JP5661553B2 - Formulations containing improved antibody molecules - Google Patents

Description

  The present invention relates to formulations containing polypeptides and / or antibodies, and in particular to stable high concentration liquid formulations containing modified anti-IL6 receptor antibodies.

  In recent years, various antibody preparations have been developed and used in practice. Many of these formulations are used for intravenous injection. In subcutaneous administration, the amount of antibody per administration is large (approximately 100 mg to 200 mg), and the volume for subcutaneous injection is generally limited. It is necessary to increase the antibody concentration in it.

  In solutions containing high concentrations of antibodies, undesirable degradation occurs, including the formation of insoluble and / or soluble aggregates. These insoluble and soluble aggregates are likely to form in the liquid state due to association of antibody molecules. If the liquid formulation is stored for a long time, deamidation of asparagine residues may result in a loss or decrease in the bioactivity of the antibody molecule. Freezing and thawing cycles also cause the formation of degraded and aggregated antibody molecules.

  Various concepts have been proposed to provide a stabilized formulation in which the reduction of the active ingredient is suppressed even after long-term storage of the formulation. Such formulations are obtained by dissolving the active ingredient and various additives in a buffer solution. There is a need to provide highly concentrated antibody-containing formulations that are inhibited from dimerization and deamidation during long-term storage, are stable, and are suitable for use in subcutaneous administration.

  Antibodies are attracting attention as pharmaceuticals because of their high stability in plasma and few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs have been developed (Non-patent Documents 1 and 2). IL-6 is a cytokine involved in various autoimmune diseases, inflammatory diseases, malignant tumors, etc. (Non-patent Document 3), and tocilizumab, a humanized anti-IL-6 receptor IgG1, is specific for IL-6 receptor To join. Tocilizumab is considered to be usable as a therapeutic agent for diseases related to IL-6 such as rheumatoid arthritis by neutralizing the biological action of IL-6 (Patent Documents 1 to 3, Non-patent Documents) Reference 4). Tocilizumab is approved in Japan as a therapeutic agent for Castleman's disease and rheumatoid arthritis (Non-patent Document 5).

  Humanized antibodies such as tocilizumab are first generation antibody drugs, and second generation antibody drugs are currently being developed that have improved first generation antibody drugs to improve efficacy, convenience, and cost. Various technologies have been developed as technologies that can be applied to second-generation antibody drugs, and technologies that improve effector function, antigen binding ability, pharmacokinetics, stability, or reduce immunogenicity risk have been reported. Has been. Technology to enhance antibody-dependent cytotoxicity (ADCC activity) or complement-dependent cytotoxicity (CDC activity) by amino acid substitution in the Fc region of IgG antibody as a method to enhance efficacy or reduce dosage Has been reported (Non-patent Document 6). Affinity maturation technique (Non-patent Document 7) has been reported as a technique for enhancing antigen binding ability and antigen neutralization ability, and introduces mutations into amino acids such as variable region complementarity determining regions (CDRs). Thus, the binding activity to the antigen can be enhanced. By enhancing the antigen binding ability, in vitro biological activity can be improved, or the dose can be reduced, and further, the in vivo efficacy can be improved (Non-patent Document 8). Currently, clinical trials of motavizumab (produced by affinity maturation), which is said to exhibit an effect superior to that of palivizumab, which is a first-generation anti-RSV antibody, are being conducted (Non-patent Document 9). An anti-IL-6 receptor antibody has been reported to have an affinity of about 0.05 nM (ie, affinity is stronger than tocilizumab) (Patent Document 4), but a human antibody or human having an affinity higher than 0.05 nM There are no reports of conjugated antibodies or chimeric antibodies.

  A problem with current antibody drugs is high production cost due to the very large amount of protein administered. In the case of tocilizumab, which is a humanized anti-IL-6 receptor IgG1 antibody, intravenous injection of about 8 mg / kg / month is also assumed (Non-patent Document 4). As for the dosage form, a subcutaneous preparation is desirable in the case of chronic autoimmune diseases. In general, it is necessary that the preparation for subcutaneous administration be a high-concentration preparation. In the case of an IgG type antibody preparation, it is generally considered that the limit is about 100 mg / mL in terms of stability. (Non-patent document 10). By increasing the plasma half-life of the antibody so that a sustained therapeutic effect can be exerted, the amount of protein administered is reduced, and by providing high stability, subcutaneous administration at a long administration interval is possible, and the cost is low. It is possible to provide a highly convenient second generation antibody drug.

  FcRn plays a major role in antibody pharmacokinetics, and IgG1 and IgG2 have the best plasma half-life, and IgG3 and IgG4 are inferior in terms of differences in plasma half-life between antibody isotypes. (Non-patent Document 11). As a method for further improving the plasma half-life of IgG1 and IgG2 antibodies having excellent plasma half-life, amino acid substitution in the constant region that enhances binding to FcRn has been reported (Non-patent Documents 12 and 13). From the viewpoint of immunogenicity, it is preferable to further improve the plasma half-life by amino acid substitution of the variable region rather than the constant region (Patent Document 5). Until now, by modifying the variable region, IL-6 receptor antibody There is no report that improved the plasma half-life.

  Another important issue in developing biopharmaceuticals is immunogenicity. Generally, mouse antibodies are reduced in immunogenicity by humanization. It is said that the risk of immunogenicity can be further reduced by using a germline sequence as a template framework for humanization (Non-patent Document 14). However, even with adalimumab, which is a fully human anti-TNF antibody, immunogenicity appears at a high frequency of 13 to 17%, and the therapeutic effect is reduced in patients with immunogenicity (non-patented References 15 and 16). Even a human antibody may have a T cell epitope in the CDR, and the T cell epitope on the CDR may cause immunogenicity. Although methods for predicting T cell epitopes in silico or in vivo have been reported (Non-patent Documents 17 and 18), the risk of immunogenicity is reduced by removing T cell epitopes predicted using these methods It is considered possible to do this (Non-patent Document 19).

  Tocilizumab which is a humanized anti-IL-6 receptor IgG1 antibody is an IgG1 antibody obtained by humanizing a mouse PM1 antibody. CDR grafting has been carried out using human sequences of NEW and REI as H and L chains, respectively, as template frameworks, but 5 amino acids remain in the framework as mouse sequences as amino acids important for activity retention ( Non-patent document 20). There have been no reports of complete humanization of mouse sequences remaining in the framework of tocilizumab, a humanized antibody, without reducing activity. In addition, the CDR sequence of tocilizumab is a mouse sequence, and, like adalimumab, there is a possibility that a T cell epitope exists in CDR, and the risk of immunogenicity cannot be denied. In clinical trials of tocilizumab, no anti-tocilizumab antibody was observed at an effective dose of 8 mg / kg, but the antibody was observed at 4 mg / kg and 2 mg / kg (Patent Document 6). . This suggests that there is room for improvement in the immunogenicity of tocilizumab. However, there were no reports on reducing the immunogenicity risk of tocilizumab by amino acid substitution.

  The isotype of tocilizumab is IgG1, but the difference in isotype is the difference in the sequence of the constant region, and the sequence of the constant region is thought to greatly affect effector function, pharmacokinetics, physical properties, etc. The selection of the sequence of the constant region is extremely important for drug development (Non-patent Document 11). In recent years, the safety of antibody drugs has become very important, and one of the causes of serious side effects seen in Phase I clinical trials of TGN1412 is the interaction (effector function) of the Fc part of the antibody with the Fcγ receptor. It is considered (Non-Patent Document 21). In antibody drugs that aim to neutralize the biological action of antigens, binding to Fcγ receptors, which are important for effector functions such as ADCC, is unnecessary, and in terms of side effects, binding to Fcγ receptors is not necessary. Rather, it may be undesirable. As a method for reducing the binding to Fcγ receptor, there is a method of changing the IgG antibody isotype from IgG1 to IgG2 or IgG4 (Non-patent Document 22). From the viewpoint of binding to Fcγ receptor I and pharmacokinetics, IgG4 It is considered that IgG2 is desirable (Non-patent Document 11). Since the isotype of tocilizumab is IgG1 and is an IL-6 receptor neutralizing antibody, it is possible that an effector function such as ADCC is not necessary, and when considering the possibility of side effects, the isotype may be IgG2.

  On the other hand, in developing antibody drugs, the physicochemical properties of the protein, especially homogeneity and stability, are extremely important, and the IgG2 isotype has significant heterogeneity derived from the disulfide bond in the hinge region. Has been reported (Non-patent Document 23). It is not easy to manufacture a large amount as a pharmaceutical while maintaining the difference in heterogeneity of the target substance / related substance derived from the disulfide bond, leading to an increase in cost. Therefore, it is desirable to be a single substance as much as possible. In addition, as a heterogeneity of the C-terminal sequence of the antibody heavy chain, a deletion of the lysine residue of the C-terminal amino acid and amidation of the C-terminal carboxyl group due to the deletion of both the C-terminal two amino acids glycine and lysine are reported. (Non-patent Document 24). In developing an IgG2 isotype antibody as a medicine, it is desirable that these heterogeneities are reduced while maintaining high stability. In order to produce a stable high-concentration subcutaneous preparation excellent in convenience, it is desirable that not only the stability is high, but also the plasma half-life is superior to IgG1, which is an isotype of tocilizumab. However, to date, we have reported a sequence of a constant region that has reduced heterogeneity for IgG2 isotype constant region antibodies, has high stability, and has a better plasma half-life than IgG1 isotype constant region antibodies. There is no.

WO 92/19759 WO 96/11020 WO 96/12503 WO 2007/143168 WO 2007/114319 WO 2004/096273

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  The object of the present invention is to modify the amino acid sequences of the variable and constant regions of tocilizumab which is a humanized anti-IL-6 receptor IgG1 antibody, thereby enhancing the pharmacokinetics while enhancing the antigen neutralizing ability. Formulation consisting of a second generation molecule superior to tocilizumab with less frequent frequency and sustained therapeutic effect, improved immunogenicity, safety and physicochemical properties (stability and homogeneity) Hereinafter, the present specification provides an “agent” or “pharmaceutical composition”.

  In addition, the present invention provides stable, and preferably suitable for use in subcutaneous administration, dimerization and deamidation during long term storage or multiple freeze / thaw cycles. Provides a high-concentration polypeptide and / or antibody-containing preparation.

  Conducted intensive research to solve the above problems and found that a stable high-concentration polypeptide and / or antibody-containing preparation can be obtained by adding the amino acid arginine or a salt thereof as a stabilizer to the solution. did.

  Hereinafter, the present invention will be described in detail. In light of the second generation molecule, which outperforms the first generation humanized anti-IL-6 receptor IgG1 antibody tocilizumab, the research focus was on obtaining a stable formulation. As a result, regarding the antibody, the present inventors have discovered a plurality of CDR mutations that improve the antigen binding ability (affinity) in the variable region of tocilizumab. By this, the present inventors succeeded in improving affinity greatly by the combination of such a mutation. The present inventors have also succeeded in improving pharmacokinetics by introducing a modification that reduces the isoelectric point of the variable region sequence. In addition, the inventors have made it possible to neutralize an antigen multiple times with one molecule of antibody by imparting pH dependency to binding to the antigen IL-6 receptor, and to improve pharmacokinetics. Successful. The present inventors also reduced the immunogenicity risk by fully humanizing mouse-derived sequences remaining in the tocilizumab framework and reducing the number of T cell epitope peptides predicted in silico in the variable region. Succeeded. Furthermore, in the constant region of tocilizumab, the present inventors reduced the binding to the Fcγ receptor lower than IgG1 to improve safety, improved the pharmacokinetics than IgG1, and further disulfide bonds in the hinge region of IgG2. We have succeeded in finding a novel constant region sequence in which the heterogeneity derived from the H chain and the heterogeneity derived from the C-terminus are reduced without reducing stability. By combining these CDR region amino acid sequence modifications, variable region amino acid sequence modifications, and constant region amino acid sequence modifications appropriately, we succeeded in creating a second generation molecule superior to tocilizumab.

The present invention has a superior ability to bind to an antigen (IL-6 receptor) by altering the amino acid sequences of the variable region and constant region of tocilizumab which is a humanized anti-IL-6 receptor IgG1 antibody, Formulations comprising humanized anti-IL-6 receptor IgG antibodies having pharmacokinetics, superior safety and physical properties (stability, uniformity), and reduced immunogenicity risks, and The present invention relates to a method for producing a pharmaceutical composition. More specifically, the following [1] to [27] are provided.
[1] (a) CDR1 including the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 including the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: 3 (VH4-M73 A polypeptide comprising CDR3 comprising the sequence of CDR3);
(B) CDR1 comprising the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: 6 (CDR3 of VH3-M73) A polypeptide comprising CDR3 comprising the sequence;
(C) CDR1 comprising the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and SEQ ID NO: 9 (CDR3 of VH5-M83) A polypeptide comprising CDR3 comprising the sequence;
(D) Includes CDR1 containing the sequence of SEQ ID NO: 10 (CDR1 of VL1), CDR2 containing the sequence of SEQ ID NO: 11 (CDR2 of VL1), and CDR3 containing the sequence of SEQ ID NO: 12 (CDR3 of VL1) A polypeptide;
(E) Includes CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 of VL3), CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR3 containing the sequence of SEQ ID NO: 15 (CDR3 of VL3) And a polypeptide; and (f) a CDR1 comprising the sequence of SEQ ID NO: 16 (CDR1 of VL5), a CDR2 comprising the sequence of SEQ ID NO: 17 (CDR2 of VL5), and a sequence of SEQ ID NO: 18 (CDR3 of VL5) A formulation comprising at least one polypeptide selected from polypeptides comprising CDR3.
[2] (a) CDR1 including the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 including the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: 3 (VH4-M73 The heavy chain variable region comprising CDR3 comprising the sequence of CDR3), and the CDR1 comprising the sequence of SEQ ID NO: 10 (CDR1 of VL1), the CDR2 comprising the sequence of SEQ ID NO: 11 (CDR2 of VL1), and SEQ ID NO: 12 An antibody comprising a light chain variable region comprising CDR3 comprising the sequence (CDR3 of VL1);
(B) CDR1 comprising the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: 6 (CDR3 of VH3-M73) A heavy chain variable region comprising CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 13 (CDR1 of VL3), a CDR2 comprising a sequence of SEQ ID NO: 14 (CDR2 of VL3), and SEQ ID NO: 15 (VL3 of An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(C) CDR1 comprising the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and SEQ ID NO: 9 (CDR3 of VH5-M83) A heavy chain variable region comprising CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 16 (CDR1 of VL5), a CDR2 comprising a sequence of SEQ ID NO: 17 (CDR2 of VL5), and SEQ ID NO: 18 (of VL5 An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(D) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1);
(E) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 20 (variable region of VH3-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 23 (variable region of VL3);
(F) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5);
(G) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1);
(H) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain comprising the sequence of SEQ ID NO: 29 (VL3); and (i) SEQ ID NO: 27 (VH5-M83) A formulation comprising at least one antibody selected from antibodies comprising a heavy chain comprising the sequence of: and a light chain comprising the sequence of SEQ ID NO: 30 (VL5).
[3] The stable preparation according to [1] or [2], comprising a histidine buffer and / or a citrate buffer.
[4] The stable preparation according to any one of [1] to [3], comprising at least one basic amino acid.
[5] comprising 1 to 500 mM histidine buffer and / or citrate buffer, 1 to 1500 mM at least one basic amino acid, 1 to 200 mg / mL antibody, and 1 to 400 mM carbohydrate. -The stable formulation as described in any one of [4].
[6] The preparation according to [4] or [5], wherein the basic amino acid is arginine.
[7] The preparation according to [5] or [6], wherein the carbohydrate is sucrose or trehalose.
[8] The preparation according to any one of [1] to [7], further comprising a surfactant.
[9] The preparation according to any one of [1] to [8], which contains a polypeptide and / or antibody in an amount of at least 10 mg / ml.
[10] The preparation according to any one of [1] to [9], comprising a polypeptide and / or antibody in an amount of at least 50 mg / ml.
[11] The preparation according to any one of [1] to [10], which contains a polypeptide and / or antibody in an amount of at least 80 mg / ml.
[12] The preparation according to any one of [1] to [11], comprising a polypeptide and / or antibody in an amount of 240 mg / ml or less.
[13] The preparation according to any one of [1] to [12], having a pH in the range of 4.5 to 7.0.
[14] The preparation according to [13], having a pH in the range of 5.5 to 6.6.
[15] The preparation according to any one of [1] to [14], which is a liquid.
[16] The preparation according to [15], which has not been lyophilized during preparation.
[17] The preparation according to any one of [1] to [16], wherein dimerization of the polypeptide molecule and / or antibody molecule is reduced.
[18] The preparation according to any one of [1] to [17], wherein dimerization of the polypeptide molecule and / or antibody molecule is inhibited.
[19] The preparation according to any one of [1] to [18] for subcutaneous administration.
[20] A method for stabilizing a solution containing an antibody, comprising the step of adding at least one basic amino acid, wherein the antibody comprises:
(A) CDR1 comprising the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 comprising the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: 3 (CDR3 of VH4-M73) A heavy chain variable region comprising a CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 10 (CDR1 of VL1), a CDR2 comprising a sequence of SEQ ID NO: 11 (CDR2 of VL1), and SEQ ID NO: 12 (of VL1 An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(B) CDR1 comprising the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: 6 (CDR3 of VH3-M73) A heavy chain variable region comprising CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 13 (CDR1 of VL3), a CDR2 comprising a sequence of SEQ ID NO: 14 (CDR2 of VL3), and SEQ ID NO: 15 (VL3 of An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(C) CDR1 comprising the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and SEQ ID NO: 9 (CDR3 of VH5-M83) A heavy chain variable region comprising CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 16 (CDR1 of VL5), a CDR2 comprising a sequence of SEQ ID NO: 17 (CDR2 of VL5), and SEQ ID NO: 18 (of VL5 An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(D) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1);
(E) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 20 (variable region of VH3-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 23 (variable region of VL3);
(F) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5);
(G) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1);
(H) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain comprising the sequence of SEQ ID NO: 29 (VL3); and (i) SEQ ID NO: 27 (VH5-M83) A method comprising: a heavy chain comprising the sequence of: and a light chain comprising the sequence of SEQ ID NO: 30 (VL5) at least one antibody selected from antibodies.
[21] A method for stabilizing an antibody during a freeze / thaw cycle of a solution containing an antibody comprising the step of adding at least one basic amino acid, wherein the antibody comprises:
(A) CDR1 comprising the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 comprising the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: 3 (CDR3 of VH4-M73) A heavy chain variable region comprising a CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 10 (CDR1 of VL1), a CDR2 comprising a sequence of SEQ ID NO: 11 (CDR2 of VL1), and SEQ ID NO: 12 (of VL1 An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(B) CDR1 comprising the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: 6 (CDR3 of VH3-M73) A heavy chain variable region comprising CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 13 (CDR1 of VL3), a CDR2 comprising a sequence of SEQ ID NO: 14 (CDR2 of VL3), and SEQ ID NO: 15 (VL3 of An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(C) CDR1 comprising the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and SEQ ID NO: 9 (CDR3 of VH5-M83) A heavy chain variable region comprising CDR3 comprising a sequence, and a CDR1 comprising a sequence of SEQ ID NO: 16 (CDR1 of VL5), a CDR2 comprising a sequence of SEQ ID NO: 17 (CDR2 of VL5), and SEQ ID NO: 18 (of VL5 An antibody comprising a light chain variable region comprising CDR3 comprising the sequence of CDR3);
(D) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1);
(E) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 20 (variable region of VH3-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 23 (variable region of VL3);
(F) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5);
(G) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1);
(H) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain comprising the sequence of SEQ ID NO: 29 (VL3); and (i) SEQ ID NO: 27 (VH5-M83) A method comprising: a heavy chain comprising the sequence of: and a light chain comprising the sequence of SEQ ID NO: 30 (VL5).

  The humanized anti-IL-6 receptor IgG antibody described above has enhanced potency and improved pharmacokinetics; thus, it can exert a long-term therapeutic effect with less administration frequency.

2 is a list of mutation sites that improve the affinity of tocilizumab for IL-6 receptor. The sequence of HCDR2 of tocilizumab is SEQ ID NO: 81; the sequence after mutation of HCDR2 (upper row) is SEQ ID NO: 82; the sequence of mutation after HCDR2 (lower row) is SEQ ID NO: 83; the sequence of HCDR3 of tocilizumab is SEQ ID NO: 84; The sequence after mutation (upper row) is SEQ ID NO: 85; the sequence after mutation of HCDR3 (lower row) is SEQ ID NO: 86; the LCDR1 sequence of tocilizumab is SEQ ID NO: 87; the sequence after mutation of LCDR1 (upper row) is SEQ ID NO: 88; The LCDR1 post-mutation sequence (bottom) is SEQ ID NO: 89; Tocilizumab LCDR3 sequence is SEQ ID NO: 90; LCDR3 post-mutation sequence (top) is SEQ ID NO: 91; LCDR3 post-mutation sequence (bottom) is SEQ ID NO: Shown in 92. It is a graph which shows the neutralization activity in BaF / gp130 of tocilizumab and RDC-23. It is a list of mutation sites that can reduce the isoelectric point of the variable region without significantly reducing the binding of tocilizumab to the IL-6 receptor. The asterisk in the figure does not affect the isoelectric point, but is a site mutated for conversion to a human sequence. Tocilizumab HFR1 sequence SEQ ID NO: 93; HFR1 mutated sequence SEQ ID NO: 94; Tocilizumab HCDR1 sequence SEQ ID NO: 95; HCDR1 mutated sequence SEQ ID NO: 96; Tocilizumab HFR2 sequence SEQ ID NO: 97; Sequence after mutation of HFR2 is SEQ ID NO: 98; Sequence of HCDR2 of tocilizumab is SEQ ID NO: 81; Sequence of mutation of HCDR2 is SEQ ID NO: 99; Sequence of HFR4 of tocilizumab is SEQ ID NO: 100; Sequence of mutation of HFR4 SEQ ID NO: 101; Tocilizumab LFR1 sequence SEQ ID NO: 102; LFR1 mutated sequence SEQ ID NO: 103; Tocilizumab LCDR1 sequence SEQ ID NO: 87; LCDR1 mutated sequence SEQ ID NO: 104; Tocilizumab LFR2 The sequence is SEQ ID NO: 105; the sequence after mutation of LFR2 is SEQ ID NO: 106; the LCDR2 sequence of tocilizumab is SEQ ID NO: 107; the sequence of mutation of LCDR2 is SEQ ID NO: 108 and 109; the LFR3 sequence of tocilizumab is SEQ ID NO: 110 ; After mutation of LFR3 SEQ ID NO: 111; Tocilizumab LFR4 sequence is shown in SEQ ID NO: 112; LFR4 post-mutation sequence is shown in SEQ ID NO: 113. It is a graph which shows the neutralization activity in BaF / gp130 of tocilizumab and H53 / L28. It is a graph which shows transition of the plasma concentration of tocilizumab and H53 / L28 after intravenous administration in mice. It is a graph which shows transition of the plasma concentration of tocilizumab and H53 / L28 after subcutaneous administration in mice. FIG. 3 is a schematic diagram showing that IgG molecules can recombine with other antigens by dissociating from membrane-type antigens in endosomes. This is a list of mutation sites that can confer pH dependence (binding at pH 7.4 and dissociating at pH 5.8) on the binding of tocilizumab to the IL-6 receptor. The HFR1 sequence of tocilizumab is SEQ ID NO: 93; the mutated sequence of HFR1 is SEQ ID NO: 114; the HCDR1 sequence of tocilizumab is SEQ ID NO: 95; the mutated sequence of HCDR1 is SEQ ID NO: 115; the LCDR1 sequence of tocilizumab is SEQ ID NO: 87; Sequence after mutation of LCDR1 is shown in SEQ ID NO: 116; LCDR2 sequence of tocilizumab is shown in SEQ ID NO: 107; Sequence after mutation of LCDR2 is shown in SEQ ID NO: 117. It is a graph which shows the neutralization activity in BaF / gp130 of tocilizumab and H3pI / L73. It is a graph which shows transition of the plasma concentration of tocilizumab and H3pI / L73 after intravenous administration in a cynomolgus monkey. It is a graph which shows transition of the plasma concentration of tocilizumab and H3pI / L73 after intravenous administration in human IL-6 receptor transgenic mice. It is a figure which shows the result of the heterogeneity evaluation derived from the C terminal of tocilizumab, tocilizumab (DELTA) K, and tocilizumab (DELTA) GK by cation exchange chromatography. It is a figure which shows the result of the evaluation of the heterogeneity derived from the disulfide bond of tocilizumab-IgG1, tocilizumab-IgG2, and tocilizumab-SKSC by cation exchange chromatography. It is a figure which shows the Tm value of each Fab domain, and the denaturation curve of each tosilizumab-IgG1, tocilizumab-IgG2, and tocilizumab-SKSC obtained by differential scanning calorimetry (DSC). FIG. 3 is a graph showing changes in plasma concentrations of tocilizumab-IgG1, tocilizumab-M44, tocilizumab-M58, and tocilizumab-M73 after intravenous administration in human FcRn transgenic mice. FIG. 6 shows neutralizing activity of tocilizumab, control, and Fv5-M83 in BaF / gp130. It is a graph which shows the neutralization activity in BaF / gp130 of tocilizumab, Fv3-M73, and Fv4-M73. It is a graph which shows transition of the plasma concentration of tocilizumab, control, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenous administration in cynomolgus monkeys. It is a graph which shows transition of the CRP density | concentration of tocilizumab, control, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenous administration in a cynomolgus monkey. It is a graph which shows transition (%) of the ratio of the free soluble IL-6 receptor after intravenous administration of tocilizumab, control, Fv3-M73, Fv4-M73, and Fv5-M83 in cynomolgus monkeys. It is a graph which shows the difference in formation of the high molecular weight component (HMW) of antibody Fv4-M73 in different preparations (A to D) over time. High molecular weight components of antibody Fv4-M73 at two time points (25 ° C) at different pH values, buffer species, and NaCl concentrations in the presence or absence of arginine (ΔHMW: increase from initial value) It is a graph which shows the difference in formation. Graph showing the difference in the formation of high molecular weight components (HMW) of antibody Fv4-M73 at one time point (25 ° C) at different pH values, buffer species, and NaCl concentrations in the presence or absence of arginine is there. High molecular weight components of antibody Fv4-M73 at three different time points (40 ° C) at different pH values, buffer species, and NaCl concentrations in the presence or absence of arginine (ΔHMW: increase from initial value) It is a graph which shows the difference in formation of. Graph showing the difference in the formation of high molecular weight components (HMW) of antibody Fv4-M73 at one time point (40 ° C) at different pH values, buffer species, NaCl concentrations in the presence or absence of arginine is there. Low molecular weight components of antibody Fv4-M73 at three different time points (40 ° C) at different pH values, buffer species, and NaCl concentrations in the presence or absence of arginine (ΔLMW: increase from initial value) It is a graph which shows the difference in formation of. FIG. 6 shows the results of anion exchange chromatography performed with a solution of antibody Fv4-M73 stored under different pH values. FIG. 6 shows the results of anion exchange chromatography performed with solutions of antibody Fv4-M73 stored under three different pH values, different concentrations of NaCl, arginine, and two different buffer species. Graph showing differences in the number of two different freeze / thaw cycles, different concentrations of NaCl, arginine, and high molecular weight components of antibody Fv4-M73 (ΔHMW: increase from initial value) for two different buffer species It is. It is a graph which shows the difference in formation of the high molecular weight component ((DELTA) HMW: increase from initial value) of antibody Fv4-M73 of three different time points (40 degreeC and 25 degreeC) about four different formulations (EH). . FIG. 6 is a graph showing the number of two different freeze / thaw cycles and the difference in formation of the high molecular weight component (ΔHMW: increase from initial value) of antibody Fv4-M73 in four different formulations (EH). It is a graph which shows the difference in formation of the high molecular weight component (ΔHMW: increase from initial value) of antibody Fv4-M73 after 3 months and 6 months at 5 ° C. for 6 different preparations. It is a graph which shows the difference in the formation of the high molecular weight component (ΔHMW: increase from the initial value) of antibody Fv4-M73 after 3 months and 6 months at -20 ° C. for 6 different preparations.

The present invention
(A) CDR1 comprising the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 comprising the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: 3 (CDR3 of VH4-M73) A polypeptide comprising CDR3 comprising the sequence;
(B) CDR1 comprising the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 comprising the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: 6 (CDR3 of VH3-M73) A polypeptide comprising CDR3 comprising the sequence;
(C) CDR1 comprising the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 comprising the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and SEQ ID NO: 9 (CDR3 of VH5-M83) A polypeptide comprising CDR3 comprising the sequence;
(D) Includes CDR1 containing the sequence of SEQ ID NO: 10 (CDR1 of VL1), CDR2 containing the sequence of SEQ ID NO: 11 (CDR2 of VL1), and CDR3 containing the sequence of SEQ ID NO: 12 (CDR3 of VL1) A polypeptide;
(E) Includes CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 of VL3), CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 of VL3), and CDR3 containing the sequence of SEQ ID NO: 15 (CDR3 of VL3) A polypeptide; and (f) a CDR1 comprising the sequence of SEQ ID NO: 16 (CDR1 of VL5), a CDR2 comprising the sequence of SEQ ID NO: 17 (CDR2 of VL5), and a sequence of SEQ ID NO: 18 (CDR3 of VL5) A preparation comprising at least one polypeptide selected from polypeptides comprising CDR3 is provided.

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

  “Formulations”, “drugs”, and “pharmaceutical compositions” as used in the present invention are prepared to be suitable for administration to animals, such as humans, directly or after reconstitution. , Means a liquid or solid formulation containing a polypeptide and / or antibody as an active ingredient. If desired, the formulation may contain pharmaceutically acceptable carriers and / or additives. For example, surfactants (eg, PEG, Tween, Pluronic), excipients, antioxidants (eg, ascorbic acid, methionine), colorants, flavoring agents, preservatives, stabilizers, Buffers, chelating agents (eg, EDTA), suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity enhancers, and flavoring agents.

  The preparation containing the polypeptide and / or antibody according to the present invention preferably does not contain a protein such as HSA (human serum albumin) or gelatin as a stabilizer.

  The preparation containing the polypeptide and / or antibody according to the present invention is preferably a polypeptide and / or at a high concentration of 10 mg / mL or more, preferably 50 mg / mL or more, more preferably 80 mg / mL or more. It is a liquid formulation containing an antibody. The concentration is 10 to 240 mg / mL, may be 10 mg / mL, or more preferably 50 mg / mL, and even more preferably 100 to 200 mg / mL.

  The liquid preparation according to the present invention is preferably prepared without performing a freeze-drying step.

  The buffer that can be used in the present invention can adjust the pH within a desired range and is pharmaceutically acceptable. In the high concentration polypeptide and / or antibody-containing preparation according to the present invention, the pH of the preparation is preferably 4.5 to 7, more preferably 5.5 to 6.6. These buffering agents are known to those skilled in the art and examples include inorganic salts such as phosphate (sodium or potassium) and sodium bicarbonate; citrate (sodium or potassium), sodium acetate, and sodium succinate And organic acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid, and gluconic acid. In addition, Tris buffer, Good buffers such as MES and MOPS, histidine (eg, histidine hydrochloride) and glycine can be used. In the high concentration polypeptide and / or antibody-containing preparation according to the present invention, the buffer is preferably a histidine buffer or a citrate buffer, and a histidine buffer is particularly preferable. The concentration of the buffer solution is generally 1 to 500 mM, preferably 5 to 100 mM, more preferably 10 to 20 mM. When a histidine buffer is used, the buffer solution preferably contains histidine at a concentration of 5-25 mM, more preferably 10-20 mM.

The preparation according to the present invention may further contain a surfactant. Typical examples of surfactants include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glycerol monocaprylate, glycerol monomyristate, and mono Glycerin fatty acid esters such as glycerol stearate; Polyglycerol fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, monostearin Polyoxyethylene sorbitan acid, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and tristearate Polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan phosphate; polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitol tetrastearate and polyoxyethylene sorbitol tetraoleate; polyoxys such as polyoxyethylene glyceryl monostearate Ethylene glycol fatty acid ester; polyethylene glycol fatty acid ester such as polyethylene glycol distearate; polyoxyethylene alkyl ether such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, and polyoxy Such as ethylene polyoxypropylene cetyl ether Polyoxyethylene polyoxypropylene alkyl ethers; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylenes such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil) Hydrogenated castor oil; polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides, eg 6-18 HLB such as polyoxyethylene octadecanamide surfactants having; anionic surfactants such as organic sodium cetyl sulfate, sodium lauryl sulfate, and C ₁₀ -C ₁₈ alkyl group such as sodium oleyl sulfate Alkyl sulfates; polyoxyethylene alkyl ether sulfates, such as polyoxyethylene lauryl sodium sulfate, wherein the average number of moles of added ethylene oxide units is 2-4 and the number of carbon atoms in the alkyl group is 10-18 ; sphingophospholipid such as sphingomyelin; natural surfactants such as lecithin and glycerophospholipids; sulfosuccinic acid alkyl salt having C ₈ -C ₁₈ alkyl group such as sulfosuccinic acid, sodium lauryl and sucrose of C ₁₂ -C ₁₈ fatty acids Sugar ester is included. These surfactants may be added individually to the formulation of the present invention, or two or more of these surfactants may be added in combination.

  Preferred surfactants are polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene alkyl ethers, particularly preferred are polysorbates 20, 21, 40, 60, 65, 80, 81 and 85, and pluronic type Surfactants, most preferred are polysorbates 20 and 80, and Pluronic F-68 (Poloxamer 188).

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

  The preparation according to the present invention may further contain not only acidic amino acids such as arginine but also amino acids such as methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, asparagine, and glutamine as stabilizers.

  In the antibody-containing preparation or antibody-containing solution preparation according to the present invention, the formation of a dimer during a freeze / thaw cycle can be inhibited by adding a carbohydrate (eg, sugar). Sugars that can be used include non-reducing oligosaccharides, for example, non-reducing disaccharides such as sucrose and trehalose, or non-reducing trisaccharides such as raffinose, with non-reducing oligosaccharides being particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

  In the antibody-containing preparation or antibody-containing solution preparation according to the present invention, the formation of multimers and degradation products during long-term storage can be inhibited by adding a carbohydrate (for example, sugar). Sugars that can 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, among which non-reducing Oligosaccharides are particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

  The sugar should be added at 0.1-500 mg / mL, preferably 10-300 mg / mL, more preferably 25-100 mg / mL.

In another aspect, the formulation according to the invention is preferably substantially composed of the following components:
(A) a modified anti-IL-6 receptor antibody;
(B) basic amino acids (eg, arginine, histidine, and / or lysine);
(C) A buffer (eg, histidine or citrate).

  Depending on the purpose, carbohydrates (eg, sugars) and / or surfactants may be included in the formulation.

  As a stabilizer, amino acids such as methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, asparagine, and glutamine may be contained.

  As used herein, the term “consisting essentially of” means that no ingredients other than those normally added to the preparation are contained, and the ingredients normally added to the preparation are: Such as suspending agents, solubilizers, tonicity agents, preservatives, adsorption inhibitors, diluents, media, pH adjusters, soothing agents, sulfur-containing reducing agents, and antioxidants , Any additional ingredients.

  Examples of suspending agents include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, powdered tragacanth, sodium carboxymethyl cellulose, and polyoxyethylene sorbitan monolaurate.

  Examples of solubilizing agents 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 preservatives include methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

  Examples of adsorption inhibitors include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, and polyethylene glycol.

Examples of sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and such C ₁ -C ₇ thioalkanic include compounds having a sulfhydryl group.

  Examples of antioxidants include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite Chelating agents such as sodium, triamyl gallate, propyl gallate, and disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate are included.

  However, the preparations (drugs, pharmaceutical compositions) according to the present invention that can be used for the prevention or treatment of IL-6 related diseases including inflammatory diseases are not limited to the above, and other conventional carriers are appropriately used. You may contain. In particular, examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride , Polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts. They 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, the anti-IL-6 receptor antibody is dissolved in an isotonic solution containing, for example, saline, glucose, or other adjuvants. Adjuvants include, for example, D-sorbitol, D-mannose, D-mannitol, and sodium chloride. In addition, suitable solubilizers such as alcohols (ethanol etc.), polyhydric alcohols (propylene glycol, PEG etc.) and nonionic surfactants (polysorbate 80 and HCO-50) may be combined.

If necessary, the polypeptide may be encapsulated in microcapsules (microcapsules made of hydroxycellulose, gelatin, poly (methyl methacrylate), etc.) or colloid drug delivery systems (liposomes, albumin microspheres). , microemulsions, nanoparticles, which may be the nanocapsules) ^{(e.g., "Remington's Pharmaceutical Science 16 th} edition", Oslo Ed. (1980) see). Furthermore, methods for preparing drugs as sustained release agents are known and these methods can be applied to the polypeptides (Langer et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12: 98-105; US Patent 3,773,919; European Patent Application (EP) 58,481; Sidman et al., Biopolymers (1983) 22: 547-56; EP 133,988 ). Furthermore, the liquid volume for subcutaneous administration can be increased by adding or mixing hyaluronidase to the drug (see eg WO 2004/078140).

  The pharmaceutical composition of the present invention may be administered orally or parenterally, but is preferably administered parenterally. In particular, the composition is administered to a patient by injection or transdermally. Injection includes systemic administration and local administration, for example, by intravenous injection, intramuscular injection, or subcutaneous injection. The composition may in particular be injected locally by intramuscular injection, at the site of treatment or at the periphery of the site. Transdermal dosage forms include, for example, ointments, gels, creams, poultices, and patches, which can be administered locally or systemically. Furthermore, the administration method can be appropriately selected depending on the age and symptoms of the patient. The dose to be administered can be selected for each administration, for example from the range of 0.0001 mg to 100 mg active ingredient per kg body weight. Alternatively, when the composition is administered to a human patient, for example, the active ingredient may be selected from the range of 0.001 to 1000 mg per kg body weight for each patient. A single administered dose preferably contains, for example, about 0.01-50 mg / kg body weight of an antibody of the invention. However, the dose of the antibody of the present invention is not limited to these doses.

  As is apparent from the results of the examples below, according to the present invention, one or more basic amino acids (arginine, histidine, and / or lysine, preferably arginine), or basic amino acids in combination with other amino acids. Can be added to the formulation to obtain a stable formulation with low antibody dimerization and deamidation during long-term storage or freeze / thaw.

  In order to evaluate the shelf life stability of high concentration antibody-containing formulations, we studied the effect of various additives by performing size exclusion chromatography tests and anion exchange chromatography tests. As a result, it was found that the amount of dimer was lower in a solution in which a high concentration of antibody was dissolved in a buffer solution containing the amino acid arginine than in a solution containing no additional arginine. These results indicate that arginine is effective as a stabilizer to inhibit antibody dimerization.

  Therefore, by adding arginine as a stabilizer, it is possible to provide a stable antibody preparation in which dimerization of the antibody is reduced.

  One embodiment of the present invention is a stable antibody-containing preparation characterized by containing an antibody and arginine in a buffer solution.

  As the arginine used in the present invention, any arginine compound itself, a derivative thereof, and a salt thereof can be used. L-arginine and its salts are preferred.

  When the preparation of the present invention contains arginine, the concentration of arginine is preferably 1 to 1500 mM, more preferably 50 to 1000 mM, more preferably 50 to 200 mM.

  In the present invention, the polypeptide is not particularly limited, but is preferably an antigen-binding substance having binding activity to human IL-6 receptor. Preferred examples of the antigen-binding substance include 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.

  In the polypeptides (a) to (f) described above, preferred examples of the polypeptides (a) to (c) include the heavy chain variable regions of antibodies, and the polypeptides (d) to (f) As a preferable example, the light chain variable region of an antibody can be mentioned.

  These variable regions can be used as part of an anti-human IL-6 receptor antibody. Anti-human IL-6 receptor antibodies using these variable regions have excellent binding activity, excellent pharmacokinetics, excellent safety, reduced immunogenicity, and / or excellent physical properties. In the present invention, excellent pharmacokinetics or improvement in pharmacokinetics is one of pharmacokinetic parameters calculated from a change over time in plasma concentration when an antibody is administered in vivo, “clearance (CL)”. Or “increase in area under the concentration curve (AUC)”, “average residence time”, or “in plasma half-life (t1 / 2)”. In the present invention, an excellent physicochemical property or an improvement in physicochemical property is not particularly limited, but means an improvement in stability, a reduction in non-uniformity, or the like.

  As a framework region (FR) of a human antibody to be linked to CDR, a CDR forming a favorable antigen binding site is selected. The FR used in the variable region of the present invention is not particularly limited, and any FR may be used, but human-derived FR is preferably used. Human-derived FRs may be those having a native sequence, and if necessary, one or more amino acids in the framework region having the native sequence are substituted so that the CDR forms an appropriate antigen-binding site. , Deletions, additions and / or insertions, etc. For example, a mutant FR sequence having a desired property can be selected by measuring and evaluating the binding activity of an antibody to an antigen using FRs substituted with amino acids (Sato, K. et al., Cancer Res. (1993)). 53, 851-856).

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

  Examples of a method for substituting one or a plurality of amino acid residues with other amino acids of interest include site-directed mutagenesis (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 DNA 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). Using this method, the desired amino acid of the antibody can be substituted with another amino acid of interest. Moreover, as a method of substituting with other amino acids, it is appropriate to use library technologies such as framework shuffling (Mol Immunol. 2007 Apr; 44 (11): 3049-60) and CDR repair (US2006 / 0122377). It is also possible to make amino acid substitutions in the framework and CDR.

Furthermore, the present invention provides a preparation comprising at least one antibody selected from the following.
(a) CDR1 having the sequence of SEQ ID NO: 1 (CDR1 of VH4-M73), CDR2 having the sequence of SEQ ID NO: 2 (CDR2 of VH4-M73), and SEQ ID NO: 3 (CDR3 of VH4-M73) A heavy chain variable region comprising CDR3 having a sequence, and a CDR1 having a sequence of SEQ ID NO: 10 (CDR1 of VL1), a CDR2 having a sequence of SEQ ID NO: 11 (CDR2 of VL1), and SEQ ID NO: 12 (of VL1 An antibody comprising a light chain variable region comprising CDR3 having the sequence of CDR3),
(b) CDR1 having the sequence of SEQ ID NO: 4 (CDR1 of VH3-M73), CDR2 having the sequence of SEQ ID NO: 5 (CDR2 of VH3-M73), and SEQ ID NO: 6 (CDR3 of VH3-M73) A heavy chain variable region comprising a CDR3 having a sequence, and a CDR1 having a sequence of SEQ ID NO: 13 (CDR1 of VL3), a CDR2 having a sequence of SEQ ID NO: 14 (CDR2 of VL3), and SEQ ID NO: 15 (of VL3 An antibody comprising a light chain variable region comprising CDR3 having the sequence of CDR3),
(c) CDR1 having the sequence of SEQ ID NO: 7 (CDR1 of VH5-M83), CDR2 having the sequence of SEQ ID NO: 8 (CDR2 of VH5-M83), and SEQ ID NO: 9 (CDR3 of VH5-M83) A heavy chain variable region comprising CDR3 having a sequence, and a CDR1 having a sequence of SEQ ID NO: 16 (CDR1 of VL5), a CDR2 having a sequence of SEQ ID NO: 17 (CDR2 of VL5), and SEQ ID NO: 18 (of VL5 An antibody comprising a light chain variable region comprising CDR3 having the sequence of CDR3),
(D) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1),
(E) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 20 (variable region of VH3-M73) and a light chain variable region comprising the sequence of SEQ ID NO: 23 (variable region of VL3),
(F) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5),
(G) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1),
(H) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain comprising the sequence of SEQ ID NO: 29 (VL3), and (i) SEQ ID NO: 27 (VH5-M83) An antibody comprising a heavy chain comprising the sequence and a light chain comprising the sequence of SEQ ID NO: 30 (VL5).

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

  As the framework region of the human antibody to be linked to the CDR of the present invention, a region in which CDR forms a favorable antigen binding site is selected. The FR used in the variable region of the present invention is not particularly limited, and any FR may be used, but human-derived FR is preferably used. Human-derived FRs may be those having a native sequence, and if necessary, one or more amino acids in the framework region having the native sequence are substituted so that the CDR forms an appropriate antigen-binding site. , Deletions, additions and / or insertions, etc. For example, a mutant FR sequence having a desired property can be selected by measuring and evaluating the binding activity of an antibody to an antigen using FRs substituted with amino acids (Sato, K. et al., Cancer Res. (1993)). 53, 851-856).

  The constant region used in the antibody of the present invention is not particularly limited, and any constant region may be used. Preferable examples of constant regions used in the antibodies of the present invention include human-derived constant regions (such as IgG1, IgG2, IgG3, IgG4, Cκ, and Cλ-derived constant regions). The constant region derived from human may be substituted, deleted, added and / or inserted with one or more amino acids. For example, as a preferred example of a human-derived constant region, in the case of a heavy chain constant region, a constant region having the amino acid sequence of SEQ ID NO: 31 (constant region of VH4-M73), SEQ ID NO: 32 (constant of VH3-M73) A constant region having the amino acid sequence of (region), a constant region having the amino acid sequence of SEQ ID NO: 33 (constant region of VH5-M83), and in the case of the light chain constant region, SEQ ID NO: 34 (VL1) A constant region having the amino acid sequence of SEQ ID NO: 35 (VL3), and a constant region having the amino acid sequence of SEQ ID NO: 36 (VL5).

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

  The corresponding variable region of these antibodies can be used as part of a molecule that reacts with the anti-human IL-6 receptor. In these variable regions, one or a plurality of amino acids (for example, within 5 amino acids, preferably within 3 amino acids) may be substituted, deleted, added and / or inserted. Examples of the method for substituting one or a plurality of amino acid residues with other amino acids of interest include the methods described above.

  The present invention also includes a polypeptide comprising the variable region described above.

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

  In these heavy chains or light chains, one or a plurality of amino acids (for example, within 10 amino acids, preferably within 5 amino acids, more preferably within 3 amino acids) may be substituted, deleted, added and / or inserted. As a method for substituting one or a plurality of amino acid residues with another amino acid of interest, for example, the above-described method can be used.

  One or more amino acid substitutions, deletions, additions and / or insertions may be made in the variable region, in the constant region, or in both the variable and constant regions. Good.

  The present invention also includes polypeptides comprising the heavy and light chains described above.

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

  A humanized antibody is also called a reshaped human antibody, which is a non-human mammal-derived complementarity determining region (CDR) grafted to the CDR of a human antibody, and its general gene Recombination techniques are also known (see European Patent Application Publication No. EP 125023, WO 96/02576).

  Specifically, for example, several DNA sequences designed to link the target CDR and the target framework region (FR) so as to have a portion overlapping both terminal regions of CDR and FR The oligonucleotide is synthesized as a primer by PCR (see the method described in WO98 / 13388). The obtained DNA is obtained by ligating with a DNA encoding a human antibody constant region or a human antibody constant region variant, then incorporating it into an expression vector, introducing this vector into a host and producing it (European patent application). See publication number EP 239400, international patent application publication number WO 96/02576).

  As the framework region of the human antibody to be linked to the CDR, a region in which the CDR forms a favorable antigen binding site is selected. If necessary, amino acid in the framework region in the variable region of the antibody may be substituted, deleted, added and / or inserted.

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

  For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2, and Cε can be used for the H chain, and Cκ and Cλ can be used for the L chain. The amino acid sequence of Cκ is shown in SEQ ID NO: 38, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 37. The amino acid sequence of Cγ1 is shown in SEQ ID NO: 40, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 39. The amino acid sequence of Cγ2 is shown in SEQ ID NO: 42, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 41. The amino acid sequence of Cγ4 is shown in SEQ ID NO: 44, and the base sequence encoding the amino acid sequence is shown in SEQ ID NO: 43.

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

  In addition, after producing a humanized antibody, amino acids in the variable region (eg, CDR, FR) or constant region may be substituted, deleted, added and / or inserted with other amino acids. The humanized antibody also includes a humanized antibody having such amino acid substitution.

  The antibody of the present invention includes not only a bivalent antibody typified by IgG but also a monovalent antibody or a multivalent antibody typified by IgM as long as it has binding activity and / or neutralizing activity to IL-6 receptor. Is also included. The multivalent antibodies of the present invention include multivalent antibodies that all have the same antigen-binding site, or multivalent antibodies that have some or all different antigen-binding sites. The antibody of the present invention is not limited to the full-length molecule of the antibody, and may be a low molecular weight antibody or a modified product thereof as long as it binds to IL-6 receptor protein.

  The low molecular weight antibody is an antibody including an antibody fragment in which a part of a full-length antibody (for example, full-length IgG) is deleted, and is not particularly limited as long as it has an IL-6 receptor binding activity and / or neutralizing activity. In the present invention, the low molecular weight antibody is not particularly limited as long as it includes a part of the full-length antibody, but preferably includes VH or VL, and particularly preferably is a low molecular weight antibody including both VH and VL. Another preferred example of the low molecular weight antibody of the present invention is a low molecular weight antibody containing the CDR of the antibody. The CDRs contained in the low molecular weight antibody may include all six CDRs of the antibody, or may include some of the CDRs.

  The low molecular weight antibody in the present invention preferably has a smaller molecular weight than the full-length antibody. For example, it may form a multimer such as a dimer, trimer, tetramer, etc. However, the molecular weight may increase.

  Specific examples of antibody fragments include, for example, Fab, Fab ′, F (ab ′) 2, and Fv. Specific examples of the low molecular weight antibody include, for example, Fab, Fab ′, F (ab ′) 2, Fv, scFv (single chain Fv), diabody, sc (Fv) 2 (single chain (Fv) 2 ) And the like. Multimers (for example, dimers, trimers, tetramers, polymers) of these antibodies are also included in the low molecular weight antibody of the present invention.

  Antibody fragments can be obtained, for example, by treating an antibody with an enzyme to produce antibody fragments. Known enzymes that produce antibody fragments include, for example, papain, pepsin, and plasmin. Alternatively, genes encoding these antibody fragments can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (eg, Co, MS 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 digestive enzyme cleaves a specific position of the antibody fragment to give an antibody fragment having a specific structure as follows. If a genetic engineering technique is used for such an enzymatically obtained antibody fragment, any part of the antibody can be deleted.

The antibody fragments obtained when the above digestive enzymes are used are as follows.
Papain digestion: F (ab) 2 or Fab
Pepsin digestion: F (ab ') 2 or Fab'
Plasmin digestion: Facb

  The low molecular weight antibody in the present invention can include an antibody fragment lacking any region as long as it has binding activity to IL-6 receptor and / or neutralizing activity.

  “Diabodies” refers to bivalent antibody fragments constructed by gene fusion (Holliger P et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404. No. 097, WO 93/11161, etc.). A diabody is a dimer composed of two polypeptide chains. Usually, in the polypeptide chain constituting the dimer, VL and VH are connected to each other by a linker in the same chain. The linker in the diabody is generally so short that VL and VH cannot bind to each other. Specifically, the amino acid residues constituting the linker are, for example, about 5 residues. Therefore, VL and VH encoded on the same polypeptide chain cannot form a single chain variable region fragment but form a dimer with another single chain variable region fragment. As a result, the diabody has two antigen binding sites.

  The scFv antibody is an antibody in which VH and VL are combined with a linker or the like to form a single chain polypeptide (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 L chain V region in scFv may be derived from any of the antibodies described herein. There is no restriction | limiting in particular in the peptide linker which connects V area | region. For example, any single chain peptide consisting of about 3 to 25 residues can be used as a linker. Specifically, for example, a peptide linker described later can be used.

The V regions of both strands can be linked by, for example, the PCR method as described above. In order to link the V regions by the PCR method, first of all, the DNA encoding the desired partial amino acid sequence is used as a template.
A DNA sequence encoding an antibody H chain or H chain V region, and a DNA sequence encoding an antibody L chain or L chain V region.

  By the PCR method using a pair of primers having sequences corresponding to the sequences at both ends of the DNA to be amplified, DNAs encoding the V regions of the H chain and the L chain are respectively amplified. Next, DNA encoding a peptide linker portion is prepared. DNA encoding a peptide linker can also be synthesized using PCR. At this time, a base sequence that can be linked to the amplification product of each V region synthesized separately is added to the 5 ′ side of the primer to be used. Next, a PCR reaction is performed using each DNA of [H chain V region DNA]-[peptide linker DNA]-[L chain V region DNA] and a primer for assembly PCR.

  The primer for assembly PCR consists of a combination of a primer that anneals to the 5 ′ side of [H chain V region DNA] and a primer that anneals to the 3 ′ side of [L chain V region DNA]. That is, the assembly PCR primer is a primer set that can amplify DNA encoding the full-length sequence of scFv to be synthesized. On the other hand, a base sequence that can be linked to each V region DNA is added to [peptide linker DNA]. As a result, these DNAs are ligated, and the full length of scFv is finally produced as an amplification product by the primers for assembly PCR. Once a DNA encoding scFv is prepared, an expression vector containing the DNA and a recombinant cell transformed with the expression vector can be obtained according to a conventional method. Further, the scFv can be obtained by culturing the resulting recombinant cells and expressing the DNA encoding the scFv.

The order of VH and VL to be combined is not particularly limited, and may be arranged in any order. For example, the following arrangement can be given.
[VH] Linker [VL]
[VL] Linker [VH]

  sc (Fv) 2 is a low molecular weight antibody in which two VHs and two VLs are combined with a linker or the like to form a single chain (Hudson et al., 1999, J Immunol. Methods; 231: 177-189 ). sc (Fv) 2 can be prepared, for example, by linking scFv with a linker.

Two VHs and two VLs are arranged in the order of VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]) starting from the N-terminal side of the single-chain polypeptide. However, the order of the two VHs and the two VLs is not particularly limited to the above arrangement, and may be arranged in any order. For example, the following arrangements can also be mentioned.
[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 VH or VL in the small molecule antibody may be substituted, deleted, added and / or inserted. Furthermore, when VH and VL are associated, as long as they have antigen-binding activity, a part of them may be deleted, or another polypeptide may be added. The variable region may be chimerized or humanized.

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

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

Examples of the amino acid sequence of the peptide linker include the following sequences.
Ser
Gly Ser
Gly Gly Ser
Ser Gly Gly
Gly Gly Gly Ser (SEQ ID NO: 45)
Ser Gly Gly Gly (SEQ ID NO: 46)
Gly Gly Gly Gly Ser (SEQ ID NO: 47)
Ser Gly Gly Gly Gly (SEQ ID NO: 48)
Gly Gly Gly Gly Gly Ser (SEQ ID NO: 49)
Ser Gly Gly Gly Gly Gly (SEQ ID NO: 50)
Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 51)
Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 52)
(Gly Gly Gly Gly Ser (SEQ ID NO: 47)) n
(Ser Gly Gly Gly Gly (SEQ ID NO: 48)) n
[N is an integer of 1 or more].

  The amino acid sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose. For example, “n” for determining the length of the above peptide linker is usually 1 to 5, preferably 1 to 3, more preferably 1 or 2.

  Synthetic compound linkers (chemical cross-linking agents) are commonly used for cross-linking peptides such as N-hydroxysuccinimide (NHS) disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS3), dithiobis (succinimidyl propionate) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis (succinimidyl succinate) (EGS), ethylene glycol Bis (sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis [2- (succinimideoxycarbonyloxy) Ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl And the like sulfone (sulfo-BSOCOES), These crosslinking agents are commercially available.

  When linking four antibody variable regions, usually three linkers are required. A plurality of linkers may be the same or different linkers may be used.

  The antibody of the present invention includes an antibody in which one or more amino acid residues are added to the amino acid sequence of the antibody. Also included are fusion proteins in which these antibodies are fused with other peptides or proteins. In the method of producing a fusion protein, a polynucleotide encoding an antibody and another peptide or a polynucleotide encoding a polypeptide are ligated in frame and the DNA is introduced into an expression vector. Any method known to those skilled in the art can be used. Other peptides or polypeptides to be subjected to fusion with the antibody of the present invention include, for example, FLAG (Hopp, TP et al., BioTechnology 6, 1204-1210 (1988)), 6 His (histidine) residues 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen Known peptides such as fragments, lck tag, α-tubulin fragment, B-tag, protein C fragment and the like can be used. Further, examples of other polypeptides to be subjected to fusion with the antibody of the present invention include GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose). Binding protein) and the like. By fusing a commercially available polynucleotide encoding these peptides or polypeptides with a polynucleotide encoding the antibody according to the present invention, and expressing the fusion polynucleotide so obtained, Can be prepared.

  The antibody described in the present invention may be a conjugated antibody bound to various molecules such as a polymer substance such as polyethylene glycol (PEG) or hyaluronic acid, a radioactive substance, a fluorescent substance, a luminescent substance, an enzyme, and a toxin. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field (for example, US 5,057,313, US 5,156,840). The “antibody” in the present invention includes these conjugated antibodies.

  The antibodies of the present invention also include antibodies with modified sugar chains.

  Furthermore, the antibody used in the present invention may be a bispecific antibody. Bispecific antibodies refer to antibodies that have variable regions that recognize different epitopes within the same antibody molecule. In the present invention, the bispecific antibody may be a bispecific antibody that recognizes different epitopes on the IL-6 receptor molecule, or one antigen binding site recognizes the IL-6 receptor and the other Bispecific antibodies whose antigen-binding sites recognize other substances can also be used. Examples of the antigen to which the other antigen-binding site of the bispecific antibody comprising the antibody of the present invention that recognizes the IL-6 receptor binds include, for example, IL-6, TNFα, TNFR1, TNFR2, CD80, CD86, CD28, CD20 , CD19, IL-1α, IL-β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxin α, lymphotoxin β , LIGHT ligand, LIGHT, VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21, IL-21R, GM-CSF, GM-CSFR, Examples include M-CSF, M-CSFR, IFN-α, VEGF, VEGFR, EGF, EGFR, CCR5, APRIL, and APRILR.

  Methods for producing bispecific antibodies are known. For example, bispecific antibodies can be produced by combining two types of antibodies with different recognition antigens. The antibody to be bound may be a ½ molecule each having an H chain and an L chain, or may be a ¼ molecule consisting only of an H chain. Alternatively, bispecific antibody-producing fused cells can be prepared by fusing hybridomas that produce different monoclonal antibodies. Furthermore, bispecific antibodies can be produced by genetic engineering techniques.

  The antibody used in the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence / absence of sugar chain, form, etc., depending on the antibody-producing cell, host or purification method described below. However, as long as the obtained antibody has a function equivalent to the antibody described in the present invention, it is considered to be included in the present invention. For example, when an antibody is expressed in a prokaryotic cell such as E. coli, a methionine residue is added to the N-terminus of the original antibody amino acid sequence. The antibodies described in the present invention also include such antibodies.

  A polypeptide such as an anti-IL-6 receptor antibody of the present invention can be produced by methods known to those skilled in the art.

  For example, based on the sequence of the obtained anti-IL-6 receptor antibody, it is possible to produce an anti-IL-6 receptor antibody using gene recombination techniques known to those skilled in the art. Specifically, on the basis of the sequence of an antibody that recognizes the IL-6 receptor, a polynucleotide encoding this antibody is constructed, introduced into an expression vector, and then expressed in an appropriate host cell. -6 receptor antibodies can be made (eg, 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 Enzymol. (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 includes the step of culturing a host cell containing a vector into which a polynucleotide encoding the polypeptide of the present invention has been introduced, (i) the polypeptide of the present invention, or (ii) the polypeptide of the present invention. A method for producing a polypeptide encoded by a gene encoding is provided.

More specifically, the present invention provides:
(A) culturing a host cell containing a vector into which a gene encoding the polypeptide of the present invention has been introduced; and (b) obtaining a polypeptide encoded by the gene. A method of making is provided.

  Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vector, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned. When using a vector for the purpose of producing the antibody described in the present invention, an expression vector is particularly useful. As an expression vector, for example, for the purpose of expression in E. coli, in addition to the characteristics that the vector is amplified in E. coli, the host is J. coli such as JM109, DH5α, HB101, XL1-Blue. Promoters that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043), or having a T7 promoter or the like is essential. As such a vector, in addition to the above vector, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Quiagen), pEGFP, or pET (in this case, BL21 expressing T7 RNA polymerase is preferred as the host) ) And the like.

  Further, the expression plasmid vector may contain a signal sequence for antibody secretion. As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) may be used when the periplasm of E. coli is produced. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli, for example, vectors for producing the antibody described in the present invention include mammalian-derived expression vectors (for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990, 18 ( 17), p5322), pEF, pCDM8), insect cell-derived expression vectors (eg, “Bac-to-BAC baculovirus expression system” (Gibco-BRL), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), Expression vectors derived from animal viruses (eg, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (eg, pZIPneo), expression vectors derived from yeast (eg, “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01) ), An expression vector derived from Bacillus subtilis (for example, pPL608, pKTH50).

  For the purpose of expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter required for the expression plasmid vector to be expressed in the cells, such as the SV40 promoter (Mulligan et al., Nature ( 1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc. More preferably, it has a gene for selecting (for example, a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.)). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , pSV2-dhfr ( "Molecular Cloning 2 ^nd edition" Cold Spring Harbor Laboratory Press, (1989 )) , etc.) was introduced, a method may be amplified using methotrexate (MTX), the expression of transient gene In the case of the purpose, there is a method of transforming with a vector having SV40 replication origin (such as pcD) using COS cells having a gene expressing SV40 T antigen on the chromosome. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, for gene copy number amplification in host cell systems, the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.

  The antibody described in the present invention thus obtained can be isolated from the inside of the host cell or outside the cell (such as a medium) and purified as a substantially pure and homogeneous antibody. Separation and purification of antibodies may be carried out using separation and purification methods used in normal antibody purification, and are not limited in any way. For example, select chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. In combination, antibodies can be separated and purified.

  Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse 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). These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Examples of the column used for affinity chromatography include a protein A column and a protein G column. Examples of the column using protein A include Hyper D, POROS, Sepharose FF (GE Amersham Biosciences). The present invention also encompasses antibodies highly purified using these purification methods.

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

Pharmaceutical Composition The present invention provides a pharmaceutical composition containing the above-mentioned polypeptide as an active ingredient. The pharmaceutical composition of the present invention can be used for diseases such as rheumatoid arthritis associated with IL-6. That is, the present invention also provides a therapeutic agent for diseases such as rheumatoid arthritis, which comprises the above-mentioned antibody as an active ingredient. Preferred examples of targeted diseases include rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile idiopathic arthritis, Castleman's disease, systemic lupus erythematosus (SLE), lupus nephritis, Crohn's disease, lymphoma, ulcerative colitis, Anemia, vasculitis, Kawasaki disease, Still's disease, amyloidosis, multiple sclerosis, transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (COPD), IgA nephropathy, Osteoarthritis, asthma, diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, renal cancer, B -cell non-Hodgkin's, pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancer cachexia, cancer nerve invasion, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed Hypersensitivity, contact dermatitis, atopic Dermatitis, mesothelioma, polymyositis, dermatomyositis, pan uveitis, anterior uveitis, middle uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative glass Examples include but are not limited to somatic retinopathy, dry eye, and postoperative inflammation.

  “Containing anti-IL-6 receptor antibody as an active ingredient” means that the anti-IL-6 receptor antibody is contained as at least one active ingredient, and does not limit the content. Moreover, the pharmaceutical composition of the present invention may contain other active ingredients in combination with the above-described polypeptides.

  The pharmaceutical composition of the present invention may be used not only for therapeutic purposes but also for preventive purposes.

  In the present invention, the amino acid contained in the amino acid sequence may be post-translationally modified. For example, the modification of N-terminal glutamine (Gln) residues to pyroglutamic acid (pGlu) residues by pyroglutamylation is well known to those skilled in the art. Of course, such post-translationally modified amino acids are included in the amino acid sequences in the present invention.

  The sugar chain that binds to the antibody according to the present invention may have any structure. The sugar chain at position 297 (EU numbering) may be of any sugar chain structure (preferably a fucosylated sugar chain), or no sugar chain may be present at that position (for example, This can be achieved by producing an antibody in E. coli or by introducing a modification such that the sugar chain does not bind to position 297 (EU numbering)).

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

  The present invention will be described below with reference to examples, but it should not be construed that the present invention is limited thereto.

[Example 1] Identification of variable region mutation sites that improve the affinity of tocilizumab for IL-6 receptor IL of tocilizumab (H chain WT-IgG1 / SEQ ID NO: 53, L chain WT-κ / SEQ ID NO: 54) In order to improve the affinity for the -6 receptor, a library in which mutations were introduced into the CDR sequence was prepared and examined. As a result of screening a library in which mutations were introduced into CDRs, mutations that improved the affinity for IL-6 receptor were found, and these were summarized in FIG. An example of a high affinity tocilizumab combining these mutations is RDC-23 (H chain RDC23H-IgG1 / SEQ ID NO: 55, L chain RDC-23L-κ / SEQ ID NO: 56). The affinity of RDC-23 for soluble IL-6 receptor and the biological activity of BaF / gp130 were compared with that of tocilizumab (see Reference Example for the method).

  The results of affinity measurement are shown in Table 1. The results of measuring the biological activity (final IL-6 concentration 30 ng / mL) by BaF / gp130 are shown in FIG. RDC-23 was found to have an approximately 60-fold improved affinity compared to tocilizumab and an approximately 100-fold improved activity as a 100% inhibitory concentration of BaF / gp130.

(Table 1)

[Example 2] Identification of mutations that improve pharmacokinetics by lowering the isoelectric point of tocilizumab In order to improve the pharmacokinetics of tocilizumab, the isoelectric point of the variable region was determined without greatly reducing the binding to the IL-6 receptor. We examined mutation sites that can be reduced. As a result of screening the variable region mutation sites inferred from the three-dimensional model of tocilizumab, we found the mutation sites that can reduce the isoelectric point of the variable region without significantly reducing the binding to the IL-6 receptor. It was summarized in 3. An example of tocilizumab with a reduced isoelectric point combining these mutations is H53 / L28 (H chain H53-IgG1 / SEQ ID NO: 57, L chain L28-κ / SEQ ID NO: 58). The affinity of H53 / L28 for soluble IL-6 receptor, biological activity by BaF / gp130, isoelectric point, and pharmacokinetics in mice were compared with that of tocilizumab (see Reference Example for method).

  The results of measuring the affinity are shown in Table 2. The result of measuring the biological activity (IL-6 final concentration 30 ng / mL) by BaF / gp130 is shown in FIG. H53 / L28 has an approximately 6-fold improvement in affinity compared to tocilizumab, indicating that the activity is improved several times as a 100% inhibitory concentration of BaF / gp130.

(Table 2)

  As a result of measuring the isoelectric point by isoelectric focusing known to those skilled in the art, the isoelectric point of tocilizumab is about 9.3, the isoelectric point of H53 / L28 is about 6.5 to 6.7, and H53 / L28 is tocilizumab The isoelectric point decreased by about 2.7. Moreover, when the theoretical isoelectric point of variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of tocilizumab was 9.20, the theoretical isoelectric point of H53 / L28 was 4.52, and H53 / L28 Compared with tocilizumab, the isoelectric point decreased by about 4.7.

  In order to evaluate the pharmacokinetics of the modified antibody H53 / L28 with a reduced isoelectric point, the pharmacokinetics of tocilizumab and H53 / L28 in normal mice were compared. Tocilizumab and H53 / L28 were administered to mice (C57BL / 6J, Charles River, Japan) at a dose of 1 mg / kg intravenously (IV) and subcutaneously (SC), and the plasma concentration was evaluated. Figure 5 shows the time course of plasma concentration after intravenous administration of tocilizumab and H53 / L28, and Figure 6 shows the time course of plasma concentration after subcutaneous administration. The pharmacokinetics obtained by WinNonlin (Pharsight) The parameters (clearance (CL), half-life (T1 / 2)) are shown in Table 3. The plasma half-life (T1 / 2) after intravenous administration of H53 / L28 was increased to about 1.3 times that of tocilizumab, and the clearance was reduced about 1.7 times. T1 / 2 after subcutaneous administration of H53 / L28 was extended to about 2 times that of tocilizumab, and the clearance was reduced about 2.1 times. Thus, it was found that pharmacokinetics can be significantly improved by lowering the isoelectric point of tocilizumab by amino acid substitution.

(Table 3)

[Example 3] Identification of mutation sites that reduce the immunogenicity of tocilizumab
Identification of mutation sites that reduce the risk of immunogenicity caused by T cell epitopes in the variable region Using TPITOPE (Methods. 2004 Dec; 34 (4): 468-75) T cell epitopes present in the variable region sequence of tocilizumab Analysis. As a result, it was predicted that there are many T cell epitopes that bind to HLA in the light chain CDR2 (there is a sequence with a high immunogenicity risk). Therefore, we examined amino acid substitutions that did not reduce stability, binding activity, and neutralization activity while reducing the immunogenicity risk of L chain CDR2 in TEPITOPE analysis.

As a result of screening, threonine of L51 (Kabat numbering, Kabat EA et al., 1991. Sequences of Proteins of Immunological Interest, NIH) of tocilizumab L chain CDR2 (SEQ ID NO: 59) was converted to glycine as follows. It was found that the risk of immunogenicity can be reduced by substituting glutamic acid for arginine (SEQ ID NO: 60) without reducing stability, binding activity, and neutralization activity.
Tocilizumab light chain CDR2 (SEQ ID NO: 59)
T cell epitope-removed tocilizumab light chain CDR2 (SEQ ID NO: 60)

[Example 4] Reduction of immunogenicity risk by complete humanization of variable region framework sequence of tocilizumab In the variable region sequence of tocilizumab, H27 of H chain FR1, H28, H29, H30 and H71 of H chain FR3 (Kabat numbering) Kabat EA et al., 1991. Sequences of Proteins of Immunological Interest (NIH)), in which mouse sequences remain in the framework sequence to maintain binding activity during the humanization of tocilizumab (Cancer Res. 1993 Feb 15; 53 (4): 851-6). Since the remaining mouse sequence can be responsible for increasing the immunogenicity risk, a study was undertaken to fully humanize the framework sequence in order to further reduce the immunogenicity risk of tocilizumab.

As a result, the H chain FR1 (SEQ ID NO: 61) of tocilizumab was replaced with the following humanized H chain FR1-A (SEQ ID NO: 62), and the H chain FR3 (SEQ ID NO: 63) was replaced with It was found that by substituting the humanized H chain FR3 (SEQ ID NO: 64), the entire framework of tocilizumab can be completely humanized without reducing stability, binding activity, and neutralization activity.
Tocilizumab H chain FR1 (SEQ ID NO: 61)
Humanized H chain FR1-A (SEQ ID NO: 62) (derived from germline IMGT hVH_4_)
Tocilizumab H chain FR3 (SEQ ID NO: 63)
Humanized H chain FR3 (SEQ ID NO: 64) (derived from Mol. Immunol. 2007, 44 (4): 412-422)

[Example 5] Identification of mutation site for improving pharmacokinetics by pH-dependent binding of tocilizumab to IL-6 receptor One of the methods for improving the pharmacokinetics of tocilizumab is that one molecule of tocilizumab contains multiple ILs. -6 is a method of modifying molecules to repeatedly bind and neutralize receptors. After binding to membrane IL-6 receptor, tocilizumab is taken into endosomes by intracellularization while bound to membrane IL-6 receptor, and then transferred to lysosome while bound to membrane IL-6 receptor. It is thought to be degraded by lysosomes. In other words, one molecule of tocilizumab normally binds to one or two membrane-type IL-6 receptors (monovalent or divalent) and is considered to be degraded by lysosomes after internalization. Tocilizumab can bind and neutralize only one or two membrane-type IL-6 receptors.

  Therefore, if pH-dependent binding tocilizumab that greatly reduces only binding under acidic conditions while maintaining binding under neutral conditions of tocilizumab can be prepared, as shown in FIG. 7, pH-dependent binding tocilizumab Dissociates from the membrane-type IL-6 receptor, which is an antigen in the endosome, and can return to plasma by binding to FcRn present in the endosome, and the pH-dependent tocilizumab returned to plasma is again membrane-type We thought it was possible to bind to IL-6 receptor. By repeating this binding in plasma and dissociation in endosomes, it is considered that one molecule of tocilizumab can repeatedly bind and neutralize multiple molecules of IL-6 receptor, which is more pH-dependent than tocilizumab. Bound tocilizumab was thought to improve pharmacokinetics.

  In order for tocilizumab to dissociate from the IL-6 receptor under acidic conditions in endosomes, the binding under acidic conditions must be significantly weaker than under neutral conditions. Since it is necessary to bind strongly to the IL-6 receptor and neutralize it on the cell surface, it is necessary to bind to the IL-6 receptor at pH 7.4, which is the cell surface pH, equal to or higher than that of tocilizumab. It has been reported that the endosomal pH is generally pH 5.5 to pH 6.0 (Nat Rev Mol Cell Biol. 2004 Feb; 5 (2): 121-32.) A pH-dependent binding tocilizumab modified to weakly bind to the IL-6 receptor at pH 6.0 would dissociate from the IL-6 receptor under acidic conditions within the endosome. That is, the pH was improved so that it strongly binds to IL-6 receptor at pH 7.4, which is the cell surface pH, and weakly binds to IL-6 receptor at pH 5.5 to pH 6.0, which is the endosomal pH. Dependent binding tocilizumab was thought to be able to bind to and neutralize multiple IL-6 receptors with a single molecule and improve pharmacokinetics.

  In order to confer pH dependence on the binding of tocilizumab to the IL-6 receptor, pKa exists in the vicinity of 6.0 to 6.5, and neutral (pH 7.4) and acidic conditions (pH 5.5 to pH 6.0). ) Was introduced into the variable region of tocilizumab, which changes the proton dissociation state. Therefore, screening was performed for the histidine introduction site of the variable region inferred from the three-dimensional structure model of tocilizumab. In addition, a library designed to randomly replace the selected variable region sequence of tocilizumab with histidine was prepared and screened. Screening was performed using as an indicator that IL-6 receptor was bound at pH 7.4 and dissociated from IL-6 receptor at pH 5.5 or 5.8, or that affinity decreased.

  As a result, we found mutation sites that can confer pH dependency (the property of binding at pH 7.4 and dissociation at pH 5.8) to the binding of tocilizumab to the IL-6 receptor. . In Figure 8, the substitution of H27 from tyrosine to histidine is a mutation in FR1 of the H chain, not the CDR, but the sequence of H27 is histidine as described in Eur. J. Immunol. 1992. 22: 1719-1728 Since it exists as a human sequence (SEQ ID NO: 65), it can be fully humanized together with Example 4 by using the following framework.

Humanized H chain FR1-B (SEQ ID NO: 65)
An example of a pH-dependent binding tocilizumab combining these mutations is H3pI / L73 (H chain H3pI-IgG1 / SEQ ID NO: 66, L chain L73-κ / SEQ ID NO: 67). Affinity of H3pI / L73 to soluble IL-6 receptor at pH 7.4, dissociation rate from membrane IL-6 receptor at pH 7.4 and pH 5.8, biological activity by BaF / gp130, and cynomolgus monkey and human Pharmacokinetics in IL-6 receptor transgenic mice was compared with tocilizumab (see Reference Example for method).

  Table 4 shows the results of measuring the affinity for soluble IL-6 receptor at pH 7.4. The results of measuring the biological activity of BaF / gp130 (IL-6 final concentration 30 ng / ml) are shown in FIG. H3pI / L73 was shown to have an affinity for soluble IL-6 receptor and an activity of BaF / gp130 at approximately pH 7.4 compared to tocilizumab.

(Table 4)

  Table 5 shows the results of measuring the dissociation rate of tocilizumab and H3pI / L73 to membrane-type IL-6 receptor at pH 7.4 and pH 5.8. H3pI / L73 showed a faster dissociation rate at pH 5.8, indicating that the pH dependence of the dissociation rate from the membrane-type IL-6 receptor was improved by about 2.6 times compared to tocilizumab.

(Table 5)

  Tocilizumab and H3pI / L73 were administered to cynomolgus monkeys at a single dose of 1 mg / kg intravenously, and changes in plasma concentrations over time were evaluated. The time course of plasma concentrations after intravenous administration of tocilizumab and H3pI / L73 is shown in FIG. As a result, H3pI / L73 significantly improved pharmacokinetics in cynomolgus monkeys compared to tocilizumab.

  Tocilizumab and H3pI / L73 were intravenously administered to human IL-6 receptor transgenic mice (hIL-6R tg mice, Proc Natl Acad Sci US A. 1995 May 23; 92 (11): 4862-6) at 25 mg / kg A single dose was administered to evaluate changes in plasma concentration over time. The time course of plasma concentrations after intravenous administration of tocilizumab and H3pI / L73 is shown in FIG. As a result, pharmacokinetics of H3pI / L73 was significantly improved in human IL-6 receptor transgenic mice compared to tocilizumab.

  The pH-dependent binding tocilizumab H3pI / L73 binds to the antigen at pH 7.4 due to the greatly improved pharmacokinetics compared to tocilizumab in cynomolgus monkeys and human IL-6 receptor transgenic mice. It was thought that it was possible to bind to and neutralize multiple IL-6 receptors with a single molecule by imparting the property of dissociating from the antigen. Moreover, it was considered that the pharmacokinetics could be further improved by imparting a stronger pH dependency to the binding to IL-6 receptor than H3pI / L73.

[Example 6] Optimization of the stationary region of tocilizumab
Reduction of H chain C-terminal heterogeneity of tocilizumab
As a heterogeneity of the H chain C-terminal sequence of IgG antibody, a deletion of lysine residue of C-terminal amino acid and amidation of C-terminal carboxyl group due to deletion of glycine and lysine, two amino acids at C-terminal, are reported (Anal Biochem. 2007 Jan 1; 360 (1): 75-83.). In tocilizumab, the main component is a sequence in which the lysine of the C-terminal amino acid present in the base sequence is deleted by post-translational modification, but the C-terminal carboxyl group due to the loss of both glycine and lysine as a secondary component in which lysine remains The amidated subcomponents of this also contribute to the heterogeneity. It is not easy to manufacture a large amount as a medicine while maintaining the difference in the heterogeneity of the target substance / related substance, leading to an increase in cost. It is desirable to be a single substance as much as possible, and it is desirable to reduce these heterogeneity in developing an antibody as a medicine. Therefore, in developing as a medicine, it is desirable that there is no heterogeneity of the H chain C terminal.

  The C-terminal amino acid was modified to reduce the heterogeneity of the C-terminal amino acid. As a result, it was found that the heterogeneity derived from the C-terminus can be avoided by deleting the lysine and glycine at the C-terminus of the H chain constant region of tocilizumab in advance. Tocilizumab, C-terminal lysine-deficient tocilizumab (tocilizumab ΔK, H chain WT-IgG1ΔK / SEQ ID NO: 68, L chain WT-κ / SEQ ID NO: 54), and C-terminal lysine and glycine-deficient tocilizumab (tocilizumab ΔGK, H Evaluation of heterogeneity of the chain WT-IgG1ΔGK / SEQ ID NO: 69, light chain WT-κ / SEQ ID NO: 54) was performed by cation exchange chromatography. ProPac WCX-10, 4 × 250 mm (Dionex) is used as the column, mobile phase A is 25 mmol / L MES / NaOH, pH 6.1, mobile phase B is 25 mmol / L MES / NaOH, 250 mmol / L NaCl, pH 6.1 was used and performed with the appropriate flow rate and gradient. The results of evaluation by cation exchange chromatography are shown in FIG. As a result, not only the C-terminal lysine of the H chain constant region but also the lysine and glycine of the C chain of the H chain constant region can be reduced for the first time by reducing the heterogeneity of the C-terminal amino acid. It was found that In human antibody constant regions IgG1, IgG2, and IgG4, all C-terminal sequences are EU numbering (see Sequences of proteins of immunological interest, see NIH Publication No.91-3242). 447th is lysine and 446th is glycine. Therefore, it was considered that the method for reducing the heterogeneity of the C-terminal amino acid found in this study can be applied to IgG2 constant region and IgG4 constant region, or variants thereof.

Reduction of heterogeneity due to disulfide bond of IgG2 isotype of tocilizumab Tocilizumab isotype is IgG1, but since tocilizumab is a neutralizing antibody, binding to Fcγ receptor is preferable when considering immunogenicity and side effects There may be no possibility. As a method for reducing the binding to the Fcγ receptor, a method of changing the IgG isotype from IgG1 to IgG2 or IgG4 (Ann Hematol. 1998 Jun; 76 (6): 231-48.) Is considered. From the viewpoint of binding and pharmacokinetics, IgG2 was considered preferable to IgG4 (Nat Biotechnol. 2007 Dec; 25 (12): 1369-72). On the other hand, in developing an antibody as a pharmaceutical, the physicochemical properties of the protein, especially homogeneity and stability, are extremely important, and the IgG2 isotype has a great deal of heterogeneity derived from the disulfide bond in the hinge region. Has been reported (J Biol Chem. 2008 Jun 6; 283 (23): 16206-15.). Maintaining the heterogeneity of target / related substance heterogeneity derived from disulfide bonds, mass production as a pharmaceutical is not easy and leads to increased costs, and it is desirable to be as single as possible . Therefore, in developing IgG2 isotype antibodies as pharmaceuticals, it is desirable to reduce heterogeneity derived from disulfide bonds without reducing stability.

  As a result of examining various variants for the purpose of reducing the heterogeneity of IgG2 isotype, EU numbering 131st cysteine and 133rd arginine present in the CH1 domain of H chain in the IgG2 constant region sequence were respectively serine. WT-SKSC (SEQ ID NO: 70), which is a constant region in which the cysteine at position 219 in the EU numbering cysteine located at the hinge upstream of the H chain is replaced with serine, is not degraded without reducing stability. It has been found that uniformity can be reduced. Tocilizumab-IgG1 (H chain WT-IgG1 / SEQ ID NO: 53, L chain WT-κ / SEQ ID NO: 54), Tocilizumab-IgG2 (H chain WT-IgG2 / SEQ ID NO: 71, L chain WT-κ / SEQ ID NO: 54) : 54) and tocilizumab-SKSC (H chain WT-SKSC / SEQ ID NO: 70, L chain WT-κ / SEQ ID NO: 54) were prepared and evaluated for heterogeneity and stability. Heterogeneity was evaluated by cation exchange chromatography. ProPac WCX-10 (Dionex) as column, 20 mM sodium acetate, pH 5.0 as mobile phase A, 20 mM sodium acetate, 1 M NaCl, pH 5.0 as mobile phase B, with appropriate flow rate and gradient It carried out using. The results of evaluation by cation exchange chromatography are shown in FIG. The stability was evaluated by evaluating the thermal denaturation intermediate temperature (Tm value) by differential scanning calorimetry (DSC) (VP-DSC, Microcal). FIG. 14 shows the DSC measurement results in 20 mM sodium acetate, 150 mM NaCl, pH 6.0, and the Tm value of the Fab domain.

As a result, it was found that tocilizumab-IgG2 has a significantly increased heterogeneity compared to tocilizumab-IgG1, but the heterogeneity can be greatly reduced by using tocilizumab-SKSC. In addition, compared to tocilizumab-IgG1, tocilizumab-IgG2 has a low stability, which is considered to be due to a hetero component in the heat denaturation peak of the Fab domain in DSC, that is, a component of a shoulder peak (Fab ^* ) with a low Tm, Tosilizumab-SKSC resulted in the disappearance of a shoulder peak with a low Tm value, which was thought to be due to the hetero component, and showed a Tm value of about 94 ° C. equivalent to the Fab domain of tocilizumab-IgG1 and tocilizumab-IgG2. SKSC was found to have high stability.

Identification of constant region mutations that improve pharmacokinetics of tocilizumab As mentioned above, C-terminal heterogeneity is reduced while maintaining high stability while reducing the binding to Fcγ receptor from IgG1, an isotype of tocilizumab It has been found that it is possible to reduce the heterogeneity of antibodies with constant nature and IgG2 isotype constant regions. Furthermore, regarding the pharmacokinetics, it is desirable that the constant region be superior to IgG1, which is an isotype of tocilizumab.

  Tocilizumab-SKSC with high stability and reduced heterogeneity for IgG2 isotype constant region antibodies to find a constant region with better plasma half-life than IgG1 isotype constant region antibodies On the other hand, as a result of screening mutation sites for the purpose of improving pharmacokinetics, EU numbering 137th glutamic acid was glycine, 138th serine was glycine, and 268 histidine was glutamine. WT-M58 in which 355th arginine was replaced with glutamine and 419th glutamine was replaced with glutamic acid, and in addition to this, 446th glycine and 447th lysine were deleted to reduce the heterogeneity of the C-terminal of the H chain. (SEQ ID NO: 72 (amino acid sequence)) was found. Furthermore, WT-M44 (SEQ ID NO: 73 (amino acid sequence)) in which 434th asparagine was substituted with alanine was prepared for IgG1. Furthermore, WT-M83 (SEQ ID NO: 74 (amino acid sequence)) in which 446th glycine and 447th lysine were deleted was prepared in order to reduce the heterogeneity of the H chain C-terminal relative to M44. Further, WT-M73 (SEQ ID NO: 75 (amino acid sequence)) in which 434th asparagine was substituted with alanine was prepared for WT-M58.

  Tocilizumab-M44 (H chain WT-M44 / SEQ ID NO: 73, L chain WT-κ / SEQ ID NO: 54), Tocilizumab-M58 (H chain WT-M58 / SEQ ID NO: 72, L chain WT-κ / SEQ ID NO: : 54) and tocilizumab-M73 (H chain WT-M73 / SEQ ID NO: 75, L chain WT-κ / SEQ ID NO: 54) were prepared and evaluated for affinity to human FcRn and pharmacokinetics by human FcRn transgenic mice (Refer to the reference example for the method).

  The evaluation of the binding of tocilizumab-IgG1, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 to human FcRn was performed by Biacore. The sex was about 2.7 times, about 1.4 times, and about 3.8 times better than tocilizumab-IgG1, respectively.

(Table 6)

  The results of pharmacokinetic evaluation of tocilizumab-IgG1, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 in human FcRn transgenic mice are shown in FIG. As shown in FIG. 15, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 were all found to have improved pharmacokinetics compared to tocilizumab-IgG1. Its pharmacokinetic improvement effect correlated with the binding ability to human FcRn. In particular, with regard to tocilizumab-M73, the plasma concentration after 28 days was improved by about 16 times compared with tocilizumab-IgG1, and therefore, an antibody having the constant region of M73 is also an antibody having the constant region of IgG1 in humans. The pharmacokinetics were considered to be significantly improved.

[Example 7] Production of fully humanized IL-6 receptor antibody with improved PK / PD Tosilizumab variants were prepared by combining multiple mutations in the variable region and constant region of tocilizumab found in the above examples. As a result of screening, as a fully humanized IL-6 receptor antibody, Fv3-M73 (H chain VH4-M73 / SEQ ID NO: 25, L chain VL1-κ / SEQ ID NO: 28), Fv4-M73 (H chain VH3 -M73 / SEQ ID NO: 26, L chain VL3-κ / SEQ ID NO: 29), Fv5-M83 (H chain VH5-M83 / SEQ ID NO: 27, L chain VL5-κ / SEQ ID NO: 30) were found.

  The affinity of the prepared Fv3-M73, Fv4-M73, and Fv5-M83 for IL-6 receptor was compared with that of tocilizumab (refer to the reference example for the method). The results of measuring the affinity of these antibodies at pH 7.4 for soluble IL-6 receptor are shown in Table 7. In addition, the neutralizing activity of BaF / gp130 was compared with tocilizumab and a control (known high affinity anti-IL-6 receptor antibody of reference example, VQ8F11-21 hIgG1 in US 2007/0280945) (refer to reference example for method). The results of measuring the biological activity of these antibodies by BaF / gp130 are shown in FIG. 16 (IL-6 final concentration 300 ng / mL: tocilizumab, control, Fv5-M83) and FIG. 17 (IL-6 final concentration 30 ng / mL: Tocilizumab, Fv3-M73, Fv4-M73). As shown in Table 7, Fv3-M73 and Fv4-M73 had an affinity about 2-3 times stronger than tocilizumab, and Fv5-M83 showed an affinity about 100 times stronger than tocilizumab (Fv5 Since it was difficult to measure affinity with -M83, affinity was measured using Fv5-IgG1 (H chain VH5-IgG1 / SEQ ID NO: 76, L chain VL5-κ / SEQ ID NO: 30) whose constant region was IgG1 The constant region is generally considered not to affect affinity). In addition, as shown in FIG. 17, Fv3-M73 and Fv4-M73 show a slightly stronger activity compared to tocilizumab, and as shown in FIG. 16, Fv5-M83 is an extremely high 50% inhibitory concentration compared to tocilizumab. Compared to a control having a strong activity and a known high-affinity anti-IL-6 receptor antibody, the neutralizing activity was about 10 times higher as a 50% inhibitory concentration.

(Table 7)

  The results of measuring the dissociation rate of tocilizumab, Fv3-M73, and Fv4-M73 from membrane-type IL-6 receptor at pH 7.4 and pH 5.8 are shown in Table 8 (see Reference Examples for methods). Fv3-M73 and Fv4-M73 were shown to have an approximately 11-fold and 10-fold improvement in the pH dependence of the dissociation rate from the membrane-type IL-6 receptor, respectively, compared to tocilizumab. Compared to H3pI / L73 in Example 5, the pH dependence of the dissociation rate is greatly improved, so the pharmacokinetics of Fv3-M73 and Fv4-M73 are significantly improved compared to H3pI / L73. It was thought that

(Table 8)

  The isoelectric point of tocilizumab, control, Fv3-M73, Fv4-M73, and Fv5-M83 was measured by isoelectric focusing by a method known to those skilled in the art. 8.4 to 8.5, Fv3-M73 is about 5.7 to 5.8, Fv4-M73 is about 5.6 to 5.7, Fv5-M83 is 5.4 to 5.5, and all antibodies have significantly reduced isoelectric points compared to tocilizumab and controls did. In addition, when the theoretical isoelectric point of variable region VH / VL was calculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectric point of tocilizumab was 9.20, the control was 7.79, Fv3-M73 was 5.49, Fv4-M73 was 5.01, Fv5- M83 was 4.27, and the isoelectric point of both antibodies was significantly reduced compared to tocilizumab and controls. Since it was shown in Example 2 that pharmacokinetics were improved by lowering the isoelectric point, Fv3-M73, Fv4-M73, and Fv5-M83 showed improved pharmacokinetics compared to tocilizumab and the control. It was thought that there was.

  T cell epitopes present in the variable region sequences of tocilizumab, Fv3-M73, Fv4-M73, and Fv5-M83 were analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, as shown in Example 3, tocilizumab was predicted to have T cell epitopes that bind many sequences to HLA, but Fv3-M73, Fv4-M73, and Fv5-M83 bind to T cell epitopes. This greatly reduced the predicted sequence. Fv3-M73, Fv4-M73, and Fv5-M83 are fully humanized with no mouse sequence remaining in the framework. These results suggested that Fv3-M73, Fv4-M73, and Fv5-M83 may have a significantly reduced immunogenicity risk compared to tocilizumab.

[Example 8] Monkey PK / PD test of fully humanized IL-6 receptor antibody Tocilizumab, control, Fv3-M73, Fv4-M73, and Fv5-M83 were administered to cynomolgus monkeys at a single dose of 1 mg / kg intravenously. The time course of plasma concentration was evaluated (see Reference Example for method). The time course of plasma concentrations after intravenous administration of tocilizumab, Fv3-M73, Fv4-M73, and Fv5-M83 is shown in FIG. As a result, Fv3-M73, Fv4-M73, and Fv5-M83 all significantly improved pharmacokinetics in cynomolgus monkeys compared to tocilizumab and controls. In particular, the pharmacokinetics of Fv3-M73 and Fv4-M73 were significantly improved compared to tocilizumab.

  Cynomolgus monkey IL-6 5 μg from day 6 to day 18 of antibody administration (day 3 to day 10 for tocilizumab) to assess how neutralized cynomolgus IL-6 receptor is / kg was subcutaneously administered daily to the back of the lumbar region, and the CRP concentration of each individual was measured 24 hours later (see Reference Examples for methods). FIG. 19 shows changes with time in the CRP concentration when each antibody was administered. In order to evaluate the degree of neutralization of cynomolgus monkey soluble IL-6 receptor, the concentration of unbound cynomolgus monkey soluble IL-6 receptor in cynomolgus monkey plasma was measured, and unbound The percentage of soluble IL-6 receptor was calculated (see Reference Example for method). FIG. 20 shows the change over time in the proportion of unbound soluble IL-6 receptor upon administration of each antibody.

  Fv3-M73, Fv4-M73, and Fv5-M83 all neutralize cynomolgus membrane IL-6 receptor more persistently compared to tocilizumab and the known high affinity anti-IL-6 receptor antibody control , The increase of CRP was suppressed for a long time. In addition, Fv3-M73, Fv4-M73, and Fv5-M83 all neutralize cynomolgus monkey soluble IL-6 receptor more persistently than tocilizumab and control, and non-binding cynomolgus monkey soluble IL -6 receptors increased for a long time. Thus, Fv3-M73, Fv4-M73, and Fv5-M83 are all superior to tocilizumab and the control in terms of the persistence of neutralization of membrane IL-6 receptor and soluble IL-6 receptor. It was found. Above all, the sustainability of neutralization of Fv3-M73 and Fv4-M73 was extremely excellent. On the other hand, Fv5-M83 suppresses CRP and non-binding cynomolgus monkey soluble IL-6 receptor lower than Fv3-M73 and Fv4-M73. Type 6 IL-6 receptor was thought to be more strongly neutralized than Fv3-M73 and Fv4-M73 and the known high affinity anti-IL-6 receptor antibody control. This is considered to be a result of reflecting in vivo the cynomolgus monkey that Fv5-M83 has a stronger affinity for the IL-6 receptor than the control and has a higher biological activity at BaF / gp130.

  Based on these findings, Fv3-M73 and Fv4-M73 have extremely long-lasting effects as anti-IL-6 receptor neutralizing antibodies compared to tocilizumab and controls, significantly reducing the administration frequency and dosage Further, it was found that Fv5-M83 is extremely excellent in the strength of action as an anti-IL-6 receptor neutralizing antibody, and also has excellent durability of action. Therefore, Fv3-M73, Fv4-M73, and Fv5-M83 are considered useful as pharmaceutical agents as IL-6 antagonists.

Example 9 Selection of Stable Buffer Species for Fv4-M73 Fv4-M73 is prepared as described above. As described in Examples 6-7, Fv4-M73 is composed of non-natural constant region M73 for improved heterogeneity, stability, safety, and pharmacokinetics, so Fv4 The stability profile of -M73 (eg, the most stable buffer species) may be different from antibodies composed of natural constant regions such as IgG1. Antibodies composed of natural IgG1 constant regions are generally reported to be most stable under histidine acetate buffer (WO / 2006/044908). Therefore, the effect of buffer species on the stability of Fv4-M73 was tested.

  Fv4-M73 was formulated in 4 different buffer formulations (as described in Table 9) having a pH of 6.0 at a final concentration of 37 mg / mL. Samples were analyzed by size exclusion and anion exchange chromatography with UV detection for 2 months under storage conditions at 40 ° C. The percentage (%) of aggregates formed in these preparations is displayed over time and is shown in FIG. An increase in the percentage of aggregates is an indicator of a decrease in antibody stability. Therefore, an increase in percentage (%) was used as an index to compare the stabilizing effects of different buffers.

  As shown in FIG. 21, the formulation containing histidine-HCl buffer (Formulation A) and the formulation containing citrate buffer (Formulation D) are the most stable, and the formulation containing acetate buffer (Formulation C) is the most stable. It was unstable. The histidine-acetate buffer (Formulation B) is slightly more unstable than the histidine-HCl buffer, which is believed to be due to the destabilizing effect of the acetate buffer.

  Thus, Fv4-M73 with a non-natural constant region is not the histidine-acetate buffer reported to be the most stable buffer species for native IgG1 antibodies, but histidine-HCl buffer and citrate. It was found to be most stable in the buffer.

Table 9 Formulations used in Example 9

Methods Size Exclusion Chromatography (SEC): Size exclusion chromatography was performed to screen antibody formulations for the presence of antibody aggregates and fragments. The sample was injected into a size exclusion G3000 SW _XL column (TOSOH). The mobile phase was 50 mM sodium phosphate and 300 mM sodium chloride (pH 7.0) flowing at a uniform concentration at a flow rate of 0.5 mL / min, and the eluted protein was detected by UV absorbance at 220 nm. The relative amounts of all protein species detected were reported as area% of product peak compared to the total area of all other detected peaks. The peak eluting earlier than the antibody monomer peak was recorded with the aggregate percentile, and the peak eluting later than the antibody monomer peak but earlier than the buffer peak was recorded with the fragment percentile.

  Sample preparation: Samples were diluted to 0.3-2 mg / mL with mobile phase and 15 microliters were injected onto the column.

  Anion exchange chromatography: Anion exchange chromatography to analyze antibody formulations for the presence of heterogeneity, in particular for the presence of deamidation (those with deamid elute as an acidic component after the main peak). The graphic was performed. The sample was injected into a DEAE-NPR column (TOSOH) at 40 ° C. The mobile phase is (A) 10 mM Tris-HCl (pH 7.5), (B) 10 mM Tris-HCl, 500 mM sodium chloride (pH 7.5), 100% mobile phase A, then 70% for 30 minutes Mobile phase A and 30% mobile phase B followed by a gradient of 100% mobile phase B for 1 minute, then maintained at a flow rate of 1.0 mL / min for 4 minutes to wash the column. The eluted protein was detected by UV absorbance at 280 nm.

  Sample preparation: Samples were diluted to 0.05-0.33 mg / mL with mobile phase and injected onto the column in a volume of 100 microliters.

Example 10 Effect of pH, NaCl, and Arginine-HCl on the stability of 100 mg / mL Fv4-M73
Fv4-M73 is formulated with different buffer formulations (described in Table 10) to a final concentration of 100 mg / mL and stored at 25 ° C and 40 ° C with UV detection for 2 months Samples were analyzed by size exclusion chromatography and anion exchange chromatography. Analyzes were also performed on freeze-thawed samples (freezing at -20 ° C for 0.5 days, thawing at room temperature for 30 minutes). The percentage of aggregate present in the formulation and the increase in percentage of aggregate from the initial value after storage for 2/4/8 weeks at 25 ° C. and 40 ° C. are shown in FIG. Shown in 25. An increase in the amount of aggregate is an indicator of a decrease in stability.

Table 10: Formulations used in Example 10

  The low molecular weight component (LMW) described in FIG. 26 is believed to be the result of direct hydrolysis of peptide bonds in a fragile spot such as the hinge region under acidic or basic conditions. This is particularly common for monoclonal antibodies in solution formulations at elevated temperatures. The acidic species eluting at about 22.5-26 minutes described in Figure 27, increased under basic conditions, is believed to be the result of non-enzymatic deamidation of the asparagine residue (a general modification of the antibody) It is done. It has been reported that this is responsible for the charge heterogeneity of monoclonal antibodies, since more acidic species are observed after incubating the antibody at high temperature and basic pH. In view of the results of FIGS. 22-26, Fv4-M73 was found to be stable in solutions with a pH of about 5.5-6.3 with respect to aggregates and degradation. With regard to the ratio of the main peak to the total peak area, optimal stability was observed at a pH of about 5.5-6.3 (Figures 27-28).

  In this study, a buffer containing 20 mM buffer and 50 mM NaCl was added by adding 100 mM NaCl to a buffer containing 20 mM buffer and 50 mM NaCl (20 mM buffer, 150 mM NaCl). An antibody solution that is as stable as (20 mM buffer, 50 mM NaCl) was obtained, suggesting that NaCl has no stabilizing effect. Addition of 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) resulted in a significant stabilizing effect of the antibody solution on aggregates From this, it was suggested that arginine-HCl has a significant stabilizing effect. As for the buffer, the solution stability was slightly better when the histidine-HCl buffer was used than the citrate buffer.

  For the freeze-thaw test (“FT” indicates the number of freeze / thaw cycles) as shown in FIG. 29, significant amounts of aggregates are observed in the formulation at lower pH or lower salt concentrations. It was. Addition of 100 mM arginine to a buffer containing 20 mM buffer and 50 mM NaCl (20 mM buffer, 50 mM NaCl, 100 mM arginine-HCl) adds 100 mM NaCl with respect to aggregate formation ( 20 mM buffer, 150 mM NaCl), suggesting that arginine-HCl has a significant stabilizing effect on freeze-thaw. Optimal stability was observed at a pH in the range of about 5.0 to 6.6 containing 100 mM arginine-HCl.

Example 11 Effect of sugar and arginine-HCl on the stability of 100 mg / mL Fv4-M73
Fv4-M73 is formulated in different buffer formulations (described in Table 11) to a final concentration of 100 mg / mL, stored at 25 ° C and 40 ° C, and size exclusion chromatography with UV detection for 2 months Samples were analyzed by chromatography and anion exchange chromatography. A predetermined number of freeze-thaw cycles (freezing at -20 ° C for 0.5 days, thawing at room temperature for 30 minutes) were also performed. The increase from the initial value of the percentage of aggregates contained in the formulation, plotted over time, is shown in FIGS. 30 and 31. An increase in the amount (%) of aggregate is an indicator of a decrease in stability.

  As shown in FIGS. 30 and 31, Formulations F and G exhibit lower stability than Formulation H, indicating that sucrose and trehalose are arginine-HCl in liquid conditions stored at 25 ° C. and 40 ° C. It was shown to have a lower stabilizing effect. On the other hand, in freeze-thaw, Formulations F and G were comparable to or higher than Formulation H, so sucrose or trehalose had a significant stabilizing effect in freeze-thaw. It was shown to show.

Table 11 Formulations used in Example 11

Example 12: Stability of 200 mg / mL Fv4-M73; effects of pH, arginine-HCl, and trehalose
Fv4-M73 was formulated to a final concentration of 200 mg / mL in 6 different formulations as described in Table 12.

Table 12 Formulations used in Example 12

  Samples were incubated at 5 ° C and -20 ° C for 3 months and 6 months, respectively. Initial, 3 month, and 6 month samples were analyzed by size exclusion chromatography with UV detection as described in Example 9. The increase from the initial percentage of aggregates in these formulations after 3 and 6 months at 5 ° C. is shown in FIG. The increase from the initial value of the percentage of aggregates in these formulations after 3 and 6 months at −20 ° C. is shown in FIG.

  As shown in FIG. 32, in solution conditions stored at 5 ° C., Fv4-M73 is clearly more stable at lower pH (most stable at pH 5.5, most unstable at pH 6.5) and more It was more stable at high arginine concentrations (most stable with 150 mM arginine-HCl, most unstable with 50 mM arginine-HCl). Addition of 50 mM trehalose showed some stabilizing effect on Fv4-M73 under solution conditions stored at 5 ° C. Since solution formulations of antibody pharmaceuticals are often stored at 5 ° C., a preferred solution formulation for Fv4-M73 contains at least 100 mM arginine-HCl with pH 5.5 to pH 6.0 histidine buffer, if necessary. Should contain additional stabilizers.

  As shown in FIG. 33, under freezing conditions stored at −20 ° C., Fv4-M73 tended to be more stable at higher pH and apparently more stable at higher arginine concentrations ( Most stable with 150 mM arginine-HCl, most unstable with 50 mM arginine-HCl). Addition of 50 mM trehalose showed a significant stabilizing effect on Fv4-M73 under freezing conditions stored at -20 ° C. The antibody drug substance is often stored in a frozen state at −20 ° C. to −70 ° C., but considering the cost of storage and shipping in the frozen state, storage at −20 ° C. is desirable. A preferred drug substance formulation for Fv4-M73 storage at −20 ° C. should contain at least 100 mM arginine-HCl along with a histidine buffer at pH 5.5 to pH 6.5 and a sugar (such as trehalose).

Reference example
Preparation of recombinant soluble human IL-6 receptor A recombinant soluble human IL-6 receptor of human IL-6 receptor, which is an antigen, was prepared as follows. J. Biochem. (1990) 108, 673-676, a soluble human IL-6 receptor consisting of the amino acid sequence from the 1st to 344th N-terminal side (Yamasaki et al., Science (1988) 241, 825-828 (GenBank # X12830)) CHO cell constant expression strain was prepared. From the culture supernatant obtained from SR344-expressing CHO cells, a soluble type was obtained by three column chromatography: Blue Sepharose 6 FF column chromatography, affinity chromatography with a column immobilized with a specific antibody against SR344, and gel filtration column chromatography. Human IL-6 receptor was purified. The fraction eluted as the main peak was used as the final purified product.

Preparation of recombinant soluble cynomolgus monkey IL-6 receptor (cIL-6R) Published rhesus monkey IL-6 receptor gene sequence (Birney et al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): Oligo DNA primer was prepared based on D556-61.), And a DNA fragment encoding the full length of cynomolgus IL-6 receptor gene was prepared by PCR using cDNA prepared from cynomolgus monkey pancreas as a template. The obtained DNA fragment was inserted into a mammalian cell expression vector, and a CHO constant expression strain (cyno.sIL-6R-producing CHO cell) was produced using this. After purifying the culture fluid of cyno.sIL-6R-producing CHO cells using HisTrap column (GE Healthcare Bioscience), it is concentrated using Amicon Ultra-15 Ultracel-10k (Millipore), and Superdex200pg16 / 60 gel filtration column (GE Health) The product was further purified by Care Bioscience, and used as a final purified product of soluble cynomolgus monkey IL-6 receptor (hereinafter cIL-6R).

Preparation of recombinant cynomolgus monkey IL-6 (cIL-6) Cynomolgus monkey IL-6 was prepared as follows. A base sequence encoding 212 amino acids registered in SWISSPROT Accession No.P79341 was created, cloned into a mammalian cell expression vector, and introduced into CHO cells to produce a constant expression cell line (cyno.IL-6 Producing CHO cells). After purifying the culture fluid of cyno.IL-6 producing CHO cells with SP-Sepharose / FF column (GE Healthcare Bioscience), using Amicon Ultra-15 Ultracel-5k (Millipore) and concentrating, Superdex75pg26 / 60 gel filtration Further purification was performed using a column (GE Healthcare Bioscience), and concentration was performed using Amicon Ultra-15 Ultracel-5k (Millipore) to obtain a final purified product of cynomolgus monkey IL-6 (hereinafter cIL-6).

Production of known high-affinity anti-IL-6 receptor antibody As a known high-affinity anti-IL-6 receptor antibody, VQ8F11-21 is a high-affinity anti-IL-6 receptor antibody described in US 2007/0280945 A1. In order to express hIgG1 (US 2007/0280945 A1, H chain amino acid sequence: SEQ ID NO: 77, L chain amino acid sequence: SEQ ID NO: 78), a mammalian cell expression vector was constructed. The antibody variable region was prepared by a PCR method (assembly PCR) combined with a synthetic oligo DNA, and IgG1 was used for the constant region. The antibody variable region and the constant region were combined by Assembly PCR method and inserted into a mammalian expression vector to prepare the intended H chain expression vector and L chain expression vector. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. Expression and purification were performed using the prepared expression vector. Expression and purification were performed by the method described in Example 1 to obtain a high affinity anti-IL-6 receptor antibody (hereinafter referred to as “control”).

Preparation, expression, and purification of tocilizumab mutants Tocilizumab mutants were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the method described in the attached instructions, and the resulting plasmid fragments were used in mammals. The target H chain expression vector and L chain expression vector were prepared by inserting into a cell expression vector. The base sequence of the obtained expression vector was determined by a method known to those skilled in the art. Antibody expression was carried out using the following method. HEK293H strain derived from human fetal kidney cancer cells (Invitrogen) is suspended in DMEM medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen), and a dish (diameter) is used at a cell density of 5-6 × 10 ⁵ cells / mL. Plate 10 mL on each dish (10 cm, CORNING), incubate overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ), and then remove the medium by suction. CHO-S-SFM-II (Invitrogen ) 6.9 mL of medium was added. The prepared plasmid was introduced into cells by the lipofection method. After collecting the obtained culture supernatant, the cells are removed by centrifugation (approximately 2000 g, 5 minutes, room temperature), and further sterilized through a 0.22 μm filter MILLEX (R) -GV (Millipore). Got. The antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose (trademark) Fast Flow (Amersham Biosciences). The purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer. The antibody concentration was calculated from the obtained value using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

Establishment of BaF3 cell line expressing human gp130
In order to obtain a cell line exhibiting IL-6-dependent proliferation, a BaF3 cell line expressing human gp130 was established as shown below.

  Full-length human gp130 cDNA (Hibi et al., Cell 1990; 63: 1149-1157 (GenBank # NM_002184)) was amplified by PCR and the DHFR gene of pCHOI (Hirata et al., FEBS Letter 1994; 356: 244-248) The expression site was removed and cloned into an expression vector pCOS2Zeo into which a Zeocin resistance gene expression site was inserted to construct pCOS2Zeo / gp130. Full-length human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1 (+) (Invitrogen) to construct hIL-6R / pcDNA3.1 (+).

10 μg of pCOS2Zeo / gp130 was mixed with BaF3 cells (0.8 × 10 ⁷ cells) suspended in PBS, and pulses were applied at a capacity of 0.33 kV and 950 μFD using Gene Pulser (Bio-Rad). BaF3 cells transfected with electroporation were cultured overnight in RPMI1640 medium (Invitrogen) containing 0.2 ng / mL mouse interleukin-3 (Peprotech) and 10% fetal bovine serum (hereinafter FBS, HyClone), and 100 ng Human gp130-expressing BaF3 cell line (R & D systems), 100 ng / mL human interleukin-6 soluble receptor (R & D systems) and RPMI1640 medium containing 10% FBS “BaF3 / gp130”) was established. Since this BaF / gp130 grows in the presence of human interleukin-6 (R & D systems) and soluble human IL-6 receptor, the growth inhibitory activity of anti-IL-6 receptor antibody (ie IL-6 receptor neutralization) Activity).

Evaluation of biological activity using human gp130-expressing BaF3 cells (BaF / gp130)
IL-6 receptor neutralizing activity was evaluated using BaF3 / gp130 showing IL-6 / IL-6 receptor-dependent proliferation. After washing BaF3 / gp130 3 times with RPMI1640 medium containing 10% FBS, human interleukin-6 (TORAY) (final concentration) of 600 ng / mL or 60 ng / mL to 5 x 10 ⁴ cells / mL 300 ng / mL or 30 ng / mL), suspended in RPMI1640 medium containing appropriate amounts of soluble human IL-6 receptor and 10% FBS, and dispensed 50 μL into each well of a 96-well plate (CORNING) . Next, the purified antibody was diluted with RPMI1640 containing 10% FBS, and 50 μL was mixed with each well. Add WST-8 reagent (Cell Counting Kit-8, Dojindo Laboratories, Inc.) diluted 2 times with PBS at 37 μC, 5% CO ₂ for 3 days at 20 μL / well. Absorbance at 450 nm (reference wavelength 620 nm) was measured using SUNRISE CLASSIC (TECAN). After culturing for 2 hours, the absorbance at 450 nm (reference wavelength: 620 nm) was measured again, and IL-6 receptor neutralizing activity was evaluated using the change in absorbance during 2 hours as an index.

Evaluation of binding to soluble human IL-6 receptor by Biacore
Kinetic analysis of antigen-antibody reaction was performed using Biacore T100 (GE Healthcare). An appropriate amount of protein A or protein A / G or anti-IgG (γ chain specific) F (ab ′) ₂ is immobilized on the sensor chip by the amine coupling method, and then the target antibody is bound at pH 7.4. Furthermore, soluble IL-6 receptor prepared at various concentrations at pH 7.4 was run as an analyte, and the interaction between the antibody and soluble human IL-6 receptor was measured. All measurements were performed at 37 ° C. From the sensorgram obtained from the measurement, the association rate constant k _a (1 / Ms) and the dissociation rate constant k _d (1 / s), which are kinetic parameters, are calculated, and K _D ( M) was calculated. Biacore T100 Evaluation Software (GE Healthcare) was used for calculation of each parameter.

Evaluation of pH-dependent dissociation into membrane-type IL-6 receptor by Biacore
Antigen-antibody reaction to membrane-type IL-6 receptor at pH 5.8 and pH 7.4 was observed using Biacore T100 (GE Healthcare). The binding to the soluble IL-6 receptor was evaluated by measuring the binding to the soluble human IL-6 receptor immobilized on the sensor chip. SR344 was biotinylated according to a method known to those skilled in the art, and biotinylated soluble human IL-6 receptor was immobilized on the sensor chip via streptavidin using the affinity between streptavidin and biotin. All measurements are performed at 37 ° C, and the mobile phase buffer is 10 mM MES pH5.8, 150 mM NaCl, 0.05% Tween20, and pH-dependent binding clones can be injected under conditions of pH7.4. After binding to soluble human IL-6 receptor (injection sample buffer is 10 mM MES pH 7.4, 150 mM NaCl, 0.05% Tween20), the pH of the mobile phase is 5.8, depending on the pH of each clone Dissociation was observed. PH 5.8 when the sample concentration is 0.25 μg / mL, bound with 10 mM MES pH 7.4, 150 mM NaCl, 0.05% Tween 20, and dissociated with 10 mM MES pH 5.8, 150 mM NaCl, 0.05% Tween 20. The dissociation rate constant (kd (1 / s)) at pH 5.8 was calculated by plotting only the dissociation phase in Biacore T100 Evaluation Software (GE Healthcare). Similarly, when the sample concentration is 0.5 μg / mL, binding with 10 mM MES pH 7.4, 150 mM NaCl, 0.05% Tween 20, and dissociation with 10 mM MES pH 7.4, 150 mM NaCl, 0.05% Tween 20 The dissociation rate constant (kd (1 / s)) at pH 7.4 was calculated by plotting only the dissociation phase at pH 7.4 using Biacore T100 Evaluation Software (GE Healthcare).

Evaluation of binding to human FcRn
FcRn is a complex of FcRn and β2-microglobulin. Oligo DNA primers were prepared based on the published human FcRn gene sequence (J. Exp. Med. (1994) 180 (6), 2377-2381). A human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) was used as a template, and a DNA fragment encoding the entire gene length was prepared by PCR using the prepared primer. Using the obtained DNA fragment as a template, a DNA fragment encoding an extracellular region (Met1-Leu290) including a signal region was amplified by PCR and inserted into an animal cell expression vector (human FcRn amino acid sequence / SEQ ID NO: 79). ). Similarly, oligo DNA primers were prepared based on the published human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. USA. (2002) 99 (26), 16899-16903). Using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as a template, a DNA fragment encoding the entire gene length was prepared by PCR using the prepared primers. Using the obtained DNA fragment as a template, the DNA fragment encoding the full length β2-microglobulin (Met1-Met119) including the signal region was amplified by PCR and inserted into a mammalian cell expression vector (human β2-microglobulin amino acid) Sequence / SEQ ID NO: 80).

  The soluble human FcRn was expressed by the following procedure. The prepared human FcRn and (human β2-microglobulin plasmid were introduced into human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) by the lipofection method using 10% fetal bovine serum (Invitrogen). After recovering the culture supernatant, purification was performed using IgG Sepharose 6 Fast Flow (Amersham Biosciences) according to the method of (J Immunol. 2002 Nov 1; 169 (9): 5171-80.), Followed by HiTrap Q. Purification was performed by HP (GE Healthcare).

Measurement of Antibody Plasma Concentration in Mice Mouse plasma antibody concentration was measured by ELISA using a method known to those skilled in the art.

Measurement of Antibody Plasma Concentration, CRP Concentration, and Unbound Soluble IL-6 Receptor by Monkey PK / PD Test Cynomolgus monkey plasma concentration was measured by a method known to those skilled in the art by ELISA.

  CRP concentration was measured with Sias R CRP (Kanto Chemical Co., Inc.) using an automatic analyzer (TBA-120FR, Toshiba Medical Systems Co., Ltd.).

The concentration of unbound soluble cynomolgus monkey IL-6 receptor in cynomolgus monkey plasma was measured as follows. By adding 30 μL of cynomolgus monkey plasma to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter cup (Millipore), all IgG type antibodies (cynomolgus IgG, anti-human) present in the plasma IL-6 receptor antibody and anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex) were adsorbed to protein A. Then, it spin-down with the high-speed centrifuge and collect | recovered pass solutions. Since the path solution does not contain anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor complex bound to protein A, the concentration of soluble cynomolgus IL-6 receptor in the protein A path solution was measured. By doing so, the concentration of unbound soluble IL-6 receptor can be measured. The soluble cynomolgus monkey IL-6 receptor concentration is determined by a method known to those skilled in the art for measuring the human IL-6 receptor concentration using the soluble cynomolgus monkey IL-6 receptor (cIL-6R) prepared above as a standard. It was measured. The ratio of unbound soluble IL-6 receptor was calculated by the following formula.

Claims (20)

(A) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73 ) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1) ;
(B) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83 ) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5) ;
(C) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1); and
(D) A preparation comprising at least one antibody selected from antibodies comprising a heavy chain comprising the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain comprising the sequence of SEQ ID NO: 30 (VL5). Histidine buffer and / or citrate buffer, the formulation of claim 1, wherein. The preparation according to claim 1 or 2 , comprising at least one basic amino acid. 1-500 mM histidine buffer and / or citrate buffer, 1-1500 mM at least one basic amino acid, 1-200 mg / mL antibody, and 1-400 mM carbohydrate. The preparation according to any one of 1 to 3 . 5. The preparation according to claim 3 or 4 , wherein the basic amino acid is arginine. 6. The preparation according to claim 4 or 5 , wherein the sugar is sucrose or trehalose. The preparation according to any one of claims 1 to 6 , further comprising a surfactant. 8. A formulation according to any one of claims 1 to 7 , comprising a polypeptide and / or antibody in an amount of at least 10 mg / ml. 9. A formulation according to any one of claims 1 to 8 , comprising a polypeptide and / or antibody in an amount of at least 50 mg / ml. 10. A formulation according to any one of claims 1 to 9 , comprising a polypeptide and / or antibody in an amount of at least 80 mg / ml. The preparation according to any one of claims 1 to 10 , comprising a polypeptide and / or antibody in an amount of 240 mg / ml or less. 12. A formulation according to any one of claims 1 to 11 having a pH in the range of 4.5 to 7.0. 13. A formulation according to claim 12 , having a pH in the range of 5.5 to 6.6. The formulation according to any one of claims 1 to 13 , which is a solution. 15. A formulation according to claim 14 , which has not been subjected to lyophilization during preparation. 16. A formulation according to any one of claims 1 to 15 , wherein dimerization of the polypeptide molecule and / or antibody molecule is reduced. The preparation according to any one of claims 1 to 16 , wherein dimerization of the polypeptide molecule and / or antibody molecule is inhibited. 18. A formulation according to any one of claims 1 to 17 for subcutaneous administration. A method of stabilizing a solution containing an antibody comprising the step of adding at least one basic amino acid, the antibody comprising:
(A) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73 ) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1) ;
(B) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83 ) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5) ;
(C) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1); and
(D) A method comprising at least one antibody selected from an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain comprising the sequence of SEQ ID NO: 30 (VL5). A method of stabilizing an antibody during a freeze / thaw cycle of a solution containing an antibody comprising the step of adding at least one basic amino acid, the antibody comprising:
(A) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 19 (variable region of VH4-M73 ) and a light chain variable region comprising the sequence of SEQ ID NO: 22 (variable region of VL1) ;
(B) an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 21 (variable region of VH5-M83 ) and a light chain variable region comprising the sequence of SEQ ID NO: 24 (variable region of VL5) ;
(C) an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain comprising the sequence of SEQ ID NO: 28 (VL1); and
(D) A method comprising at least one antibody selected from an antibody comprising a heavy chain comprising the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain comprising the sequence of SEQ ID NO: 30 (VL5).

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