, C γ 1 , C γ 2, C γ 3, C γ 4, C mu, C δ , C α 1 , C α 2, C epsilon may be used for the H chain, and C κ and C λ may be used for the L chain. The amino acid sequence of C κ is shown in SEQ ID NO: 38, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 37. The amino acid sequence of C γ 1 is shown in SEQ ID NO: 40, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 39. The amino acid sequence of C γ 2 is shown in SEQ ID NO: 42, and the nucleotide sequence encoding this 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, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 43.

Also, the human antibody C region can be modified to improve antibody stability or antibody production stability. A human antibody of any isotype, for example, IgG, IgM, IgA, IgE or IgD, can be used to humanize the antibody. However, the present invention preferably uses IgG. IgG1, IgG2, IgG3, IgG4, etc. can be used as IgG.

The variable regions (e.g., CDRs and FRs) of the humanized antibody and the amino acid of the constant region can be deleted, added, deleted, and/or substituted into an amino acid after preparation. The antibody of the present invention also includes such a humanized antibody comprising an amino acid substitution or the like.

The antibody of the present invention includes not only a bivalent antibody represented by IgG but also a monovalent antibody, and a multivalent antibody represented by IgM as long as it has IL-6 receptor binding activity and/or neutralizing activity. The multivalent antibody of the present invention includes a multivalent antibody having the same antigen binding site, and a multivalent antibody having all or part of antigen binding sites. The antibody of the present invention includes not only an intact antibody molecule but also a minibody and a modified product thereof as long as it binds to the IL-6 receptor protein.

A mini-antibody system comprises an antibody lacking an antibody fragment lacking a portion of an intact antibody (eg, intact IgG, etc.), and as long as an antibody fragment having IL-6 receptor binding activity and/or neutralizing activity and comprising a portion lacking an intact antibody, That is, it is not particularly limited. The micro-antibody of the present invention is not particularly limited as long as it has a part of an intact antibody. However, such mini-antibodies preferably comprise VH or VL, more preferably VH and VL. Other preferred antibodies of the invention include, for example, a minibody comprising an antibody CDR. These mini-antibodies may comprise all or a portion of the six CDRs of the antibody.

The mini-antibody of the present invention preferably has a molecular weight smaller than that of the intact antibody. However, such mini-antibodies can form multimers, such as dimers, trimers or tetramers, and thus their molecular weight will sometimes be greater than intact antibodies.

In particular, antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv. Meanwhile, the mini-antibody includes, for example, Fab, Fab', F(ab')2, Fv, svFc (single-chain Fv), diabody, and sc(Fv)2 (single-chain (Fv) 2). Polymers of such antibodies (e.g., dimers, trimers, tetramers, and polymers) are also included in the minibody of the present invention.

Antibody fragments can be obtained by, for example, treating an antibody with an enzyme to produce an antibody fragment. Enzymes which produce antibody fragments are known, including, for example, papain, trypsin, and plasmin. Alternatively, it can be constructed by introducing a gene encoding this antibody fragment into a expression vector and expressing it in an appropriate host cell (see, for example, Co, M. S. et al., J. Immunol. (1994) 152 , 2968-2976; Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496; Pluckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669; Bird, RE et al., TIBTECH (1991) 9, 132-137.

The enzyme is cleaved at a specific portion of the antibody fragment to produce an antibody fragment of a specific structure as shown below. Genetic engineering techniques can be applied to antibody fragments obtained by this enzyme method to remove any part of the antibody.

The antibody fragments obtained using the above decomposition enzymes are as follows:

Papaya Enzyme Digestion: F(ab)2 or Fab

Trypsin digestion: F(ab') 2 or Fab'

Cytosolic digestion: Facb

The minibody of the present invention includes an antibody fragment lacking any region as long as it has IL-6 receptor binding activity and/or neutralizing activity.

"diabody" means a bivalent antibody fragment constructed by gene fusion (Holliger P et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; EP 404,097; WO 93/11161 Wait). The double-antibody system consists of two dimers consisting of multiple peptide chains. For each of the multi-peptide chains forming the dimer, VL and VH are generally linked by a linker of the same chain. In general, the linkers in the diabody are so short that VL and VH cannot bind to each other. In detail, the number of amino acid residues constituting the linker is, for example, about 5 residues. Thus, VL and VH encoded on the same multi-peptide do not form a single-chain variable region fragment and will form a dimer with another single-chain variable region fragment. As a result, this diabody has two antigen-binding sites.

ScFv antibody is a single-chain polypeptide produced by linking VH and VL with a linker or the like (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883; Plueckthun" The Pharmacology of Monoclonal Antibodies" Vol. 113, eds., Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)). The H chain V region and the L chain V region of scFv can be derived from any of the antibodies described herein. The peptide linker for linking the V region is not particularly limited. For example, any single-stranded peptide comprising about 3 to 25 residues can be used as a linker. In detail, the peptide linker or the like described below can be used.

The V-region of the two strands can be linked by, for example, the above PCR. First, the DNA encoding the complete amino acid sequence of one of the DNAs shown below or the desired partial amino acid sequence is used as a template, and the V region is linked by PCR: a DNA sequence encoding the H chain or H chain V region of the antibody, and A DNA sequence encoding the L chain or L chain V region of an antibody.

The DNA encoding the V region of the H chain or the L chain is amplified by PCR using a pair of primers containing the corresponding sequences at both ends of the DNA to be amplified. Then, DNA encoding the linker moiety of this peptide can be prepared. The DNA encoding the peptide linker can also be synthesized by PCR. The nucleotide sequence which can be used to join the separately synthesized V region amplification products is added to the 5' end of the primer to be used. Then, PCR was carried out using each DNA of [H chain V region DNA]-[peptide peptide DNA]-[L chain V region DNA] and a combination PCR primer.

This combined PCR primer includes a set of primers, one of which binds to the 5' end of the [H chain V region DNA] and the other primer binds to the 3' end of the [L chain V region DNA]. In other words, the combined PCR primer is a set of primers that can be used to amplify the DNA encoding the full length sequence of the scFv to be synthesized. At the same time, a nucleic acid sequence which can be used to link the DNA of each V region is added to [peptide peptide DNA]. These DNAs are then ligated and the complete scFv is generated using the combined PCR primers as the final amplification product. Once the DNA encoding the scFv is produced, a expression vector comprising such DNA can be obtained by a conventional method, and recombinant cells expressing the shape transformation of the vector can be obtained. Further, this scFv can be obtained by expressing the scFv-encoding DNA by culturing the obtained recombinant cells.

The order of the VH and VL to be linked is not particularly limited and may be arranged in any order. The possible arrangements are as follows:

[VH] link [VL]

[VL] link [VH]

Sc(Fv)2 is a single-chain minibody formed by linking two VHs and two VLs using a linker or the like (Hudson et al., 1999, J Immunol. Methods 231: 177-189). Sc(Fv)2 can be produced, for example, using a linker-linked scFv.

Preferably, two VHs and two VLs of an antibody can be arranged in the order of VH, VL, VH and VL from the N-terminus of the single-stranded multi-peptide ([VH] linker [VL] linker [VH] Linker [VL]). However, the order of 2 VHs and 2 VLs is not limited to the above arrangement, and may be arranged in any order. Examples of arrangements are as follows:

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

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

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

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

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

The amino acid sequence of the VH or VL of a minibody can include substitutions, deletions, additions, and/or insertions. Further, as long as the combination of VH and VL has an antigen-binding activity, a part of it may be deleted or another multi-peptide may be added. Moreover, such variable regions can be chimeric or humanized.

In the present invention, a linker which can be used to link an antibody variable region includes any peptide linker which can be introduced by genetic engineering, and a synthetic linker, for example, Protein Engineering, (1996) 9(3), 299-305. Link.

A preferred linker in the present invention is a peptide linker. The length of the peptide linker is not particularly limited, and those skilled in the art can appropriately select the length depending on the purpose. Typical lengths are from 1 to 100 amino acids, preferably from 3 to 50 amino acids, more preferably from 5 to 30 amino acids, more preferably from 12 to 18 amino acids (for example, 15 Amino acid).

For example, an amino acid sequence for a peptide linker includes the following sequences:

Ser

Gly Ser

Gly Gly Ser

Ser Gly Gly

Gly Gly Gly Ser (sequence identification number 45)

Ser Gly Gly Gly (sequence identification number 46)

Gly Gly Gly Gly Ser (sequence identification number 47)

Ser Gly Gly Gly Gly (sequence identification number 48)

Gly Gly Gly Gly Gly Ser (sequence identification number 49)

Ser Gly Gly Gly Gly Gly (Sequence ID 50)

Gly Gly Gly Gly Gly Gly Ser (Sequence ID 51)

Ser Gly Gly Gly Gly Gly Gly (Sequence ID 52)

(Gly Gly Gly Gly Ser (sequence number 47))n

(Ser Gly Gly Gly Gly (sequence number 48))n

Wherein n is an integer of 1 or more.

The amino acid sequence of the peptide linker can be suitably selected according to the use by those skilled in the art. For example, the "n" which determines the length of the peptide linker is usually 1 to 5, preferably 1 to 3, and more preferably 1 to 2.

Synthetic linkers (chemical crosslinkers), including crosslinkers routinely used to crosslink peptides, for example, N-hydroxysuccinimide (NHS), diammonium imine suberate (DSS) ), bis(sulfosuccinimide) suberate (BS3), disulfide (amber succinimide propionate) (DSP), disulfide bis(sulfosuccinimide propionic acid) Ester) (DTSSP), ethylene glycol bis(succinimide succinate) (EGS), ethylene glycol bis(sulfosuccinimide succinate) (sulfo-EGS), diammonium Imino tartaric acid ester (DST), bis sulfosuccinimide tartaric acid ester (sulfo-DST), bis[2-ammonium iminooxycarbonyloxy]ethyl]anthracene (sulfo-BSOCOES). Such crosslinkers are commercially available.

In general, three linkers are required to link four antibody variable regions. These majority links can be the same or different linkers.

The antibody of the present invention also includes an antibody in which one or more amino acids are added to the amino acid sequence of the antibody. Furthermore, the antibody of the present invention also includes a fusion protein in which the above antibody is fused to another peptide or protein. The fusion protein can be prepared by ligating a polynucleotide encoding an antibody and a polynucleotide encoding another peptide or a multi-peptide into a framework, and expressing the DNA in a host. This vector. Techniques known to those skilled in the art can be used. A peptide or polypeptide fused to an antibody of the present invention may be a known peptide, for example, FLAG (Hopp, T. P et al., BioTechnology 6, 1204-1210 (1988)); 6xHis, by 6 Composition of histidine residues; 10x His; influenza virus lectin (HA), human c-myc fragment, VSV-GP fragment, p18 HIV fragment, T7-tag, HSV-tag, E-tag, SV40 T Antigen fragment, lck tag, α-tubulin fragment, B-tag, and protein C fragment. Polypeptides fused to the antibodies of the present invention, for example, GST (glutathione-S-transferase), HA (influenza lectin), immunoglobulin constant region, β-galactosidase, and MBP ( Maltose binds to protein). Commercially available polynucleotides encoding such peptides or peptides can be fused to polynucleotides encoding the antibodies of the invention. The fused polypeptide can be prepared by expressing the fusion polynucleotide thus obtained.

Further, the antibody of the present invention may also be a conjugated antibody linked to various molecules, such as polymers, including polyethylene glycol (PEG) and hyaluronic acid; radioactive substances; fluorescent substances; luminescent substances; enzymes, And toxins. Such conjugated antibodies can be obtained by chemically modifying the obtained antibodies. Methods of antibody modification have been established in the art (see, for example, US 5,057,313, US 5,156,840). "Antibody" of the invention also includes such conjugated antibodies.

Further, the antibody of the present invention includes an antibody having an altered sugar chain.

Further, the antibody used in the present invention may be a bispecific antibody. A bispecific anti-system refers to a variable region on the same antibody molecule that recognizes different epitopes. The bispecific antibody of the present invention may be a bispecific antibody that recognizes different epitopes on the IL-6 receptor molecule, or one of the antigen binding sites recognizes the IL-6 receptor and the other antigen binding site recognizes another A bispecific antibody to a substance. An antigen that binds to another antigen-binding site of a bispecific antibody comprising an IL-6 receptor recognition antibody of the present invention, including, for example, IL-6, TNFα, TNFR1, TNFR2, CD80, CD86, CD28, CD20, CD19 , IL-1 alpha, IL-beta, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, Blys, lymphotoxin alpha, 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-CSF, IFN-α, VEGF, VEGFR, VEEGF, EGFR, CCR5, APRIL, and APRILR.

Methods for producing bispecific antibodies are known. Bispecific antibodies can be prepared, for example, by linking two types of antibodies that recognize different antigens. The antibody to be linked may be a half molecule, each comprising an H chain and an L chain, or a quarter molecule, including only one H chain. Alternatively, fusion cells producing bispecific antibodies can be prepared by fusion to produce fusion tumors of different monoclonal antibodies. In addition, bispecific antibodies can be produced using genetic engineering techniques.

As described below, the antibody used in the present invention may differ depending on the purification method or the cell or host used to produce the antibody in the amino acid sequence, molecular weight, isoelectric point, presence/absence of a sugar chain, and form. However, as long as the resulting antibody is functionally equivalent to the antibody of the present invention, it should be considered as being included in the present invention. For example, when an antibody is expressed in a prokaryotic cell such as E. coli, methionine is added to the N-terminus of the original antibody amino acid sequence. Such antibodies are also included in the antibodies of the invention.

The multipeptide or the like of the anti-IL-6 receptor antibody of the present invention can be produced by a method known to those skilled in the art.

The anti-IL-6 receptor antibody can be prepared based on the sequence of the obtained anti-IL-6 receptor antibody by genetic recombination techniques known to those skilled in the art. In particular, an anti-IL-6 receptor antibody can be constructed by constructing a polynucleotide encoding the antibody by recognizing the sequence of the antibody according to the IL-6 receptor, inserting the polynucleotide into a expression vector, and then appropriately Prepared for expression in host cells (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun , A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymo 1. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663- 669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

Accordingly, the present invention provides (i) a method of producing a multi-peptide of the present invention, or (ii) a method of producing a multi-peptide, which is encoded by a gene encoding a multi-peptide of the present invention, wherein The method comprises the steps of: cultivating a host cell comprising a vector into which a polynucleotide encoding the polypeptide of the present invention is introduced.

In particular, the invention provides a method of producing a multi-peptide of the invention comprising the steps of:

(a) cultivating a host cell, the host cell comprising a vector into which a gene encoding the polypeptide of the present invention is introduced;

(b) obtaining a multi-peptide encoded by this gene.

Examples of vectors include: M13 type vector, pUC type vector, pBR322, pBluescript, and pCR-Script. Alternatively, when the target is sub-selected and excised, the vectors other than the above include: pGEM-T, pDIRECT, and pT7. Expression vectors are especially useful for the production of the antibodies of the invention. For example, when the expression vector is for expression in E. coli, the vector should be characterized by amplification in E. coli. Further, when the host is Escherichia coli such as JM109, DH5α, HB101 or XL1-Blue, it is necessary to have a promoter capable of efficiently expressing it in Escherichia coli, for example, a 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), T7 promoter, and the like. In addition to the above, such vectors include pGEX-5X-1 (Pharmacia), "QIAexpress system" (Quiagen), pEGFP, and pET (in this case, the host is preferably BL21, which expresses T7 RAN polymerase).

Also, the expression plastid vector can comprise a sequence of messages directed against antibody secretion. For the message sequence for antibody secretion, the pe1B message sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) can be used to provide production in the extracellular matrix of E. coli. The vector can be introduced into the host cell using, for example, a calcium chloride method or an electroporation method.

In addition to vectors for E. coli, vectors for producing the antibodies of the present invention include, for example, mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. (1990) 18(17)). , p5322), pEF and pCDM8), insect cell-derived expression vectors (such as "Bac-to-BAC baculovirus expression system" (Gibco-BRL) and pBacPAK8), plant-derived expression vectors (eg, pMH1 and pMH2), animals Viral-derived expression vectors (eg, pHSV, pMV, and pAdexLcw), retroviral-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01) And Bacillus subtilis-derived expression vectors (eg, pPL608 and pKTH50).

When expressing a plastid vector for expression in animal cells such as CHO, COS and NIH3T3, it is necessary to have a promoter for expression in such cells, for example, the SV40 promoter (Mulligan et al., Nature (1979) 277, 108). , MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, or CMV promoter. More preferably, this vector has a gene for selecting cells transformed by morphogenetic ( For example, a drug resistance gene, which is allowed to be distinguished by a drug (neomycin, G418, etc.) vectors having such characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Furthermore, when the target stably expresses the gene and amplifies the gene copy number in the cell, a method can be used to introduce a vector having a DHFR gene into the CHO cell lacking the nucleic acid synthesis pathway to compensate for this deficiency (for example, pSV2-dfhr ("Molecular Cloning 2 ^nd edition" Cold Spring Harbor Laboratory Press, (1989)), and this vector is amplified using methotrexate (MTX). Also, when the target is a transitional gene expression, a method can be used COS cells having a gene expressing the SV 40 T antigen on their chromosome are transformed with a vector having an SV40 origin of replication (pcD, etc.). Replication derived from polyomavirus, adenovirus, bovine papilloma virus (BPV), etc. can be used. In order to amplify the number of gene copies in the host cell strain, the expression vector may comprise an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, Escherichia coli, and guanine-guanine phosphate. The ribosyltransferase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, and the like are used as selection markers.

The antibody of the present invention obtained can be isolated from the host cell or from the outside of the cell (medium, etc.) and purified into a substantially pure and homogeneous antibody. Such antibodies can be purified using conventional isolation and methods for antibody purification, without limitation. For example, the antibody can be appropriately selected and combined by column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis, isoelectric polymerization, dialysis, Recrystallization, etc., separation and purification.

Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Such chromatography can be carried out using liquid chromatography, for example, HPLC, FPLC. A column for affinity chromatography, including a proteinA column, and a proteinG column. Examples of proteinA columns are used, including HyperD, POROS, and Sepharose FF (GE Amersham Biosciences). The present invention also encompasses antibodies that are highly purified using such purification methods.

The IL-6 receptor binding activity of the obtained antibody can be measured by a method known to those skilled in the art. Methods for determining the antigen binding activity of an antibody include, for example, an enzyme-linked immunosorbent assay (ELISA), an enzyme immunoassay (EIA), a radioimmunoassay (RIA), and a fluorescent antibody method. For example, when an enzyme immunoassay is used, a sample containing an antibody such as a purified antibody and a culture supernatant of an antibody-producing cell is added to a flat plate coated with an antigen. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added. And these flats are incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added, and the absorbance is measured to evaluate the antigen binding activity.

Pharmaceutical composition

The present invention provides a pharmaceutical composition comprising the above polypeptide as an active ingredient. The pharmaceutical composition of the present invention can be used in an IL-6-related disease such as rheumatoid arthritis. Accordingly, the present invention also provides an agent for treating a disease such as rheumatoid arthritis, which comprises one of the above antibodies as an active ingredient. Examples of preferred target diseases include, but are not limited to, rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile idiopathic arthritis, Castleman disease, systemic erythema Lupus (SLE), lupus nephritis, Crohn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, Still's disease, amyloidosis, multiple sclerosis, colonization, age-related retina Plaque degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardium Infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin's lymphoma, pancreatic cancer, lung cancer, esophageal cancer, rectal cancer, cancer cachexia, cancer nerve invasion, Myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed allergy, contact dermatitis, allergic dermatitis, mesothelium Tumor, polymyositis, dermatomyositis, total uveitis, anterior uveitis, uvitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry Eye disease, and inflammation after surgery.

The phrase "containing a primary antibody against IL-6 receptor as an active ingredient" means that a primary antibody comprising an anti-IL-6 receptor is contained as at least one active ingredient, but the content thereof is not particularly limited. Further, the pharmaceutical composition of the present invention may be combined with the above-mentioned multi-peptide and other active ingredients.

The pharmaceutical composition of the present invention can be used for therapeutic purposes as well as for prophylactic use.

The amino acid included in the amino acid sequence of the present invention can be modified after translation. For example, modification of the N-terminal glutamine (Gln) residue to pyroglutamic acid (pGlu) residues using pyroglutamine guanamine is a method well known to those skilled in the art. Naturally, such post-translationally modified amino acids are also included in the amino acid sequences of the invention.

The sugar chain which binds to the antibody of the present invention may be of any structure. The 297 (EU numbering) sugar chain may be any sugar chain structure (preferably fucosylated sugar chain) or may have no sugar chain at this position (for example, by producing antibodies in E. coli, or by borrowing The change was introduced to make the 297-position (EU numbering) sugar-free chain binding).

All prior art cited herein is incorporated herein by reference.

Example

Hereinafter, the present invention will be described by way of examples, but it should be understood that the invention is not limited thereto.

Example 1

Identification of the mutation site of the variable region for enhancing the affinity of TOCILIZUMAB for IL-6 receptor

Construction of a gene pool into which mutated CDR sequences have been introduced, and analysis to improve the affinity of TOCILIZUMAB (H chain WT-IgG1/SEQ ID NO: 53; L chain WT-κ/SEQ ID NO: 54) for IL-6 receptor . Screening of CDR mutation libraries to identify mutations that improve the affinity for the IL-6 receptor. This mutation is shown in Figure 1. The combination of these mutations results in a high affinity TOCILIZUMAB, for example, RDC-23 (H chain RDC23H-IgG1/SEQ ID NO: 55; L chain RDC-23L-κ/SEQ ID NO: 56). The affinity for the soluble IL-6 receptor was compared between RDC-23 and TOCILIZUMAB, and the biological activity confirmed using BaF/gp130 (see Reference Example of this method).

The results of the affinity measurement are shown in Table 1. The biological activity results confirmed by BaF/gp130 (the final concentration of IL-6 was 30 ng/ml) are shown in Fig. 2. The results showed that the affinity of RDC-23 was about 60 times higher than that of TOCILIZUMAB, and the activity expressed by the 100% inhibitory concentration of BaF/gp130 was about 100 times higher.

Example 2

Identification of mutations for improving the pharmacokinetics of TOCILIZUMAB by reducing the isoelectric point

To improve the pharmacokinetics of TOCILIZUMAB, a check was made to identify mutation sites that would reduce the isoelectric point of the variable region but did not significantly reduce binding to the IL-6 receptor. Mutation sites in the variable region were screened according to the predictors of the three-dimensional structural model of TOCILIZUMAB to identify mutation sites that would reduce the isoelectric point of the variable region but did not significantly reduce binding to the IL-6 receptor. These are shown in Figure 3. Combining these mutations results in a TOCILIZUMAB having a reduced isoelectric point, including, for example, H53/L28 (H chain H53-IgG1/SEQ ID NO: 57; L chain L28-κ/SEQ ID NO: 58). Comparing the affinity for IL-6 receptor, the isoelectric point, the pharmacokinetics in mice, and the biological activity confirmed using BaF/gp130 between H53/L28 and TOCILIZUMAB (see Reference Example of the method) ).

The results of the affinity measurement are shown in Table 2. The results of the biological activity obtained using BaF/gp130 (the final concentration of IL-6 was 30 ng/ml) are shown in Fig. 4. The results showed that the affinity for H53/L28 was about 6 times higher than that of TOCILIZUMAB, and the activity expressed by the 100% inhibitory concentration of BaF/gp130 was about several times higher.

The isoelectric point results confirmed by isoelectric point electrophoresis known to those skilled in the art show that the isoelectric points of TOCILIZUMAB and H53/L28 are about 9.3, and 6.5 to 6.7, respectively. Therefore, the isoelectric point of H53/L28 is reduced by about 2.7 compared to TOCILIZUMAB. Further, the theoretical isoelectric point of the variable region of VH/VL was calculated using GENETYX (GENETYX CORPORATION). The results show that the theoretical isoelectric points of TOCILIZUMAB and H53/L28 are 9.20 and 4.52, respectively. Therefore, the isoelectric point of H53/L28 is reduced by about 4.7 compared to TOCILIZUMAB.

To assess the pharmacokinetics of the altered antibody H53/L28 with reduced isoelectric point, the pharmacokinetics of TOCILIZUMAB and H53/L28 in normal mice were compared. A single dose of TOCILIZUMAB or H53/L28 at 1 mg/kg was administered intravenously (IV) or subcutaneously (SC) to mice (C57BL/6J; Charles River Japan, Inc.) to assess temporal changes in plasma concentrations. The temporal changes in plasma concentrations after intravenous administration or subcutaneous administration of TOCILIZUMAB and H53/L28 are shown in Fig. 5 and Fig. 6 , respectively. The pharmacokinetic parameters (clearance, CL) and half-life (T1/2) obtained using WinNonlin (Pharsight) are shown in Table 3. The plasma half-life (T1/2) after intravenous administration of H53/L28 was extended to TOCILIZUMAB. About 1.3 times, and the clearance rate was reduced by about 1.7 times. The T1/2 increase after H53/L28 subcutaneous administration was about 2 times that of TOCILIZUMAB, and the clearance rate was reduced by about 2.1 times. Therefore, it was found that it can be substituted by amino acid. The isoelectric point of TOCILIZUMAB is lowered, thereby significantly improving pharmacokinetics.

Example 3

Identification of mutation sites that reduce the immunogenicity of TOCILIZUMAB

Identification of mutations that reduce the risk of immunogenicity of T cell epitopes present in the variable region

The T cell epitopes present in the variable regions of the TOCILIZUMAB sequence were analyzed using TEPITOPE (Methods. 2004 Dec; 34(4): 468-75). As a result, the L chain CDR2 is predicted to have a number of T cell epitopes that bind to HLA (i.e., sequences with a high risk of immunogenicity). Therefore, TEPITOPE analysis was performed to examine amino acid substitutions that would reduce the immunogenicity risk of the L chain CDR2 without reducing stability, binding activity or neutralizing activity.

As described below, the screening results demonstrate that the sulphate of L chain CDR2 (SEQ ID NO: 59) L51 of TOCILIZUMAB can be replaced by glycine, and the arginine of L53 is glutamic acid (SEQ ID NO: 60). Reduced immunogenicity without reducing stability, binding activity or neutralizing activity (Kabat's numbering; Kabat et al., (1991) Sequence of Proteins of Immunological Interest, NIH).

TOCILIZUMAB L-chain CDR2 (SEQ ID NO: 59)

The TOCILIZUMAB L chain CDR2 removes the T cell epitope (SEQ ID NO: 60).

Example 4

Reduces the risk of immunogenicity by completely humanizing the variable region framework sequence of TOCILIZUMAB

For the humanization of TOCILIZUMAB, some mouse sequences were retained in the framework sequence to maintain binding activity (Cancer Res. 1993 Feb 15; 53(4): 851-6). These sequences are H27, H28, H29, H30 in the H chain FR1 of the variable region sequence of TOCILIZUMAB and H71 in the H chain FR3 (Kabat's numbering; Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, NIH)). The retained mouse sequence is a potential cause of increased risk of immunogenicity. Therefore, we assessed whether the framework sequence can be fully humanized to further reduce the immunogenic risk of TOCILIZUMAB.

The results show that the complete framework of TOCILIZUMAB can be replaced by the H-chain FR1 (SEQ ID NO: 61) of TOCILIZUMAB as the following humanized H-chain FR1-A (SEQ ID NO: 62), and substituted H-chain FR3 (SEQ ID NO: 63) ) is a humanized H chain FR3 (SEQ ID NO: 64) which is fully humanized but does not reduce stability, binding activity or neutralizing activity.

TOCILIZUMAB H chain FR1 (sequence identification number 61)

Humanized H chain FR1-A (SEQ ID NO: 62) (derived from the germ cell line IMGT hVH_4)

TOCILIZUMBA H chain FR3 (sequence identification number 63)

Humanized H chain FR3 (SEQ ID NO: 64) (derived from Mol. Immunol. 2007. 44(4): 412-422)

Example 5

Link to the IL-6 receptor based on the pH dependence of TOCILIZUMAB to identify mutations that improve pharmacokinetics

One of the methods for improving the pharmacokinetics of TOCILIZUMAB is to modify the molecule such that a single molecule of TOCILIZUMAB repeatedly binds and neutralizes several molecules of IL-6 receptor. It is hypothesized that when TOCILIZUMAB is linked to the membrane-type IL-6 receptor, TOCILIZUMAB is linked to the membrane-type IL-6 receptor, enters the intracellular inner capsule via internalization, and is then linked to the membrane-type IL-6 receptor. The state is transferred to the lysate and decomposed by the lysate. In particular, one molecule of TOCILIZUMAB is typically linked to one or two membrane-type IL-6 receptor molecules (in a monovalent or bivalent manner) and decomposed in lysosomes after internalization. Therefore, one molecule of TOCILIZUMAB can only bind and neutralize one or two molecules of the membrane type IL-6 receptor.

Therefore, the inventors of the present invention thought whether it is possible to create a combination of TOCILIZUMAB in a pH-dependent manner, wherein the binding of TOCILIZUMAB can be maintained under neutral conditions, and under acidic conditions, the connection is significantly reduced, and the TOCILIZUMAB can be combined in a pH-dependent manner. It is isolated from the membrane type IL-6 receptor (antigen) in the inner capsule and returned to the plasma by ligating the FcRn present in the inner capsule, as shown in FIG. Once returned to the plasma, the TOCILIZUMAB bound in a pH dependent manner can again be ligated to the membrane type IL-6 receptor. Using this repeated binding in plasma and separation in the inner capsule, it is envisaged that one molecule of TOCILIZUMAB can repeatedly bind/neutralize several molecules of IL-6 receptor. Therefore, TOCILIZUMAB combined in a pH-dependent manner is presumed to have better pharmacokinetics than TOCILIZUMAB.

TOCILIZUMAB isolated from the IL-6 receptor under acidic conditions in the inner vesicles, its binding must be significantly attenuated compared to under neutral conditions. On the cell surface, strong IL-6 receptor binding is required for neutralization; therefore, at the cell surface pH, pH 7.4, the binding of the antibody to the IL-6 receptor must be equal or stronger than TOCILIZUMAB. It has been reported that the pH of the inner capsule is generally 5.5 to 6.0 (Nat Rev Mol Cell Biol. 2004 Feb; 5(2): 121-32). Therefore, if the pH-dependently bound TOCILIZUMAB is modified to be weakly linked to the IL-6 receptor at pH 5.5 to 6.0, it is predicted that the inner capsule will be isolated from the IL-6 receptor under acidic conditions. In particular, if the pH-dependent TOCILIZUMAB is modified to pH 7.4, it is the cell surface pH, strongly linked to the IL-6 receptor, and at the pH of the inner capsule, pH 5.5-6.0, weakly Linked to the IL-6 receptor, one molecule of TOCILIZUMAB can bind and neutralize several molecules of IL-6 receptor, and the pharmacokinetics can be improved accordingly.

One of the possible ways to provide pH dependence in combination with TOCILIZUMAB to IL-6 receptor is to introduce a histidine residue in the variable region of TOCILIZUMAB because the histidine residue has a pKa of about 6.0 to 6.5 and its protons The separation state changes between neutral (pH 7.4) and acidity (pH 5.5 to 6.0). Therefore, according to the three-dimensional structural model of TOCILIZUMAB, screening was performed for identifying the site where histidine was introduced in the variable region. In addition, the selected variable region sequences of TOCILIZUMAB were randomly substituted with histidine to design a library of screenings. The screening system was used to bind to the IL-6 receptor at pH 7.4 and to isolate or decrease affinity from the IL-6 receptor at pH 5.5 to 5.8 as an indicator.

As a result, the inventors of the present invention found that the pH-dependent (binding at pH 7.4, separation at pH 5.8) binding to the mutation site of TOCILIZUMAB to IL-6 receptor is shown in Fig. 8. In Figure 8, the tyrosine acid of H27 is substituted with histidine, which is a mutation of the H chain FR1, not in the CDR. However, as described in Eur. J. Immunol. (1992) 22:1719-1728, the sequence having a histidine acid at H27 is a human sequence (SEQ ID NO: 65). Therefore, the antibody can be completely humanized by combining Example 4 using the following framework.

Humanized H chain FR1-B (sequence identification number 65)

Combinations of mutations, including, for example, H3pI/L73 (H chain H3pI-IgG1/SEQ ID NO: 66; L chain L73-κ/SEQ ID NO: 67) can produce TOCILIZUMAB with pH-dependent binding properties. H3pI/L73 and TOCILIZUMAB affinity for soluble IL-6 receptor at pH 7.4, rate of isolation from membrane type IL-6 receptor at pH 7.4 and pH 5.8, biological activity using BaF/gp130, and The pharmacokinetics in cynomolgus monkey and human IL-6 receptor gene-transferred mice were compared (see Reference Example of this method).

The results of the affinity test against soluble IL-6 receptor at pH 7.4 are shown in Table 4. The results of the biological activity obtained using BaF/gp130 (final IL-6 concentration of 30 ng/ml) are shown in Fig. 9. These results show that H3pI/L73 is comparable to TOCILIZUMAB in affinity for soluble IL-6 receptor at pH 7.4 and activity on BaF/gp130.

The rate of separation of TOCILIZUMAB or H3pI/L73 from the membrane type IL-6 receptor at pH 7.4 and pH 5.8 is shown in Table 5. Compared to TOCILIZUMAB, the separation rate of H3pI/L73 at pH 5.8 was faster, and the pH dependence of the rate of separation from the membrane type IL-6 receptor was increased by about 2.6-fold.

A single dose of TOCILIZUMAB or H3pI/L73 was administered intravenously to cynomolgus monkeys at 1 mg/kg to assess temporal changes in plasma concentrations. The temporal change in plasma concentration of TOCILIZUMAB or H3pI/L73 after intravenous administration is shown in FIG. The results showed that the pharmacokinetics of H3pI/L73 was significantly improved in cynomolgus monkeys compared to TOCILIZUMAB.

A single dose of TOCILIZUMAB or H3pI/L73 was administered intravenously at 25 mg/kg to human IL-6 receptor gene-transforming mice (hIL-6R tg mice; Proc Natl Acad Sci USA. 1995 May 23; 92(11) : 4862-6) to assess the temporal change in plasma concentration. The temporal change in plasma concentration of TOCILIZUMAB or H3pI/L73 after intravenous administration is shown in FIG. The results showed that the pharmacokinetics of H3pI/L73 was significantly improved compared to TOCILIZUMAB in human IL-6 receptor gene-transferred mice.

H3pI/L73, a TOCILIZUMAB with pH-dependent binding properties, showed significant improved pharmacokinetics compared to TOCILIZUMAB in cynomolgus monkey and human IL-6 receptor gene-transferred mice. This represents the ability to bind and neutralize several molecules of the IL-6 receptor using a single molecule by providing the property of binding to the antigen at pH 7.4 and isolation from the antigen at pH 5.8. It is also believed that the pharmacokinetics can be further improved by making the IL-6 receptor binding more pH dependent than H3pI/L73.

Example 6

Optimization of the TOCILIZUMAB constant region

Reduce the heterogeneity of the C-terminus of the TOCILIZUMAB H chain

It has been reported that the heterogeneity of the H-terminal C-terminal sequence of IgG antibodies, due to the deletion of the two C-terminal amino acids, glycine and lysine, the removal of the C-terminal amino acid lysine residues and the amidation C Terminal carboxyl group. (Anal Biochem. 2007 Jan 1;360(1):75-83). Further, in TOCILIZUMAB, the main component is a sequence in which the C-terminal amino acid lysine in the nucleotide sequence is deleted by post-translational modification; however, the amide acid retention in the minor component is secondary. The C-terminal carboxyl group in the component also contributes to heterogeneity due to the deletion of both glycine and lysine. It is not easy and costly to desire to maintain the heterogeneity of the target substance/related substance in the production room by mass production of the drug. If it is possible to develop an antibody as a pharmaceutical, it is a single substance and the heterogeneity is reduced. Therefore, when the antibody is developed as a pharmaceutical, it is preferred that the H-chain C-terminal heterogeneity does not exist.

The C-terminal amino acid is altered to reduce C-terminal amino acid heterogeneity. The results show that the heterogeneity of the C-terminal derivation can be avoided by deleting the amino acid and glycine residues at the C-terminus of the H chain constant region of TOCILIZUMAB from the nucleotide sequence in advance. The following heterogeneity was evaluated by cation exchange chromatography: TOCILIZUMAB, TOCILIZUMAB lacking a C-terminal lysine residue (TOCILIZUMABδK: H chain WT-IgGIδK/SEQ ID NO: 68; L chain WT-κ/SEQ ID NO: 54), lacking TOCILIZUMAB (TOCILIZUMABδGK: H chain WT-IgG1δGK/SEQ ID NO: 69; L chain WT-κ/SEQ ID NO: 54) at the C-terminus of the amino acid and glycine residues. A ProPac WCX-10 4x250 mm (Dionex) column was used; mobile phase A used 25 mmol/L MES/NaOH (pH 6.1) and mobile phase B used 25 mmol/L MES/NaOP, 250 mmol/L NaCl (pH 6.1). Use the appropriate flow rate and gradient. The evaluation results obtained by cation exchange chromatography are shown in Fig. 12. The results show that the C-terminal amino acid heterogeneity can be reduced by pre-delete the amino acid and glycine residues at the C-terminus of the constant region of the H chain from the nucleotide sequence, rather than merely deleting the H chain constant region in advance. The amino acid residue at the C-terminus. The C-terminal sequences of all human antibody IgG1, IgG2, and IgG4 constant regions have lysine and glycine at positions 447 and 446, respectively, according to the EU numbering method (see Sequences of proteins of immunological interest, NIH Publication No. 91-3242). acid. Therefore, the method for reducing the heterogeneity of the C-terminal amino acid found in this study is expected to be used in the IgG2 and IgG4 constant regions and variants thereof.

Reduce the heterogeneity of disulfide bond derivation in the IgG2 isoform TOCILIZUMAB

The isoform of TOCILIZUMAB is IgG1. Since TOCILIZUMAB is a neutralizing antibody linked to an Fc gamma receptor, it may be disadvantageous from the viewpoint of immunogenicity and adverse effects. A possible method for reducing Fcγ receptor binding is to convert an IgG antibody isoform from IgG1 to IgG2 or IgG4 (Ann Hematol. 1998 Jun; 76(6): 231-248). From the viewpoint of Fcγ receptor I binding and pharmacokinetics, IgG2 is more desirable than IgG4 (Nat Biotechnol. 2007 Dec; 25(12): 1369-72). At the same time, the physical and chemical properties of proteins, especially homogeneity and stability, are very important when developing antibodies for pharmaceuticals. The IgG2 isoform is reported to have very high heterogeneity due to the disulfide bond in the hinge region (J Biol Chem. 2008 Jun 6; 283(23): 16206-15). The heterogeneity derived from the disulfide bond associated with the mass production of the drug while maintaining the target substance/related substance between productions is not easy and costly. Therefore, as much as possible, it is desirable to be a single substance. Therefore, when the IgG2 isotype antibody is developed as a pharmaceutical, it is preferred to reduce the heterogeneity derived from the disulfide bond without lowering the stability.

Various variants were evaluated for the use of reducing the heterogeneity of IgG2 isoforms. As a result, it was found that the WT-SKSC constant region (SEQ ID NO: 70) can reduce the heterogeneity without lowering the stability, wherein the IgG2 constant region sequence H chain CH1 region at position 131 of the cysteine residue and 133 positions The arginine residues (EU numbering method) are each substituted with a serine and an lysine, and the cysteine residue at position 219 in the upper hinge of the H chain is substituted with a serine. Preparation of 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/sequence Identification 54) and TOCILIZUMAB-SKSC (H-chain WT-SKSC/SEQ ID NO: 70; L-chain WT-κ/SEQ ID NO: 54) and used to assess heterogeneity and stability. Heterogeneity was assessed by cation exchange chromatography. A ProPac WCX-10 (Dionex) column was used: mobile phase A using 20 mM sodium acetate (pH 5.0) and mobile phase B using 20 mM sodium acetate, 1 M NaCl (pH 5.0). Use the appropriate flow rate and gradient. The evaluation results obtained by cation exchange chromatography are shown in FIG. The stability was evaluated based on the intermediate temperature in the thermal denaturation (Tm value) determined by differential scanning calorimetry (DSC) (VP-DSC: Microcal). The results of DSC measurement of Tm values in 20 mM sodium acetate, 150 mM NaCl, pH 6.0 and Fab regions are shown in FIG.

The results show that the heterogeneity of TOCILIZUMAB-IgG2 is significantly increased compared to TOCILIZUMAB-IgG1; however, the heterogeneity can be significantly reduced by conversion to TOCILIZUMAB-SKSC. Further, compared with TOCILIZUMAB-IgG1, the DSC of TOCILIZUMAB-IgG2 has a low-stability shoulder (Fab*) component, that is, a low Tm, in the heat-denatured peak of the Fab region, which is presumed to be due to a heterogeneous component. However, when converted to TOCILIZUMAB-SKSC, the shoulder (low Tm) due to the heterogeneous component disappeared, and the Tm value was about 94 ° C, which was equivalent to the Fab region of TOCILIZUMAB-IgG1 and TOCILIZUMAB-IgG2. Therefore, TOCILIZUMAB-SKSC shows high stability.

Identification of pharmacokinetically modified mutation sites in the constant region of TOCILIZUMAB

As described above, it is achievable from IgG1, which is a homolog of TOCILIZUMAB, which reduces C-terminal heterogeneity and reduces the heterogeneity of the antibody constant region of the IgG2 isoform while reducing binding to the Fcγ receptor and maintaining high stability. Further, it is preferred that the constant region has superior pharmacokinetic properties as compared with the IgG1, TOCILIZUMAB isoform.

In order to find a constant region having a superior plasma half-life than an antibody having an IgG1 isoform constant region, screening was performed to identify a mutation site for improving the pharmacokinetics of TOCILIZUMAB-SKSC having the same IgG2 isoform as described above. High stability associated with antibody in the constant region and reduced heterogeneity. As a result, WT-M58 (SEQ ID NO: 72 (amino acid sequence)) was found in which glutamic acid at position 137 of the EU number was substituted with glycine and leucine at position 138 was replaced with WT-SKSC. For glycine, the histidine acid at position 268 is substituted with glutamine, the arginine at position 355 is substituted with glutamine, and the glutamine at position 419 is substituted with glutamic acid, of which 446 is glycine and The tyrosine acid at position 447 was deleted to reduce the heterogeneity of the C-terminus of the H chain. Further, WT-M44 (SEQ ID NO: 73) (amino acid sequence) was prepared, and the aspartic acid substituted at position 434 was substituted with respect to IgG1. Alanine. Further, the 446-glycolic acid and the 447-amino acid were removed from M44 to produce WT-M83 (SEQ ID NO: 74 (amino acid sequence)) to reduce the heterogeneity of the C-terminus of the H chain. WT-M73 (SEQ ID NO: 75 (amino acid sequence)) was produced by substituting 428-position of aspartic acid of WT-M58 with alanine.

Preparation of TOCILIZUMABM-44 (H chain WT-M44/SEQ ID NO: 73; L chain WT-κ/SEQ ID NO: 54), TOCILIZUMABM-58 (H chain WT-M58/SEQ ID NO: 58/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), and evaluated for humans using human FcRn gene-transferred mice FcRn and pharmacokinetics (see reference examples of the method).

Binding of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58 and TOCILIZUMAB-M73 to human FcRn was assessed using Biacore. As shown in Table 6, TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73 were respectively superior to the binding of TOCILIZUMAB-IgG1 by about 2.7 times, 1.4 times, and 3.8 times.

The pharmacokinetics of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73 in human FcRn gene-transferred mice were evaluated. The result is shown in Fig. 15. As shown in Figure 15, better pharmacokinetics were shown compared to TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, and TOCILIZUMAB-M73. The effect of improved pharmacokinetics is related to the ability to bind to human FcRn. In particular, the concentration of TOCILIZUMAB-M73 in plasma after 28 days was about 16-fold better than that of TOCILIZUMAB-IgG1. Therefore, it is speculated that an antibody having a constant region of M73 has significantly improved pharmacokinetic properties in humans as compared with an antibody having an IgG1 constant region.

Example 7

Preparation of fully humanized IL-6 receptor antibodies with improved PK/PD

The TOCILIZUMAB variant was prepared by combining multiple mutations of the variable and constant regions of TOCILIZUMAB found in the above examples. The fully humanized IL-6 receptor antibody found in many screens is: 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) and Fv5-M83 (H chain VH5-M83/SEQ ID NO: 27; L chain VL5-κ/SEQ ID NO: 30).

The affinity of the prepared Fv-M73, Fv4-M73, Fv5-M83 for the IL-6 receptor and the affinity of TOCILIZUMAB for the IL-6 receptor were compared (see Reference Example of the method). The affinity of these antibodies for the soluble IL-6 receptor at pH 7.4 is shown in Table 7. Further, the BaF/gp130 neutralizing activity of the same with TOCILIZUMAB and the control group (the high-affinity anti-IL-6 receptor described in the known Reference Examples, and the VQ8F11-21 hIgG1 described in US2007/0280945) was compared (see the present invention). Reference example). The results of determining the biological activities of these antibodies using BaF/gp130 are shown in Figure 16 (TOCILIZUMAB, control group, Fv5-M83 final IL-6 concentration of 300 ng/ml) and Figure 17 (TOCILIZUMAB, Fv3-M73, The final IL-6 concentration of Fv4-M73 was 30 ng/ml). As shown in Table 7, Fv3-M73 and Fv4-M73 have about 2 to 3 times higher affinity than TOCILIZUMAB, and Fv5-M83 is about 100 times more affinity than TOCILIZUMAB (due to difficulty in determining the affinity of Fv5-M83, Affinity (H chain VH5-IgG1/SEQ ID NO: 76; L chain VL5-κ/SEQ ID NO: 30) was determined using Fv5-IgG1, and Fv5-M83 has a constant region of IgG1 form; this constant region is generally considered to have affinity no effect). As shown in Fig. 17, Fv3-M73 and Fv4-M73 were slightly more active than TOCILIZUMAB. As shown in Figure 16, Fv5-M83 is very active, at 50% inhibition concentration, 100 times higher than TOCILIZUMAB. Fv5-M83 also showed about 10-fold higher neutralizing activity than the control group (known high-affinity anti-IL-6 receptor antibody) at a 50% inhibitory concentration.

The rate of separation of TOCILIZUMAB, Fv3-M73 and Fv4-M73 from membrane-type IL-6 receptors at pH 7.4 and pH 5.8 was determined. As shown in Table 8 (see Reference Example of the Method), the pH dependence of the separation rate of Fv3-M73 and Fv4-M73 from the membrane-type IL-6 receptor was improved by about 11-fold and 10-fold compared to TOCILIZUMAB. . The pH dependence of the separation rate of H3pI/L73 as described in Example 5 was considerably improved, indicating that the pharmacokinetics of Fv3-M73 and Fv4-M73 were significantly improved compared to H3pI/L73.

The isoelectric point of TOCILIZUMAB, control, Fv3-M73, Fv4-M73, Fv5-M83 was determined by isoelectric focusing electrophoresis using methods known to those skilled in the art. The results show that the isoelectric point of TOCILIZUMAB is about 9.3; the isoelectric point of the control group 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; Fv5-M83 The isoelectric point is about 5.4~5.5. Therefore, each antibody has a significantly lower isoelectric point than TOCILIZUMAB and the control group. Further, the theoretical isoelectric point of the variable region VH/VL is calculated by GENETYX (GENETYX CORPORATION). The results showed that the isoelectric point of TOCILIZUMAB was about 9.20; the isoelectric point of the control group was 7.79; the isoelectric point of Fv3-M73 was 5.49; the isoelectric point of Fv4-M73 was 5.01; and the isoelectric point of Fv5-M83 was 4.27. Therefore, each antibody has a significantly lower isoelectric point than TOCILIZUMAB and the control group. It was shown by Example 2 that the pharmacokinetics were improved by lowering the isoelectric point, so that Fv3-M73, Fv4-M73 and Fv5-M83 were considered to have improved pharmacokinetics compared to TOCILIZUMAB and the control group.

T cell epitopes of 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, it was predicted that TOCILIZUMAB has a T cell epitope, many of which bind to HLA, as shown in Example 3. Conversely, the number of sequences predicted to bind to the T cell epitope was significantly reduced in Fv3-M73, Fv4-M73, and Fv5-M83. Furthermore, the framework of Fv3-M73, Fv4-M73 or Fv5-M83 does not have a mouse sequence and is therefore fully humanized. These indicate that the immunogenic risk of Fv3-M73, Fv4-M73 and Fv5-M83 may be significantly reduced compared to TOCILIZUMAB.

Example 8

PK/PD test of fully humanized IL-6 receptor antibody in monkeys

TOCILIZUMAB, control group, Fv3-M73, Fv4-M73 and Fv5-M83 were administered intravenously at a dose of 1 mg/kg to cynomolgus monkeys to assess temporal changes in plasma concentrations (see Reference Examples of the Method). The plasma concentration changes of TOCILIZUMAB, Fv3-M73, Fv4-M73 and Fv5-M83 after intravenous injection are shown in Fig. 18. The results showed that Fv3-M73, Fv4-M73 and Fv5-M83 showed significantly improved pharmacokinetics in cynomolgus monkeys compared to TOCILIZUMAB and the control group. Among them, Fv3-M73 and Fv4-M73 showed highly improved pharmacokinetics compared to TOCILIZUMAB.

The potency of each antibody to neutralize the membrane type cynomolgus IL-6 receptor was assessed. After antibody administration (TOCILIZUMAB from day 3 to day 10), cynomolgus IL-6 was administered subcutaneously at 5 μg/kg per day from day 6 to day 18, and was determined 24 hours later. CRP concentration of each animal (see reference example of the method). The time change of the CRP concentration after administration of each antibody is shown in Fig. 19. To assess the potency of each antibody to neutralize the soluble cynomolgus IL-6 receptor, the plasma concentration of the free soluble cynomolgus IL-6 receptor in cynomolgus monkeys was determined and the percentage of free soluble IL-6 receptor was calculated. (See the reference example of the method). The percentage change in the percentage of free soluble IL-6 receptor after administration of each antibody is shown in FIG.

Fv3-M73, Fv4-M73 and Fv5-M83 neutralize the membrane-type cynomolgus IL-6 receptor in a more sustained manner than TOCILIZUMAB and the control group (known high-affinity anti-IL-6 receptor antibodies), And the increase in CRP is inhibited for a long period of time. In addition, Fv3-M73, Fv4-M73 and Fv5-M83 neutralized the free soluble cynomolgus IL-6 receptor in a more sustained manner than TOCILIZUMAB and the control group, and inhibited free soluble cynomolgus IL over a longer period of time. -6 receptors increase. These findings demonstrate that Fv3-M73, Fv4-M73 and Fv5-M83 are superior to TOCILIZUMAB and the control group in terms of sustained neutralization of membrane type and soluble IL-6 receptor. Among them, Fv3-M73 and Fv4-M73 are significantly superior in terms of sustained neutralization. At the same time, Fv5-M83 inhibited CRP and free soluble cynomolgus IL-6 receptor more strongly than Fv3-M73 and Fv4-M73. Therefore, Fv5-M83 is considered to be stronger than Fv3-M73, Fv4-M73 and the control group (known high-affinity anti-IL-6 receptor antibody) in neutralizing the membrane type and soluble IL-6 receptor. It is believed that the results in these cynomolgus monkeys reflect the stronger affinity of Fv5-M83 for IL-6 receptor and the stronger biological activity of Fv5-M83 in the BaF/gp139 test line relative to the control group.

These findings represent that Fv3-M73 and Fv4-M73 have a highly superior persistence as an anti-IL-6 antibody neutralizing antibody compared to TOCILIZUMAB and the control group, and thus can significantly reduce the dose and frequency of administration. Further, Fv5-M83 proved to be remarkably superior in terms of the activity intensity and strength persistence of the neutralizing antibody against the IL-6 receptor. Therefore, Fv3-M73, Fv4-M73, and Fv5-M83 are expected to be useful as pharmaceutical IL-6 antagonists.

Example 9

Choose a stable buffer type for Fv4-M73

Fv4-M73 was prepared as described above. Since Fv4-M73, as in Examples 6-7, consists of the non-native constant region M73 to improve heterogeneity, stability, safety and pharmacokinetics, the stability profile of Fv4-M73 (eg, most stable buffer species) It may be different from an antibody consisting of a natural constant region such as IgG1. Antibodies consisting of the native IgG1 constant region are reported to be most stable in histidine-acetate buffer (WO/2006/044908). Therefore, the effect of the buffer type on the stability of Fv4-M73 was determined.

Fv4-M73 was formulated in 4 different buffer formulations of pH 6.0 to give a final concentration of 37 mg/mL (as described in Table 9). The samples were stored under storage conditions at 40 ° C for more than 2 months, analyzed by UV, and analyzed by size exclusion chromatography and anion exchange chromatography. The percentage of agglomerates formed in these formulations is shown in Figure 21 as time. . The percentage increase in agglutination represents an indicator of the decrease in stability of the antibody. Therefore, the percentage increase is used as an indicator for comparing the stabilizing effects of different buffers.

As shown in Figure 21, the formulation with histidine-HCl buffer (Formulation A) and citrate buffer (Formulation D) was the most stable, while the formulation with acetate buffer (Formulation C) was the most unstable. Histidine-acetate (Formulation B) is slightly less stable than histidine-HCl buffer, presumably due to destabilization of acetate buffer.

Therefore, it was found that Fv4-M73 with a non-native constant region is most stable in histidine-HCl buffer and citrate buffer, instead of the reported histidine-acetate buffer as a native IgG1 antibody. The most stable type of buffer.

method

Size Exclusion Chromatography (SEC) : Size exclusion chromatography was performed to screen for antibody formulations in which antibody agglutinates and fragments were present. The sample injection size is excluded from the G3000 SW _XL column (TOSOH). The mobile phase was 50 mM sodium phosphate, 300 mM sodium chloride (pH 7.0), and flowed at a gradient of 0.5 mL/min. The extracted protein detected UV absorbance at 200 nm. The relative amount of any protein species detected is expressed as a percentage of the total area of the product peak relative to all other detected peaks. The peak extracted earlier than the peak of the antibody monomer was recorded as the agglutination percentile, and the peak extracted later than the peak of the antibody monomer but earlier than the peak of the buffer was recorded as the percentile of the fragment.

Sample preparation: The sample was diluted to 0.3 to 2 mg/mL with the mobile phase and 15 μL was injected into the column.

Anion exchange chromatography: Anion exchange chromatography was performed to analyze the presence of heterogeneous antibody formulations, especially the presence of deamidation (de-amidated by acidic species after the main peak). The sample was injected into a DEAE-NPR column (TOSOH) at 40 °C. The mobile phase (A) was 10 mM Tris-HCl (pH 7.5), B), 10 mM Tris-HCl, 500 mM sodium chloride (pH 7.5), the gradient was 100% mobile phase A, and 70% mobile phase A and 30 minutes later. 30% of phase B was moved, then 100% of phase B was moved 1 minute, then the column was washed at a flow rate of 1.0 mL/min for 4 minutes. The extracted protein was tested for UV absorbance at 280 nm.

Sample preparation: The sample was diluted to 0.05-0.33 mg/mL with the mobile phase and 100 μL was injected into the column.

Example 10

Effect of pH, NaCl and arginine-HCl on the stability of Fv4-M73 at 100 mg/mL

Formulation of Fv4-M73 in different buffer formulations (as shown in Table 10, to a final concentration of 100 mg/mL, and by size exclusion chromatography and anion exchange chromatography, stored at 25 and 40 °C by UV detection) A 2-month sample and also tested frozen-thawed samples (freezing at -20 °C for 0.5 days and then thawing at room temperature for 30 minutes. Percentage of agglutinate present in the formulation, and 2/4/ at 25 and 40 °C) The percentage of agglomerates increased after 8 weeks compared to the initial, as shown in Figures 22-25. The increase in agglutination was used as an indicator of the decrease in stability.

Actual value (pH)

The low molecular weight species (LMW) in Fig. 26 is presumed to be a result of a weak point such as a hinge portion directly hydrolyzing a peptide bond under acidic or basic conditions. This is especially true for single antibody in liquid formulations at elevated temperatures. The acidic species was extracted at about 22.5 to 26 minutes, which increased under alkaline conditions and is shown in Figure 27. It is speculated that due to the non-enzymatic deamination of the indoleamine residue (common antibody modification), it was reported. By incubating the antibody at a high temperature at an alkaline pH, it is observed that the more acidic species are involved in the charge heterogeneity of the individual antibodies. Considering the results of Figures 22-26, it was found that Fv4-M73 is stable in solution with respect to agglutination and decomposition at a pH of about 5.5 to 6.3. It was observed that the pH of about 5.5 to 6.3 is the optimum stability with respect to the ratio of the main peaks relative to the total peak area (Fig. 27-28).

In the present study, 100 mM NaCl was added to a buffer containing 20 mM buffer and 50 mM NaCl (20 mM buffer, 150 mM NaCl), which was equivalent to containing 20 mM buffer and 50 mM NaCl (20 mM buffer, 50 mM NaCl). A stable antibody solution indicates that NaCl has no stabilizing effect. Adding 100 mM arginine to a buffer containing 20 mM buffer and 50 mM NaCl (20 mM buffer, 50 mM NaCl, and 100 mM arginine-HCl) showed significant stabilization of the antibody solution relative to agglutination, representing spermine Acid-HCl has a remarkable stabilizing effect. In the case of buffers, the stability of the solution using the histidine-HCl buffer was slightly better than that of the citrate buffer.

Regarding the freeze-thaw study ("FT" represents the number of freeze/thaw cycles), as shown in Figure 29, a significant amount of agglutinate was observed in formulations of lower or lower salt concentrations. Adding 100 mM arginine to a buffer containing 20 mM buffer and 50 mM NaCl (20 mM buffer, 50 mM NaCl, 100 mM arginine-HCl) showed that in the formation of agglutination, compared to the addition of 100 mM NaCl (20 mM buffer, 150 mM NaCl) was more effective, indicating that arginine-HCl has a significant stabilizing effect on freeze-thaw. The optimum stability was observed at a pH of about 5.0 to 6.6 containing 100 mM arginine-HCl.

Example 11

The effect of sugar and arginine-HCl on Fv4-M73 at 100 mg/mL

Fv4-M73 was formulated in 4 different buffer formulations to a final concentration of 100 mg/mL (as described in Table 11). These samples were stored under storage conditions of 25 and 40 ° C for more than 2 months, analyzed by UV, analyzed by size exclusion chromatography and anion exchange chromatography, and subjected to a specific number of freeze/thaw cycles ( It was frozen at -20 degrees for 0.5 days and thawed at room temperature for 30 minutes). The percentage of agglutinate contained in the formulation was plotted from time to time and is shown in Figures 30 and 31. The percentage increase in the amount of agglomerates is an indicator of the decrease in stability.

As shown in Figures 30 and 31, Formulations F and G provided less stability than Formulation H, indicating that sucrose and trehalose were smaller than arginine-HCl in liquid conditions maintained at 25 and 40 °C. Stabilization, while freeze-thaw, Formulations F and G provide comparable or higher stability than Formulation H, indicating that sucrose or trehalose has a significant stabilizing effect on freeze-thaw.

Example 12

Stability of Fv4-M73 at 200mg/mL: pH, arginine-HCl and trehalose

Fv4-M73 was formulated in 6 different formulations as described in Table 12, with a final concentration of 200 mg/mL.

The samples were each incubated at 5 ° C and -20 ° C for 3 months and 6 months, and the 3 month sample and the 6 month sample were analyzed by size exclusion chromatography as described in Example 9, and detected by UV. The percentage of agglomerate increase from the beginning in this formulation at 5 ° C after 3 months and 6 months, as shown in Figure 32. The percentage of agglomerate increase from the initial in this formulation at -20 °C after 3 months and 6 months, as shown in Figure 33.

As shown in Figure 32, Fv4-M73 is apparently stable at lower pH (most stable at pH 5.5, most unstable at pH 6.5) and higher arginine at liquid conditions maintained at 5 °C. The concentration is relatively stable (most stable at 150 mM arginine-HCl and most unstable at 50 mM arginine-HCl). The addition of 50 mM trehalose showed some stabilizing effect on Fv4-M73 stored in liquid conditions at 5 °C. Since the pharmaceutical liquid formulation of the antibody is usually stored at 5 ° C, the preferred liquid formulation of Fv4-M73 should contain at least 100 mM arginine-HCl and histidine buffer pH 5.5 ~ pH 6.0, and if necessary additional Stabilizer.

As shown in Figure 33, Fv4-M73 was stable at higher pH at -20 °C, and was significantly stable at higher concentrations of arginine (most stable at 150 mM arginine-HCl, at 50 mM arginine-HCl is the most unstable). The addition of 50 mM trehalose at -20 ° C under cryopreservation conditions showed a significant stabilizing effect on Fv4-M73. A large number of antibodies are often stored in -20 to -70 ° C in a frozen state. Consider the storage and shipping costs in the frozen state, and it is expected to be stored at -20 °C. A preferred bulk formulation for the storage of Fv4-M73 at -20 ° C should contain at least 100 mM arginine-HCl and histidine buffer pH 5.5 to pH 6.5 and sugar (eg trehalose).

Reference embodiment

Preparation of soluble recombinant human IL-6 receptor

A soluble recombinant human IL-6 receptor that is a human IL-6 receptor that is an antigen is produced as follows. Produces a structurally soluble human with amino acid sequence 1 to position 344 from J. Biochem. (1990) 108, 673-676 (Yamasaki et al., Science (1988) 241, 825-828 (GenBank * X12830) CHO cell line of IL-6 receptor. The soluble human IL-6 receptor was cultured from CHO cells expressing SR344 and purified by 3-column chromatography: Blue Sepharose 6 FF column chromatography, using immobilization The column of the antibody of SR344 was subjected to affinity chromatography and gel filtration column chromatography, and the fraction extracted from the main peak was used as a final purified sample.

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

Preparation of oligo DNA primers based on the revealed gene sequence of the rhesus monkey IL-6 receptor (Birney et al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue: D556-61) Using this primer, a DNA fragment encoding the IL-6 receptor gene of the whole cynomolgus monkey was prepared by PCR using cDNA prepared from the pancreas of cynomolgus monkey. The obtained DNA fragment was inserted into a mammalian expression vector, and This vector was used to prepare a stable CHO cell line (cyno.sIL-6R-producing CHO cell line). The cyno.sIL-6R-producing CHO cell line was purified using a HisTrap column (GE Healthcare Bioscience) and Amicon Ultra-15 Ultracel. -10 k (Millipore) was concentrated, and further purified by Superdex 200pg16/60 gel column (GE Healthcare Bioscience) to obtain a sample of the finally purified 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 deposited in SWISSPROT accession number P79341 was prepared and cloned into a mammalian expression vector. The resulting vector was introduced into CHO cells to prepare a stable expression cell strain (cynoIL-6 producing CHO cell strain). The medium in which the cynoIL-6-producing CHO cell strain was purified using an SP-Sepharose/FF column (GE Healthcare Bioscience), and concentrated with Amicon Ultra-15 Ultracel-5k (Millipore). Further, it was purified by Superdex 200pg16/60 gel column (GE Healthcare Bioscience), and then concentrated by Amicon Ultra-15 Ultracel-5k (Millipore) to obtain a finally purified cynomolgus IL-6 sample (hereinafter referred to as cIL-6). ).

Preparation of known high affinity anti-IL-6 receptor antibodies

A mammalian cell expression vector was constructed to express VQ8F11-21 hIgG1, VQ8F11-21 hIgG1 as a known high affinity anti-IL-6 receptor antibody. VQ8F11-21 hIgG1 is described in US2007/0280945 A1 (US2007/0280945 A1; the amino acid sequences of the H and L chains, respectively, listed in SEQ ID NO: 77 and 78). The antibody variable region was constructed by PCR using a combination of synthetic oligo DNA (combined PCR), and IgG1 was used as a constant region. The antibody variable region and the constant region are combined together by a combination PCR and inserted into a mammalian expression vector to construct a expression vector for the H chain and the L chain of interest. The nucleotide sequence of the resulting expression vector is determined by methods known to those skilled in the art. The expression vector was constructed as described in Example 1, and the high-affinity anti-IL-6 receptor antibody (hereinafter referred to as "control group") was expressed and purified.

Preparation, performance and refinement of TOCILIZUMAB variants

The TOCILIZUMAB variant was prepared according to the instruction manual attached to the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The obtained plasmid fragment is inserted into a mammalian cell expression vector to construct a expression vector for the H chain and the L chain of interest. The nucleotide sequence of the resulting expression vector is confirmed by methods known to those skilled in the art. The antibody is expressed as follows. Human embryonic kidney cancer-derived HEK293H cell line (Invitrogen) was suspended in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). The cells were seeded at a cell density of 5-6 x ^{10 5} cells/ml with 10 mL each in a petri dish (10 cm in diameter; Corning) for adhering cells, and cultured in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for one full day and one All night. Then, the medium was removed by suction, and 6.9 mL of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plastids were introduced into the cells by lipofection. The collected culture supernatant was centrifuged (about 2000 g, 5 min, room temperature) to remove the cells and passed through a 0.22 μm filter MILLEX. -GV (Millipore) was filtered to remove bacteria to obtain a supernatant. The antibodies obtained from the culture supernatant using ^{rProtein A Sepharose TM Fast Flow (Amersham} Biosciences) known to those skilled in the art of refining methods. In order to determine the concentration of the purified antibody, the absorbance at 280 nm was measured using a spectroscopic spectrometer. The absorbance coefficient was calculated by the PACE method (Protein Science 1995; 4: 2411-2423), and the antibody concentration was calculated from the determined value.

Establishment of human gp130-expressing BaF3 cell line

A BaF3 cell line expressing human gp130 was established using the procedure described below to obtain a cell line that proliferated in an IL-6-dependent manner.

The full-length human gp130 cDNA was amplified by PCR (Hibi et al., Cell (1990) 63:1149-1157 (GenBank #NM_002184), and cloned into the expression vector pCOS2Zeo to construct pCOS2Zeo/gp130. pCOS2Zeo is a expression vector. The DHFR gene expression region was removed from pCHOI (Hirata et al., FEBS Letter (1994) 456: 244-248) and inserted into the expression region of the Zeocin anti-drug gene. The full-length human IL-6R cDNA was amplified by PCR, and It was cloned into pcDNA3.1(+) (Invitrogen) to construct hIL-6R/pcDNA3.1(+).

10 μg of pC0S2Zeo/gp130 was mixed with BaF3 cells (0.8× ^{10 7} cells) suspended in PBS, and then pulsed with Gene Pulser (Bio-Rad) at 0.33 kV and 950 micro FD. BaF cells having a gene introduced by electroporation were cultured in RPMI 1640 medium (Invitrogen) supplemented with 0.2 ng/ml mouse IL-3 (Peprotech) and 10% fetal bovine serum (hereinafter referred to as FBS, HyClone). Day and night, and build a performance by adding RPMI 1640 medium supplemented with 100 ng/ml human IL-6 (R&D systems), 100 ng/ml human IL-6 soluble receptor (R&D systems) and 10% FBS BaF3 cell line of human gp130 (hereinafter referred to as "BaF3/gp130"). This BaF/gp130 proliferates in the presence of human IL-6 (R&D systems) and soluble human IL-6 receptors and can be used to assess the growth inhibitory activity of anti-IL-6 receptor antibodies (or IL-6 receptor neutralizing activity). ).

Evaluation of biological activity by BaF cell line (BaF/gp130) expressing human gp130

The IL-6 receptor neutralizing activity was evaluated using BaF3/gp130 which was proliferated in an IL-6/IL-6 receptor-dependent manner. After washing 3 times with RPMI 1640 supplemented with 10% FBS, BaF/gp130 was supplemented with RPMI1640 supplemented with 600 ng/ml or 60 ng/ml human IL-6 (TORAY), appropriate amount of soluble human IL-6 receptor and 10% FBS. Suspended to 5x10 ⁴ cells/ml (final concentration of 300 ng/ml or 30 ng/ml). The cell suspension was dispensed (50 microliter/well) on a 96 well plate (CORNING). The purified antibody was then diluted with RPMI 1640 containing 10% FBS and added to each well (50 microliter/well). The cells were incubated for 3 days at 37 ° C under 5% CO ₂ . WST-8 reagent (Cell Counting Kit-8; Dojindo Laboratories) was diluted 2-fold in PBS. When 20 μL of the drug was added to each well, the absorbance at 450 nm (reference wavelength: 620 nm) was measured using SUNRISE CLASSIC (TECAN). After 2 hours of incubation, the absorbance was measured at 450 nm (reference wavelength: 620 nm). The IL-6 receptor neutralizing activity was evaluated using the change in absorbance as an index during 2 hours.

Binding to soluble human IL-6 receptor based on Biacoare assay

Antigen-antibody reaction kinetics were analyzed using Biacore T100 (GE Healthcare). The antibody of interest is bound to the wafer at pH 7.4 by immobilizing an appropriate amount of protein A or protein A/G or anti-IgG (gamma chain specificity) F(ab') 2 on an induction wafer by an amine coupling method. The soluble human IL-6 receptor antibody interaction was determined by running on the wafer at pH 7.4 adjusted to various concentrations of soluble IL-6 receptor as an analyte. All assays were performed at 37 °C. From the measured spectrum, the kinetic parameters were calculated, combined with the rate constant k _a (1/Ms) and the dissociation rate constant k _d (1/s). Then, based on this rate constant, K _D (M) is determined. The respective parameters were determined using Biacore T100 Evaluation Software (GE Healthcare).

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

The antigen-antibody interaction with the membrane type IL-6 receptor was observed at pH 5.8 and pH 7.4 using Biacore T100 (GE Healthcare). Binding to the membrane-type IL-6 receptor was assessed by assessing binding to the soluble human IL-6 receptor immobilized on the sensing wafer. SR344 is biotinylate by methods known to those skilled in the art. Based on the affinity between biotin and Streptavidin, the biotinylated soluble human IL-6 receptor is immobilized on a sensing wafer via streptavidin. All assays were performed at 37 °C. The mobile phase buffer was 10 mM MES (pH 5.8), 150 mM NaCl, and 0.05% Tween20. A pH-binding clone was shown and injected at pH 7.4 to connect to the soluble human IL-6 receptor (injected sample buffer was 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05). % Tween20). Then, the pH-dependent dissociation of each of the colonies was observed at pH 5.8, which is the pH of the mobile phase. The dissociation rate constant (kd(1/s)) at pH 5.8 was calculated using only Biacore T100 Evaluation Software (GE Healthcare) by only meeting the dissociation phase at pH 5.8. The sample concentration is 0.25 μg/ml. The binding was carried out in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20, and dissociation was carried out at 10 mM MES (pH 5.8), 150 mM NaCl, and 0.05% Tween20. Similarly, the dissociation rate constant (kd (1/s)) at pH 7.4 was calculated by only meeting the dissociated phase at pH 7.4. The sample concentration is 0.5 microgramme/ml. The binding was carried out in 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween 20, and dissociation was carried out at 10 mM MES (pH 7.4), 150 mM NaCl, and 0.05% Tween20.

Assessing the binding of human FcRn

FcRn is a complex of FcRn and β2-microglobulin. Oligo DNA primers were prepared according to the disclosed human FcRn gene sequence (J. Exp. Med. (1994) 180(6): 2377-2381). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and prepared primers. Using the obtained DNA fragment as a template, a fragment encoding the extracellular region containing the message region (Met1-Leu290) was PCR-amplified, and an animal cell expression vector (amino acid of human FcRn listed in SEQ ID NO: 79) was inserted. sequence). Similarly, oligo DNA primers were prepared in accordance with the disclosed human β2-microglobulin gene sequence (Proc. Nat1. Acad. Sci. USA (2002) 99(26): 16899-16903). A DNA fragment encoding the entire gene was prepared by PCR using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as a template and prepared primer. Using the DNA fragment obtained as a template, a DNA fragment encoding the information region (Met1-Met119) encoding intact β2-microglobulin was amplified by PCR and inserted into a mammalian cell expression vector (listed in SEQ ID NO: 80). The amino acid sequence of human β2-microglobulin).

The soluble human FcRn was expressed in the following procedure. The plastids constructed for human FcRn and β2-microglobulin were introduced into cells of human embryonic kidney cancer-derived cell line HEK293H (Invitrogen) by lipofection using 10% FBS. The culture supernatant obtained was collected, and the FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) according to the method described in J. Immunol. 2002 Nov1; 169(9): 5171-80, and further using HiTrap Q HP (GE Healthcare). refined.

Determine the concentration of antibodies in mouse plasma

The concentration of antibody in mouse plasma is determined by ELISA according to methods known to those skilled in the art.

The antibody concentration and CRP concentration in monkey plasma were confirmed by PK/PD test. Free soluble IL-6 receptor

Plasma concentrations in cynomolgus monkeys were confirmed by ELISA using methods known to those skilled in the art.

The concentration of CRP was determined using an automated analyzer (TBA-120FR; Toshiba Medical Systems Co.) using Cias R CRP (KANTO CHEMICAL CO., Inc.).

The plasma concentration of free soluble cynomolgus IL-6 receptor in cynomolgus monkeys was confirmed by the following procedure. All IgG-type antibodies (cynomolgus monkey IgG, in plasma) were prepared by loading 30 μL of cynomolgus monkey plasma in a 0.22 μM filter bowl (Millipore) with an appropriate amount of rProtein A Sepharose Fast Flow resin (GE Healthcare). The anti-human IL-6 receptor antibody, and the anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex) are adsorbed to protein A. The solution in the cup was then transferred using high speed centrifugation to collect the passed solution. The resulting solution contained no protein A-linked anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex. Therefore, the free soluble IL-6 receptor concentration can be determined by measuring the concentration of the soluble cynomolgus IL-6 receptor in the solution of ProteinA. The concentration of soluble cynomolgus IL-6 receptor is confirmed using a method known to those skilled in the art for determining the concentration of soluble human IL-6 receptor. The soluble cynomolgus IL-6 receptor (cIL-6R) prepared as described above was used as a standard. The percentage of free soluble IL-6 receptor is calculated by the following formula.

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Figure 1 shows a table of mutation sites that improve the affinity of TOCILIZUMAB for IL-6 receptor. The HCDR2 sequence of TOCILIZUMAB is shown in SEQ ID NO: 81; the mutated HCDR2 sequence (listed above) is shown in SEQ ID NO: 82; the mutated HCDR2 sequence (below) is shown in SEQ ID NO: 83; the HCDR3 sequence of TOCILIZUMAB is shown in SEQ ID NO: No. 84; the mutated HCDR3 sequence (listed above) is shown in SEQ ID NO: 85; the mutated HCDR3 sequence (below) is shown in SEQ ID NO: 86; the LCDR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 87; the mutated LCDR1 sequence (listed above) is shown in sequence identification number 88; the mutated LCDR1 sequence (below) is shown in SEQ ID NO: 89; the LCDR3 sequence of TOCILIZUMAB is shown in SEQ ID NO: 90; the mutated LCDR3 sequence (listed above) is shown in sequence identification. No. 91; the mutated LCDR3 sequence (below) is shown in SEQ ID NO:92.

Figure 2 shows the neutralization activity of TOCILIZUMAB and RDC-23 in BaF/gp130.

Figure 3 shows a table of mutation sites capable of reducing the isoelectric point of the variable region without significantly reducing the binding of TOCILIZUMAB to IL-6 receptor. The asterisk in the figure represents a mutation site that has no effect on the isoelectric point but is mutated in order to transform into a human sequence. The HFR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 93; the mutated HFR1 sequence is shown in SEQ ID NO: 94; the HCDR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 95; the mutated HCDR1 sequence is shown in SEQ ID NO: 96; the HFR2 sequence of TOCILIZUMAB Shown in SEQ ID NO: 97; the mutated HFR2 sequence is shown in SEQ ID NO: 98; the HCDR2 sequence of TOCILIZUMAB is shown in SEQ ID NO: 81; the mutated HCDR2 sequence is shown in SEQ ID NO: 99; the HFR4 sequence in TOCILIZUMAB is shown in SEQ ID NO: No. 100; the mutated HFR4 sequence is shown in SEQ ID NO: 101; the LFR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 102; the mutated LFR1 sequence is shown in SEQ ID NO: 103; the LCDR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 87; The latter LCDR1 sequence is shown in sequence identification number 104; the LFR2 sequence of TOCILIZUMAB is shown in sequence identification number 105; the LFR2 sequence after mutation is shown in sequence identification number 106; the LCDR2 sequence of TOCILIZUMAB is shown in sequence identification number 107; the LCDR2 sequence after mutation Displayed in sequence identification numbers 108 and 109; the LFR3 sequence of TOCILIZIMAB is shown in sequence identification number 110; After the LFR3 sequence is shown in SEQ ID NO 111; TOCILIZUMAB of LFR4 sequence is shown in SEQ ID NO 112; LFR4 after the mutant sequence is shown in SEQ ID NO 113.

Figure 4 shows the neutralizing activity of TOCILIZUMAB and H53/L28 in BaF/gp130.

Figure 5 shows the temporal changes in plasma concentrations of TOCILIZUMAB and H53/L28 in mice after intravenous administration.

Figure 6 shows the temporal changes in plasma concentrations of TOCILIZUMAB and H53/L28 in mice after subcutaneous administration.

Figure 7 illustrates that an IgG molecule can recombine to another antigen by isolation from a membrane type antigen in an endosome.

Figure 8 shows a mutant site (binding to pH 7.4, isolated at pH 5.8) that provides pH dependence of TOCILIZUMAB binding to the IL-6 receptor. The HFR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 93; the mutated HFR1 sequence is shown in SEQ ID NO: 114; the HCDR1 sequence of TOCILIZUMAB is shown in SEQ ID NO: 95; the mutated HCDR1 sequence is shown in SEQ ID NO: 115; the LCDR1 sequence of TOCILIZUMAB Displayed in SEQ ID NO: 87; the mutated LCDR1 sequence is shown in SEQ ID NO: 116; the LCDR2 sequence of TOCILIZUMBA is shown in SEQ ID NO: 107; and the mutated LCDR2 sequence is shown in SEQ ID NO: 117.

Figure 9 shows the TOCILIZUMAB and H3pI/L73 neutralizing activities in BaF/gp130.

Figure 10 shows the temporal changes in plasma concentrations of TOCILIZUMAB and H3pI/L73 in cynomolgus monkeys after intravenous administration.

Figure 11 shows the temporal changes in plasma concentrations of TOCILIZUMAB and H3pI/L73 in human IL-6 receptor gene-transferred mice after intravenous administration.

Figure 12 shows the results of evaluating the C-terminally derived heterogeneity of TOCILIZUMAB, TOCILIZUMAB δ K, TOCILIZUMAB δ GK using cation exchange chromatography.

Figure 13 shows the results of evaluating the heterogeneity of disulfide bond-derived TOCILIZUMAB-IgG1, TOCILIZUMAB IgG-2, TOCILIZUMAB SKSC using cation exchange chromatography.

Figure 14 shows denaturation curves of TOCILIZUMAB-IgG1, TOCILIZUMAB IgG-2, TOCILIZUMAB SKSC obtained using a differential scanning calorimeter (DSC), and Tm values for each Fab region.

Figure 15 shows the temporal changes in plasma concentrations of TOCILIZUMAB-IgG1, TOCILIZUMAB-M44, TOCILIZUMAB-M58, TOCILIZUMAB-M73 in human FcRn transgenic mice after intravenous administration.

Figure 16 shows the neutralizing activity of TOCILIZUMAB, control and Fv5-M83 in BaF/gp130.

Figure 17 shows the neutralizing activity of TOCILIZUMAB, Fv3-M73, Fv4-M73 in BaF/gp130.

Figure 18 shows the temporal changes in plasma concentrations of TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 in cynomolgus monkeys after intravenous administration.

Figure 19 shows the temporal changes in CRP concentrations of TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 in cynomolgus monkeys after intravenous administration.

Figure 20 shows the time course of the percentage of free soluble IL-6 receptors in TOCILIZUMAB, control, Fv3-M73, Fv4-M73 and Fv5-M83 in cynomolgus monkeys after intravenous administration.

Figure 21 shows the difference in high molecular weight species (HMW) of the different formulations (A-D) over time to form the antibody Fv4-M73.

Figure 22 shows the high molecular weight species (δ HMW: increased from the beginning) at different pH values, buffer type, NaCl concentration, with or without arginine at two time points (25 ° C) to form antibody Fv4-M73 The difference in value).

Figure 23 shows the difference in high molecular weight species (HMW) of the antibody Fv4-M73 formed at two time points (25 ° C) at different pH values, buffer species, NaCl concentration, and with or without arginine.

Figure 24 shows the high molecular weight species of the antibody Fv4-M73 at different pH values, buffer species, NaCl concentration, and with or without arginine at three different time points (40 ° C) (δHMW: from the start The difference in value added).

Figure 25 shows the difference in high molecular weight species (HMW) of the antibody Fv4-M73 formed at different time points, buffer species, NaCl concentration, and with or without arginine at one time point (40 °C).

Figure 26 shows the high molecular weight species of the antibody Fv4-M73 at different pH values, buffer species, NaCl concentration, and with or without arginine at three different time points (40 ° C) (δLMW: from the start The difference in value added).

Figure 27 shows the results of anion exchange chromatography performed on a solution of the antibody Fv4-M73 stored at different pH values.

Figure 28 shows the results of anion exchange chromatography of a solution of antibody Fv4-M73 stored at different pH values, different NaCl concentrations, arginine and two different buffers.

Figure 29 shows the difference in the formation of high molecular weight substances (δHMW: increasing from the initial value) of antibody Fv4-M73 for different numbers of freeze/thaw cycles, different NaCl concentrations, arginine, and 2 different buffers. .

Figure 30 shows the difference in the formation of high molecular weight species (δHMW: increasing value from the initial) of antibody Fv4-M73 for 3 different time points (at 40 ° C and 25 ° C) and 4 different formulations (E-H).

Figure 31 shows the difference in high molecular weight species (δHMW: increasing from the initial value) of antibody Fv4-M73 for 2 different cycles of freezing/thawing cycles and 4 different formulations (E-H).

Figure 32 shows the difference in high molecular weight species (δHMW: increasing from the initial value) of the antibody Fv4-M73 formed by 6 different formulations after 3 months and 6 months at 5 °C.

Figure 33 shows the difference in high molecular weight species (δHMW: increasing from the initial value) of the antibody Fv4-M73 formed by 6 different formulations after 3 months and 6 months at -20 °C.

Claims (20)

A pharmaceutical formulation comprising at least one antibody selected from the group consisting of: (a) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 20 (VH3-M73 a variable region) comprising the sequence of SEQ ID NO: 23 (variable region of VL3); and (b) an antibody comprising a heavy chain and a light chain comprising the sequence of SEQ ID NO:26 (VH3-M73), the light chain comprises the sequence of sequence identification number 29 (VL3). A stable pharmaceutical formulation as claimed in claim 1 comprising a histidine and/or citrate buffer. A stable pharmaceutical formulation as claimed in claim 1 or 2, comprising at least one cationic amino acid. A stable pharmaceutical formulation according to claim 1 or 2, comprising 1 to 500 mM histidine and/or citrate buffer, 1 to 1500 mM of at least one cationic amino acid, and 1 to 200 mg/mL of antibody. And 1~400mM carbohydrates. The pharmaceutical formulation of claim 4, wherein the cationic amino acid is arginine. The pharmaceutical formulation of claim 4, wherein the carbohydrate is sucrose or trehalose. For example, the pharmaceutical formulation of claim 1 or 2 further comprises a surfactant. For example, the pharmaceutical formula of claim 1 or 2, wherein the doctor The pharmaceutical formulation contains at least 10 mg/ml of the multipeptide and/or antibody. The pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation contains at least 50 mg/ml of the multipeptide and/or antibody. A pharmaceutical formulation according to claim 1 or 2, wherein the pharmaceutical formulation contains at least 80 mg/ml of the multipeptide and/or antibody. The pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation contains less than or equal to 240 mg/ml of the multipeptide and/or antibody. For example, the pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation has a pH in the range of 4.5 to 7.0. For example, the pharmaceutical formulation of claim 12, wherein the pharmaceutical formulation has a pH in the range of 5.5 to 6.6. A pharmaceutical formulation as claimed in claim 1 or 2 wherein the formulation is a liquid. The pharmaceutical formulation of claim 14 wherein the pharmaceutical formulation is not lyophilized during the preparation of the formulation. The pharmaceutical formulation of claim 1 or 2 wherein the dimerization of the multipeptide and/or antibody molecule is reduced. The pharmaceutical formulation of claim 1 or 2 wherein the dimerization of the multi-peptide and/or antibody molecule is inhibited. The pharmaceutical formulation of claim 1 or 2, wherein the pharmaceutical formulation is for subcutaneous administration. A method of stabilizing a solution containing an antibody comprising adding at least one cationic amino acid, wherein the antibody is at least one antibody selected from the group consisting of: (a) an antibody comprising a heavy chain variable region comprising a sequence of SEQ ID NO: 20 (variable region of VH3-M73), and a light chain variable region comprising a sequence of SEQ ID NO: 23 (variable region of VL3); and (b) an antibody comprising a heavy chain and a light chain comprising the sequence of SEQ ID NO: 26 (VH3-M73), the light chain comprising the sequence Sequence of identification number 29 (VL3). A method for stabilizing an antibody, comprising a method of adding at least one cationic amino acid in a solution of a freeze/thaw cycle, wherein the antibody is selected from at least one of the following antibodies: (a) an antibody comprising a heavy chain variable region And a light chain variable region comprising the sequence of SEQ ID NO: 20 (variable region of VH3-M73) comprising the sequence of SEQ ID NO: 23 (variable region of VL3) And (b) an antibody comprising a heavy chain comprising a sequence of SEQ ID NO: 26 (VH3-M73), and a light chain comprising the sequence of SEQ ID NO: 29 (VL3).