RU2565809C2 - Pharmaceutical formulation containing advanced antibody molecules - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is a pharmaceutical formulation exhibiting neutralising activity on IL-6 receptor, having pH from 5.5 to 6.6 and containing histidine and/or citrate buffer, and at least one antibody specified in an antibody containing a heavy chain variable region including CDR1 comprising HDHAWS sequence, CDR2 comprising FISYSGITNYNPSLQG sequence, and CDR3 comprising SLARTTAMDY sequence, and a light chain variable region including CDR1 comprising QASTDISSHLN sequence, CDR2 comprising YGSHLLS sequence, and CDR3 comprising GQGNRLPYT sequence, and its versions containing the same hypervariable regions in the concentration of 10-240 mg/ml. What is presented is a method for stabilising the antibody in the process of cycles of freezing/thawing of an antibody-containing solution, wherein the above method involves adding arginine with arginine taken in the concentration of 1-1500 mM; the solution has pH from 5.0 to 6.6, wherein the above antibody is taken in the concentration of 10-240 mg/ml and is one of the antibodies specified above. What is disclosed is a method for stabilising the solution having pH from 5.5 to 6.6 and containing the antibody, wherein the above method involves adding arginine. Arginine is taken in the concentration of 1-1500 mM, wherein the above antibody is taken in the concentration of 10-240 mg/ml and is one of the antibodies specified above.

EFFECT: invention enables producing the pharmaceutical solution of anti-IL-6 receptor antibody aggregation stable in storage.

16 cl, 33 dwg, 12 tbl, 12 ex

Description

FIELD OF THE INVENTION

The present invention relates to a composition containing a polypeptide and / or antibody, in particular, to a stable liquid composition containing a modified antibody against IL-6 receptor in high concentration.

State of the art

In recent years, various antibody compositions have been developed, and these compositions have been used for practical purposes. Many of these formulations have been used for intravenous injection. In this case, it is necessary to obtain such antibody-containing compositions for subcutaneous injection, which would have a high concentration of antibodies in the liquid (from about 100 mg to 200 mg), and their volume for subcutaneous injection would be mainly limited.

In solutions containing a high concentration of antibodies, unwanted decomposition occurs, which leads to the formation of insoluble and / or soluble aggregates. Such insoluble and soluble aggregates are likely to form in the liquid state, due to the association of antibody molecules. In the case of storing the liquid composition for a long period of time, the biological activity of antibody molecules may be lost or reduced, due to the deamidation of aspartic residues. Conducting cycles of freezing and thawing also leads to the formation of split and aggregated antibody molecules.

Various ideas have been proposed for the preparation of a stabilized composition in which the loss of the active component is reduced after storage of the composition, even over a long period of time. Such formulations are prepared by dissolving the active component and various additives in a buffer solution. Therefore, there is a need to obtain a highly concentrated antibody-containing composition in which dimerization and deamidation are inhibited during long-term storage, and which is stable and can be used for subcutaneous administration.

Antibodies are of particular interest as pharmaceuticals because they have high plasma stability and do not cause significant side effects. Such antibodies include a number of commercially available pharmaceutically acceptable antibodies such as IgG and many pharmaceutically acceptable antibodies that are currently under development (non-patent documents 1 and 2). IL-6 is a cytokine that causes various autoimmune diseases, inflammatory diseases, malignant tumors, and the like. (non-patent document 3). Tocilizumab (TOCILIZUMAB), i.e., a humanized IgG1 antibody against IL-6 receptor, specifically binds to IL-6 receptor. It is believed that tocilizumab can be used as a therapeutic agent for the treatment of IL-6-associated diseases, such as rheumatoid arthritis, as this antibody neutralizes the biological activity of IL-6 (patent documents 1-3 and non-patent document 4). In Japan, tocilizumab was approved for use as a therapeutic agent for the treatment of Castleman disease and rheumatoid arthritis (Non-Patent Document 5).

Humanized antibodies, such as tocilizumab, are pharmaceutically acceptable first-generation antibodies. Pharmaceutically acceptable second-generation antibodies are currently at the research stage to improve the efficacy, ease of use and lower cost of pharmaceutically acceptable first-generation antibodies. Various technologies are currently being developed that can be used to produce pharmaceutically acceptable second generation antibodies. There are descriptions of methods for enhancing effector function, increasing antigen-binding ability, improving the pharmacokinetic properties and increasing the stability of these antibodies, as well as methods to reduce the risk of immunogenicity. It has been reported that such methods of increasing drug efficacy or reducing the dose are methods for increasing antibody-dependent cell-mediated cytotoxic activity (ADCC activity) or complement-dependent cytotoxic activity (CDC activity) carried out by amino acid substitution in the Fc region of an IgG antibody (non-patent document 6). It has also been reported that affinity maturation (Non-Patent Document 7) is a method of increasing antigen binding capacity or antigen neutralizing ability. Such a technology allows enhancing antigen binding activity by introducing amino acid mutations into the hypervariable region (complementarity determining region (CDR)) of the variable region or the like. An increase in antigen binding capacity leads to an increase in biological activity in vitro or allows a reduction in the administered dose, and also leads to an increase in in vivo efficacy (Non-Patent Document 8). Clinical trials are currently underway to characterize the Motavizumab antibody (obtained by affinity maturation), which is thought to be much more potent than Palivizumab, i.e. a pharmaceutically acceptable first-generation anti-RSV antibody (non-patent document 9). An anti-IL-6 receptor antibody with an affinity of approximately 0.05 nM (i.e., with an affinity greater than that of tocilizumab) is also described in the literature (Patent Document 4). However, in the literature there are no data on human, humanized or chimeric antibodies, the affinity of which exceeds 0.05 nM.

The problem that is currently faced by specialists in the preparation of pharmaceutically acceptable antibodies is the high cost of their production associated with the introduction of very large quantities of protein. So, for example, the dose of tocilizumab, namely, a humanized IgG1 antibody against IL-6 receptor, for intravenous injection is approximately 8 mg / kg / month (Non-Patent Document 4). For the treatment of chronic autoimmune diseases, subcutaneous administration is the preferred form of administration of the composition. In General, it is necessary that the compositions for subcutaneous administration had a high concentration of antibodies. In terms of stability or the like, the dose limit for an IgG type antibody is usually about 100 μg / ml (Non-Patent Document 10). Inexpensive and convenient pharmaceutical formulations based on second-generation antibodies, which can be administered subcutaneously for longer periods of time, can be obtained by increasing the half-life of the antibody in plasma in order to increase the duration of its therapeutic effect and thereby reduce the amount of administration protein, as well as by giving this antibody high stability.

FcRn is closely related to the pharmacokinetics of antibodies. Regarding differences in the half-life of antibodies of IgG1 and IgG2 isotypes in plasma, it is known that IgG1 and IgG2 have a significantly longer plasma half-life than IgG3 and IgG4 (Non-Patent Document 11). As reported, a method for even longer plasma half-lives of IgG3 and IgG4 antibodies, which already have a sufficiently long plasma half-life, is to replace amino acids in the constant region, leading to increased levels of binding to FcRn (non-patent documents 12 and 13). From the point of view of immunogenicity, a further increase in plasma half-life was achieved by replacing amino acids, preferably in the variable, but not in the constant region (Patent Document 5). However, at present, there is no data on increasing the half-life of antibodies against IL-6 receptor in plasma, achieved by modifying the variable region.

Another important problem faced by experts in the field of development of biopharmaceuticals is immunogenicity. In general terms, the immunogenicity of murine antibodies can be reduced by humanizing antibodies. Moreover, it is assumed that when the antibody is humanized, the risk of developing immunogenicity can be further reduced by using the germline lineage sequence as a matrix (Non-Patent Document 14). However, even Adalimumab, i.e. a fully human anti-TNF antibody, showed a high level (13% -17%) of the development of immunogenicity, and it was found that in patients who had immunogenicity, the therapeutic effect was low (non-patent documents 15 and 16). T cell epitopes may even be present in the CDRs of human antibodies, and such T cell epitopes in CDRs may cause immunogenicity. In silico and in vitro methods for predicting T cell epitopes are described in the literature (Non-Patent Documents 17 and 18). It is believed that the risk of developing immunogenicity can be reduced by removing T-cell epitopes predicted by such methods (Non-Patent Document 19).

Tocilizumab, i.e., a humanized anti-IL-6 receptor antibody, is an IgG1 antibody obtained by humanizing a murine PM1 antibody. CDR attachment is carried out using the human NEW and REI sequences as the framework matrix for the H and L chains, respectively; however, the five amino acids of the mouse sequence in the framework region have been retained as essential amino acids to maintain activity (Non-Patent Document 20). At present, there is no data on the complete humanization of the remaining mouse sequence in the skeleton region of the humanized tocilizumab antibody without reducing its activity. In addition, the tocilizumab CDR sequence is a murine sequence, and such a sequence, like adalimumab, may have T-cell epitopes in the CDR, which may cause a risk of immunogenicity. In clinical trials of tocilizumab, anti-tocilizumab antibodies were not detected at an effective dose of 8 mg / kg, but they were observed at doses of 2 mg / kg and 4 mg / kg (patent document 6). This suggests that the immunogenicity of tocilizumab may be further reduced. However, in the literature there is no evidence of a decrease in the risk of developing tocilizumab immunogenicity during amino acid substitution.

The isotype of tocilizumab is IgG1. Differences in isotypes are determined by differences in the sequences of the constant region. Since it is assumed that the sequence of the constant region has a significant effect on effector function, pharmacokinetics, physical properties of antibodies, etc., the choice of the sequence of the constant region is very important for the development of pharmaceutical compositions based on antibodies (non-patent document 11). In recent years, the safety of antibody-based pharmaceutical formulations has become important. The interaction of the Fc portion of the antibody and the Fcγ receptor (effector function) can lead to serious side effects in clinical trials of TGN1412 phase I (non-patent document 21). In order to obtain antibody-based pharmaceutical preparations in order to neutralize the biological activity of the antigen, binding to the Fcγ receptor, which plays an important role in the effector functions of such ADCCs, is not necessary. Fcγ receptor binding may even be undesirable in terms of side effects. A method of reducing the level of binding to the Fcγ receptor is used to change the isotype of an IgG antibody from IgG1 to IgG2 or IgG4 (non-patent document 22). IgG2 is more preferred than IgG4 in terms of pharmacokinetics and binding to Fcγ receptor I (Non-Patent Document 11). Tocilizumab is an antibody that neutralizes the IL-6 receptor, and its isotype is IgG1. Thus, in terms of minimizing possible side effects, IgG2 may be the preferred isotype because it does not require effector functions such as ADCC.

Nevertheless, in the development of pharmaceutical preparations based on antibodies, the physicochemical properties of proteins, in particular, their homogeneity and stability, are very important. The IgG2 isotype has been reported to have significant heterogeneity due to the presence of disulfide bonds in its hinge region (Non-Patent Document 23). The complexity and high cost of large-scale industrial production of pharmaceutical preparations based on such antibodies with the preservation of the desired substances / related substances is associated with their heterogeneity due to the difference in disulfide bonds in different products. Thus, if possible, it is advisable to use one substance. In addition, it was reported that the heterogeneity of the C-terminal sequences of the antibody H-chain is due to deletion of the C-terminal lysine amino acid residue and amidation of the C-terminal carboxyl group as a result of deletion of two C-terminal amino acids, glycine and lysine (non-patent document 24). When developing antibodies of the IgG2 isotype, which can be used as pharmaceuticals, it is necessary to strive to reduce such heterogeneity and maintain high stability. To obtain convenient stable stable formulations having a high concentration and intended for subcutaneous administration, it is preferable that such formulations not only have high stability, but also have a plasma half-life longer than the half-life of IgG1, which is an isotype of tocilizumab. However, there is currently no data on the sequences of the constant region of antibodies with a constant region of the IgG2 isotype, which have reduced heterogeneity, high stability and a half-life in plasma exceeding the half-life of antibodies with a constant region of the IgG1 isotype.

List of cited patent documents

PDD 1: WO 92/19759.

PDD 2: WO 96/11020.

PDD 3: WO 96/12503.

PDD 4: WO 2007/143168.

PDD 5: WO 2007/114319.

PDD 6: WO 2004/096273.

Non-Patent Documents:

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NAP 3: Nishimoto N, Kishimoto T., Interleukin 6: from bench to bedside., Nat Clin Pract Rheumatol. 2006 Nov; 2 (11): 619-26.

NAP 4: Maini RN, Taylor PC, Szechinski J, Pavelka K, Broll J, Balint G, Emery P, Raemen F, Petersen J, Smolen J, Thomson D, Kishimoto T; CHARISMA Study Group., Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist, Tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate., Arthritis Rheum. 2006 Sep; 54 (9): 2817-29.

NAP 5: Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda Y, Sasaki T, Nishioka K, Hara M, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S , Kodama F, Nakahara H, Hagihara K, Yoshizaki K, Kishimoto T. Humanized anti-interleukin-6 receptor antibody treatment of multicentric Castleman disease. Blood. 2005 Oct 15; 106 (8): 2627-32.

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NAP 12: Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1; 176 (1): 346-56.

NAP 13: Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES., Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul; 15 (7): 637-40.

NAP 14: Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods 2005 May; 36 (1): 35-42.

NAP 15: Bartelds GM, Wijbrandts CA, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA, Tak P, Wolbink GJ. Clinical response to adalimumab: The relationship with anti-adalimumab antibodies and serum adalimumab concentrations in rheumatoid arthritis. Ann Rheum Dis. 2007 Mar 9; [publication of the European application is in print].

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Description of the invention

An object of the present invention is to provide pharmaceutical formulations (which may also be referred to as “agents” or “pharmaceutical compositions”) containing second-generation molecules having improved properties compared to the humanized IgG1 antibody against IL-6 receptor, namely, tocilizumab, by modifying the amino acid sequences of the variable and constant regions of tocilizumab in order to increase its antigen-neutralizing ability and improve pharmacokinetic properties, with the next achievement of a prolonged therapeutic effect with a lower frequency of administration of these formulations, as well as a decrease in immunogenicity, increased safety and improved physicochemical properties (increased stability and homogeneity).

In addition, the present invention relates to compositions containing high concentrations of the polypeptide and / or antibody, where in these compositions, dimerization and deamidation are inhibited during long-term storage or during multiple cycles of freezing / thawing, and where such compositions are stable and, preferably, suitable for subcutaneous administration.

To solve the above problems, intensive studies were carried out, as a result of which it was found that a stable composition containing high concentrations of the polypeptide and / or antibody can be obtained by adding arginine or its salt as a stabilizer to the amino acid solution.

The present invention is described in detail below. The authors of the present invention conducted special studies in order to obtain a pharmaceutical composition that is stable due to the presence of second-generation molecules that have better properties compared to the humanized IgG1 antibody against IL-6 receptor, namely tocilizumab first generation. Therefore, upon receipt of the antibody in question, the authors of the present invention introduced many CDR mutations into the variable regions of tocilizumab, improving its ability to bind (affinity) to the antigen. Thus, the authors of the present invention were able to significantly increase the affinity of this antibody using a combination of such mutations. The authors of the present invention also managed to improve the pharmacokinetic properties by introducing modifications that reduce the value of the isoelectric point of the sequence of the variable region. The authors of the present invention have also been able to improve the pharmacokinetic properties by providing pH-dependent binding to the IL-6 receptor antigen, as a result of which one antibody molecule can repeatedly neutralize the antigen. In addition, the authors of the present invention were able to reduce the risk of developing immunogenicity by completely humanizing the mouse sequences present in the tocilizumab framework region and reducing the number of T-cell epitope peptides in the variable regions predicted in silico. In addition, the authors of the present invention were also able to identify new constant region sequences for the tocilizumab constant region that reduce the level of binding to the Fcγ receptor, increase safety, improve pharmacokinetic properties compared to IgG1 sequences and reduce the heterogeneity associated with the presence of disulfide bonds in the hinge region IgG2 also reduces the heterogeneity associated with the C-terminus of the H chain without compromising stability. The authors of the present invention were able to obtain second-generation molecules with improved properties compared with tocilizumab, by appropriately combining these modifications of the amino acid sequence in CDR, in variable regions and in constant regions.

The present invention relates to pharmaceutical compositions that contain a humanized IgG antibody against the IL-6 receptor, with increased ability to bind to the antigen (IL-6 receptor), improved pharmacokinetic properties, increased safety and improved physical properties (increased stability and homogeneity), as well as have a reduced risk of developing immunogenicity, where these pharmaceutical compositions are obtained by modifying the amino acid sequences of variable and constant nt areas of the humanized IgG1 antibody against IL-6 receptor, namely, tocilizumab; as well as to methods for producing said pharmaceutical compositions. More specifically, the present invention relates to:

(1) a pharmaceutical composition comprising at least one polypeptide selected from:

(a) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 1 (CDR1 VH4-M73), CDR2 containing the sequence of SEQ ID NO: 2 (CDR2 VH4-M73), and CDR3 containing the sequence of SEQ ID NO: 3 (CDR3 VH4-M73);

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

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

(d) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 10 (CDR1 VL1), CDR2 containing the sequence of SEQ ID NO: 11 (CDR2 VL1), and CDR3 containing the sequence of SEQ ID NO: 12 (CDR3 VL1);

(e) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 VL3), CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 VL3), and CDR3 containing the sequence of SEQ ID NO: 15 (CDR3 VL3); and

(f) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 16 (CDR1 VL5), CDR2 containing the sequence of SEQ ID NO: 17 (CDR2 VL5), and CDR3 containing the sequence of SEQ ID NO: 18 (CDR3 VL5);

(2) a pharmaceutical composition comprising at least one antibody selected from:

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

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

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

(d) an antibody that contains the variable region of the heavy chain containing the sequence of SEQ ID NO: 19 (variable region VH4-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 22 (variable region VL1);

(e) an antibody that contains the variable region of the heavy chain containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 23 (variable region VL3);

(f) an antibody that contains the variable region of the heavy chain containing the sequence of SEQ ID NO: 21 (variable region VH5-M83), and the variable region of the light chain containing the sequence of SEQ ID NO: 24 (variable region VL5).

(g) an antibody that contains a heavy chain containing the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain containing the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody that contains a heavy chain comprising the sequence of SEQ ID NO: 26 (VH5-M73) and a light chain comprising the sequence of SEQ ID NO: 29 (VL3);

(i) an antibody that contains a heavy chain comprising the sequence of SEQ ID NO: 27 (VH4-M83) and a light chain comprising the sequence of SEQ ID NO: 30 (VL5);

(3) a stable pharmaceutical composition (1) or (2) containing histidine and / or citrate buffer;

(4) a stable pharmaceutical composition according to (1) to (3), containing at least one cationic amino acid;

(5) a stable pharmaceutical composition according to any one of (1) to (4), comprising 1-500 mM histidine and / or citrate buffer, 1-1500 mM at least one cationic amino acid, 1-200 mg / ml of antibody, and 1-400 mm carbohydrate;

(6) the composition according to paragraphs (4) or (5), where the cationic amino acid is arginine;

(7) the composition according to paragraphs (5) or (6), where the carbohydrate is sucrose or trehalose;

(8) a composition according to any one of paragraphs (1) to (7), which also contains a surfactant;

(9) a composition according to any one of paragraphs (1) to (8), comprising a polypeptide and / or antibody in an amount of at least 10 mg / ml;

(10) a composition according to any one of paragraphs (1) to (9), comprising a polypeptide and / or antibody in an amount of at least 50 mg / ml;

(11) a composition according to any one of paragraphs (1) to (10), comprising a polypeptide and / or antibody in an amount of at least 80 mg / ml;

(12) a composition according to any one of paragraphs (1) to (11), comprising a polypeptide and / or antibody in an amount of 240 mg / ml or less;

(13) the composition according to any one of paragraphs (1) to (12), having a pH in the range from 4.5 to 7.0;

(14) the composition according to paragraph (13), having a pH in the range from 5.5 to 6.6;

(15) the composition according to any one of paragraphs (1) to (14), wherein said composition is a liquid;

(16) the composition according to paragraph (15), which, during its preparation, was not subjected to lyophilization;

(17) a formulation according to any one of (1) to (16), wherein the degree of dimerization of the polypeptide and / or antibody molecules has been reduced;

(18) a composition according to any one of (1) to (17), wherein the dimerization of the polypeptide and / or antibody molecules has been inhibited;

(19) the composition according to any one of paragraphs (1) to (18), intended for subcutaneous administration;

(20) to a method for stabilizing a solution containing an antibody, wherein said method comprises adding at least one cationic amino acid, wherein said antibody is at least one antibody selected from:

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

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

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

(d) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 19 (variable region VH4-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 22 (variable region VL1);

(e) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 23 (variable region VL3);

(f) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 21 (variable region VH5-M83), and the variable region of the light chain containing the sequence of SEQ ID NO: 24 (variable region VL5);

(g) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain containing the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain containing the sequence of SEQ ID NO: 29 (VL3); and

(i) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 27 (VH5-M83) and a light chain containing the sequence of SEQ ID NO: 30 (VL5);

(21) to a method for stabilizing an antibody during a freeze / thaw cycle of a solution containing an antibody, wherein said method comprises adding at least one cationic amino acid, and wherein said antibody is at least one antibody selected from:

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

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

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

(d) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 19 (variable region VH4-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 22 (variable region VL1);

(e) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 23 (variable region VL3);

(f) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 21 (variable region VH5-M83), and the variable region of the light chain containing the sequence of SEQ ID NO: 24 (variable region VL5);

(g) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain containing the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain containing the sequence of SEQ ID NO: 29 (VL3); and

(i) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 27 (VH5-M83), and a light chain containing the sequence of SEQ ID NO: 30 (VL5).

The above-described humanized IgG antibodies against IL-6 receptor have increased efficiency and improved pharmacokinetic properties, and therefore they have a prolonged therapeutic effect at a lower frequency of administration.

A brief description of the graphic material

Figure 1 presents a list of mutation sites that increase the affinity of tocilizumab for IL-6 receptor. The tocilizumab HCDR2 sequence is presented in SEQ ID NO: 81; the HCDR2 sequence obtained after the introduction of the mutation (top line) is presented in SEQ ID NO: 82; the HCDR2 sequence obtained after the introduction of the mutation (bottom line) is presented in SEQ ID NO: 83; the tocilizumab HCDR3 sequence is presented in SEQ ID NO: 84; the HCDR3 sequence obtained after the introduction of the mutation (top line) is presented in SEQ ID NO: 85; the sequence of HCDR3 obtained after the introduction of the mutation (bottom line) is presented in SEQ ID NO: 86; the tocilizumab LCDR1 sequence is presented in SEQ ID NO: 87; the LCDR1 sequence obtained after the introduction of the mutation (top line) is presented in SEQ ID NO: 88; the LCDR1 sequence obtained after the introduction of the mutation (bottom line) is presented in SEQ ID NO: 89; the tocilizumab LCDR3 sequence is presented in SEQ ID NO: 90; the LCDR3 sequence obtained after the introduction of the mutation (top line) is presented in SEQ ID NO: 91; and the LCDR3 sequence obtained after the introduction of the mutation (bottom line) is presented in SEQ ID NO: 92.

2 is a graph illustrating the neutralizing activity of tocilizumab and RDC-23 in BaF / gp130.

Figure 3 presents a list of mutation sites that can reduce the value of the isoelectric point of the variable region without significantly reducing the level of binding of tocilizumab to the IL-6 receptor. Asterisks at mutation sites mean a site that does not affect the magnitude of the isoelectric point, but which has been mutated to turn this sequence into a human sequence. The tocilizumab HFR1 sequence is presented in SEQ ID NO: 93; the HFR1 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 94; the tocilizumab HCDR1 sequence is presented in SEQ ID NO: 95; the HCDR1 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 96; the tocilizumab HFR2 sequence is presented in SEQ ID NO: 97; the HFR2 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 98; the tocilizumab HCDR2 sequence is set forth in SEQ ID NO: 81; the HCDR2 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 99; the tocilizumab HFR4 sequence is set forth in SEQ ID NO: 100; the HFR4 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 101; the tocilizumab LFR1 sequence is set forth in SEQ ID NO: 102; the LFR1 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 103; the tocilizumab LCDR1 sequence is presented in SEQ ID NO: 87; the sequence of LCDR1 obtained after the introduction of the mutation is presented in SEQ ID NO: 104; the tocilizumab LFR2 sequence is presented in SEQ ID NO: 105; the LFR2 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 106; the tocilizumab LCDR2 sequence is presented in SEQ ID NO: 107; the LCDR2 sequences obtained after the introduction of the mutation are presented in SEQ ID NO: 108 and 109; the tocilizumab LFR3 sequence is set forth in SEQ ID NO: 110; the LFR3 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 111; the tocilizumab LFR4 sequence is presented in SEQ ID NO: 112, and the LFR4 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 113.

In FIG. 4 is a graph illustrating the neutralizing activity of tocilizumab and H53 / L28 in BaF / gp130.

In FIG. 5 is a graph illustrating the plasma concentrations of tocilizumab and H53 / L28 in mice versus time following intravenous administration.

In FIG. 6 is a graph illustrating the plasma concentrations of tocilizumab and H53 / L28 in mice versus time following subcutaneous administration.

In FIG. 7 schematically shows that an IgG molecule can again bind to another antigen as a result of dissociation from a membrane-type antigen in the endosome.

In FIG. Figure 8 shows a list of mutation sites that can give tocilizumab a pH-dependent ability to bind to the IL-6 receptor (binding at pH 7.4 and dissociation at pH 5.8). The tocilizumab HFR1 sequence is presented in SEQ ID NO: 93; the HFR1 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 114; the tocilizumab HCDR1 sequence is presented in SEQ ID NO: 95; the HCDR1 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 115; the tocilizumab LCDR1 sequence is presented in SEQ ID NO: 87; the sequence of LCDR1 obtained after the introduction of the mutation is presented in SEQ ID NO: 116; the tocilizumab LCDR2 sequence is presented in SEQ ID NO: 107, and the LCDR2 sequence obtained after the introduction of the mutation is presented in SEQ ID NO: 117.

In FIG. 9 is a graph illustrating the neutralizing activity of tocilizumab and H3pI / L73 in BaF / gp130.

In FIG. 10 is a graph illustrating the concentration of tocilizumab and H3pI / L73 in plasma of dog-like monkeys versus time after intravenous administration.

In FIG. 11 is a graph illustrating the plasma concentrations of tocilizumab and H3pI / L73 in mice transgenic to the human IL-6 receptor versus time following intravenous administration.

In FIG. 12 is a graph illustrating the results of the analysis of the C-terminal heterogeneity of tocilizumab (TOCILIZUMAB), TOCILIZUMABΔK, and TOCILIZUMABΔGK by cation exchange chromatography.

In FIG. 13 is a graph illustrating the results of analysis of the tosylizumab-IgG1, tocilizumab-IgG2, and tocilizumab-SKSC heterogeneity associated with a disulfide bond using cation exchange chromatography.

In FIG. 14 is a graph illustrating denaturation curves for tocilizumab-IgG1, tocilizumab-IgG2, and tocilizumab-SKSC obtained by differential scanning calorimetry (DSC), and Tm for each Fab domain.

In FIG. 15 is a graph illustrating the plasma concentrations of tocilizumab-IgG1, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 in mice transgenic for human FcRn versus time following intravenous administration.

In FIG. 16 is a graph illustrating the neutralizing activity of tocilizumab, control, and Fv5-M83 in BaF / gp130.

In FIG. 17 is a graph illustrating the neutralizing activity of tocilizumab, Fv3-M73 and Fv4-M73 in BaF / gp130.

In FIG. Figure 18 is a graph illustrating the concentrations of tocilizumab, controls, Fv3-M73, Fv4-M73 and Fv5-M83 in dog-like monkeys versus time after intravenous administration.

In FIG. 19 is a graph illustrating CRP concentration for tocilizumab, control, Fv3-M73, Fv4-M73 or Fv5-M83 in dog-like monkeys versus time after intravenous administration.

In FIG. 20 is a graph illustrating the percentage of free soluble IL-6 receptor in dog-like monkeys versus time after intravenous administration of tocilizumab, control, Fv3-M73, Fv4-M73, or Fv5-M83.

In FIG. 21 is a graph illustrating differences in the formation of high molecular weight (HMW) Fv4-M73 antibodies versus time for different formulations (A-D).

In FIG. 22 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: higher than baseline) Fv4-M73 antibodies at two points in time (at 25 ° C) at different pH values, in different buffers, at different concentrations of NaCl and in the presence or in lack of arginine.

In FIG. 23 is a graph illustrating differences in the formation of high molecular weight (HMW) Fv4-M73 antibodies at one point in time (at 25 ° C.) at various pH values, in different buffers, at different concentrations of NaCl and in the presence or absence of arginine.

In FIG. 24 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: above baseline) Fv4-M73 antibodies at three points in time (at 40 ° C) at different pH values, in different buffers, at different concentrations of NaCl and in the presence or in lack of arginine.

In FIG. 25 is a graph illustrating differences in the formation of high molecular weight (HMW) Fv4-M73 antibodies at one point in time (at 40 ° C.) at various pH values, in different buffers, at different NaCl concentrations and in the presence or absence of arginine.

In FIG. 26 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: above baseline) Fv4-M73 antibodies at three points in time (at 40 ° C) at different pH values, in different buffers, at different concentrations of NaCl and in the presence or in lack of arginine.

In FIG. 27 is a diagram illustrating the results of anion exchange chromatography performed on Fv4-M73 antibody solutions that can be stored at various pH values.

In FIG. 28 is a graph showing the results of anion exchange chromatography performed on Fv4-M73 antibody solutions that can be stored at three different pH values, at different concentrations of NaCl, arginine, and two different buffers.

In FIG. 29 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: above baseline) Fv4-M73 antibodies for two different freeze / thaw cycles at different concentrations of NaCl, arginine, and two different buffers.

In FIG. 30 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: higher than baseline) Fv4-M73 antibodies at three different time points (at 40 ° C and 25 ° C) and for four different compositions (E-H).

In FIG. 31 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: higher than baseline) Fv4-M73 antibodies over two different freeze / thaw cycles and for four different formulations (E-H).

In FIG. 32 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: above baseline) Fv4-M73 antibodies for six different compositions after storage for three and six months at 5 ° C.

In FIG. 33 is a graph illustrating differences in the formation of high molecular weight (delta-HMW: higher than baseline) Fv4-M73 antibodies for six different compositions after storage for three and six months at -20 ° C.

Description of the invention

The present invention relates to a pharmaceutical composition comprising at least one polypeptide selected from:

(a) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 1 (CDR1 VH4-M73), CDR2 containing the sequence of SEQ ID NO: 2 (CDR2 VH4-M73), and CDR3 containing the sequence of SEQ ID NO: 3 (CDR3 VH4-M73);

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

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

(d) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 10 (CDR1 VL1), CDR2 containing the sequence of SEQ ID NO: 11 (CDR2 VL1), and CDR3 containing the sequence of SEQ ID NO: 12 (CDR3 VL1);

(e) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 VL3), CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 VL3), and CDR3 containing the sequence of SEQ ID NO: 15 (CDR3 VL3); and

(f) a polypeptide comprising CDR1 containing the sequence of SEQ ID NO: 16 (CDR1 VL5), CDR2 containing the sequence of SEQ ID NO: 17 (CDR2 VL5), and CDR3 containing the sequence of SEQ ID NO: 18 (CDR3 VL5).

The polypeptides and antibodies of the invention can be prepared by standard methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA).

The terms "pharmaceutical composition", "agent" and "pharmaceutical composition" used in the present invention, mean a liquid or solid composition containing polypeptides and / or antibodies as active (s) component (s), which are prepared so that they can be administered to an animal, such as a human, immediately or after breeding. If necessary, such a composition may contain pharmaceutically acceptable carriers and / or additives. For example, such a composition may contain detergents (e.g., PEG, tween, pluronic), excipients, antioxidants (e.g., ascorbic acid, methionine), colorants, flavors, preservatives, stabilizers, buffering agents, chelating agents (e.g. EDTA), suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow stimulants and flavoring agents.

A composition comprising the polypeptide and / or antibody of the invention preferably does not contain HSA (human serum albumin) and gelatin proteins used as stabilizing agents.

The composition comprising the polypeptide and / or antibody of the invention is preferably a liquid pharmaceutical composition comprising the polypeptide and / or antibody in a high concentration of at least 10 mg / ml, preferably at least 50 mg / ml, and more preferably not less than 80 mg / ml. Typically, this concentration is from 10 to 240 mg / ml and can be 10 mg / ml, or more preferably 50 mg / ml, and even more preferably from 100 to 200 mg / ml.

The liquid pharmaceutical composition according to the invention is preferably obtained without lyophilization step (s).

A buffering agent that can be used in the present invention is an agent that can adjust the pH in the desired range and is pharmaceutically acceptable. In a composition containing high concentrations of the polypeptide and / or antibody of the invention, the pH of the composition is preferably 4.5-7, and more preferably 5.5-6.6. Such buffering agents are known to those skilled in the art, and examples of these agents are inorganic salts, such as phosphoric acid salts (sodium or potassium salts) and sodium bicarbonate; organic acid salts such as citric acid salts (sodium or potassium salts), sodium acetate and sodium succinate; and acids, such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid and gluconic acid. In addition, Tris buffers, Good's buffers such as MES and MOPS, histidine (e.g., histidine hydrochloride salt) and glycine can also be used. In a composition containing high concentrations of the polypeptide and / or antibody of the invention, the preferred buffer is histidine or citrate buffer, with histidine buffer being particularly preferred. The concentration of the buffer solution is usually from 1 to 500 mm, preferably from 5 to 100 mm, and more preferably from 10 to 20 mm. In the case of using a histidine buffer, the buffer solution contains histidine in a concentration of preferably 5-25 mM, and more preferably 10-20 mM.

The composition according to the invention may also contain a surfactant. Typical examples of surfactants are nonionic surfactants, for example, sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate; glycerol fatty acid esters such as glycerol monocaprilate, glycerol monomyristate and glycerol monostearate; polyglycerol fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitan tetrastearate and polyoxyethylene sorbitan tetraoleate; polyoxyethylene glycerol fatty acid esters such as polyoxyethylene glyceryl monostearate; polyethylene glycol fatty acid esters such as polyethylene glycol distearate; polyoxyethylene alkyl esters such as polyoxyethylene lauryl ether; polyoxyethylene and polyoxypropylene alkyl ethers such as polyoxyethylene and polyoxypropylene glycol ether, polyoxyethylene and polyoxypropylene propyl ether and polyoxyethylene and polyoxypropylene cetyl ether; polyoxyethylene alkyl phenyl ethers such as polyoxyethylene nonyl phenyl ether; polyoxyethylene cured castor oils, such as polyoxyethylene castor oil and polyoxyethylene cured castor oil (polyoxyethylene hydrogenated castor oil); polyoxyethylated beeswax derivatives such as beeswax based on polyoxyethylene sorbitol; polyoxyethylated lanolin derivatives, such as polyoxyethylated lanolin; surfactants having HLB values of 6-18, such as amides of polyoxyethylene and fatty acids, for example, polyoxyethylene octadecanamide; anionic surfactants, for example, alkyl sulfate salts having a C ₁₀ -C ₁₈ alkyl group, such as sodium cetyl sulfate, sodium lauryl sulfate and sodium oleyl sulfate; polyoxyethylene alkyl ester sulfate salts in which the average molar number of attached ethylene oxide units is 2-4 and the number of carbon atoms in the alkyl group is 10-18, such as polyoxyethylene sodium lauryl sulfate; alkyl sulfosuccinate salts having a C ₈ -C ₁₈ alkyl group such as sodium lauryl sulfosuccinate; natural surfactants such as lecithin and glycerophospholipids; sphingophospholipids, such as sphingomyelin; and sucrose esters of C ₁₂ -C ₁₈ fatty acids. Such surfactants can be separately added to the composition according to the invention, or two or more of these surfactants can be added in combination with each other.

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

The amount of surfactant (s) added to the antibody of the invention is usually 0.0001-10% (w / v), preferably 0.001-5%, and more preferably 0.005-3%.

The composition according to the invention may also contain acidic amino acids, such as arginine, as well as methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, asparagine and glutamine, where these amino acids are used as a stabilizing agent.

In antibody-containing formulations or in an antibody-containing solution of the formulations of the invention, dimer formation during freeze / thaw cycles can be inhibited by the addition of carbohydrate (s) (e.g., sugar (s)). Sugars that can be used are non-reducing oligosaccharides, for example non-reducing disaccharides, such as sucrose and trehalose, or non-reducing trisaccharides, such as raffinose, and non-reducing oligosaccharides are particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

In antibody-containing formulations or in an antibody-containing solution of the formulations of the invention, the formation of multimers and cleavage products during long-term storage can be inhibited by the addition of carbohydrate (s) (e.g., sugar (s)). Sugars that can be used are a number of sugar alcohols, such as mannitol and sorbitol; and non-reducing oligosaccharides, for example, non-reducing disaccharides, such as sucrose and trehalose, or non-reducing trisaccharides, such as raffinose, and non-reducing oligosaccharides are particularly preferred. Preferred non-reducing oligosaccharides are non-reducing disaccharides, more preferably sucrose and trehalose.

Sugars should be added in an amount of 0.1-500 mg / ml, preferably 10-300 mg / ml, and more preferably 25-100 mg / ml.

In another aspect of the invention, the composition according to the invention preferably consists mainly of the following components, such as:

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

B) a cationic amino acid (e.g., arginine, histidine and / or lysine);

C) buffering agent (s) (e.g. histidine or citrate).

For the purposes of the present invention, carbohydrate (s) (e.g., sugars) and / or surfactant (s) may also be included in the composition.

Amino acids such as methionine, glycine, alanine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine can also be included as a stabilizing agent.

As used herein, the term “essentially consisting of” means that the compositions may contain a component that is not usually present in such compositions, and such components are usually optional additional components described below, such as suspending agents, solubilizing agents, isotonicizing agents, preservatives, adsorption inhibitors, diluents, carriers, pH adjusting agents, emollients, sulfur-containing reducing agents and antioxidants.

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

Examples of solubilizing agents are polyoxyethylated hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil and fatty acid ethyl esters.

Examples of isotonicity agents are sodium chloride, potassium chloride and calcium chloride.

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

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

Examples of the sulfur-containing reducing agents are compounds having a sulfhydryl group (s) (s) such as N-acetylcysteine, N-atsetilgomotsistein, tioktinovaya acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione and C ₁ -C ₇ -thioalkanes.

Examples of antioxidants are erythorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole, alpha-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl diacetate, and ammonium triethyl diacetate (EDTA), sodium pyrophosphate and sodium metaphosphate.

However, the composition (agent, pharmaceutical composition) according to the invention, which can be used to prevent or treat IL-6-associated diseases, including inflammatory diseases, has a composition that is not limited to the above components, and such a composition may also contain other appropriate standard carriers. In particular, examples of such carriers are light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium-containing carmellose, sodium-containing carmellose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, diethylaminoacetate, polyvinyl acetalone, polyvinyl geridyl perinolide 60 polyoxyethylene hydrogenated castor oil, sucrose, carboxymethyl cellulose, corn starch and inorganic acid salts. These compositions may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin and immunoglobulin; and amino acids. When preparing aqueous solutions for injection, antibodies against the IL-6 receptor are dissolved, for example, in isotonic solutions containing saline, glucose or other adjuvants. Adjuvants are, for example, D-sorbitol, D-mannose, D-mannitol and sodium chloride. In addition, appropriate solubilizing agents, for example, alcohol (ethanol, etc.), polyhydric alcohol (propylene glycol, PEG, etc.), and nonionic surfactants (Polysorbate 80 and HCO-50) can be combined.

If necessary, the polypeptides can be encapsulated in microcapsules (microcapsules obtained from hydroxycellulose, gelatin, polymethylmethacrylate, etc.), or they can be included in the colloidal drug delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules etc.) (see., e.g., «Remington's Pharmaceutical Science 16 ^th edition», Oslo Ed. (1980)). In addition, methods for preparing agents used as sustained release agents are known to those skilled in the art, and such agents can be applied to polypeptides (Langer et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer Chem. Tech. (1982) 12: 98-105; US Pat. No. 3,773,919; European Patent Application (EP) No. 58481; Sidman et al., Biopolymers (1983) 22: 547-56; EP No. 133988). In addition, the volume of liquid for subcutaneous administration can be increased by adding hyaluronidase to the agent or mixing it with the agent (see, for example, WO 2004/078140).

The pharmaceutical compositions of the invention can be administered orally and parenterally, preferably parenterally. In particular, these compositions may be administered to a patient by injection or transdermally. Such injections are, for example, systemic and local injections administered intravenously, intramuscularly or subcutaneously, and the like. The compositions can be injected locally in the area to be treated, or in peripheral areas, in particular by intramuscular injection. Dosage forms for transdermal administration are, for example, ointments, gels, creams, poultices and patches, which can be administered topically or systemically. In addition, administration methods may be appropriately selected depending on the age of the patient and the symptoms of the disease. The administered dose may, for example, be in the range from 0.0001 mg to 100 mg of the active ingredient per kg of body weight for each administration. Alternatively, when the compositions are administered to a human, the dose of the active ingredient may be, for example, in the range of 0.001 to 1000 mg per kg body weight for each patient. The dose for a single administration preferably contains, for example, an antibody according to the invention in an amount of about 0.01 to 50 mg / kg body weight. However, the dose of an antibody of the invention is not limited to these doses.

In accordance with the present invention, as shown by the results described in the following examples, a stable composition can be obtained in which the degree of dimerization and deamidation of the antibody during long-term storage or during the freezing / thawing cycle is low, where the specified composition is obtained by adding one or several cationic amino acids (arginine, histidine and / or lysine, preferably arginine), or a cationic amino acid in combination with another amino acid.

To assess the stability of the composition containing a high concentration of antibodies during storage, the authors of the present invention conducted studies of the effects of various additives by performing tests using size exclusion chromatography and anion exchange chromatography. As a result, it was found that in solutions with a high concentration of antibodies dissolved in a buffer solution containing the arginine amino acid, the number of dimers was lower than in solutions that did not contain additional arginine. The results showed that arginine is effective as a stabilizer for inhibiting antibody dimerization.

Thus, by adding arginine as a stabilizer, a stable antibody composition can be obtained in which the degree of antibody dimerization is reduced.

One embodiment of the invention is a stable antibody-containing composition, characterized in that it contains the antibody and arginine in a buffered solution.

As the arginine used in the present invention, any arginine compound per se, its derivatives and its salts can be used. Preferred are L-arginine and its salts.

If the composition according to the invention contains arginine, the concentration of arginine is preferably 1-1500 mm, more preferably 50-1000 mm, and most preferably 50-200 mm.

The polypeptides of the invention are not particularly limited, however, preferably, they are antigen binding molecules having binding activity to the human IL-6 receptor. Such antigen binding molecules are preferably, for example, antibody heavy chain variable regions (VH), antibody light chain variable regions (VL), antibody heavy chains, antibody light chains, and antibodies.

Of the above polypeptides (a) to (f), preferred examples of the antibody heavy chain variable regions are polypeptides (a) to (c), and preferred examples of the antibody light chain variable regions are polypeptides (d) to (f).

Such variable regions can be used as part of an antibody against the human IL-6 receptor. Antibodies against the human IL-6 receptor that utilize such a variable region have enhanced binding activity, excellent pharmacokinetic properties, a high degree of safety, reduced immunogenicity and / or improved physicochemical properties. In the present invention, “excellent pharmacokinetic properties” or “improved pharmacokinetic properties” means any of such properties as: a decrease in the level of clearance (CL), an increase in the area under the curve (AUC), an increase in the average residence time, and an increase in the half-life in plasma (t1 / 2) ”, where these properties are pharmacokinetic parameters calculated on the basis of the residence time of the antibody at a given concentration in plasma after its introduction into the body. In the present invention, “excellent physicochemical properties” or “improved physicochemical properties” means, but is not limited to, increased stability, reduced heterogeneity, or the like.

Frame regions of a human antibody (FR) binding to CDRs are chosen such that the CDR forms the desired antigen binding site. The FRs used for the variable regions of the invention are not particularly limited, and any FR can be used, however, human FRs are preferred. Human FRs having a natural sequence may be used. Alternatively, if necessary, substitutions, deletions, additions and / or insertions or the like can be introduced into the frame region having a natural sequence. one or more amino acids, so that the CDR can form an adequate antigen binding site. Mutant FR sequences possessing desired properties can be selected, for example, by measuring and evaluating the activity of antigen binding to an antibody having FR with amino acid substitutions (Sato, K. et al., Cancer Res. (1993) 53, 851-856) .

In addition, in the CDR sequence described above, one or more amino acids can be replaced, deleted, added and / or inserted. It is preferable that the CDR sequence, after substitution, deletion, addition and / or insertion of one or more amino acids, has activity equivalent to that of the unmodified CDR sequence, i.e., binding activity and neutralizing activity, as well as corresponding stability and immunogenicity and / or appropriate pharmacokinetic properties. The number of amino acids to be replaced, deleted, added and / or inserted is not specifically limited; however, the number of such amino acids is preferably three or less amino acids, more preferably two or less amino acids, and even more preferably one amino acid per CDR.

Methods for replacing one or more amino acid residues with other amino acids of interest are, for example, 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 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 mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, TA (1985) Rapid and efficient site-specific mutagen esis without phenotypic selection. Proc Natl Acad Sci U. S. A. 82, 488-492). This method can be used to replace the desired amino acids in an antibody with other amino acids of interest. In addition, using libraries, amino acids in the framework regions and CDRs can be replaced by other appropriate amino acids by methods, such as chain permutation in the framework region (Mol. Immunol. 2007 Apr; 44 (11): 3049-60) and CDR repair (US 2006/0122377).

The present invention also relates to a pharmaceutical composition comprising at least one antibody selected from:

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

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

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

(d) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 19 (variable region VH4-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 22 (variable region VL1);

(e) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and the variable region of the light chain containing the sequence of SEQ ID NO: 23 (variable region VL3);

(f) an antibody that includes the variable region of the heavy chain containing the sequence of SEQ ID NO: 21 (variable region VH5-M83), and the variable region of the light chain containing the sequence of SEQ ID NO: 24 (variable region VL5);

(g) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 25 (VH4-M73) and a light chain containing the sequence of SEQ ID NO: 28 (VL1);

(h) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 26 (VH3-M73) and a light chain containing the sequence of SEQ ID NO: 29 (VL3); and

(i) an antibody that includes a heavy chain containing the sequence of SEQ ID NO: 27 (VH5-M83), and a light chain containing the sequence of SEQ ID NO: 30 (VL5).

The antibodies described above can be used as antibodies against the human IL-6 receptor, which have enhanced binding activity, excellent pharmacokinetic properties, a high degree of safety, reduced immunogenicity and / or improved physicochemical properties.

The frame regions of a human antibody for attachment to the CDRs of the invention are selected such that the CDRs form the desired antigen binding site. The FRs used for the variable regions of the invention are not particularly limited, and any FR can be used, however, human FRs are preferred. In this case, human FRs having a natural sequence can be used. Alternatively, if necessary, substitutions, deletions, additions and / or insertions or the like can be introduced into the frame region having a natural sequence. one or more amino acids so that the CDRs can form an adequate antigen binding site. Mutant FR sequences possessing desired properties can be selected, for example, by measuring and evaluating the activity of antigen binding to an antibody having FR with amino acid substitutions (Sato, K. et al., Cancer Res. (1993) 53, 851-856) .

Moreover, the constant regions used for the antibodies of the invention are not particularly limited, and any constant region can be used. Preferred constant regions used for antibodies according to the invention are, for example, human constant regions (constant regions derived from IgG1, IgG2, IgG3, IgG4, Cκ, Cλ and the like). In human constant regions, one or more amino acids may be substituted, deleted, added and / or inserted. Preferred human heavy chain constant regions are, for example, constant regions containing the amino acid sequence of SEQ ID NO: 31 (constant region VH4-M73), constant regions containing the amino acid sequence of SEQ ID NO: 32 (constant region VH3-M73), constant regions containing the amino acid sequence of SEQ ID NO: 33 (constant region VH5-M83), and preferred human constant regions of the light chain are, for example, constant regions containing the amino acid after ovatelnost SEQ ID NO: 34 (VL1), constant regions comprising the amino acid sequence of SEQ ID NO: 35 (VL3), and constant regions comprising the amino acid sequence of SEQ ID NO: 36 (VL5).

In addition, in the CDR sequence described above, one or more amino acids can be replaced, deleted, added and / or inserted. It is preferable that the CDR sequence, after substitution, deletion, addition and / or insertion of one or more amino acids, has an activity equivalent to the activity that it had before replacement, deletion, addition and / or insertion of a CDR sequence, i.e., binding activity and neutralizing activity, as well as appropriate stability and immunogenicity and / or appropriate pharmacokinetic properties. The number of amino acids to be replaced, deleted, added and / or inserted is not specifically limited; however, it is preferable that such amino acids be three or less, more preferably two or less, and even more preferably one amino acid per CDR.

The corresponding variable regions of these antibodies can be used as part of a molecule that reacts with the human IL-6 receptor. These variable regions may also contain substitutions, deletions, additions and / or insertions of one or more amino acids (for example, five amino acids or less, preferably three amino acids or less). Methods for replacing one or more amino acid residues with other amino acids of interest are, for example, the methods described above.

The present invention also relates to polypeptides containing the variable regions described above.

The corresponding heavy chains and light chains of these antibodies can be used as part of a molecule that reacts with the human IL-6 receptor. Antibodies against the human IL-6 receptor, which use such heavy and light chains, have increased binding activity, excellent pharmacokinetic properties, a high degree of safety, reduced immunogenicity and / or improved physicochemical properties.

These heavy and light chains may also contain substitutions, deletions, additions and / or insertions of one or more amino acids (for example, ten amino acids or less, preferably five amino acids or less, and more preferably three amino acids or less). Methods for replacing one or more amino acid residues with other amino acids of interest are, for example, the methods described above.

In variable regions, in constant regions, or in both regions, substitutions, deletions, additions and / or insertions of one or more amino acids can be made.

The present invention also relates to polypeptides containing the heavy and light chains described above.

Antibodies according to the invention are preferably humanized antibodies.

Humanized antibodies are also called "reconstructed human antibodies." Such a humanized antibody is prepared by attaching a hypervariable region (CDR) derived from a non-human mammal to the CDR of a human antibody. Standard genetic recombination methods used to obtain such antibodies are also known to those skilled in the art (see European Patent Application No. EP 125023 and WO 96/02576).

In particular, for example, a DNA sequence designed so that a CDR of interest is linked to a framework region of interest (FR) is synthesized by PCR using several oligonucleotides that have overlapping portions with CDR and FR ends and are used as primers (see the method described in WO 98/13388). A humanized antibody is obtained by ligation of the obtained DNA with DNA encoding a constant region of a human antibody or a modified constant region of a human antibody; embedding said DNA in an expression vector; and administering this vector to the host to produce antibodies therein (see European Patent Application No. EP 239400 and International Patent Application Publication No. WO 96/02576).

Frame regions of a human antibody that binds to CDRs are selected such that the CDRs form the desired antigen binding site. If necessary, amino acid substitutions, deletions, additions and / or insertions can be introduced into the framework region of the variable region of the antibody.

As a constant region of a humanized antibody, a constant region of a human antibody or a modified constant region of a human antibody can be used in which one or more amino acid substitutions, deletions, additions and / or insertions can be made.

So, for example, C-gamma 1, C-gamma 2, C-gamma 3, C-gamma 4, C-mu, C-delta, C-alpha 1, C-alpha 2 and C-epsilon can be used for H -chains, while C-kappa and C-lambda can be used for the L-chain. The amino acid sequence of C-kappa is shown in SEQ ID NO: 38, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 37. The amino acid sequence of C-gamma 1 is shown in SEQ ID NO: 40, and the nucleotide sequence encoding this the amino acid sequence shown in SEQ ID NO: 39. The C-gamma 2 amino acid sequence is shown in SEQ ID NO: 42, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 41. Amino acid a different C-gamma 4 sequence is shown in SEQ ID NO: 44, and the nucleotide sequence encoding this amino acid sequence is shown in SEQ ID NO: 43.

In addition, the C regions of a human antibody can be modified in order to increase the stability of the antibody or the stability of the antibody during its production. Human antibodies of any isotype, such as IgG, IgM, IgA, IgE or IgD, can be used to humanize the antibodies, however, IgG is preferably used in the present invention. As IgG, IgG1, IgG2, IgG3, IgG4 or the like can be used.

After antibody production, amino acids in the variable region (e.g., CDR and FR) and in the constant region of the humanized antibody can be deleted, added, inserted and / or replaced. Antibodies according to the invention are also humanized antibodies that contain amino acid substitutions and the like.

Antibodies according to the invention are not only divalent antibodies, which are IgG, but also monovalent antibodies and multivalent antibodies, which are IgM, provided that they have IL-6 receptor binding activity and / or neutralizing activity. The multivalent antibodies of the invention are multivalent antibodies in which all antigen binding sites are identical, and multivalent antibodies in which all or some of the antigen binding sites are different from each other. Antibodies according to the invention are not only whole antibody molecules, but also mini-antibodies and their modified products, provided that they bind to the IL-6 receptor protein.

Miniantibodies are antibodies containing an antibody fragment in which there is no part of the whole antibody (for example, whole IgG or the like), and such antibodies are not particularly limited, provided that they have binding activity to the IL-6 receptor or neutralizing activity , and contain a fragment of the antibody in which there is no part of the whole antibody (for example, whole IgG or the like). The mini-antibodies of the invention are not particularly limited so long as they contain a portion of the whole antibody. However, the mini-antibodies preferably contain VH or VL, particularly preferably VH and VL. Other preferred mini-antibodies according to the invention are, for example, mini-antibodies containing CDR antibodies. Miniantibodies may contain all six CDR antibodies or some of these CDRs.

The mini-antibodies of the invention preferably have a lower molecular weight than whole antibodies. However, miniantibodies can form multimers, for example, dimers, trimers or tetramers, and therefore their molecular weight sometimes exceeds the molecular weight of whole antibodies.

In particular, antibody fragments are, for example, Fab, Fab ', F (ab') ₂ and Fv. In addition, mini-antibodies are, for example, Fab, Fab ', F (ab') ₂ , Fv, scFv (single chain Fv), diantibodies and sc (Fv) ₂ (single chain (Fv) ₂ ). In accordance with the present invention, mini-antibodies are also multimers (for example, dimers, trimers, tetramers and polymers) of these antibodies.

Antibody fragments can be obtained, for example, by treating antibodies with enzymes to produce antibody fragments. Enzymes that are known to be useful for producing antibody fragments are, for example, papain, pepsin and plasmin. Alternatively, a gene encoding such an antibody fragment can be constructed, introduced into an expression vector, and expressed in appropriate host cells (see, for example, 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).

Hydrolyzing enzymes cleave an antibody fragment at specific sites, resulting in antibody fragments with the specific structures described below. For deletion of any part of the antibody in order to obtain such enzymatically produced antibody fragments, genetic engineering methods can be used.

Antibody fragments obtained using the above hydrolyzing enzymes are the following fragments:

Papain hydrolysis: F (ab) ₂ or Fab

Pepsin hydrolysis: F (ab ') ₂ or Fab'

Plasmin hydrolysis: Facb

Mini-antibodies according to the invention are antibody fragments in which there is no region, provided that they have IL-6 receptor binding activity and / or neutralizing activity.

The term “diantibody” means a fragment of a divalent antibody constructed by linking genes (Holliger P. et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448; EP 404097; WO 93/11161 and the like. ) Diantibodies are dimers consisting of two polypeptide chains. In each of the polypeptide chains forming the dimer, VL and VH are usually linked via a linker in the same chain. In general terms, the linker in the dienter is short enough, and therefore VL and VH cannot communicate with each other. In particular, the number of amino acid residues constituting the linker is, for example, approximately five residues. Thus, the encoded VL and VH present on the same polypeptide cannot form a single chain fragment of the variable region, but will form a dimer with another single chain fragment of the variable region. As a result, such a diantibody has two antigen-binding sites.

ScFv antibodies are single chain polypeptides obtained by linking VH and VL via a linker or the like. (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883; Pluckthun “The Pharmacology Monoclonal Antibodies” Vol. 113, eds., Resenburg & Moore, Springer Verlag, New York, pp. 269-315, (1994)). The V region of the H chain and the V region of the L chain of scFv can be derived from any antibody described herein. The peptide linker for the binding of V regions is not particularly limited. So, for example, any single-stranded peptide containing from about 3 to 25 residues can be used as a linker. In particular, peptide linkers described below or the like can be used.

The V regions of two strands can be coupled, for example, by PCR described above. First, to bind V regions using PCR, DNA encoding a full-sized amino acid sequence or the desired incomplete amino acid sequence encoded by one of the DNAs presented below is used as a matrix, namely:

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

a DNA sequence encoding an L chain or V region of an antibody L chain.

DNAs encoding the V region of an H chain or L chain are amplified by PCR using a pair of primers containing sequences corresponding to the two ends of the amplified DNA. Then get DNA encoding part of the peptide linker. DNA encoding a peptide linker can also be synthesized by PCR. The nucleotide sequence that can be used to attach separately synthesized amplification products of the V region is attached to the 5'-end of the primers used. Then, PCR is performed using each DNA in the chain [DNA of the V region of the H chain] - [DNA of the peptide linker] - [DNA of the V region of the L chain] and PCR primers are assembled.

The assembled PCR primers contain a combination of a primer that hybridizes to the 5 ′ end of the molecule [H chain V region DNA] and a primer that hybridizes to the 3 ′ end of the [L chain V region DNA] molecule. In other words, the assembled PCR primers are a set of primers that can be used to amplify DNA encoding the full-length sequence synthesized by scFv. In addition, nucleotide sequences that can be used to attach to each DNA of the V region are attached to the molecule [peptide linker DNA]. Then these DNAs are attached to each other, after which all scFv is ultimately produced as an amplification product using assembled PCR primers. After the production of scFv-coding DNAs, expression vectors containing these DNAs can be obtained, and recombinant cells transformed with these expression vectors can be produced using standard methods. Then scFv can be obtained by expression of scFv-coding DNA by culturing the obtained recombinant cells.

The order of attachment of VH and VL has no specific restrictions, and therefore they can be arranged in any order. Examples of this arrangement are presented below:

[VH] -linker- [VL]

[VL] -linker- [VH].

sc (Fv) ₂ is a single chain mini-antibody obtained by attaching two VH and two VL using linkers and the like. (Hudson et al., 1999, J. Immunol. Methods 231: 177-189). sc (Fv) ₂ can be obtained, for example, by attaching scFv using a linker.

Preferably, two VH and two VL antibodies are arranged in the following order: VH, VL, VH and VL ([VH] -linker- [VL] -linker- [VH] -linker- [VL]), starting at the N-terminus of the single chain polypeptide, however, the arrangement of two VH and two VL is not limited to the above order, and they can be arranged in any order. Examples of this arrangement are presented below:

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

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

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

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

[VL] -linker- [VH] -linker- [VL] -linker- [VH].

The amino acid sequence of the VH or VL miniantibodies may comprise substitutions, deletions, additions and / or insertions. In addition, if the collected VH and VL have antigen-binding activity, some of them may be deleted, or other polypeptides may be added. In addition, the variable regions may be chimeric or humanized.

In the present invention, linkers that can be used to bind the variable regions of an antibody are any peptide linkers that can be introduced by genetic engineering and synthetic linkers, for example, linkers described in Protein Engineering (1996) 9 (3), 299 -305.

Preferred linkers according to the invention are peptide linkers. The length of the peptide linkers is not particularly limited, and one skilled in the art can independently choose the length of the linker depending on the purpose of its use. The length of the linker is usually from one to 100 amino acids, preferably from 3 to 50 amino acids, more preferably from 5 to 30 amino acids, particularly preferably from 12 to 18 amino acids (for example, 15 amino acids).

So, for example, the amino acid sequences for peptide linkers are 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 ,

where n is an integer of 1 or more.

Amino acid sequences of peptide linkers can be appropriately selected by the person skilled in the art depending on the goals. So, for example, the above number "n", which determines the length of the peptide linker, is usually 1-5, preferably 1-3, and more preferably 1 or 2.

Synthetic linkers (chemical cross-linking agents) are, for example, cross-linking agents commonly used for cross-linking peptides, for example, N- hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS3), dithiobisimpropion (succinium) (DSP), dithiobis (sulfosuccinimidyl propionate) (DTSSP), bis (succinimidyl succinate) ethylene glycol (EGS), bis (sulfosuccinimidyl succinate) ethylene glycol (sulfo-EGS), disuccinimidyl succinate (DST), disuccinimidosulfate (DST), dis bis [2- (succinimidohydroxycarbonyloxy) ethyl] sulfone (BSOCOES); and bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl] sulfone (sulfo-BSOCOES). Such cross-linking agents are commercially available.

In general terms, three linkers are required to bind the four variable regions of an antibody. Many of these linkers may be the same or different.

Antibodies according to the invention are also antibodies in the amino acid sequence of which one or more amino acid residues have been added. In addition, the antibodies of the invention are also hybrid proteins in which the above antibody is linked to another peptide or protein. A fusion protein can be obtained by ligating a polynucleotide encoding an antibody of the invention and a polynucleotide encoding another peptide or polypeptide while maintaining a reading frame, introducing such a hybrid into the expression vector and expressing it in the host. Moreover, methods known to those skilled in the art can be applied. The peptide or polypeptide hybridizing with the antibody of the invention can be a known peptide, for example, FLAG (Hopp, TP et al., BioTechnology 6, 1204-1210 (1988)), 6 × His, consisting of six His residues (histidines) , 10 × His, influenza virus (HA) hemagglutinin, human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7 tag, HSV tag, E tag, SV40 T-antigen fragment, lck tag, α fragment β-tubulin, a B-label and a protein fragment C. Polypeptides forming a hybrid with the antibodies of the invention are, for example, GST (glutathione S-transferase), HA (influenza virus hemagglutinin), constant region of immunoglobulin, β-galactosidase and MBP (protein binding to maltose). Commercially available polynucleotides encoding such peptides or polypeptides may be fused to a polynucleotide encoding an antibody of the invention. A fusion polypeptide can be obtained by expressing the fusion polynucleotide thus obtained.

In addition, the antibodies of the invention can also be conjugated antibodies bound to various molecules, such as polymers, including polyethylene glycol (PEG) and hyaluronic acid; radioactive substances; fluorescent substances; luminescent substances; enzymes and toxins. Such conjugated antibodies can be obtained by chemical modification of the produced antibodies. Antibody modification methods are well known in the art (see, for example, US Pat. Nos. 5,057,313 and 5,156,840). The term "antibodies" according to the invention also includes these conjugated antibodies.

In addition, antibodies according to the invention are antibodies with modified sugar chains.

In addition, the antibodies used in the present invention may be bispecific antibodies. The term “bispecific antibody” means an antibody having variable regions recognizing different epitopes in the same antibody molecule. The bispecific antibody of the invention may be a bispecific antibody that recognizes various epitopes on an IL-6 receptor molecule, or a bispecific antibody in which one of the antigen binding sites recognizes the IL-6 receptor and the other antigen binding site recognizes another substance. Examples of antigens that bind to another antigen-binding site of a bispecific antibody that includes an antibody of the invention recognizing IL-6 receptor are IL-6, TNF-alpha, 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 alpha, lymphotoxin beta, ligand LIGHT, LIGHT , VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21, IL-21R, GM-CSF, GM-CSFR, M-CSF, M -CSFR, IFN-alpha, VEGF, VEGFR, EGF, EGFR, CCR5, APRIL and APRILR.

Methods for producing bispecific antibodies are known in the art. Bespecifically antibodies can be obtained, for example, by binding of two types of antibodies that recognize different antigens. Such binding antibodies may be half of the molecule, each of which contains an H chain and an L chain, or a quarter of a molecule containing only one H chain. Alternatively, fusion cells producing bispecific antibodies can be obtained by fusion of hybridomas producing various monoclonal antibodies. In addition, bespecifically antibodies can be obtained by genetic engineering.

As described below, the antibodies of the invention may differ in their amino acid sequences, molecular weights, isoelectric points, presence / absence of sugar chains and in their conformation, depending on the purification method or the type of cell or host used to produce the antibodies. However, the present invention includes any antibody produced, provided that it is functionally equivalent to the antibody of the invention. So, for example, if an antibody according to the invention is expressed in prokaryotic cells, for example, in Escherichia coli , then a methionine residue is attached to the N-terminus of the amino acid sequence of the original antibody. Such antibodies are also antibodies of the invention.

Anti-IL-6 receptor antibody polypeptides and the like. according to the invention can be obtained by methods known in the art.

An anti-IL-6 receptor antibody can be obtained, for example, by known genetic recombination methods based on the sequence of the obtained anti-IL-6 receptor antibody. In particular, an anti-IL-6 receptor antibody can be obtained by constructing an antibody-encoding polynucleotide based on the sequence of an antibody recognizing an IL-6 receptor, inserting the polynucleotide into an expression vector and expressing it in an appropriate host cell (see, for example, Co , MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird , RE & Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

Thus, the present invention relates to methods for producing (i) a polypeptide according to the invention or (ii) a polypeptide encoded by a gene encoding a polypeptide according to the invention, where these methods include the step of culturing a host cell containing a vector into which a polynucleotide encoding a polypeptide according to invention.

More specifically, the present invention relates to methods for producing a polypeptide according to the invention, wherein said method comprises the steps of:

(a) culturing a host cell containing a vector into which a gene encoding a polypeptide of the invention is inserted; and

(b) obtaining a polypeptide encoded by this gene.

Examples of vectors are vectors of the type M13, vectors of the type pUC, pBR322, pBluescript and pCR-Script. Alternatively, if it is necessary to subclone and cut cDNA, then in addition to the above vectors, other vectors can be used, including, for example, pGEM-T, pDIRECT and pT7. Expression vectors are particularly suitable for the production of antibodies according to the invention. So, for example, if an expression vector is used for expression in E. coli , then such a vector must have distinctive features that allow this vector to amplify in E. coli . In addition, if the host is an E. coli strain such as JM109, DH5α, HB101 or XL1-Blue, it is important that such a vector contains a promoter that allows the vector to be expressed efficiently in E. coli , for example, the lacZ promoter (Ward et al ., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al ., Science (1988) 240, 1041-1043), T7 promoter or the like . These vectors, in addition to the above vectors, are pGEX-5X-1 (Pharmacia), QIAexpress system (Quiagen), pEGFP and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).

In addition, plasmid expression vectors may contain signal sequences for antibody secretion. For the production of E. coli in the periplasm, the signal sequence pelB can be used as the signal sequence for antibody secretion (Lei, SP et al ., J. Bacteriol. (1987) 169, 4379). The vectors can be introduced into host cells, for example, by methods using calcium chloride or by electroporation.

In addition to vectors for E. coli , vectors for producing antibodies according to the invention are, for example, mammalian expression vectors (e.g. pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. (1990) 18 (17), p5322), pEF and pCDM8), insect cell expression vectors (eg, Bac-to-BAC baculovirus expression system (Gibco-BRL) and pBacPAK8), plant expression vectors (eg pMH1 and pMH2), animal virus expression vectors (eg pHSV, pMV and pAdexLcw), expression vectors of retroviruses (eg, pZIPneo), yeast expression vectors (eg, “ex pressures in Pichia "(Invitrogen), pNV11 and SP-Q01) and expression vectors of Bacillus subtilis (e.g. pPL608 and pKTH50).

If a plasmid expression vector is used for expression in animal cells, such as CHO, COS, and NIH3T3 cells, then it must have the promoter necessary for expression in these cells, for example, the SV40 promoter (Mulligan et al ., Nature (1979) 277, 108 ), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al ., Nucleic Acids Res. (1990) 18, 5322) or the CMV promoter. An even more preferred vector is a vector having a gene for selecting transformed cells (for example, a drug resistance gene that can be identified using an appropriate agent (neomycin, G418 or the like). Vectors having such properties are, for example , pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

In addition, if it is necessary to stably express genes and to amplify the number of copies of genes in cells, then a method can be applied in which CHO cells deficient in the cascade of nucleic acid synthesis reactions are introduced together with a vector having a DHFR gene that compensates for such a deficiency (for example , pSV2-dhfr (“Molecular Cloning 2nd edition” Cold Spring Harbor Laboratory Press, (1989)), and then this vector is amplified using methotrexate (MTX). In addition, if transient gene expression is necessary, then the method can be applied, in which COS cells carrying on their the omosome, the gene expressing the SV40 T antigen is transformed with a vector carrying the SV40 origin of replication (pcD, etc.) Replication origin derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can be used. , for amplification of a number of copies of genes in host cell lines, expression vectors may contain an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthinguanine phosphoribosyl transferase gene (Ecogpt), dihydrofolate reductase gene (dhfr), etc. as a selective marker.

The obtained antibodies according to the invention can be isolated from host cells or their environment (medium or the like) and purified to obtain essentially pure and homogeneous antibodies. Antibodies can be isolated and purified by standard methods of separation and purification of antibodies, which do not have any restrictions. For example, antibodies can be isolated and purified using specifically selected and combined methods, such as column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis, isoelectric focusing, dialysis recrystallization and the like

Chromatographic methods are, for example, 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). Such chromatographic methods can be carried out using liquid chromatography, for example, HPLC and HPLC. The columns used for affinity chromatography are protein A columns and G protein columns. Examples of protein A columns are Hyper D, POROS, and Sepharose FF columns (GE Amersham Biosciences). The present invention also includes highly purified antibodies obtained by these purification methods.

The binding activity of the obtained antibodies to the IL-6 receptor can be measured by methods known in the art. Methods for measuring the antigen-binding activity of antibodies are, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and methods using fluorescent antibodies. For example, when using an enzyme-linked immunosorbent assay, antibody-containing samples, such as purified antibodies and supernatants of antibody-producing cell cultures, are added to the antigen-coated plates. Then a “second” antibody labeled with an enzyme such as alkaline phosphatase is added and the plates are incubated. After washing, an enzyme substrate such as p-nitrophenyl phosphate is added and the absorbance is read to evaluate antigen binding activity.

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositions containing the above polypeptide as an active ingredient. The pharmaceutical compositions of the invention can be used to treat IL-6-associated diseases, such as rheumatoid arthritis. Thus, the present invention also relates to agents for treating diseases, such as rheumatoid arthritis, wherein said agents contain the above antibody as an active ingredient. Preferred examples of diseases that can be treated in accordance with the present invention include, but are not limited to, 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, diseases associated with transplantation, age-related macular degeneration, nkylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetic nephropathy, graft versus host disease (GVHD), endometriosis, hepatitis (NASH), myocardial infarction, septicler sclerosclerosis , osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, non-Hodgkin’s B-cell lymphoma, pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cachexia in cancer, neuroinvasion in cancer, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, panuveitis, uveitis of the anterior lobe, intermediate uveitis, scleritis, dermatitis, dermatitis, niteritis, niteritis, skiteritis, , proliferative retinopathy of the vitreous body, dry eye syndrome and postoperative inflammation.

The expression “contains an anti-IL-6 receptor antibody as an active ingredient” means that the agent contains an anti-IL-6 receptor antibody as at least one of the active ingredients in an amount that is not specifically limited. In addition, the pharmaceutical compositions of the invention may contain other active ingredients in combination with the polypeptides described above.

The pharmaceutical compositions according to the invention can be used not only for therapeutic purposes, but also for prophylactic purposes.

Amino acids contained in the amino acid sequences of the invention can be post-translationally modified. Thus, for example, the modification of the N-terminal glutamic (Gln) residue to form pyroglutamic acid (pGlu), carried out by pyroglutamation, is well known to those skilled in the art. It goes without saying that such post-translationally modified amino acids are included in the amino acid sequences of the invention.

In addition, the sugar chains associated with the antibodies of the invention may have any structure. The sugar chain at position 297 (according to the European Numbering System (EU)) may have any structure (preferably a fucosylated sugar chain), or the sugar chain may not be linked to an antibody (for example, this can be achieved by producing antibodies in Escherichia coli or by introducing a modification in which the sugar chain does not attach at position 297, in accordance with EU).

All documents cited herein are incorporated into this description by reference.

Examples

The following is a more detailed description of the present invention with reference to examples, which should not be construed as limiting the present invention.

Example 1

Identification of mutation sites in variable regions in order to increase the affinity of tocilizumab in relation to the receptor of IL-6

A library of CDR sequences was constructed in which mutations were introduced and which were analyzed for their ability to increase the affinity of tocilizumab (wild-type H chains (WT) -IgG1 / SEQ ID NO: 53; wild-type (WT) -appa L chains / SEQ ID NO: 54) with respect to the IL-6 receptor. CDR mutation library screening revealed mutations that increase IL-6 receptor affinity. These mutations are shown in FIG. 1. The combination of these mutations yielded high affinity tocilizumab, such as RDC-23 (H chain RDC23H-IgG1 / SEQ ID NO: 55; L chain RDC-23L-kappa / SEQ ID NO: 56). A comparison was made of the affinities for the soluble IL-6 receptor and the biological activities of RDC-23 and tocilizumab determined using BaF / gp130 (see comparative examples for this method).

The result of the affinity measurement is presented in Table 1. The result of determining the biological activity using BaF / gp130 (final concentration of IL-6 was 30 ng / ml) is shown in FIG. 2. The results showed that, compared with tocilizumab, the affinity of RDC-23 was about 60 times higher, and the activity, expressed as the concentration giving 100% inhibition of BaF / gp130, was about 100 times higher.

Table 1 k _a (1 / ms) k _d (1 / s) K _D (M) Tocilizumab 4.9E + 05 2,0E-03 4.0E-09 RDC-23 6.4E + 05 4.3E-05 6,7E-11

Example 2

Identification of mutations contributing to the improvement of the pharmacokinetic properties of tocilizumab by reducing its isoelectric point

To improve the pharmacokinetic properties of tocilizumab, a study was conducted which aims to identify mutation sites that reduce the isoelectric point of the variable regions without significantly reducing the level of binding to IL-6 receptor. Screening of mutation sites in the variable regions, which were predicted based on models of the three-dimensional structure of tocilizumab, revealed mutation sites that contribute to a decrease in the isoelectric point of the variable regions without significantly reducing its binding to the IL-6 receptor. The data are presented in FIG. 3. The combination of these mutations allowed to obtain tocilizumab with a lower isoelectric point, including, for example, H53 / L28 (H chain H53-IgG1 / SEQ ID NO: 57; L chain L28-kappa / SEQ ID NO: 58). A comparison was made of the affinities for the soluble IL-6 receptor, the values of isoelectric points, pharmacokinetic properties in mice and biological activities for H53 / L28 and tocilizumab determined using BaF / gp130 (see comparative examples for this method).

The result of the affinity measurement is presented in Table 2. The result of determining the biological activity using BaF / gp130 (final concentration of IL-6 was 30 ng / ml) is shown in FIG. 4. The results showed that, compared with tocilizumab, the affinity of H53 / L28 was approximately six times higher, and the activity, expressed as the concentration giving 100% inhibition of BaF / gp130, was approximately several times higher.

table 2 k _a (1 / ms) k _d (1 / s) K _D (M) Tocilizumab 4.9E + 05 2,0E-03 4.0E-09 H53 / L28 7.6E + 05 5,2E-04 6.8E-10

The results of determining the value of the isoelectric point by the known method of electrophoresis with isoelectric focusing showed that the isoelectric points of tocilizumab and H53 / L28 were approximately 9.3 and 6.5-6.7, respectively. Thus, the isoelectric point H53 / L28 was approximately 2.7 lower than the isoelectric point of tocilizumab. In addition, the theoretical value of the isoelectric point of the VH / VL variable regions was calculated using GENETYX (GENETYX CORPORATION). The result showed that the theoretical isoelectric points of tocilizumab and H53 / L28 were 9.20 and 4.52, respectively. Thus, the H53 / L28 isoelectric point was approximately 4.7 lower than the tocilizumab isoelectric point.

To evaluate the pharmacokinetic properties of the modified H53 / L28 antibody, which has a lower isoelectric point, a comparison was made of the pharmacokinetic properties of tocilizumab and H53 / L28 in normal mice. To evaluate the time-dependent plasma dependence of tocilizumab or H53 / L28, mice (C57BL / 6J; Charles River Japan, Inc.) were given a single dose of tocilizumab or H53 / L28 in an amount of 1 mg / kg intravenously (iv) or subcutaneously (sc) . The time dependence of the concentration of tocilizumab and H53 / L28 in plasma after intravenous or subcutaneous administration is shown in FIG. 5 and 6, respectively. The pharmacokinetic parameters (clearance (CL) and half-life (T _1/2 )) obtained using WinNonlin (Pharsight) are presented in Table 3. The plasma half-life (T _1/2 ) of H53 / L28 after intravenous administration is approximately 1 , 3 times the half-life of tocilizumab, and clearance decreased by about 1.7 times. The half-life (T _1/2 ) of H53 / L28 after subcutaneous administration was approximately two times greater than the half-life of tocilizumab, and clearance decreased by approximately 2.1 times. Thus, it was found that the pharmacokinetic properties can be significantly improved by decreasing the isoelectric point of tocilizumab after the introduction of amino acid substitutions.

Table 3 i.v. s.c. CL,
ml / h / kg T _1/2 ,
day CL / F
ml / h / kg T _1/2 ,
day Tocilizumab 0.177 18.5 0.18 14.7 H53 / L28 0.102 23.5 0,086 29.7

Example 3

Identification of mutation sites contributing to the reduction of tocilizumab immunogenicity

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

T cell epitopes present in the tocilizumab variable region sequence were analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, it has been suggested that CDR2 L chains have many T cell epitopes that must bind to HLA (that is, this chain has a sequence responsible for a high risk of immunogenicity). Thus, a TEPITOPE assay was performed to evaluate amino acid substitutions that would reduce the risk of immunogenicity of the CDR2 L chain but would not reduce stability, binding activity, or neutralizing activity.

As described below, screening results have demonstrated that the risk of immunogenicity can be reduced without compromising stability, binding activity or neutralizing activity by replacing threonine at position L51 (Kabat numbering; Kabat EA et al ., (1991) Sequences Proteins Immunological Interest, NIH) ) CDR2 of the L chain (SEQ ID NO: 59) of tocilizumab on glycine and arginine at position L53 on glutamic acid (SEQ ID NO: 60):

Tocilizumab L chain CDR2 (SEQ ID NO: 59); and

Tocilizumab L-chain CDR2 with deleted T-cell epitopes (SEQ ID NO: 60).

Example 4

Reducing the risk of immunogenicity by fully humanizing the frame sequences of the tocilizumab variable region .

In the method of humanizing tocilizumab, some mouse sequences were left in the frame sequence in order to maintain binding activity (Cancer Res. 1993 Feb 15; 53 (4): 851-6). Such sequences are H27, H28, H29 and H30 in FR1 H chain and H71 in FR3 H chain (Kabat numbering; Kabat EA et al ., (1991) Sequences of Proteins of Immunological Interest, NIH) in the variable sequence areas of tocilizumab. Mouse sequences that have been preserved may increase the risk of immunogenicity. Thus, an analysis was performed to determine if a fully humanized framework sequence could further reduce the risk of tocilizumab immunogenicity.

The results showed that the entire frame sequence of tocilizumab can be fully humanized without compromising stability, binding activity or neutralizing activity by replacing the tocilizumab FR1 H chain (SEQ ID NO: 61) with humanized FR1-A H chain (SEQ ID NO: 62 ) below and replacing the FR3 H chain (SEQ ID NO: 63) with a humanized FR3 H chain (SEQ ID NO: 64) below:

Tocilizumab H chain FR1 (SEQ ID NO: 61);

humanized FR1-A H chain (SEQ ID NO: 62) (derived from the germ line of IMGT hVH_4);

Tocilizumab H3 FR3 (SEQ ID NO: 63);

humanized FR3 H chain (SEQ ID NO: 64) (prepared as described in Mol. Immunol. 2007, 44 (4): 412-422).

Example 5

Identification of mutation sites in order to improve the pharmacokinetic properties, based on the pH-dependent binding of tocilizumab to IL-6 receptor

One way to improve the pharmacokinetic properties of tocilizumab is to improve the properties of the molecule so that one tocilizumab molecule can bind several times to the IL-6 receptor and neutralize several molecules of the IL-6 receptor. It has been suggested that after binding to the membrane type IL-6 receptor, tocilizumab is absorbed by the intracellular endosomes by internalization upon binding to the membrane type IL-6 receptor, and then, upon binding to the membrane type IL-6 receptor, it is transferred to lysosomes and decomposes these lysosomes. In particular, one tocilizumab molecule usually binds to one or two membrane-type IL-6 receptor molecules (via a monovalent or divalent mechanism) and decomposes in lysosomes after internalization. Therefore, one tocilizumab molecule can bind to only one or two membrane-type IL-6 receptor molecules and neutralize only one or two of these molecules.

Thus, the authors of the present invention believe that if it is possible to obtain tocilizumab, which binds depending on the pH, where the indicated binding of tocilizumab is maintained under neutral conditions, but is significantly reduced under acidic conditions, then tocilizumab, which binds depending on the pH, will dissociate from membrane-type receptor (antigen) IL-6 in the endosomes and return to plasma due to binding to FcRn present in the endosomes, as illustrated in FIG. 7. After returning to plasma, tocilizumab, the binding of which is pH dependent, can again bind to the membrane-type IL-6 receptor. Obviously, as a result of re-binding in plasma and dissociation in endosomes, one tocilizumab molecule can bind several times to several IL-6 receptor molecules / neutralize several IL-6 receptor molecules. Thus, it is believed that tocilizumab, the binding of which depends on pH, has improved pharmacokinetic properties compared with standard tocilizumab.

In order for tocilizumab to dissociate from the IL-6 receptor under acidic conditions in the endosome, binding under acidic conditions should be significantly weaker than under neutral conditions. On the cell surface, strong binding to the IL-6 receptor is necessary to neutralize, and therefore, at pH 7.4, which is the pH of the cell surface, the antibody must bind to the IL-6 receptor with the same strength as tocilizumab, or more . It has been reported that the pH in the endosome is usually 5.5-6.0 (Nat. Rev. Mol. Cell Biol. 2004 Feb; 5 (2): 121-32). Thus, if tocilizumab, the binding of which depends on pH, was modified in order to weaken its binding to the IL-6 receptor at a pH of 5.5-6.0, then it can be assumed that it will dissociate from the IL-6 receptor under acidic conditions in endosomes. In particular, if tocilizumab, the binding of which is pH dependent, has been modified in order to enhance its binding to the receptor at pH 7.4, which is the cell surface pH, and in order to weaken its binding to the IL-6 receptor at pH 5.5 -6.0, which is an endosomal pH, then one tocilizumab molecule can bind to several molecules of the IL-6 receptor and neutralize several molecules of the IL-6 receptor, and therefore it can have improved pharmacokinetic properties.

A possible method of reporting tocilizumab's ability to pH-dependently bind to the IL-6 receptor is the introduction of histidine residues into the variable region of tocilizumab, since the pKa of the histidine residue is approximately 6.0-6.5, and its proton dissociation levels under neutral conditions (pH 7, 4) and in acidic conditions (pH 5.5-6.0) are different. Thus, a screening was performed to identify histidine administration sites in variable regions based on a three-dimensional structural model of tocilizumab. In addition, arbitrary histidine substitutions were made in the selected sequences of the tocilizumab variable region in order to construct a screening library. Screening was performed by binding to IL-6 receptor at pH 7.4 and dissociation from IL-6 receptor, or by reducing affinity at pH 5.5-5.8, where these parameters are used as indicators.

As a result, the authors of the present invention have identified mutation sites that impart tocilizumab the ability to pH-dependently bind to IL-6 receptor (the ability to bind at pH 7.4 and dissociate at pH 5.8). The results are presented in FIG. 8. In FIG. 8, the replacement of tyrosine with histidine at position H27 is a modification in the FR1 H chain, but not in the CDR. However, as described in Eur. J. Immunol. (1992) 22: 1719-1728, the sequence with histidine at position H27 is the human sequence (SEQ ID NO: 65). Thus, this antibody can be fully humanized in the following frame sequence in combination with the humanization described in Example 4.

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

A combination of mutations, including, for example, H3pI / L73 (H3pI-IgG1 H chain / SEQ ID NO: 66; L73 Kappa L chain / SEQ ID NO: 67), can give Tocilizumab pH-dependent binding. H3pI / L73 and tocilizumab were compared by their affinity for the soluble IL-6 receptor at pH 7.4, the dissociation rate from the membrane-type IL-6 receptor at pH 7.4 and pH 5.8, and the biological activity evaluated using BaF / gp130 , and pharmacokinetic properties in dog-like monkeys and mice transgenic for the human IL-6 receptor (see comparative examples for this method).

The results of the affinity binding assay for soluble IL-6 receptor at pH 7.4 are shown in Table 4. The results of the biological activity assays using BaF / gp130 (final IL-6 concentration = 30 ng / ml) are shown in FIG. 9. These results showed that the affinity of binding of H3pI / L73 to the soluble IL-6 receptor at pH 7.4 and activity measured using BaF / gp130 were comparable with the indicated parameters for tocilizumab.

Table 4 k _a (1 / ms) k _d (1 / s) K _D (M) Tocilizumab 5.1E + 05 1,0E-03 2,1E-09 H3pI / L73 5.4E + 05 7.4E-04 1,4E-09

The results of measuring the dissociation rate of tocilizumab or H3pI / L73 from the membrane-type IL-6 receptor at pH 7.4 and pH 5.8 are presented in table 5. Compared to tocilizumab, the dissociation rate of H3pI / L73 at pH 5.8 was higher, and The dissociation rate from the membrane-type receptor IL-6 depending on the pH was approximately 2.6 times higher.

Table 5 pH 7.4
k _a (1 / s) pH 5.8
k _d (1 / s) K _{d (pH5.8)} / K _{d (pH7.4)}
pH dependence Tocilizumab 2,5E-04 2,5E-04 1.00 H3p1 / L73 2.6E-04 6,7E-04 2.59

To evaluate the time dependence of the concentration of tocilizumab or H3pI / L73 in plasma, dog-like monkeys were given a single dose of tocilizumab or H3pI / L73 in an amount of 1 mg / kg. The plasma concentration of tocilizumab or H3pI / L73 versus time after intravenous administration is shown in FIG. 10. The results showed that in dog-like monkeys, the pharmacokinetic properties of H3pI / L73 were significantly better than the pharmacokinetic properties of tocilizumab.

To evaluate the plasma concentration of tocilizumab or H3pI / L73 versus time for mice transgenic to the human IL-6 receptor (hIL-6R transgenic mice; Proc Natl Acad Sci USA. 1995 May 23; 92 (11): 4862-6) , a single dose of tocilizumab or H3pI / L73 of 25 mg / kg was intravenously administered. The plasma concentration of tocilizumab or H3pI / L73 versus time after intravenous administration is shown in FIG. 11. The results showed that in mice transgenic to the human IL-6 receptor, the pharmacokinetic properties of H3pI / L73 were significantly better than the pharmacokinetic properties of tocilizumab.

H3pI / L73, which is tocilizumab, capable of pH-dependent binding, showed significantly better pharmacokinetic properties in dog-like monkeys and in mice transgenic to the human IL-6 receptor compared to tocilizumab. This suggests that one molecule can bind to several molecules of the IL-6 receptor and neutralize several molecules of the IL-6 receptor, giving it the ability to bind to antigen at pH 7.4 and to dissociate from antigen at pH 5.8. It is also believed that the pharmacokinetic properties can be further improved by reporting an even more pronounced pH-dependent binding of IL-6 receptor than is observed with H3pI / L73.

Example 6

Optimization of the constant region of tocilizumab

Decreased heterogeneity of the C-terminus of the tocilizumab H chain

In addition, it was reported that the heterogeneity of the C-terminal sequences of the antibody H chain is due to deletion of the C-terminal lysine amino acid residue and amidation of the C-terminal carboxyl group as a result of deletion of two C-terminal amino acids, glycine and lysine (Anal. Biochem. 2007 Jan. 1; 360 (1): 75-83). In addition, in tocilizumab, the main component is the sequence in which the C-terminal amino acid lysine was deleted in the nucleotide sequence as a result of post-translational modification; however, subcomponents in which lysine is retained and subcomponents in which the C-terminal carboxyl group is amidated as a result of deletion of glycine and lysine are also signs of heterogeneity. The complexity and high cost of large-scale industrial production of pharmaceutical preparations based on such antibodies with the preservation of the desired substances / related substances is associated with the heterogeneity of various products. If possible, it is desirable to use a single substance, and when developing antibodies used as pharmaceuticals, such a substance should have reduced heterogeneity. Thus, in the development of antibodies used as pharmaceuticals, it is preferable that the C-terminal heterogeneity of the H chain is absent.

To reduce the heterogeneity of the C-terminal amino acids, such C-terminal amino acids have been modified. The data obtained showed that C-terminal heterogeneity can be prevented by a preliminary deletion made in the nucleotide sequence to remove lysine and glycine residues at the C-terminus of the constant region of the tocilizumab H chain. Tocilizumab lacking a C-terminal lysine residue (TOCILIZUMABΔK: H chain WT-IgG1ΔK / SEQ ID NO: 68; L chain WT-kappa / SEQ ID NO: 54), and tocilizumab lacking a C-terminal lysine and glycine residues (TOCILIZUMABΔGK: H chain WT-IgG1ΔGK / SEQ ID NO: 69; L chain WT-kappa / SEQ ID NO: 54) were evaluated for heterogeneity by cation exchange chromatography. A ProPac WCX-10 column, 4 × 250 mm (Dionex) was used, where mobile phase A was 25 mmol / L MES / NaOH (pH 6.1) and mobile phase B was 25 mmol / L MES / NaOH, 250 mmol / L NaCl (pH 6.1). Chromatography was performed at an appropriate flow rate and in an appropriate gradient. The results of the analysis carried out by cation exchange chromatography are shown in FIG. 12. The results showed that the heterogeneity of the C-terminal amino acids can be reduced by a preliminary deletion made in the nucleotide sequence to remove lysine and glycine residues at the C-terminus of the constant region of the H chain, but not by preliminary deletion of only the lysine residue at C- end of the constant region of the H chain. All C-terminal sequences of the constant region of human IgG1, IgG2 and IgG4 antibodies contain lysine and glycine at positions 447 and 446, respectively, according to the European numbering system (see Sequences of proteins of immunological interest, NIH Publication No.91-3242). Therefore, it is suggested that the method for reducing the heterogeneity of C-terminal amino acids found in this study can also be applied to the constant regions of IgG2 and IgG4 and their variants.

Decreased heterogeneity due to disulfide bonds in tocilizumab of the IgG2 isotype

The isotype of tocilizumab is IgG1. Since tocilizumab is a neutralizing antibody, binding to the Fcγ receptor may not be desirable in terms of producing immunogenicity and side effects. A possible way to reduce Fc-gamma receptor binding is to convert the IgG antibody isotype from IgG1 to IgG2 or IgG4 (Ann Hematol. 1998 Jun; 76 (6): 231-48). In terms of the level of binding to the Fcγ receptor I and pharmacokinetics, it is believed that IgG2 is more preferred than IgG4 (Nat. Biotechnol. 2007 Dec; 25 (12): 1369-72). In addition, the physicochemical properties of proteins, and in particular, homogeneity and stability, are very important in the development of antibodies as pharmaceuticals. An IgG2 isotype antibody has been reported to be highly heterogeneous due to disulfide bonds in the hinge region (J. Biol. Chem. 2008 Jun. 6; 283 (23): 16206-15). The complexity and high cost of large-scale industrial production of pharmaceutical preparations based on such antibodies while maintaining the desired substances / related substances is associated with heterogeneity due to the difference in disulfide bonds in different products. Thus, whenever possible, it is desirable to use a single substance. So, for example, when developing IgG2 isotype antibodies as pharmaceuticals, it is preferable that the heterogeneity due to disulfide bonds be reduced, but so that this does not lead to a decrease in stability.

To reduce the heterogeneity of antibodies of the IgG2 isotype, various options were evaluated. As a result, it was found that heterogeneity can be reduced without compromising stability using the WT-SKSC constant region (SEQ ID NO: 70), where in the IgG2 constant region sequences the cysteine residue is at position 131 and the arginine residue is at position 133 (in accordance with European by numbering) in the CH1 domain, the H chains were replaced by serine and lysine, respectively, and the cysteine residue at position 219 (according to European numbering) in the upper hinge region of the H chain was replaced by serine. Tocilizumab-IgG1 (H chain WT-IgG1 / SEQ ID NO: 53; L chain WT-kappa / SEQ ID NO: 54), tocilizumab IgG2 (H chain WT-IgG2 / SEQ ID NO: 71; L- WT-kappa chain / SEQ ID NO: 54) and tocilizumab-SKSC (H-chain WT-SKSC / SEQ ID NO: 70; WT-kappa L chain / SEQ ID NO: 54) were obtained and analyzed for heterogeneity and stability . Heterogeneity was evaluated by cation exchange chromatography. A ProPac WCX-10 column (Dionex) was used, where the mobile phase A was 20 mM sodium acetate (pH 5.0) and the mobile phase B was 20 mM sodium acetate, 1M NaCl (pH 5.0). Chromatography was performed at an appropriate flow rate and in an appropriate gradient. The results of the analysis carried out by cation exchange chromatography are shown in FIG. 13. Stability was evaluated based on the intermediate temperature during thermal denaturation (Tm values) determined using differential scanning calorimetry (DSC) (VP-DSC; Microcal). DSC measurements in 20 mM sodium acetate, 150 mM NaCl, pH 6.0, and Tm values of the Fab domain are shown in FIG. fourteen.

The results showed that the heterogeneity of tocilizumab-IgG2 markedly increased compared to tocilizumab-IgG1, however, the heterogeneity can be significantly reduced by conversion to tocilizumab-SKSC. In addition, when compared with tocilizumab-IgG1, DSC of tocilizumab-IgG2 gave a peak component of the shoulder (Fab *) with low stability, i.e. with low Tm, at the peaks for thermal denaturation of the Fab domain, which is presumably due to the presence of a heterogeneous component. However, upon conversion to tocilizumab-SKSC, the shoulder of the peak (with a low Tm), which is believed to be due to the presence of a heterogeneous component, disappeared, and the Tm value was approximately 94 ° C and was equivalent to the Tm value of the Fab domain of tocilizumab-IgG1 and tocilizumab -IgG2. Thus, it was found that tocilizumab-SKSC has high stability.

Identification of mutation sites that improve pharmacokinetic properties in the constant region of tocilizumab

As described above, based on IgG1, which is the isotype of tocilizumab, a decrease in C-terminal heterogeneity and a decrease in the heterogeneity of antibodies with constant regions of the IgG2 isotype can be achieved, as well as a decrease in the level of binding to the Fcγ receptor and maintaining high stability. In addition, it is preferable that the constant region has improved pharmacokinetic properties compared with IgG1, which is an isotype of tocilizumab.

To identify constant regions that have longer plasma half-lives than the constant regions of IgG1 isotype antibodies, screening was performed to identify mutation sites in order to improve the pharmacokinetic properties of tocilizumab-SKSC, which has high stability and lower heterogeneity compared to antibodies containing constant regions of the IgG2 isotype, as mentioned above. The result was an antibody WT-M58 (SEQ ID NO: 72 (amino acid sequence)), in which, compared to WT-SKSC, the glutamic acid at position 137, according to European numbering, was replaced by glycine, the serine at position 138 was replaced glycine, histidine at position 268 was replaced by glutamine, arginine at position 355 was replaced by glutamine, glutamine at position 419 was replaced by glutamic acid, and glycine at position 446 and lysine at position 447 were deleted to reduce the heterogeneity of the C-terminus of the H chain. In addition, WT-M44 (SEQ ID NO: 73 (amino acid sequence)) was obtained in which, compared to IgG1, asparagine at position 434 was replaced with alanine. In addition, WT-M83 (SEQ ID NO: 74 (amino acid sequence)) was obtained by deletion of glycine at position 446 and lysine at position 447 of the M44 antibody in order to reduce the heterogeneity of the C-terminus of the H chain. In addition, the WT-M73 antibody (SEQ ID NO: 75 (amino acid sequence)) was produced by replacing asparagine at position 434 with alanine in WT-M58.

Tocilizumab-M44 (H chain WT-M44 / SEQ ID NO: 73; L chain WT-kappa / SEQ ID NO: 54), tocilizumab-M58 (H chain WT-M58 / SEQ ID NO: 72; L- WT-kappa chain / SEQ ID NO: 54) and tocilizumab-M73 (H-chain WT-M73 / SEQ ID NO: 75; WT-kappa L chain / SEQ ID NO: 54) were obtained and evaluated for human binding affinity FcRn and pharmacokinetic properties in mice transgenic for human FcRn (see comparative examples for this method).

The binding of tocilizumab-IgG1, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 to human FcRn was evaluated using Biacore. As shown in table 6, the binding level of tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 was approximately 2.7 times, 1.4 times and 3.8 times higher than the binding level of tocilizumab-IgG1, respectively.

Table 6 KD (μm) tocilizumab-IgG1 1,62 tocilizumab-M44 0.59 tocilizumab-M58 1.17 tocilizumab-M73 0.42

Tocilizumab-IgG1, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73 were evaluated for pharmacokinetic properties in mice transgenic for human FcRn. The results are presented in FIG. 15. It has been found that all antibodies, namely, tocilizumab-M44, tocilizumab-M58 and tocilizumab-M73, have improved pharmacokinetic properties compared to tocilizumab-IgG1, as shown in FIG. 15. The effect of improving pharmacokinetic properties correlates with the ability to bind to human FcRn. In particular, the plasma concentration of tocilizumab-M73 after 28 days was approximately 16 times higher than the concentration of tocilizumab-IgG1. Thus, it has also been suggested that antibodies having the M73 constant region have significantly better pharmacokinetic properties in humans compared to antibodies having the IgG1 constant region.

Example 7

Obtaining fully humanized antibodies against IL-6 receptor with improved PK / PD properties

Tocilizumab variants were obtained by combining multiple mutations in the variable and constant regions of tocilizumab identified as described above in the examples. The fully humanized anti-IL-6 receptor antibodies identified by various screening methods are: Fv3-M73 (VH4-M73 H chain / SEQ ID NO: 25; VL1 Kappa L chain / SEQ ID NO: 28), Fv4- M73 (H chain VH3-M73 / SEQ ID NO: 26; L chain VL3-Kappa / SEQ ID NO: 29) and Fv5-M83 (H chain VH5-M83 / SEQ ID NO: 27; L chain VL5 Kappa / SEQ ID NO: 30).

The affinities of the obtained Fv3-M73, Fv4-M73 and Fv5-M83 against the IL-6 receptor were compared with the affinity of tocilizumab (see comparative example for this method). The affinities of these antibodies to the soluble IL-6 receptor, determined at pH 7.4, are presented in Table 7. In addition, their BaF / gp130-neutralizing activities were compared with the corresponding activities of tocilizumab and control (a known high-affinity anti-IL-6 receptor antibody described in a comparative example, and VQ8F11-21 hIgG1 described in US application 2007/0280945) (see comparative example for this method). The results obtained in determining the biological activity of these antibodies using BaF / gp130 are shown in FIG. 16 (tocilizumab, control antibody, and Fv5-M83 with a final IL-6 concentration of 300 ng / ml) and FIG. 17 (tocilizumab, Fv3-M73 and Fv4-M73 with a final IL-6 concentration of 30 ng / ml). As shown in Table 7, the affinity of Fv3-M73 and Fv4-M73 was approximately 2–3 times higher than the affinity of tocilizumab, and the affinity of Fv5-M83 was approximately 100 times higher than the affinity of tocilizumab (due to the difficulty in measuring the affinity of Fv5-M83, such affinity was determined using Fv5-IgG1 (H chain VH5-IgG1 / SEQ ID NO: 76; L chain VL5-kappa / SEQ ID NO: 30), which had a constant region like IgG1; moreover, it is generally believed that the constant region does not have any effect on affinity). As shown in FIG. 17, Fv3-M73 and Fv4-M73 had slightly higher activity than tocilizumab. As shown in FIG. 16, Fv5-M83 had a very high activity, which was more than 100 times higher than the activity of tocilizumab in terms of 50% inhibitory concentration. Fv5-M83 also had a neutralizing activity that was about 10 times higher than the neutralizing activity of the control antibody in terms of 50% inhibitory concentration (known high affinity anti-IL-6 receptor antibody).

Table 7 k _a (1 / ms) k _d (1 / s) K _D (M) Tocilizumab 4.0E + 05 1,1E-03 2.7E-09 Fv3-m73 8.5E + 05 8,7E-04 1,0E-09 Fv4-m73 7.5E + 05 1,0E-03 1,4E-09 Fv5-m83 1,1E + 06 2.8E-05 2,5E-11

The dissociation rate of tocilizumab, Fv3-M73 and Fv4-M73 from the membrane-type IL-6 receptor was determined at pH 7.4 and 5.8. As the results shown in table 8 showed (see a comparative example for this method), the pH-dependent dissociation rate of Fv3-M73 and Fv4-M73 from the membrane-type IL-6 receptor was approximately 11 times and 10 times higher, respectively, compared to tocilizumab. A significant increase in the pH-dependent dissociation rate with respect to H3pI / L73 described in Example 5 suggests that the pharmacokinetic properties of Fv3-M73 and Fv4-M73 were significantly better than the pharmacokinetic properties of H3pI / L73.

Table 8 pH 7.4
k _a (1 / s) pH 5.8
k _d (1 / s) K _{d (pH5.8)} / K _{d (pH7.4)}
pH dependence Tocilizumab 2,5E-04 2,5E-04 1.00 Fv3-m73 4.9E-04 5,3E-03 10.88 Fv4-m73 5,1E-04 5,1E-03 10.06

The isoelectric points of tocilizumab control, Fv3-M73, Fv4-M73 and Fv5-M83 were determined using isoelectric focusing electrophoresis using a method known to those skilled in the art. The results showed that the isoelectric point was approximately 9.3 for tocilizumab; approximately 8.4-8.5 for control; about 5.7-5.8 for Fv3-M73; about 5.6-5.7 for Fv4-M73; and 5.4-5.5 for the Fv5-M83. Thus, each of these antibodies had a significantly lower isoelectric point compared to the isoelectric point for tocilizumab and control. In addition, the theoretical value of the isoelectric point of the VH / VL variable regions was calculated using GENETYX (GENETYX CORPORATION). The results showed that the theoretical value of the isoelectric point was 9.20 for tocilizumab; 7.79 for control; 5.49 for Fv3-M73; 5.01 for Fv4-M73; and 4.27 for the Fv5-M83. Thus, each antibody had a significantly lower value of the isoelectric point compared with the values for tocilizumab and control. Since, as shown in Example 2, the pharmacokinetic properties were improved by decreasing the isoelectric point, it is obvious that the pharmacokinetic properties of Fv3-M73, Fv4-M73 and Fv5-M83 were better than the pharmacokinetic properties of tocilizumab and control.

T cell epitopes in the tocilizumab variable region sequence, Fv3-M73, Fv4-M73 or Fv5-M83 were analyzed using TEPITOPE (Methods. 2004 Dec; 34 (4): 468-75). As a result, it was suggested that tocilizumab has T-cell epitopes, many of which can bind to HLA, as described in Example 3. In contrast, the number of sequences in Fv3-M73, Fv4-M73 and Fv5-M83, which, as was predicted to bind to T-cell epitopes, significantly reduced. In addition, the Fv3-M73, Fv4-M73, or Fv5-M83 framework region does not have a mouse sequence and is fully humanized. It has been suggested that the risk of immunogenicity in Fv3-M73, Fv4-M73 and Fv5-M83 can be significantly reduced compared to tocilizumab.

Example 8

Analysis of pharmacokinetic / pharmacodynamic properties (FC / PD) of fully humanized antibodies against IL-6 receptor in monkeys

Each antibody, such as tocilizumab, control antibody, Fv3-M73, Fv4-M73, and Fv5-M83, was administered intravenously to dog-like monkeys at a single dose of 1 mg / kg to evaluate the plasma concentration of these antibodies over time (see comparative example for this method). The plasma concentrations of tocilizumab, Fv3-M73, Fv4-M73 and Fv5-M83 depending on the time after intravenous administration are shown in FIG. 18. The results showed that each of the antibodies, namely, Fv3-M73, Fv4-M73 and Fv5-M83, had significantly improved pharmacokinetic properties in dog-like monkeys compared with the pharmacokinetic properties of tocilizumab and a control antibody. Among these antibodies, Fv3-M73 and Fv4-M73 had much better pharmacokinetic properties compared with tocilizumab.

The efficacy of each antibody in neutralizing membrane-type IL-6 receptor in dog-like monkeys was evaluated. Dog-like monkey IL-6 was injected subcutaneously into the lower back of the animal at a concentration of 5 μg / kg every day from day 6 to day 18 after administration of the antibody (day 3 - day 10 for tocilizumab), and after 24 hours the CRP concentration in each animal was determined ( see comparative example for this method). CRP concentrations versus time after administration of each antibody are shown in FIG. 19. To evaluate the effectiveness of each antibody in neutralizing soluble IL-6 receptor in dog-like monkeys, the concentration of free soluble IL-6 receptor in the plasma of dog-like monkeys was determined and the percentage of free soluble IL-6 receptor was calculated (see comparative example for this method). The percent free IL-6 soluble receptor versus time after administration of each antibody is shown in FIG. twenty.

Each of Fv3-M73, Fv4-M73 and Fv5-M83 had a longer neutralizing effect on membrane-type IL-6 receptor in dog-like monkeys and inhibited an increase in CRP level over a longer period of time compared to tocilizumab and control (known high affinity anti-IL-6 receptor antibody). In addition, each of Fv3-M73, Fv4-M73 and Fv5-M83 had a longer neutralizing effect on the soluble IL-6 receptor in dog-like monkeys and inhibited the increase in the level of free soluble IL-6 receptor in dog-like monkeys for a longer period time compared with tocilizumab and control. The data obtained showed that all antibodies, namely, Fv3-M73, Fv4-M73 and Fv5-M83, had a longer neutralizing effect on membrane-type IL-6 receptor and soluble IL-6 receptor compared with tocilizumab and control. Of these antibodies, Fv3-M73 and Fv4-M73 had a particularly long neutralizing effect. In addition, Fv5-M83 inhibited CRP and the free soluble receptor of IL-6 dog-like monkeys to a higher degree than Fv3-M73 and Fv4-M73. Thus, it can be considered that Fv5-M83 has a stronger neutralizing effect on membrane-type IL-6 receptor and soluble IL-6 receptor than Fv3-M73, Fv4-M73 antibodies and control antibody (a known high-affinity anti-IL receptor antibody -6). The in vivo results obtained for dog-like monkeys showed that Fv5-M83 has a higher affinity for the IL-6 receptor and stronger biological activity in the BaF / gp130 assay system compared to the control.

The results suggest that Fv3-M73 and Fv4-M73, compared with tocilizumab and a control antibody, have significantly longer activity as antibodies that neutralize the IL-6 receptor, and therefore they can be administered at a much lower dose and with a lower frequency. In addition, it was demonstrated that Fv5-M83, used as an antibody that neutralizes the receptor of IL-6, has much greater activity and retains this activity for a longer period of time. Thus, it is contemplated that antibodies Fv3-M73, Fv4-M73, and Fv5-M83 can be used as pharmaceutical antagonists of IL-6.

Example 9

Selection of stabilizing buffers for the Fv4-M73

Fv4-M73 was prepared as described above. Since Fv4-M73 consists of an unnatural constant region of M73, in order to reduce heterogeneity and increase stability and safety and improve pharmacokinetic properties, as described in examples 6-7, a stability profile (such as that achieved using buffers having the highest stabilizing effect) Fv4-M73, which would differ from the stability profile of the antibody, consisting of a natural constant region, such as the constant region of IgG1. An antibody consisting of the natural constant region of IgG1 has been reported to be essentially the most stable in the presence of histidine acetate buffer (WO / 2006/044908). Therefore, the effect of buffers on the stability of the Fv4-M73 was tested.

Composition Fv4-M73 was obtained with a final concentration of 37 mg / ml in four different buffer solutions (as described in table 9) having a pH of 6.0. Samples were analyzed by size exclusion chromatography and anion exchange chromatography with UV detection for two months storage at 40 ° C. Over the entire period, the percentage of aggregates formed in these compositions was recorded, and the values obtained are presented in FIG. 21. An increase in the percentage of aggregates is an indicator of a decrease in antibody stability. Therefore, an increase in the percentage of aggregates was used as an indicator to compare the stabilization effect achieved using various buffers.

As shown in FIG. 21, the formulations containing the histidine-HCl buffer (composition A) and the citrate buffer (composition D) were the most stable, and the composition containing the acetate buffer (composition C) was the most unstable. The histidine acetate buffer (composition B) was slightly more unstable than the histidine HCl buffer, which was probably due to the destabilizing effect of the acetate buffer.

Thus, it was found that the Fv4-M73 composition containing the unnatural constant region is most stable in the histidine-HCl buffer and in the citrate buffer, but not in the histidine-acetate buffer, which was reported to be the buffer with the highest stabilizing effect on the natural IgG1 antibody.

Table 9
The compositions used in example 9 mAb Conc. mg / ml Structure Buffer NaCl pH Fv4-m73 37 BUT 20 mM histidine-HCl 150 mm 6.0 AT 20 mM histidine acetate FROM 20 mM acetate D 20 mm citrate

Methods

Size exclusion chromatography (SEC): Size exclusion chromatography was performed to screen for antibody-containing formulations for the presence of aggregates and antibody fragments. Samples were injected into a G3000 SWXL exclusion column (TOSOH). The mobile phase is 50 mM sodium phosphate, 300 mM sodium chloride (pH 7.0), and isocratic elution was carried out at a flow rate of 0.5 ml / min. The eluted protein was detected by UV absorption at 220 nm. The relative amount of any protein was determined as the percentage of the peak area of the product relative to the area of all other detected peaks. The peak eluting earlier than the peak of the antibody monomer was recorded as the percentile of the aggregates, and the peaks eluting later than the peak of the antibody monomer, but earlier than the peak of the buffer, was recorded as the percentile of the fragments.

Sample preparation: Samples were diluted with the mobile phase to a concentration of 0.3-2 mg / ml, and 15 microliters of the sample were injected into the column.

Anion-exchange chromatography: Anion-exchange chromatography was performed to analyze the composition of the antibody for heterogeneity, and in particular for deamidation (compounds with deamidation were eluted as acid molecules after the main peak). Samples were injected into a DEAE-NPR (TOSOH) column 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), and elution was performed in a gradient of 100% mobile phase A, then 70% mobile phase A and 30% mobile phase B for 30 minutes, and then 100% mobile phase B for 1 minute, after which the sample was held for 4 minutes to rinse the column at a flow rate of 1.0 ml / min. The eluted protein was detected by UV absorption at 280 nm.

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

Example 10

The effect of pH, NaCl and arginine-HCl on the stability of Fv4-M73 at 100 mg / ml

Fv4-M73 was prepared at a final concentration of 100 mg / ml in various buffer solutions (described in Table 10), and these samples were analyzed by size exclusion chromatography and anion exchange chromatography with UV detection for a two-month period when stored at 25 ° C and 40 ° C, and also subjected to a freeze-thaw cycle (freeze for 0.5 days at -20 ° C and thaw for 30 minutes at room temperature). The percentage of aggregates present in the formulations and the percentage increase in the number of aggregates compared to the initial number of aggregates after storage for two / four / eight weeks at 25 ° C and 40 ° C are shown in FIG. 22-25. An increase in the number of units is an indicator of a decrease in stability.

Table 10
The compositions used in example 10 Buffer 20 mM histidine-HCl 20 mm citrate NaCl 50 mm 150 mm 50 mm 50 mm 150 mm 50 mm Arginine-NS - - 100 mm - - 100 mm Desired pH 4,5 4.3 4.3 4.0 4.3 4.3 4,5 5,0 4.9 4.9 5,0 4.8 4.9 4.8 5.5 5,4 5,4 5,4 5,4 5,4 5,4 6.0 6.0 5.8 5.8 6.0 5.9 5.7 6.5 6.4 6.3 6.3 6.2 6.2 6.2 7.0 6.6 6.7 6.6 6.5 - 6.8

Actual value (pH)

It has been suggested that the low molecular weight compounds (LMW) described in FIG. 26 are the result of direct hydrolysis of the peptide bonds in areas of weak bonds, such as a hinge region under acidic or basic conditions. This is especially true for monoclonal antibodies in liquid formulations at high temperatures. It has been suggested that acid molecules eluting in about 22.5-26 minutes and over a longer period of time under basic conditions, as described in FIG. 27 are formed as a result of the nonenzymatic deamidation of aspartic residues (in antibodies with standard modification), which were reported to be responsible for the heterogeneity of the charge of monoclonal antibodies in the analysis of more acid molecules after incubation of the antibodies in alkaline pH at elevated temperatures. In analyzing the results presented in FIG. 22-26, it was found that Fv4-M73 is stable in solution at a pH of about 5.5-6.3 with respect to aggregation and decomposition. Optimum stability in terms of the ratio of the main peak to the total peak area was observed at a pH of about 5.5-6.3 (Fig. 27-28).

In this study, adding 100 mM NaCl to a buffer containing 20 mM buffering agent and 50 mM NaCl (20 mM buffer, 150 mM NaCl) resulted in the formation of the same stable antibody solution as a buffer containing 20 mM buffering agent and 50 mM NaCl (20 mM buffer, 50 mM NaCl), which suggests that NaCl does not have a stabilizing effect. The addition of 100 mM arginine to a buffer containing 20 mM buffering agent and 50 mM NaCl (20 mM buffer, 50 mM NaCl and 100 mM arginine-HCl) had a significant stabilizing effect on the antibody solution from the point of view of aggregation, suggesting that arginine-HCl has a significant stabilizing effect. As for the buffering agents, the stability of the solution was slightly improved using the histidine-HCl buffer, but not the citrate buffer.

When conducting a freeze-thaw test (“FT” means the number of freeze / thaw cycles) illustrated in FIG. 29, a significant number of aggregates were observed at a lower pH or at a lower salt concentration in these formulations. Adding 100 mM arginine to a buffer containing 20 mM buffering agent and 50 mM NaCl (20 mM buffer, 50 mM NaCl and 100 mM arginine-HCl) was more effective in reducing aggregation than adding 100 mM NaCl (20 mM buffer , 150 mM NaCl), which suggests that arginine-HCl has a significant stabilizing effect after a freeze-thaw cycle. Optimum stability was observed at a pH of about 5.0-6.6 in a buffer containing 100 mM arginine-HCl.

Example 1 1

The effect of sugar and arginine-HCl on the stability of Fv4-M73 at 100 mg / ml

Fv4-M73 was prepared at a final concentration of 100 mg / ml in various buffer solutions (described in Table 11), after which these samples were analyzed by size exclusion chromatography and anion exchange chromatography with UV detection for a two-month period when stored at 25 ° C and 40 ° C, and subjected to a certain number of freeze-thaw cycles (freeze for 0.5 days at -20 ° C and thaw for 30 minutes at room temperature). Graphs were constructed for the percentage of aggregates present in the compositions and the percentage increase in aggregates compared to the initial number of aggregates versus time, and these graphs are shown in FIG. 30 and 31. An increase in the number of aggregates (in percent) is an indicator of a decrease in stability.

As shown in FIG. 30 and 31, compositions F and G had less stability than composition H, which indicated that sucrose and trehalose had a less stabilizing effect than arginine-HCl in a liquid composition under storage at 25 ° C and 40 ° C, and during freeze-thaw cycles, compositions F and G had the same stability as composition H, or greater stability, which indicates that sucrose or trehalose have a significant stabilizing effect on freezing / thawing.

Table 11
The compositions used in example 11 mAb Conc. mg / ml Structure Filler Buffer pH Fv4-m73 one hundred E absent 20 mm histidine-HCl,
50 mM NaCl 6.5 F 100 mM sucrose G 100 mM trehalose H 100 mM arginine-HCl

Example 12

Stability Fv4-M73 at 200 mg / ml; effect of pH, arginine- HCl and trehalose

Fv4-M73 with a final concentration of 200 mg / ml was prepared in the form of six different formulations described in table 12.

Table 12
The compositions used in example 12 mAb Conc. mg / ml Structure Buffer Arg-hcl
(mm) Filler pH Fv4-m73 200 one 20 mm
histidine one hundred - 5.5 2 6.0 3 6.5 four fifty 6.0 5 150 6 one hundred 50 mM trehalose

Samples were incubated at 5 ° C and at -20 ° C for three months and six months, respectively. Initial samples, three-month samples and six-month samples were analyzed by size exclusion chromatography with UV detection, as described in Example 9. The percentage increase in the number of aggregates at 5 ° C compared to the initial level in these compositions after storage for three months and six months is presented in FIG. 32. The percentage increase in the number of aggregates compared with the initial level at -20 ° C in these formulations after storage for three months and six months is shown in FIG. 33.

As shown in FIG. 32, the composition of Fv4-M73, when stored in a liquid state at 5 ° C, was more stable at a lower pH (most stable at pH 5.5 and least stable at pH 6.5) and more stable at a higher concentration of arginine (the most stable at 150 mM arginine-HCl and the least stable at 50 mM arginine HCl). The addition of 50 mM trehalose had some stabilizing effect on Fv4-M73 when stored in a liquid state at 5 ° C. Since liquid pharmaceutical antibody formulations are often stored at 5 ° C, the preferred Fv4-M73 liquid formulation should contain at least 100 mM arginine-HCl with histidine buffer at pH 5.5-6.0 and, if necessary, an additional stabilizer .

As shown in FIG. 33, the composition of Fv4-M73, when stored frozen at -20 ° C, showed greater stability at a higher pH and significantly higher stability at a higher concentration of arginine (the highest stability at 150 mM arginine-HCl and the lowest stability at 50 mM arginine-HCl). The addition of 50 mM trehalose had a significant stabilizing effect on Fv4-M73 when stored frozen at -20 ° C. Antibodies in large quantities are often stored frozen at temperatures from -20 ° C to -70 ° C. As for the costs of storage and transportation in the frozen state, it is desirable that such storage be carried out at -20 ° C. Large volumes of the Fv4-M73 formulation when stored at -20 ° C should preferably contain at least 100 mM arginine-HCl with histidine buffer at pH 5.5-6.5 and sugar (such as trehalose).

Comparative examples

Obtaining soluble recombinant human receptor IL-6

A soluble recombinant human IL-6 receptor derived from the human IL-6 receptor, which is an antigen, was prepared as described below. A CHO cell line was obtained constitutively expressing a soluble human IL-6 receptor containing a sequence starting from the first N-terminal amino acid and ending with the 344th amino acid, as described in J. Biochem. (1990) 108, 673-676 (Yamasaki et al., Science (1988) 241, 825-828 (GenBank # X12830)). The soluble human IL-6 receptor was purified from the culture supernatant of SR344-expressing CHO cells by three types of column chromatography: Blue Sepharose 6 FF Sepharose column chromatography, column affinity chromatography with SR344 specific antibody immobilized on it, and gel column chromatography -filtration. Fractions eluting as the main peak were used as the final purified sample.

Obtaining soluble recombinant receptor for IL-6 dog-like monkeys (cIL-6R)

Oligo DNA primers were prepared based on the described gene sequence for the rhesus monkey IL-6 receptor (Birney et al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D556-61). A DNA fragment having the full length gene of the dog-like monkey receptor IL-6 was obtained by PCR using primers, and cDNA isolated from the pancreas of the dog-like monkeys was used as a template. The obtained DNA fragment was inserted into the expression vector of mammalian cells, and stable expression of the indicated fragment in the CHO cell line (cyno.sIL-6R-producing CHO cell line) was achieved using this vector. The culture medium of dog-like monkey sIL-6R-producing CHO cells was purified on a HisTrap column (GE Healthcare Bioscience), and then concentrated using an Amicon Ultra-15 Ultracel-10k adsorbent (Millipore). The final purified sample of the soluble dog-monkey monkey IL-6 receptor (hereinafter referred to as cIL-6R) was obtained after further purification by gel filtration on a Superdex200pg16 / 60 column (GE Healthcare Bioscience).

Obtaining recombinant dog-like monkey IL-6 (cIL-6)

Dog-like monkey IL-6 was prepared by the method described below. A nucleotide sequence encoding 212 amino acids was obtained and deposited in SWISSPROT reg. No. P79341, and then this sequence was cloned into the expression vector of mammalian cells. The resulting vector was introduced into CHO cells in order to obtain a stable expression cell line (IL-6R-producing dog-like monkey CHO cell line). The culture medium of the dog-like monkey IL-6R-producing CHO cell line was purified on an SP-Sepharose / FF column (GE Healthcare Bioscience) and then concentrated using an Amicon Ultra-15 Ultracel-5k adsorbent (Millipore). The final purified sample of dog-like monkey IL-6 (designated cIL-6R hereinafter) was obtained after further purification by gel filtration on a Superdex75pg26 / 60 column (GE Healthcare Bioscience), followed by concentration with Amicon Ultra-15 Ultracel-5k adsorbent (Millipore).

Obtaining a known high-affinity antibody against IL-6 receptor

The mammalian cell expression vector was designed to express VQ8F11-21 hIgG1, a known high-affinity anti-IL-6 receptor antibody. The VQ8F11-21 hIgG1 antibody is described in US patent application 2007/0280945 A1 (US 2007/0280945 A1; the amino acid sequences of the H chain and L chain are shown in SEQ ID NOs: 77 and 78, respectively). The variable region of the antibody was constructed by PCR using a combination of synthetic oligo DNA (obtained by PCR assembly), and IgG1 was used for the constant region. The variable and constant regions of the antibody were combined by PCR assembly and then inserted into the mammalian expression vector in order to construct expression vectors for the H chain and L chain of interest. The nucleotide sequences of the resulting expression vectors were determined by a method known to those skilled in the art. The high affinity anti-IL-6 receptor antibody (hereinafter referred to as the “control antibody”) was expressed and purified using the constructed expression vectors by the method described in Example 1.

Obtaining, expression and purification of tocilizumab variants

Tocilizumab variants were obtained using the QuikChange site-directed mutagenesis kit (Stratagene) by the method described in the instructions attached to the manual. The resulting plasmid fragments were inserted into the expression vectors of mammalian cells in order to construct expression vectors for the H chains and L chains of interest. The nucleotide sequences of the resulting expression vectors were determined by a method known to those skilled in the art. Antibodies were expressed by the method described below. HEK293H cell lines derived from cancer cells of the kidneys of a human embryo (Invitrogen) were suspended in DMEM medium (Invitrogen) to which 10% fetal bovine serum (Invitrogen) was added. Cells were plated in a volume of 10 ml per dish in adhesive cell plates (10 cm diameter; CORNING) at a cell density of 5-6 × 10 ⁵ cells / ml and cultured in a CO2 incubator (37 ° C, 5% CO ₂ ) for one day and one night. Then the medium was removed by suction and 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added. The resulting plasmid was introduced into the cells by lipofection. The resulting culture supernatants were collected, centrifuged (approximately 2000g, 5 minutes at room temperature) to remove cells and sterilized by filtration through a 0.22 μm MILLEX (R) -GV (Millipore) filter to give supernatants. Antibodies were isolated from the obtained culture supernatants by a known method using recombinant protein A - Sepharose ^TM Fast Flow (Amersham Biosciences). To determine the concentration of purified antibodies, the optical density was measured at a wavelength of 280 nm using a spectrophotometer. Antibody concentrations were determined based on values calculated using the absorption coefficients measured by the PACE method (Protein Science 1995; 4: 2411-2423).

Obtaining a cell line BaF3 expressing human gp130

The BaF3 cell line expressing human gp130 was obtained in accordance with the procedure described below, resulting in the production of a cell line proliferating depending on IL-6.

Full length human gp130 cDNA (Hibi et al., Cell (1990) 63: 1149-1157 (GenBank # NM_002184)) was amplified by PCR and cloned into the pCOS2Zeo expression vector to construct pCOS2Zeo / gp130. pCOS2Zeo is an expression vector constructed by removing the expression region of the DHFR gene from pCHOI (Hirata et al., FEBS Letter (1994) 356: 244-248) and embedding the expression region of the zeocin resistance gene. The full length cDNA of human IL-6R was amplified by PCR and cloned into pcDNA3.1 (+) (Invitrogen) to give the hIL-6R / pcDNA3.1 (+) construct.

10 micrograms of pCOS2Zeo / gp130 were mixed with BaF3 cells (0.8 × 10 ⁷ cells) suspended in PBS, and then 0.33 kV and 950 μF pulses were applied using a Gene Pulser (Bio-Rad) device. BaF3 cells having an electroporated gene were cultured for one day and one night in RPMI 1640 medium (Invitrogen), to which 0.2 ng / ml mouse interleukin-3 (Peprotech) and 10% fetal bovine serum were added ( hereinafter referred to as FBS, HyClone) and then selected by adding RPMI 1640 medium to which 100 ng / ml human interleukin-6 (R&D systems), 100 ng / ml soluble human interleukin-6 receptor (R&D systems) and 10% were added FBS to produce a BaF3 cell line expressing human gp130 (hereinafter referred to as “BaF3 / gp130”). These BaF / gp130 cells proliferate in the presence of human interleukin-6 (R&D systems) and a soluble human IL-6 receptor, and therefore they can be used to evaluate the growth-inhibitory activity (or IL-6 receptor neutralizing activity) of an anti-IL receptor antibody -6.

Assessment of biological activity using BaF3 cells expressing human gp130 (BaF / gp130)

The activity aimed at neutralizing the IL-6 receptor was evaluated using BaF3 / gp130 cells, which proliferate depending on the level of IL-6 / IL-6 receptor. After three washes with RPMI1640 medium, to which 10% FBS was added, BaF3 / gp130 cells at a density of 5 × 10 ⁴ cells / ml were suspended in RPMI1640 medium, into which 600 ng / ml or 60 ng / ml human interleukin-6 was added ( TORAY) (at a final concentration of 300 ng / ml or 30 ng / ml), the corresponding amount of soluble human IL-6 receptor and 10% FBS. Cell suspensions were dispensed (50 microliters / well) into 96-well plates (CORNING). The purified antibodies were then diluted with RPMI1640 medium containing 10% FBS and added to each well (50 microliters / well). Cells were cultured at 37 ° C in an atmosphere of 5% CO ₂ for three days. Reagent WST-8 (cell counting kit - 8; Dojindo Laboratories) was diluted twice with PBS. Then, 20 microliters of the indicated reagent were immediately added to each well, and the absorbance was measured at a wavelength of 450 nm (reference wavelength: 620 nm) on a SUNRISE CLASSIC (TECAN) device. After culturing for two hours, the absorbance was again measured at a wavelength of 450 nm (reference wavelength: 620 nm). The IL-6 receptor neutralizing activity was evaluated over two hours by the change in optical density used as an indicator.

Biacore Soluble Human IL-6 Receptor Assay

The kinetics of the antigen-antibody reaction were analyzed using a Biacore T100 (GE Healthcare). The interaction of the antibody with the soluble human IL-6 receptor was evaluated by immobilizing the appropriate amount of protein A or protein A / G or anti-IgG F (ab ') ₂ (specific for the gamma chain) on a sensor chip by the amine binding method of interest antibodies on the chip at pH 7.4, and assay for the action of the soluble IL-6 receptor, used at various concentrations as an analyte at pH 7.4 and immobilized on the chip. All measurements were performed at 37 ° C. Kinetic parameters, association rate constants ka (1 / s) and dissociation rate constants kd (1 / s) were calculated from sensorograms constructed from the measurement results. Then, based on the rate constants of association and dissociation, K _D (M) was determined. The corresponding parameters were determined using the Biacore T100 Evaluation Software (GE Healthcare) computer program.

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

Monitoring the antigen-antibody reaction with the membrane-type IL-6 receptor at pH 5.8 and pH 7.4 was performed using Biacore T100 (GE Healthcare). Membrane-type IL-6 receptor binding was analyzed by evaluating the binding of a soluble human IL-6 receptor immobilized on a sensor chip. SR344 was biotinylated by a method known to those skilled in the art. Biotinylated soluble human IL-6 receptor was immobilized on a sensor chip using streptavidin based on the affinity of binding of biotin and streptavidin. All measurements were performed at 37 ° C. The buffer for the mobile phase was 10 mM MES (pH 5.8), 150 mM NaCl and 0.05% Tween 20. A clone detecting pH-dependent binding was introduced at pH 7.4 to bind to the soluble human receptor IL-6 (sample injection buffer was 10 mM MES (pH 7.4), 150 mM NaCl and 0.05% Tween 20). Then, pH-dependent dissociation of each clone was monitored at pH 5.8, which corresponds to the pH of the mobile phase. The dissociation rate constant (Kd (1 / s)) at pH 5.8 was calculated using the Biacore T100 Evaluation Software (GE Healthcare) by plotting a curve only for the dissociation phase at pH 5.8. The concentration of the sample was 0.25 microgram / ml. The binding was carried out in 10 mm MES (pH 7.4), 150 mm NaCl and 0.05% tween 20, and dissociation was carried out in 10 mm MES (pH 5.8), 150 mm NaCl and 0.05% tween 20. Similar Thus, the dissociation rate constant (Kd (1 / s)) at pH 7.4 was calculated using the Biacore T100 Evaluation Software (GE Healthcare) by plotting the curve for the dissociation phase only at pH 7.4. The concentration of the sample was 0.5 microgram / ml. Binding was carried out in 10 mM MES (pH 7.4), 150 mM NaCl and 0.05% Tween 20, and dissociation was carried out in 10 mM MES (pH 7.4), 150 mM NaCl and 0.05% Tween 20.

Assessment of binding to human FcRn

FcRn is a complex of FcRn and β2-microglobulin. Oligo DNA primers were prepared based on the described sequence of the human FcRn gene (J. Exp. Med. (1994) 180 (6): 2377-2381). The DNA fragment corresponding to the full-sized gene was obtained by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as a template and the resulting primers. The obtained DNA fragment used as a template and encoding the extracellular domain containing the signal region (Met1-Leu290) was amplified by PCR and inserted into the animal cell expression vector (the amino acid sequence of human FcRn is shown in SEQ ID NO: 79). Similarly, oligo DNA primers were prepared based on the described human β2-microglobulin gene sequence (Proc. Natl. Acad. Sci. USA. (2002) 99 (26): 16899-16903). The DNA fragment corresponding to the full-sized gene was obtained by PCR and using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH), serving as a template, and the obtained primers. The obtained DNA fragment used as a template and encoding a full-sized β2-microglobulin containing the signal region (Met1-Met119) was amplified by PCR and inserted into the expression vector of mammalian cells (the amino acid sequence of human β2-microglobulin is presented in SEQ ID NO: 80).

Soluble human FcRn was expressed in accordance with the following procedure. Plasmids designed for human FcRn and β2-microglobulin were introduced by lipofection into cells of the HEK293H cell line derived from cancer cells of the kidneys of a human embryo (Invitrogen) using 10% FBS (Invitrogen). The resulting culture supernatant was collected, and FcRn was purified using IgG-Sepharose 6 Fast Flow (Amersham Biosciences) by the method described in J. Immunol. 2002 Nov 1; 169 (9): 5171-80, and then again purified on a HiTrap Q HP column (GE Healthcare).

Determination of antibody concentration in mouse plasma

The concentration of antibodies in the plasma of mice was determined using ELISA method known in the art.

Monkey PK / PD test to determine antibody concentration, CRP concentration, and free soluble IL-6 receptor in monkey plasma

The concentration of antibodies in the plasma of dog-like monkeys was determined using ELISA method known to specialists.

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

The concentration of free soluble IL-6 receptor in the plasma of dog-like monkeys was determined by the method described below. All IgG antibodies (IgG of dog-like monkeys, anti-human IL-6 receptor antibody and complex “anti-human IL-6 receptor antibody-soluble dog-like monkey receptor IL-6” complex) in plasma were adsorbed onto protein A by loading 30 microliters of plasma of dog-like monkeys onto the corresponding amount of resin, namely, recombinant protein A-sepharose Fast Flow (GE Healthcare), dried in a 0.22 μm filter with a cap (Millipore). Then, the solution in the filter with the cap was centrifuged in a high speed centrifuge to collect the solution passing through the centrifuge. The missed solution did not contain a complex protein A-bound antibody against the human IL-6 receptor, a soluble dog-like monkey receptor IL-6. Therefore, the concentration of the free soluble IL-6 receptor can be determined by measuring the concentration of the soluble IL-6 receptor of dog-like monkeys in a solution passing through protein A. The concentration of the soluble IL-6 receptor of dog-like monkeys was determined by a known method for measuring the concentration of soluble human IL-6 receptor. The soluble monkey-like receptor IL-6 receptor (cIL-6R) obtained as described above was used as the standard. The percentage of free soluble IL-6 receptor was calculated by the following formula:

The concentration of free soluble IL-6 receptor after administration of antibodies

――――――――――――――――――――――――――――――――― × 100

Concentration of soluble IL-6 receptor prior to antibody administration

Claims (16)

1. The pharmaceutical composition having a neutralizing activity against the receptor of IL-6, having a pH in the range from 5.5 to 6.6 and containing, at a concentration of 10-240 mg / ml, at least one antibody selected from:
(i) an antibody that contains a heavy chain variable region comprising CDR1 containing the sequence of SEQ ID NO: 4 (CDR1 VH3-M73), CDR2 containing the sequence of SEQ ID NO: 5 (CDR2 VH3-M73), and CDR3 containing the sequence SEQ ID NO: 6 (CDR3 VH3-M73), and the variable region of the light chain comprising CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 VL3), CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 VL3), and CDR3, containing the sequence of SEQ ID NO: 15 (CDR3 VL3); and
(ii) an antibody that consists of two heavy chains with a variable region containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and two light chains with a variable region containing the sequence of SEQ ID NO: 23 (variable region VL3) ; and
(iii) an antibody that consists of two heavy chains comprising the sequence of SEQ ID NO: 26 (VH3-M73) and two light chains comprising the sequence of SEQ ID NO: 29 (VL3),
and histidine and / or citrate buffer. 2. The pharmaceutical composition according to claim 1, containing at least one cationic amino acid. 3. The pharmaceutical composition according to claim 1, containing 1-500 mM histidine and / or citrate buffer, 1-1500 mM at least one cationic amino acid, 10-240 mg / ml antibodies and 1-400 mm carbohydrate. 4. The composition according to p. 2, where the cationic amino acid is arginine. 5. The composition according to p. 3, where the carbohydrate is sucrose or trehalose. 6. The composition according to p. 1, which also contains a surfactant. 7. The composition according to p. 1, containing the antibody in an amount of 50-240 mg / ml. 8. The composition according to p. 1, containing the antibody in an amount of 80-240 mg / ml. 9. The composition according to p. 1, containing the antibody in an amount of 240 mg / ml 10. The composition according to p. 1, where the specified composition is a liquid. 11. The composition according to p. 10, which, in the process of its preparation, was not subjected to lyophilization. 12. The composition according to p. 1, where the degree of dimerization of the antibody molecules has been reduced. 13. The composition of claim 1, wherein the dimerization of the antibody molecules has been inhibited. 14. The composition according to p. 1, intended for subcutaneous administration. 15. The method of stabilizing antibodies in the process of freezing / thawing a solution containing the antibody, where the specified method includes the addition of arginine, while arginine is in a concentration of 1-1500 mm, the solution has a pH in the range from 5.0 to 6.6 where the specified antibody is at a concentration of 10-240 mg / ml and is at least one antibody selected from:
(i) an antibody that contains a heavy chain variable region comprising CDR1 containing the sequence of SEQ ID NO: 4 (CDR1 VH3-M73), CDR2 containing the sequence of SEQ ID NO: 5 (CDR2 VH3-M7 3), and CDR3 containing the sequence of SEQ ID NO: 6 (CDR3 VH3-M73), and the variable region of the light chain comprising CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 VL3); CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 VL3), and CDR3 containing the sequence of SEQ ID NO: 15 (CDR3 VL3);
(ii) an antibody that consists of two heavy chains with a variable region containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and two light chains with a variable region containing the sequence of SEQ ID NO: 23 (variable region VL3) ;
(iii) an antibody that consists of two heavy chains containing the sequence of SEQ ID NO: 26 (VH3-M73), and two light chains containing the sequence of SEQ ID NO: 29 (VL3). 16. A method of stabilizing a solution having a pH in the range of 5.5 to 6.6 containing an antibody, wherein said method comprises adding arginine, wherein arginine is in a concentration of 1-1500 mM, wherein said antibody is in a concentration of 10-240 mg / ml and is at least one antibody selected from:
(i) an antibody that contains a heavy chain variable region comprising CDR1 containing the sequence of SEQ ID NO: 4 (CDR1 VH3-M73), CDR2 containing the sequence of SEQ ID NO: 5 (CDR2 VH3-M73), and CDR3 containing the sequence SEQ ID NO: 6 (CDR3 VH3-M73), and the variable region of the light chain comprising CDR1 containing the sequence of SEQ ID NO: 13 (CDR1 VL3), CDR2 containing the sequence of SEQ ID NO: 14 (CDR2 VL3), and CDR3, containing the sequence of SEQ ID NO: 15 (CDR3 VL3);
(ii) an antibody that consists of two heavy chains with a variable region containing the sequence of SEQ ID NO: 20 (variable region VH3-M73), and two light chains with a variable region containing the sequence of SEQ ID NO: 23 (variable region VL3) ; and
(iii) an antibody that consists of two heavy chains containing the sequence of SEQ ID NO: 26 (VH3-M73), and two light chains containing the sequence of SEQ ID NO: 29 (VL3).

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