Advanced antibody molecules

FIELD: medicine.

SUBSTANCE: there are offered: IL-6 receptor antibody, a coding gene, a vector and a host cell for producing the antibody, a method for producing the antibodies, and a pharmaceutical composition for treating IL-6-related diseases containing the antibody.

EFFECT: use of the invention provides new humanised IL-6 receptor antibodies that can find further application in therapy of the IL-6-mediated diseases.

8 cl, 22 dwg, 8 tbl, 9 ex

 

The scope to which the invention relates

The present invention relates to pharmaceutical compositions containing the antibody against the receptor of IL-6, used as an active ingredient, and to methods of producing these compositions, etc.

Prior art

Antibodies are of particular interest as pharmaceutical agents, since they have a high stability in plasma and do not cause significant side effects. Such antibodies include a number of commercially available pharmaceutically acceptable antibody type IgG and many pharmaceutically acceptable antibodies, which are currently at the stage of testing (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. humanitariannet IgG1 antibody against the receptor for IL-6, specifically binds to the receptor of IL-6. 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 will neutralize the biological activity of IL-6 (patent documents 1-3 and non-patent document 4). In Japan tocilizumab was allowing the Yong for use as a therapeutic agent for the treatment of diseases of Kalman and rheumatoid arthritis (non-patent document 5).

Humanized antibody such as tocilizumab, are pharmaceutically acceptable antibody of the first generation. Pharmaceutically acceptable antibody of the second generation are now at the stage of research in order to improve the efficiency and ease of use, and cost reduction pharmaceutically acceptable antibody of the first generation. Currently developed various technologies that can be applied to obtain pharmaceutically acceptable antibody of the second generation. There are descriptions of the methods enhance effector function, enhance ability to bind to the antigen, improved pharmacokinetic properties and stability these antibodies, and methods to reduce the risk of immunogenicity. As reported, such methods increase the effectiveness of the drug or dose reduction are methods of enhancing antibody-dependent cell-mediated cytotoxic activity (ADCC activity) or complement-dependent cytotoxic activity (CDC activity), conducted by the amino acid substitutions in the Fc-region of IgG antibodies (non-patent document 6). It was also reported that the method of increasing antigennegative ability or antigen-neutralizing ability is affinity maturation (C the patent document 7). This technology allows you to boost antigennegative activity by introducing amino acid mutations in the hypervariable region (complementarity-determining region (CDR)of the variable region or the like, the Improvement antigennegative ability leads to increased biological activity ofin vitroor reduces the input dose and increases the efficiency of thein vivo(non-patent document 8). Currently, clinical trials are conducted for characterization of antibodies motavizumab (Motavizumab) (obtained by affinity maturation), which, as expected, has a much higher efficiency than palivizumab (Palivizumab), i.e. pharmaceutically acceptable anti-RSV antibody of the first generation (non-patent document 9). The literature also described antibody against the receptor of IL-6 with an affinity of about 0.05 nm (i.e. with affinity greater than the affinity of tocilizumab are reviewed) (patent document 4). However, in the literature there are no data on human, humanized or chimeric antibodies, affinity which is greater than 0.05 nm.

The challenge currently facing professionals in obtaining pharmaceutically acceptable antibodies are high cost of production associated with the introduction of very large quantities of protein. T is to, for example, the dose of tocilizumab are reviewed, namely gumanitarnogo IgG1 antibodies against the receptor for IL-6, for intravenous injection is approximately 8 mg/kg/month (non-patent document 4). For the treatment of chronic autoimmune diseases preferred form of injection is a subcutaneous injection. In General, you want the drugs for subcutaneous injection had a high concentration. From the point of view of stability, etc. is the maximum dose for antibody type IgG is typically about 100 μg/ml (non-patent document 10). Inexpensive and easy to use pharmaceutical drugs based on antibodies of the second generation, which can be injected subcutaneously for longer periods of time, can be obtained by extending the half-life of antibodies in the plasma in order to prolong its therapeutic effect, thereby reducing the amount of input protein, as well as by mail to such high antibody stability.

FcRn is closely associated with the pharmacokinetics of the antibody. With regard to differences in time half-life of antibody isotypes IgG1 and IgG2 in plasma, it is known that IgG1 and IgG2 have significantly longer half-life in plasma than IgG3 and IgG4 (non-patent document 11). As reported, the method further increase the half-life of antibody IgG3 and IgG4 in the square is the ZMA, already have a fairly long half-life in plasma, is the replacement of amino acids in the constant region, resulting in increased binding to FcRn (non-patent documents 12 and 13). From the point of view of immunogenicity further increase the time half-life in plasma was achieved by replacing amino acid is preferably variable, but not in the constant region (patent document 5). However, at present there are no data about the longer half-life of antibodies against the receptor of IL-6 in plasma, and it does so by modifying variable regions.

Another important problem faced by specialists in the development of Biopharmaceuticals is immunogenicity. In General, the immunogenicity of murine antibodies can be reduced by "humanization" of antibodies. It is assumed that when the humanization of antibodies, the risk of immunogenicity can be further reduced by using a frame sequence of the germ line as a matrix (non-patent document 14). However, even adalimumab (Adalimumab), that is, a fully human anti-TNF antibody was detected high (13%-17%) development of immunogenicity, and it was found that patients who were observed immunogenicity, therapeutic effect was n skim (non-patent documents 15 and 16). T-cell epitopes can be present even in the CDR of a human antibody, and such T-cell epitope in CDR can cause immunogenicity. Methodsin silicoandin vitrofor predicting T-cell epitopes described in the literature (non-patent documents 17 and 18). It is assumed that the risk of immunogenicity can be reduced by removal of T-cell epitopes predicted by such methods (non-patent document 19).

Tocilizumab, i.e. humanitariannet antibody against the receptor of IL-6, is an IgG1 antibody, obtained by humanizing mouse antibodies PM1. Joining CDR carried out using human sequences NEW and REI as a frame matrix for circuits H and L, respectively; however, five amino acids of the mouse sequence in the frame region were retained as essential amino acids to maintain activity (non-patent document 20). Currently there are no data on the full humanization of the rest of the murine sequence in the frame region gumanitarnogo antibody tocilizumab are reviewed without reducing its activity. In addition, the CDR sequence of tocilizumab are reviewed is the mouse sequence, and this sequence, like adalimumab, may have a T-cell epitope in CDR, which may cause the risk of development and is monogenist. In clinical trials of tocilizumab are reviewed antibodies against the tocilizumab are reviewed not detected when the 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 are reviewed can be further reduced. However, in the literature there are no data on the reduction of the risk of immunogenicity of tocilizumab are reviewed at amino acid replacement.

The isotype of tocilizumab are reviewed are IgG1. Differences in the isotypes are determined by the differences in the sequences of the constant region. Since it is assumed that the sequence of the constant region has a significant impact on effector function, pharmacokinetics, physical properties of antibodies and the like, the choice of the sequence of the constant region is very important for the development of pharmaceutical drugs based on antibodies (non-patent document 11). In recent years, the safety of pharmaceutical drugs based on antibodies is of paramount importance. The interaction of the Fc part of the antibody and Fcγ-receptor (effector function) can cause serious side effects in clinical trials of TGN1412 phase I (non-patent document 21). To obtain pharmaceutical drugs based on antibodies to neutralize the biological activity of the antigen binding Fcγ, which plays an important role in effector functions such ADCC, is not necessary. The binding to Fcγ receptor may even be undesirable from the viewpoint of side effects. A method of reducing binding to Fcγ receptor is used to change the isotype of IgG antibodies with IgG1 to IgG2 or IgG4 (non-patent document 22). IgG2 is preferable IgG4, from the viewpoint of pharmacokinetics and binding to Fcγ receptor I (non-patent document 11). Tocilizumab is an antibody neutralizing the receptor for IL-6, and its isotype is IgG1. Thus, from the point of view of minimizing possible side effects, IgG2 may be preferred by isotype, because his actions do not require effector functions such as ADCC.

However, during the development of pharmaceutical drugs based on antibodies is very important physico-chemical properties of proteins, and in particular, their homogeneity and stability. It was reported that the IgG2 isotype has 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 on the basis of such antibodies with maintaining the desired substances/related substances is due to their heterogeneity,due to the difference disulfide bonds in various products. Thus, if possible, it is desirable to use one substance. In addition, it was reported that the heterogeneity of the C-terminal sequences of the H-chain antibodies is due to a deletion of the C-terminal lysine amino acid residue and the amidation of the C-terminal carboxyl group in the deletion of two C-terminal amino acids, glycine and lysine (non-patent document 24). When developing antibodies isotype IgG2 as pharmaceuticals, it is preferable to reduce this heterogeneity and to maintain a high stability. To get comfortable in the use of stable preparations having a high concentration and which is intended for subcutaneous injection, it is preferable that these drugs had not only high stability, but also had a half-life in plasma is greater than the half-life of IgG1, which is an isotype of tocilizumab are reviewed. However, at present there are no data on the sequences of the constant region of the antibody with a constant region of IgG2 isotype, having reduced heterogeneity, high stability and half-life in plasma is greater than the half-life of the antibody with a constant region of the IgG1 isotype.

Previous documents related to the present invention are set forth below:

[Previous document]

[Patent document Prov.]

[Patent document 1] WO 92/19759

[Patent document 2] WO 96/11020

[Patent document 3] WO 96/12503

[Patent document 4] WO 2007/143168

[Patent document 5] WO 2007/114319

[Patent document 6] WO 2004/096273

[Patent documents]

[Non-patent document 1] Janice M. Reichert, Clark J. Rosensweig, Laura B. Faden & Matthew C. Dewitz, Monoclonal antibody successes in the clinic, Nature Biotechnology 23, 1073-1078 (2005).

[Non-patent document 2] A.K. Pavlou, Belsey M.J., therapeutic antibodies market to 2008., Eur J Pharm Biopharm. 2005 Apr; 59(3):389-96.

[Non-patent document 3] Nishimoto n, Kishimoto T., Interleukin 6: from bench to bedside., Nat Clin Pract Rheumatol. 2006 Nov; 2(11):619-26.

[Non-patent document 4] Maini R.N., 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.

[Non-patent document 5] N. Nishimoto, 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.

[Non-patent document 6] S.J. Kim, Y. Park, H.J. Hong, Antibody engineering for the development of therapeutic antibodies., Mol Cells. 2005 Aug 31; 20(1):17-29. Review.

[Non-patent document 7] A. Rothe, Hosse R.J., Power B.E. Ribosome display for improved biotherapeutic molecules. Expert Opin Biol Ther. 2006 Feb; 6(2):177-87.

[Non-patent document 8] A. Rajpal, Beyaz N., L. Haber, Cappuccilli G., Yee H., Bhatt R.R., Takeuchi, T., Lerner R.A., Crea R.,A general method for greatly improving the affinity of antibodies by using combinatorial libraries., Proc Natl Acad Sci U S A. 2005 Jun 14; 102(24):8466-71. Epub 2005 Jun 6.

[Non-patent document 9] H. Wu, Pfarr D.S., Johnson, S., Brewah Y.A., Woods, R., Patel N.K., White W.I., Young J.F., Kiener P.A. Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract. J Mol Biol. 2007, 368, 652-665.

[Non-patent document 10] S.J. Shire, Shahrokh z, Liu J. Challenges in the development of high protein concentration formulations. J Pharm Sci. 2004 Jun; 93(6):1390-402.

[Non-patent document 11] Salfeld J.G. Isotype antibody selection in engineering.Nat Biotechnol. 2007 Dec; 25(12):1369-72.

[Non-patent document 12] Hinton P.R., Xiong J.M., Johlfs .G., M.T. Tang, S. Keller, Tsurushita N., An engineered human IgG1 antibody with longer serum half-life., J Immunol. 2006 Jan 1; 176(1):346-56.

[Non-patent document 13] Ghetie V., Popov S., Borvak j, Radu C., Matesoi D., Medesan, C., R.J. Ober, E.S. Ward, Increasing the serum persistence of an IgG fragment by random mutagenesis., Nat Biotechnol. 1997 Jul; 15(7):637-40.

[Non-patent document 14] W.Y. Hwang, Almagro J.C., Buss T.N., P. Tan, J. Foote Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods. 2005 May; 36(1):35-42.

[Non-patent document 15] Bartelds G.M., Wijbrandts CA, M.T. Nurmohamed, Staple, S., W.F. Lems, Aarden L., Dijkmans B.A., P. Tak, Wolbink G.J. Clinical response to adalimumab: The relationship with anti-adalimumab antibodies and serum adalimumab concentrations in rheumatoid arthritis. Ann Rheum Dis. 2007 Mar 9; [Epub ahead of print].

[Non-patent document 16] Bender N.K., Heilig C.E., Droll B., J. Wohlgemuth, Armbruster F.P., B. Heilig Immunogenicity, efficacy and adverse events of adalimumab in RA patients. Rheumatol Int. 2007 Jan; 27(3):269-74.

[Non-patent document 17] Van Walle I., Gansemans y, P.W. Parren, Stas P., Lasters I. Immunogenicity screening in protein drug development. Expert Opin Biol Ther. 2007 Mar; 7(3):405-18.

[Non-patent document 18] Jones T.D., Phillips WJ, Smith B.J., Bamford CA., Nayee P.D., Baglin T.P., Gaston J.S., Baker M.P. Identification and removal of a promiscuous CD4+ T cell an epitope from the C1 domain of factor VIII. J Thromb Haemost. 2005 May; 3(5):991-1000.

[Non-patent document 19] Chirino AJ, Ary M.L., S.A. Marshall gold, moor mud the immunogenicity of protein therapeutics. Drug Discov Today. 2004 Jan 15; 9(2):82-90.

[Non-patent document 20] K. Sato, M. Tsuchiya, J. Saldanha, Koishihara y, Ohsugi Y., Kishimoto, T., Bendig M.M. Reshaping a human antibody to inhibit the interleukin 6-dependent tumor cell growth. Cancer Res. 1993 Feb 15; 53(4):851-6.

[Non-patent document 21] Strand V., Kimberly R., J.D. Isaacs Biologic therapies in rheumatology: lessons learned future directions. Nat Rev Drug Discov. 2007 Jan; 6(1):75-92.

[Non-patent document 22] J.E. Gessner, H. Heiken, Tamm, A., Schmidt R.E. The IgG Fc receptor family. Ann Hematol. 1998 Jun; 76(6):231-48.

[Non-patent document 23] T.M. Dillon, Ricci M.S., Vezina, C., Flynn G.C., Liu Y.D., Rehder D.S., M. Plant, Henkle B., Li Y., Deechongkit, S., B. varnum poor, Wypych J., Balland, A., P.V. Bondarenko Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J Biol Chem. 2008 Jun 6; 283(23):16206-15.

[Non-patent document 24] K.A. Johnson, Paisley-Flango K., Tangarone B.S., T.J. Porter, Rouse J.C. Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal Biochem. 2007 Jan 1; 360(1):75-83.

Description of the invention

[Problems that can be solved by the present invention]

The present invention can be implemented on the basis of the above facts. The aim of the present invention to provide pharmaceutical compositions containing the molecules of the second generation with improved properties compared to humanized IgG1 antibody against the receptor for IL-6, namely t is ciliolabrum, by modification of the amino acid sequences of the variable and constant regions of tocilizumab are reviewed in order to improve its antigen-neutralizing capacity and improve pharmacokinetic properties, with the subsequent achievement of a prolonged therapeutic effect with less frequency of such compositions, and reduced immunogenicity, increased security and improved physico-chemical properties (stability and homogeneity) (such pharmaceutical compositions may also be referred to as "means" or "drugs"). Another objective of the present invention is to develop methods of producing the above pharmaceutical compositions.

[Means of solving problems]

The authors present invention special studies were conducted in order to obtain molecules of the second generation, with better properties compared with humanized IgG1 antibody against the receptor for IL-6, namely, p tocilizumab are reviewed first generation, by modification of the amino acid sequences of the variable and constant regions of tocilizumab are reviewed to improve its effectiveness and improve pharmacokinetic properties in order to achieve a prolonged therapeutic effect at a lower frequency of its introduction, and reduce immunogenicity, is ysenia its security and improve its physico-chemical properties (stability and homogeneity). As a result, the authors of the present invention was introduced many CDR mutations in the variable regions of tocilizumab are reviewed, improving its ability to communicate (affinity) with the antigen. Thus, the authors of the present invention significantly increased the affinity of this antibody using a combination of such mutations. The authors present invention also managed to improve pharmacokinetic properties by introducing modifications that reduce the value of the isoelectric point of the sequence variable regions. The authors present invention also managed to improve pharmacokinetic properties by providing a pH-dependent binding to the antigen receptor of IL-6, resulting in one molecule antibodies can significantly neutralize the antigen. In addition, the authors present invention was able to reduce the risk of immunogenicity by full humanization of murine sequences present in the frame region of tocilizumab are reviewed, and the reduction of peptide T-cell epitopes in the variable regions predicted byin silico. In addition, the authors present invention was also able to identify new sequence of the constant region to the constant region of tocilizumab are reviewed, which reduce the level of binding to Fcγ receptor, increase safety, improve Farmak the kinetic properties compared with the sequences of IgG1 and reduce heterogeneity, associated with disulfide bonds in the hinge region of IgG2, as well as reduce the heterogeneity associated with the end of the H-chain without compromising stability. The authors of the present invention was able to obtain molecules of the second generation with improved properties compared with tocilizumab are reviewed, by appropriately combining these modifications of amino acid sequences in the CDRs, variable regions and constant regions.

The present invention relates to pharmaceutical compositions that contain humanitariannet IgG antibody against the receptor of IL-6 with enhanced ability to bind to the antigen (receptor IL-6), improved pharmacokinetic properties, enhanced security and improved physical properties (increased stability and homogeneity), and also have a reduced risk of immunogenicity, where these pharmaceutical compositions are obtained by modification of the amino acid sequences of the variable and constant regions gumanitarnogo IgG1 antibodies against the receptor for IL-6, namely tocilizumab are reviewed; and to methods of obtaining the above pharmaceutical compositions. More specifically, the present invention relates:

[1] any polypeptide selected from:

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

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

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

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

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

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

[2] any antibody selected from:

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

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

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

[3] for any variable region selected from:

(a) variable regions of the heavy chain that contains the sequence of SEQ ID NO: 19 (variable regions VH4-M73);

(b) variable regions of the heavy chain that contains the sequence of SEQ ID NO: 20 (variable regions VH3-M73);

(c) variabel the Oh region of the heavy chain, contains the sequence of SEQ ID NO: 21 (variable regions VH5-M83);

(d) variable region of the light chain containing the sequence of SEQ ID NO: 22 (variable regions VL1);

(e) variable region of the light chain containing the sequence of SEQ ID NO: 23 (variable regions VL3); and

(f) variable region of the light chain containing the sequence of SEQ ID NO: 24 (variable regions VL5);

[4] any antibody selected from:

(a) antibodies, which contains a variable region heavy chain comprising the sequence of SEQ ID NO: 19 (variable region VH4-M73), and variable region light chain comprising the sequence of SEQ ID NO: 22 (variable region VL1);

(b) antibodies, which contains a variable region heavy chain comprising the sequence of SEQ ID NO: 20 (variable region VH3-M73), and variable region light chain comprising the sequence of SEQ ID NO: 23 (variable region VL3); and

(c) antibody, which contains a variable region heavy chain comprising the sequence of SEQ ID NO: 21 (variable region VH5-M83), and variable region light chain comprising the sequence of SEQ ID NO: 24 (variable region VL5);

[5] to any heavy chain or light chain selected from:

(a) a heavy chain containing the sequence of SEQ ID NO: 25 (VH4-M73);

(b) a heavy chain containing the series is here SEQ ID NO: 26 (VH3-M73);

(c) a heavy chain containing the sequence of SEQ ID NO: 27 (VH5-M83);

(d) the light chain containing the sequence of SEQ ID NO: 28 (VL1);

(e) the light chain containing the sequence of SEQ ID NO: 29 (VL3); and

(f) a light chain containing the sequence of SEQ ID NO: 30 (VL5);

[6] any antibody selected from:

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

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

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

[7] to gene, codereuse polypeptide according to any one of paragraphs[1]-[6];

[8] to the vector carrying the gene according to item [7];

[9] to the cell master, the carrier vector according to item [8];

[10] the method of producing the polypeptide according to any one of paragraphs [1] - [6] by culturing the host cell according to item [9]; and

[11] the pharmaceutical composition containing the polypeptide according to any one of items [1]to[6] or the polypeptide obtained by the method according to item [10].

[Implementation of the present invention]

Humanized IgG antibody against the receptor for IL-6, obtained by the methods according to the invention, adut increased efficiency and improved pharmacokinetic properties, and so they have prolonged therapeutic effect at a lower frequency injection.

Brief description of the graphical material

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

Figure 2 presents gr the FIC, illustrating the neutralizing activity of tocilizumab are reviewed and RDC-23 BaF/gp130.

Figure 3 presents a list of sites of mutations that can reduce the value of the isoelectric point of the variable regions without significant reduction in the level of binding of tocilizumab are reviewed with receptors IL-6. Asterisks at the sites of mutations mean a website that does not affect the value of the isoelectric point, but was mutated to convert this sequence in the human sequence. The sequence of HFR1 of tocilizumab are reviewed is presented in SEQ ID NO: 93; a sequence of HFR1, obtained after the introduction of the mutations presented in SEQ ID NO: 94; HCDR1 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 95; HCDR1 sequence obtained after the introduction of the mutations presented in SEQ ID NO: 96; a sequence HFR2 of tocilizumab are reviewed is presented in SEQ ID NO: 97; a sequence HFR2 obtained after the introduction of the mutations presented in SEQ ID NO: 98; a HCDR2 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 81; a HCDR2 sequence obtained after the introduction of mutations, presented in SEQ ID NO: 99; sequence HFR4 of tocilizumab are reviewed is presented in SEQ ID NO: 100; sequence HFR4 obtained after the introduction of the mutations presented in SEQ ID NO: 101; sequence LFR1 of tocilizumab are reviewed is presented in SEQ ID NO: 102; the sequence LFR1 obtained after the introduction of th is purpose, presented in SEQ ID NO: 103; a LCDR1 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 87; a LCDR1 sequence obtained after the introduction of the mutations presented in SEQ ID NO: 104; sequence LFR2 of tocilizumab are reviewed is presented in SEQ ID NO: 105; a sequence LFR2 obtained after the introduction of the mutations presented in SEQ ID NO: 106; the LCDR2 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 107; LCDR2 sequence obtained after the introduction of the mutations presented in SEQ ID NO: 108 and 109; the sequence LFR3 of tocilizumab are reviewed is presented in SEQ ID NO: 110; sequence LFR3 obtained after the introduction of the mutations presented in SEQ ID NO: 111; sequence LFR4 of tocilizumab are reviewed is presented in SEQ ID NO: 112, and the sequence LFR4 obtained after the introduction of the mutations presented in SEQ ID NO: 113.

In Fig. 4 presents a graph illustrating the neutralizing activity of tocilizumab are reviewed and H53/L28 in BaF/gp130.

In Fig. 5 presents a graph illustrating the concentration of tocilizumab are reviewed and H53/L28 in the plasma of mice depending on time after intravenous injection.

In Fig. 6 presents a graph illustrating the concentration of tocilizumab are reviewed and H53/L28 in the plasma of mice depending on time after subcutaneous injection.

In Fig. 7 schematically shows that the IgG molecule can re-connect with another antigen in the dissociation of the antigen membrane-type endo is ome.

In Fig. 8 presents a list of sites of mutations that can inform the tocilizumab are reviewed the ability of pH-dependent binding with the receptor of IL-6 for binding at pH 7.4 and dissociation at pH 5.8). The sequence of HFR1 of tocilizumab are reviewed is presented in SEQ ID NO: 93; a sequence of HFR1, obtained after the introduction of the mutations presented in SEQ ID NO: 114; HCDR1 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 95; HCDR1 sequence obtained after the introduction of the mutations presented in SEQ ID NO: 115; a LCDR1 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 87; a LCDR1 sequence obtained after the introduction of the mutations presented in SEQ ID NO: 116; a LCDR2 sequence of tocilizumab are reviewed is presented in SEQ ID NO: 107, and the LCDR2 sequence obtained after the introduction of mutations presented in SEQ ID NO: 117.

In Fig. 9 presents a graph illustrating the neutralizing activity of tocilizumab are reviewed and H3pI/L73 in BaF/gp130.

In Fig. 10 presents a graph illustrating the concentration of tocilizumab are reviewed and H3pI/L73 plasma abacadabra monkeys depending on time after intravenous injection.

In Fig. 11 presents a graph illustrating the concentration of tocilizumab are reviewed and H3pI/L73 in the plasma of mice transgenic for the human receptor, IL-6, depending on time after intravenous injection.

In Fig. 12 is a diagram illustrating the results of the analysis of C-Kon the eve of the heterogeneity of tocilizumab are reviewed (TOCILIZUMAB), TOCILIZUMABΔK and TOCILIZUMABΔGK carried out using cation exchange chromatography.

In Fig. 13 is a diagram illustrating the results of the analysis associated by a disulfide bond heterogeneity of tocilizumab are reviewed-IgG1, tocilizumab are reviewed-IgG2 and tocilizumab are reviewed-SKSC carried out using cation exchange chromatography.

In Fig. 14 is a diagram illustrating denaturation curves for tocilizumab are reviewed-IgG1, tocilizumab are reviewed-IgG2 and tocilizumab are reviewed-SKSC, obtained by differential scanning calorimetry (DSC), and the value of Tm for each Fab-domain.

In Fig. 15 presents a graph illustrating the concentration of tocilizumab are reviewed-IgG1, tocilizumab are reviewed-M44, tocilizumab are reviewed-M58 and tocilizumab are reviewed-M73 in plasma in mice transgenic for human FcRn, depending on time after intravenous injection.

In Fig. 16 presents a graph illustrating the neutralizing activity of tocilizumab are reviewed, control, and Fv5-M83 in BaF/gp130.

In Fig. 17 presents a graph illustrating the neutralizing activity of tocilizumab are reviewed, Fv3-M73, and Fv4-M73 in BaF/gp130.

In Fig. 18 presents a graph illustrating the concentration of tocilizumab are reviewed, control, Fv3-M73, Fv4-M73, and Fv5-M83 at abacadabra monkeys depending on time after intravenous injection.

In Fig. 19 shows a graph illustrating the concentration of CRP for tocilizumab are reviewed, control, Fv3-M73, Fv4-M73 or Fv5-M83 abacadabra the monkeys depending on time after intravenous injection.

In Fig. 20 shows a graph illustrating the percentage of free soluble receptor of IL-6 in abacadabra monkeys depending on time after intravenous tocilizumab are reviewed, control, Fv3-M73, Fv4-M73 or Fv5-M83.

In Fig. 21 shows a graph illustrating the inhibitory effect of tocilizumab are reviewed and Fv4-M73 on the production of MCP-1 from synovial cells of a person suffering from rheumatoid arthritis (RA).

In Fig. 22 shows a graph illustrating the inhibitory effect of tocilizumab are reviewed and Fv4-M73 on the production of VEGF from synovial cells of a person suffering from rheumatoid arthritis (RA).

The method of implementation of the present invention

The present invention relates to the following polypeptides (a)-(f):

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

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

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

(d) the polypeptide comprising CR1, contains the sequence of SEQ ID NO: 10 (CDR1 VL1), CDR2-containing sequence of SEQ ID NO: 11 (CDR2 VL1), and CDR3-containing sequence of SEQ ID NO: 12 (CDR3 VL1);

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

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

The polypeptides according to the invention have no particular restrictions, but, preferably, they are antigennegative molecules with activity aimed at binding with the receptor of IL-6. Such antihistamine molecules are, for example, variable regions of heavy chain antibodies (VH), variable region light chain antibody (VL), the heavy chain antibody, a light chain antibodies and antibodies.

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

These variable regions can be used as part of antibodies against the human receptor of IL-6. Antibodies against ilovecosmo receptor IL-6, using this variable region, have a high binding activity, excellent pharmacokinetic properties, high security, reduced immunogenicity and/or improved physical and chemical properties. In the present invention "excellent pharmacokinetic properties" or "improved pharmacokinetic properties" means any of the properties, such as: lower level clearance (CL)", increasing the "area under the curve (AUC)", increasing the "average residence time" and increasing the "time-life in plasma (t1/2), where these properties are the pharmacokinetic parameters derived from the plasma concentration time with the introduction of antibodies in the body. In the present invention "excellent physico-chemical properties" or "improved physico-chemical properties" means, but is not limited to, increased stability, reduced heterogeneity or similar

Skeleton human antibodies (FR), communicating with CDR, are chosen so that the CDR was formed into a desirable antigennegative website. FR used for variable regions according to the invention, no specific limitation, and can be used any FR; however, preferred is a human FR. Can be used in owani human FR, having a natural sequence. Alternatively, if necessary, in the frame area having a natural sequence can be introduced substitutions, deletions, additions and/or insertions or the like of one or several amino acids, so that the CDR was able to form an adequate antigennegative website. Mutant sequence FR having the desired properties can be selected, for example, by measuring and evaluating the binding activity of the antigen with the antibody having the FR amino acid substitutions (Sato, K.et al., Cancer Res. (1993) 53, 851-856).

In addition, the CDR sequence, described above, can be replaced deleterows, added and/or is embedded one or more amino acids. In this case, preferably, the CDR sequence, after substitutions, deletions, additions and/or insertions of one or several amino acids, had activity equivalent to the activity of the unmodified sequence, CDR, i.e. binding activity and neutralizing activity, as well as have appropriate stability and immunogenicity and/or appropriate pharmacokinetic properties. The number of amino acids, which must be replaced, deleterow, added and/or built, has no particular restriction; however, the number of such amino acids is preferably three or less, valueproposition two or less, and even more preferably one amino acid CDR.

Methods of replacing one or more amino acid residues other interest amino acids 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, M.J., and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 100, 468-500; Kramer, W., Drutsa, V., Jansen, H.W., Kramer, B., Pflugfelder, M., and Fritz, H.J. (1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456; Kramer W., and Fritz H.J. (1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367; Kunkel, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U. S. A. 82, 488-492). This method can be applied for replacement of the necessary amino acids in the antibody other interest amino acids. In addition, the amino acids in the framework regions and CDR can be replaced by the corresponding amino acids by methods using libraries such as permutation circuits in a frame region (Mol. Immunol. 2007 Apr; 44(11):3049-60) and repair CDR (US 2006/0122377).

The present invention also relates to the following antibodies (a)to(c):

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

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

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

The antibodies described above can be used as antibodies against the human receptor of IL-6 with enhanced binding activity, excellent pharmacokinetic properties, high security, low what monogenist and/or improved physical and chemical properties.

Skeleton human antibodies that bind to the CDR according to the invention, are chosen so that the CDR was formed into a desirable antigennegative website. FR used for variable regions according to the invention, no specific limitation, and can be used any FR, however, preferred are human FR. This can be used by the human FR, having a natural sequence. Alternatively, if necessary, in the frame area having a natural sequence can be introduced substitutions, deletions, additions and/or insertions or the like of one or several amino acids, so that the CDR was able to form an adequate antigennegative website. Mutant sequence FR having the desired properties can be selected, for example, by measuring and evaluating the binding activity of the antigen with the antibody having the FR amino acid substitutions (Sato, K.et al., Cancer Res. (1993) 53, 851-856).

While constant region used for antibody according to the invention, no specific limitation, and can be used any constant region. Preferred constant regions used for antibody according to the invention are, for example, a human constant region (constant region derived from IgG1, IgG2, IgG3, IgG4, Cκ, Cλ and p). In human constant regions can be replaced, deleterow, added and/or is embedded one or more amino acids. Preferred human constant regions of the heavy chain are, for example, a constant region containing the amino acid sequence of SEQ ID NO: 31 (constant region VH4-M73), constant region containing the amino acid sequence of SEQ ID NO: 32 (const region VH3-M73), constant region 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, a constant region containing the amino acid sequence of SEQ ID NO: 34 (VL1), a constant region containing the amino acid sequence of SEQ ID NO: 35 (VL3), and a constant region containing the amino acid sequence of SEQ ID NO: 36 (VL5).

In addition, the CDR sequence, described above, can be replaced deleterows, added and/or is embedded one or more amino acids. In this case, preferably, the CDR sequence, after substitutions, deletions, additions and/or insertions of one or several amino acids, had activity equivalent to the activity of the unmodified sequence, CDR, i.e. binding activity and neutralizing activity, and so the e appropriate stability and immunogenicity and/or appropriate pharmacokinetic properties. The number of amino acids, which must be replaced, deleterow, added and/or built, has no particular restriction; however, preferably, such amino acids was three or less, more preferably two or less, and still more preferably one amino acid CDR.

Amino acids may also be replaced deleterows, added and/or embedded methods described above.

The present invention also relates to the following variable fields (a)-(f):

(a) variable regions of the heavy chain that contains the sequence of SEQ ID NO: 19 (variable regions VH4-M73);

(b) variable regions of the heavy chain that contains the sequence of SEQ ID NO: 20 (variable regions VH3-M73);

(c) variable regions of the heavy chain that contains the sequence of SEQ ID NO: 21 (variable regions VH5-M83);

(d) variable region of the light chain containing the sequence of SEQ ID NO: 22 (variable regions VL1);

(e) variable region of the light chain containing the sequence of SEQ ID NO: 23 (variable regions VL3); and

(f) variable region of the light chain containing the sequence of SEQ ID NO: 24 (variable regions VL5).

Variable regions described above can be used as part of antibodies against the human receptor of IL-6. Antibodies against the human receptor, IL-6, in which use is are these variable regions, they have an increased binding activity, excellent pharmacokinetic properties, high security, reduced immunogenicity and/or improved physical and chemical properties.

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

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

In addition, the present invention relates to the following antibodies (a)to(c):

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

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

(c) the antibody that contains a variable region heavy the th chain, including the sequence of SEQ ID NO: 21 (variable region VH5-M83), and variable region light chain comprising the sequence of SEQ ID NO: 24 (variable region VL5).

Variable regions described above can be used as part of antibodies against the human receptor of IL-6. Antibodies against the human receptor, IL-6, which use such variable regions, have increased binding activity, excellent pharmacokinetic properties, high security, reduced immunogenicity and/or improved physical and chemical properties.

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

While constant region used for antibody according to the invention, no specific limitation, and can be used any constant region. Preferred constant regions used for antibody according to the invention are, for example, a human constant region (constant region PR is coming from IgG1, IgG2, IgG3, IgG4, κ chain, chain λ and the like). In human constant regions can be replaced, deleterow, added and/or is embedded one or more amino acids. Preferred human constant regions of the heavy chain are, for example, a constant region containing the amino acid sequence of SEQ ID NO: 31 (constant region VH4-M73), constant region containing the amino acid sequence of SEQ ID NO: 32 (const region VH3-M73), constant region 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, a constant region containing the amino acid sequence of SEQ ID NO: 34 (VL1), a constant region containing the amino acid sequence of SEQ ID NO: 35 (VL3), and a constant region containing the amino acid sequence of SEQ ID NO: 36 (VL5).

The present invention also applies to the following heavy or light chains (a)-(f):

(a) a heavy chain containing the sequence of SEQ ID NO: 25 (VH4-M73);

(b) a heavy chain containing the sequence of SEQ ID NO: 26 (VH3-M73);

(c) a heavy chain containing the sequence of SEQ ID NO: 27 (VH5-M83);

(d) the light chain containing the sequence of SEQ ID NO: 28 (VL1);

(e) the light chain containing the sequence of SEQ ID NO: 29 (VL3); and

(f) light chain, contains the sequence of SEQ ID NO: 30 (VL5).

Heavy and light chains described above, can be used as part of antibodies against the human receptor of IL-6. Antibodies against the human receptor, IL-6, which use such heavy and light chains, have high binding activity, excellent pharmacokinetic properties, high security, reduced immunogenicity and/or improved physical and chemical properties.

Heavy and light chains described above, 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 by three amino acids or less). Methods of replacing one or more amino acid residues other interest amino acids are, for example, the methods described above.

In variable regions, constant regions, or in those and other areas can be substitutions, deletions, additions and/or insertions of one or several amino acids.

The present invention also applies to the following antibodies (a)to(c):

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

(b) anti is Elo, which contains a heavy chain comprising the sequence of SEQ ID NO: 26 (VH3-M73), and light chain comprising the sequence of SEQ ID NO: 29 (VL3); and

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

The above-described antibodies are antibodies against the human receptor, IL-6, which have high binding activity, excellent pharmacokinetic properties, high security, reduced immunogenicity and/or improved physical and chemical properties.

The above-described antibodies may also contain substitutions, deletions, additions and/or insertions of one or more amino acids (e.g., 20 amino acids or less, preferably ten amino acids or less, and more preferably five amino acids or less). Methods of replacing one or more amino acid residues other interest amino acids are, for example, the methods described above.

In variable regions, constant regions, or in those and other areas can be substitutions, deletions, additions and/or insertions of one or several amino acids.

Antibodies according to the invention are preferably humanized antibodies.

Humanized antibodies are also referred to the as "reconstructed human antibodies". This humanitariannet antibody is produced by attaching a hypervariable region (CDR)derived from a mammal, not a man, to a CDR of a human antibody. Standard methods of genetic recombination, used to produce such antibodies, are also known in the art (see European patent application number EP 125023 and WO 96/02576).

In particular, for example, a DNA sequence designed to interest CDR was associated with interest frame region (FR), synthesized by PCR conducted using several oligonucleotides that have an overlapping portion with the ends of the CDR and FR and used as primers (see the method described in WO 98/13388). Humanitariannet antibody is produced by ligating the resulting DNA with DNA encoding a constant region of a human antibody or a modified constant region of human entitle; embedding the specified DNA expression vector; and the introduction of this vector to the owner to produce his antibody (see European patent application number EP 239400 and publication of International patent application no WO 96/02576).

Skeleton human antibodies that bind to the CDR, are chosen so that the CDR was formed into a desirable antigennegative website. Optionally, in karasouli variable regions of the antibodies can be introduced amino acid substitutions, deletions, additions and/or insertions.

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

For example, Cγ1, Cγ2, Cγ3, Cγ4, Cµ, Cδ, Cα1, Cα2, and by cε can be used for the H-chain, and Cκ and Cλ can be used for the L-chain. Amino acid sequence Cκ presented in SEQ ID NO: 38, and the nucleotide sequence encoding this amino acid sequence represented in SEQ ID NO: 37. Amino acid sequence Cγ1 presented in SEQ ID NO: 40, and the nucleotide sequence encoding this amino acid sequence represented in SEQ ID NO: 39. Amino acid sequence Cγ2 presented in SEQ ID NO: 42, and the nucleotide sequence encoding this amino acid sequence represented in SEQ ID NO: 41. Amino acid sequence Cγ4 presented in SEQ ID NO: 44, and the nucleotide sequence encoding this amino acid sequence represented in SEQ ID NO: 43.

In addition, human antibodies can be modified in order to increase the stability of the antibody or stability of the antibody when e is about producing. For humanization of antibodies can be used by the human antibodies of all isotypes, such as IgG, IgM, IgA, IgE or IgD, however, in the present invention preferably uses IgG. As IgG can be used IgG1, IgG2, IgG3, IgG4 or other

After obtaining antibodies amino acids in the variable regions (e.g., CDR and FR) and constant region gumanitarnogo antibodies can be deleterows, added, built, and/or replaced. Antibodies according to the invention also include humanized antibodies, which contain amino acid substitutions, etc.

Antibodies according to the invention are not only bivalent antibodies, representing IgG, but also monovalent antibodies and multivalent antibodies, representing IgM, provided that they possess the activity of the receptor binding of IL-6 and/or neutralizing activity. Multivalent antibodies according to the invention are multivalent antibodies, in which all antigennegative sites are identical, and multivalent antibodies in which some or all antigennegative sites differ 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 are associated with the protein receptor of IL-6.

Mini-antibodies are antibodies, including fragments of antibodies, in which no part of a whole antibody (for example, whole IgG or the like), and such antibodies have no particular restrictions, provided that they possess the activity of the receptor binding of IL-6 or neutralizing activity, and contain a fragment antibodies in which no part of a whole antibody (for example, whole IgG or the like). Mini-antibodies according to the invention have no particular restrictions, provided that they contain a part of the whole antibody. However, mini-antibodies preferably contain a VH or VL, and particularly preferably VH and VL. Other preferred mini-antibodies according to the invention are, for example, mini-antibodies containing the CDRs of the antibody. Mini-antibodies can contain all six CDRs of the antibodies or some of these CDR.

Mini-antibodies according to the invention preferably have a lower molecular weight than whole antibodies. However, mini-antibodies can form multimer, for example, dimers, trimers or tetramer, and therefore their molecular weight is sometimes greater than the molecular weight of whole antibodies.

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

Antibody fragments can be obtained, for example, by treating the antibody enzymes by producing fragments of antibodies. Enzymes, which are known, can be used to produce fragments of antibodies are, for example, papain, pepsin, and plasmin. Alternative gene encoding such an antibody fragment may be constructed, introduced into the expression vector and expressed in the appropriate cells of the host (see, for example, Co, M. S.et al., J. Immunol. (1994) 152, 2968-2976; Better, M. &Horwitz, A. H. 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, R. E.et al., TIBTECH (1991) 9, 132-137).

Gidrolizuemye enzymes break down the fragment antitela in specific sites, resulting in formation of antibody fragments with specific structures described below. For deletions, any part of the antibodies can be used in methods of genetic engineering that can be applied to such enzyme produced fragments of antibodies.

Fragments of antibodies obtained using the above gidroliznaya enzymes are the following pieces:

Hydro is of papain: F(ab) 2or Fab

Hydrolysis by pepsin: F(ab')2or Fab'

Hydrolysis by plasmin: Facb

Mini-antibodies according to the invention are antibody fragments lacking any region, provided that they possess the activity of the receptor binding of IL-6 and/or neutralizing activity.

The term "venticello" means a bivalent fragment antibodies constructed by concatenating genes (Holliger P.et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; EP 404097; WO 93/11161, etc). Venticelli are dimers consisting of two polypeptide chains. In each of the polypeptide chains forming dimer, VL and VH are usually linked by the linker in the same chain. In General, the linker in denticle is quite short, and therefore VL and VH can not 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, are present on the same polypeptide, can't form a single-chain fragment variable region, and will form a dimer with another single-chain fragment variable region. As a result, this dianthicola has two antigenspecific site.

The scFv antibodies are single-chain polypeptides, obtained by joining VH and VL via a linker or the like (Hston, J. S.et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883; Pluckthun “The Pharmacology of Monoclonal Antibodies” Vol. 113, eds., Resenburg & Moore, Springer Verlag, New York, pp. 269-315, (1994)). V region of H chain and the V region of the L-chain scFv can occur from any of the antibodies described in this application. The peptide linker for linking the V-regions has no specific limitations. So, for example, the linker can be any single-stranded peptide containing from about 3 to 25 residues. In particular, can be used peptide linkers described below, or other

V-area of the two chains can be attached, for example, by using PCR, as described above. First, to link V-regions using PCR as the template using a DNA encoding a full-sized amino acid sequence or the desired partial amino acid sequence encoded by one of the DNA, presented below, namely a DNA sequence that encodes the H chain or the V region of the H-chain antibodies, and DNA encoding the L chain or the V region of the L-chain antibodies.

DNA encoding the V region of the H-chain or L-chain, amplified by PCR using pairs of primers containing sequences corresponding to the two ends amplificare DNA. Then get a DNA encoding part of the peptide linker. DNA encoding a peptide linker can also be synthesisof is on by PCR. The nucleotide sequence, which can be used to attach a separately synthesized products of amplification of the V-region, attached to the 5'-end of primers used. This was followed by PCR using each DNA chain [DNA V-N-chain]-[DNA peptide linker]-[DNA V-region L-chain] and assemble PCR primers.

Collected PCR primers contain a combination of primer, which hybridizes with the 5'-end of the molecule [DNA V-N-chains], and primer, which hybridizes with 3'-end of the molecule [DNA V-region L-chain]. In other words, the collected PCR primers are a set of primers that can be used for amplification of DNA encoding the full-size sequence, synthesized scFv. In addition, nucleotide sequence, which can be used to join each DNA V-region, attached to the molecule [DNA peptide linker]. Then these DNA attached to each other, after which the entire scFv, ultimately, is produced as the product of amplification using the collected PCR primers. After production of scFv-encoding DNA can be obtained expression vectors containing the DNA, and can be produced recombinant cells transformed with these expression vectors, with application of the standard is x methods. Then scFv can be obtained by the expression of scFv-encoding DNA by culturing the obtained recombinant cells.

The procedure for joining VH and VL has no specific limitation, and therefore, they can be in any order. Examples of such locations are presented below:

[VH]-linker-[VL]

[VL]-linker-[VH].

sc(Fv)2represents a single-stranded mini-antibody obtained by joining two VH and two VL using linkers etc. (Hudsonet al., 1999, J. Immunol. Methods 231:177-189). sc(Fv)2can be obtained, for example, by attaching scFv using the linker.

Preferably two VH and two VL of the antibody are as follows: VH, VL, VH, and VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]), starting from N-end single-chain polypeptide, however, the order of the two VH and two VL is not limited to the above order, and they can appear in any order. Examples of such locations 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]

Amino acid sequence of VH or VL mini-antibodies may contain substitutions, deletions, additions and/or insertions. In addition, if collected VH and VL have antigennegative the th activity, a part of them can be demeterova, or can be added to other polypeptides. In addition, the variable regions can be chimeric or humanitarianism.

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

Preferred linkers according to the invention are peptide linkers. The length of the peptide linkers has no particular restrictions, and the person skilled in the art can choose the length of the linker depending on the purpose of its application. 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, and particularly preferably 12 to 18 amino acids (for example, 15 amino acids).

So, for example, amino acid sequences of peptide linkers include the following sequence:

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 I NO: 47]) n

(Ser Gly Gly Gly Gly [SEQ ID NO: 48])n,

where n is an integer of 1 or more.

Amino acid sequence of peptide linkers can be appropriately selected by a specialist, depending on their goals. So, for example, above the number "n"which indicates the length of the peptide linker, usually equal to 1-5, preferably 1-3, more preferably 1 or 2.

Synthetic linkers (chemical cross-binding agents are, for example, cross-communicating agents commonly used for cross-linking peptides, for example,N-hydroxysuccinimide (NHS), disuccinimidyl (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylester) (DSP), dithiobis(sulfosuccinimidyl) (DTSSP), bis(Succinimidyl) of ethylene glycol (EGS), bis(sulfosuccinimidyl) of ethylene glycol (sulfo-EGS), disuccinimidyl (DST), desulfobacteriaceae (sulfo-DST), bis[2-(succinyldicholine)ethyl]sulfon (BSOCOES), and bis[2-(sulfosuccinimidyl)ethyl]sulfon (sulfo-BSOCOES). Such cross-communicating agents are commercially available.

In General terms, to link the four variable regions of the antibodies to three linker. Many of these linkers may be the same or different.

Antibodies according to the invention are antibodies, amino acid sequence which have been added one or more amino acid residues. In addition, the antibodies according to the invention are hybrid proteins in which the above-described antibody linked to another peptide or protein. The hybrid protein can be obtained by ligating polynucleotide encoding the antibody according to the invention, and polynucleotide encoding another peptide or polypeptide with preservation of the reading frame, the introduction of such a hybrid expression vector and its expression in the host. This can be applied to methods known in the art. A peptide or polypeptide, forming a hybrid with the antibody according to the invention may be a known peptide, for example, FLAG (Hopp, T. P.et al., BioTechnology 6, 1204-1210 (1988)), 6×His consisting of six His residues (histidine), 10×His, the hemagglutinin of influenza virus (HA), a fragment of the human c-myc, fragment, VSV-GP fragment p18HIV, T7-tag, HSV-tag, E-tag, a fragment of the T-antigen of SV40, lck tag, a fragment of α-tubulin, B-tag, and a fragment of protein C Polypeptides, forming a hybrid with the antibodies according to the invention are, for example, GST (glutathione-S-transferase), HA (hemagglutinin of influenza virus), the constant region of immunoglobulins, β-galactosidase and MBP (protein binding to maltose). Commercial the Eski available polynucleotide, encoding such peptides or polypeptides, may form a hybrid with polynucleotides coding for the antibody according to the invention. The hybrid polypeptide can be obtained by the expression thus obtained hybrid polynucleotide.

In addition, the antibodies according to the invention may also be conjugated to antibodies associated with 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 produced antibodies. Methods of modification of antibodies are well known in the art (see, for example, U.S. patent No. 5057313 and 5156840). The term "antibody" according to the invention also includes the conjugated antibodies.

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

In addition, the antibodies used in the present invention, can be bespecifically antibodies. The term "bespecifically antibody" means an antibody with variable regions that recognize different epitopes of the same molecule antibodies. Bespecifically antibody according to the invention can be bespecifically antic the lo, recognize different epitopes on the receptor molecule IL-6, or bespecifically antibody in which one of antigenspecific sites recognizes the receptor for IL-6, and the other antigennegative site recognizes another substance. Examples of antigens that bind with other antigennegative site especifismo antibody, which comprises the antibody according to the invention that recognizes the receptor for IL-6, are IL-6, TNF-α, TNFR1, TNFR2, CD80, CD86, CD28, CD20, CD19, IL-1α, IL-β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxin-α, lymphotoxin-β 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 of producing bespecifically antibodies known in the art. Bespecifically antibodies can be obtained, for example, by linking the two types of antibodies that recognize different antigens. These bound antibodies can be half of the molecule, each of which contains the H-chain and L-chain, or a quarter of a molecule containing only one N-chain. Alternative merged cells producing bespecifically antibodies can be obtained by merging the hybridomas producing different monoclonal antibodies. In addition, bespecifically antibodies can be obtained by genetic engineering methods.

As described below, the antibodies according to the invention may vary according to the AMI amino acid sequences, molecular mass, isoelectric point, the presence/absence of sugar chains, and conformation, depending on the cleaning method or type of cell or host used to produce antibodies. However, the present invention includes any of the obtained antibody, provided that it is functionally equivalent to the antibody according to the invention. For example, if the antibody according to the invention is expressed in prokaryotic cells, for example, inEscherichia colito N-end amino acid sequence of the original antibody attached meinenemy balance. Such antibodies are antibodies according to the invention.

Polypeptides antibodies against the receptor for IL-6, etc. according to the invention can be obtained by methods known to experts.

Antibody against the receptor of IL-6 can be obtained, for example, known methods of genetic recombination on the basis of the sequence obtained antibodies against the receptor for IL-6. In particular, the antibody against the receptor of IL-6 can be obtained by constructing the antibody-coding polynucleotide on the basis of sequence antibodies that recognize the receptor for IL-6, embed this polynucleotide in the expression vector and its expression in the appropriate cell host (see, for example, Co, M. S.et al., J. Immunol. (1994 152, 2968-2976; Better, M. and Horwitz, A. H., 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, R. E. & Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

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

More specifically, the present invention relates to methods of producing the polypeptide according to the invention, where the method involves the following stages:

(a) culturing the host cell containing the vector, which is embedded in the gene encoding the polypeptide according to the invention; and

(b) obtaining the polypeptide encoded by that gene.

Examples of vectors are vectors of type M13 vectors of type pUC, pBR322, pBluescript, and pCR-Script. Alternatively, if you want to subclinical and cut cDNA, in addition to the above vectors can be used and other vectors, including, for example, pGEM-T, pDIRECT, and pT7. Expression vectors are particularly suitable for producing antibodies according to the invention. For example, if the expression vector is used to expre is in these E. colisuch a vector must have distinctive features that this vector to amplificates inE. coli. In addition, if the owner is theE. colisuch as JM109, DH5α, HB101, or XL1-Blue, it is important that this vector contains a promoter that allows this vector efficiently expressed inE. colifor example, lacZ promoter (Wardet al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Betteret al., Science (1988) 240, 1041-1043), T7 promoter or the like Such 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 the RNA polymerase T7).

In addition, plasmid expression vectors can contain a signal sequence for secretion of antibodies. For producing periplasmE. colias a signal sequence for secretion of antibodies, can be used to signal the pelB sequence (Lei, S. P.et al., J. Bacteriol. (1987) 169, 4379). Vectors can be introduced into cells of the host, for example, methods using calcium chloride or electroporation methods.

In addition to vectors forE. coli, vectors for producing antibodies according to the invention are, for example, expression vectors mammals (for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. (1990) 18(17), p5322), pEF, and pCDM8), expression ve the Torah insect cells (for example, baculovirus expression system Bac-to-BAC" (Gibco-BRL) and pBacPAK8), expression vectors of plants (for example, pMH1 and pMH2), expression vectors of animal viruses (for example, pHSV, pMV, and pAdexLcw), expression vectors retroviruses (for example, pZIPneo), expression vectors, yeast (for example, "set for expression inPichia" (Invitrogen), pNV11, and SP-Q01), and expression vectorsBacillus subtilis(for example, pPL608 and pKTH50).

If the plasmid expression vector used for expression in animal cells such as CHO cells, COS, and NIH3T3, it should have a promoter necessary for expression in such cells, for example, SV40 promoter (Mulliganet al., Nature (1979) 277, 108), the promoter MMLV-LTR, EF1α promoter (Mizushimaet al., Nucleic Acids Res. (1990) 18, 5322), or CMV. Another preferred vector is a vector having a gene for selection of transformed cells (for example, a gene of resistance to the drug, which can be identified using the appropriate tools (neomycin, G418, or the like). Vectors having such properties are, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

In addition, if you want to stably Express the genes and to amplify the number of copies of genes in cells, the method can be applied, in which CHO cells, deficient in cascade reactions for the synthesis of nucleic acids, are administered together with the vector in the region have the DHFR gene, which compensates for this 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 necessary transient expression of a gene, the method can be applied, in which COS cells, bearing on its chromosome a gene expressing T-antigen SV40 transform the vector carrying the origin of replication of SV40 (pcD etc). Can be used originy replication originating from polyomavirus, adenovirus, virus, bovine papillomavirus (BPV), etc. in Addition, amplification of the number of gene copies in the lines of the host cells, expression vectors can contain a gene for aminoglycoside transferase (APH)gene timedancing (TK), gene kantenwein-phosphoribosyltransferaseE. coli(Ecogpt)gene dihydrofolate-reductase (dhfr), etc. is used as a selective marker.

The obtained antibodies according to the invention can be isolated from host cells or their environment (environment or the like) and purified with obtaining 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, ultra is filtering, salting out, precipitation with a solvent, solvent extraction, distillation, immunoprecipitation, polyacrylamide gel electrophoresis with LTOs, isoelectric focusing, dialysis, recrystallization, etc.

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. Marshaket al., Cold Spring Harbor Laboratory Press, 1996). Such chromatographic methods can be carried out using liquid chromatography, for example, HPLC and GEHB. Columns used for affinity chromatography are columns with protein a and column G-protein. Examples of columns with protein And are speakers with Hyper D, POROS and separate FF (GE Amersham Biosciences). The present invention also includes antibodies to the high degree of purification obtained by these methods of cleaning.

The binding activity of the obtained antibodies to the receptor of IL-6 can be measured by methods known in the art. Measurement methods antigennegative 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 used in the research Institute for enzyme immunoassay, in tablets, coated antigen, add the antibody-containing samples, such as purified antibodies and supernatant cultures antibody-producing cells. Then add the second antibody labeled with an enzyme such as alkaline phosphatase, and tablets incubated. After wash add substrate of the enzyme, such as p-nitrophenylphosphate, and read the optical density to estimate antigennegative activity.

The pharmaceutical composition

The present invention also relates to pharmaceutical compositions containing the above-described polypeptide as an active ingredient. The pharmaceutical compositions according to the invention can be used for the treatment of IL-6-associated diseases, such as rheumatoid arthritis. Thus, the present invention also relates to a means for treating diseases, such as rheumatoid arthritis, where these tools 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, a disease of Caslano, systemic lupus erythematosus (SLE), lupus nephritis, a disease of the Rhone, lymphoma, ulcerative colitis, anemia, vasculitis, disease Kawasaki, of still's disease, amyloidosis, multiple sclerosis, a disease associated with transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetic nephropathy, graft-versus-host (GVHD), endometriosis, hepatitis (NASH), myocardial infarction, atherosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin lymphoma, pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cachexia in cancer, neuroinvasive in cancer, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, proveit, uveitis anterior lobe, intermediate uveitis, scleritis, keratitis, inflammation of the eyes narit optic nerve, diabetic retinopathy, proliferative retinopathy vitreous body, the syndrome of "dry eye" and postoperative inflammation.

The expression "contains antibody against the receptor of IL-6 as an active ingredient"means, this tool contains an antibody against the receptor of IL-6 as at least one of the active ingredients in quantity without a specific limitation. In addition, the pharmaceutical compositions according to the invention can 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 preventive purposes.

The polypeptides according to the invention can be obtained by standard methods (see, for example, the guide Remington''s Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). If necessary, such polypeptides may contain pharmaceutically acceptable carriers and/or additives. For example, they may contain detergents (e.g., PEG and twin), fillers, antioxidants (e.g. ascorbic acid), colorants, flavors, preservatives, stabilizers, sautereau substances (for example, phosphoric acid, citric acid and other organic acids), hepatoblastoma substances (e.g., EDTA), suspendresume agents, agents, giving isotonicity, binders, dezintegriruetsja substances, oil, stimulants fluidity and corrigentov. However, the agents according to the invention, used for the prevention or treatment of vos is Alitalia diseases, not limited to the above-mentioned agents and may optionally contain other standard media. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium-containing carmellose, sodium-containing carmellose, hydroxypropylcellulose, hypromellose, diethylaminoacetate polyvinylacetate, polyvinylpyrrolidone, gelatin, triglyceride fatty acids with medium chain length, gidrirovannoe a polyoxyethylene castor oil 60, sucrose, carboxymethylcellulose, corn starch and inorganic salts. They may also contain other low molecular weight polypeptides; proteins, such as serum albumin, gelatin and immunoglobulin; and amino acids. Upon receipt of aqueous solutions for injection, antibodies against the receptor for IL-6 is dissolved, for example, 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, and the like), polyhydric alcohols (propylene glycol, PEG, etc.) and nonionic surfactants (Polysorbate 80 and HCO-50) can be combined with each other.

If necessary, the polypeptides can be encapsu iravani in microcapsules (microcapsules, made from hydrocellulose, gelatin, poly(methylaniline) and the like), or they can be included in the colloidal system for drug delivery (liposomes, albumen microspheres, microemulsions, nanoparticles, nanocapsules and the like (see, for example, “Remington''s Pharmaceutical Science 16th edition”, Oslo Ed. (1980)). In addition, experts of the known methods of obtaining drugs in the form of sustained-release preparations, and such methods can be applied to obtain polypeptides (Langeret al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12:98-105; U.S. patent No. 3773919; European Patent Application (EP) No. 58481; Sidmanet al., Biopolymers (1983) 22:547-56; EP No. 133988). In addition, the volume of liquid for subcutaneous injection can be increased by adding hyaluronidase to the tool or mixing with it (see, for example, WO 2004/078140).

The pharmaceutical compositions according to the invention can be administered orally or parenterally, and preferably parenterally. In particular, these compositions can be administered to the patient by injection or percutaneous. Such injections are, for example, systemic and local injections, administered intravenously, intramuscularly or subcutaneously, etc. of the Composition can be injected locally in the area under treatment, or in the peripheral region, in particular, by intramuscular injection. Medication is nymi forms for percutaneous introduction are for example, ointments, gels, creams, poultices and plasters, which can be entered locally or systemically. In addition, the routes of administration can be appropriately selected depending on the patient's age and symptoms. Injected dose may, for example, be in the range from 0.0001 mg to 100 mg of active ingredient per kg of body weight for each injection. Alternatively, when the introduction of the compositions of the person, the dose of active ingredient may comprise, for example, in the range from 0.001 to 1000 mg per kg of body weight for each patient. The dose for a single administration preferably contains, for example, the antibody according to the invention in quantities of from about 0.01 to 50 mg/kg of body weight. However, the dosage of the antibodies according to the invention is not limited to these doses.

Amino acids in the amino acid sequences according to the invention, can be excision of modified (for example, specialists are well known modification of N-terminal glutamine education pyroglutamic acid, by means of programiirovaniya). Needless to say that such excision of modified amino acids included in the amino acid sequences according to the invention.

In addition, the sugar chain-related antibodies according to the invention, may be part of the structure. Sugar chain at position 297 (in accordance with the European numbering system (EU)may have any structure (preferably fokusirovanie sugar chain, or the sugar chain may be linked to an antibody (for example, this can be achieved by producing antibodies inEscherichia colior by introducing a modification in which a sugar chain attached at position 297, in accordance with the EU).

All documents cited here are entered into the present description by reference.

Examples

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

Was designed library of CDR sequences that have been introduced mutations that were analyzed for their ability to increase the affinity of tocilizumab are reviewed (H-chain wild-type (WT)-IgG1/SEQ ID NO: 53; L-chain wild-type (WT)-Kappa/SEQ ID NO: 54) in relation to the receptor of IL-6. Screening of libraries of mutations in the CDR revealed mutations that increase the affinity of the receptor for IL-6. These mutations are shown in Fig. 1. The combination of these mutations was possible to obtain high-affinity tocilizumab, such as RDC-23 (H-chain RDC23H-IgG/SEQ ID NO: 55; L-chain RDC-23L-Kappa/SEQ ID NO: 56). A comparison was made between affinely to the soluble receptor of IL-6 and of the biological activities of the RDC-23 and tocilizumab are reviewed, defined using BaF/gp130 (see comparative examples for this method).

The result of measuring the affinity is presented in table 1. The result of the determination of biological activity conducted using BaF/gp130 (the final concentration of IL-6 was 30 ng/ml), shown in Fig. 2. The results showed that, compared with tocilizumab are reviewed affinity RDC-23 was approximately 60-fold higher and the activity expressed as the concentration that gives 100% inhibition BaF/gp130, was higher by about 100 times.

Table 1
ka(1/MS)kd(1/s)KD(M)
Tocilizumab4,9E+052,0E-034,0E-09
RDC-236,4E+054,3E-056,7E-11

[Example 2]. Identification of mutations that contribute to improving the pharmacokinetic properties of tocilizumab are reviewed, by reducing the magnitude of his and elektricheskoi point

To improve the pharmacokinetic properties of tocilizumab are reviewed study was conducted, the purpose of which is to identify the sites of mutations contributing to the reduction of the isoelectric point of the variable regions without significant reduction in the level of the receptor binding of IL-6. Screening site mutations in the variable regions that were predicted based on models of three-dimensional structure of tocilizumab are reviewed, identified mutation sites, contributing to the reduction of the isoelectric point of the variable regions without significantly decreasing its binding to the receptor of IL-6. The data shown in Fig. 3. The combination of these mutations allowed to receive tocilizumab with a reduced value of the 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 between affinely to the soluble receptor of IL-6, the values of isoelectric points, pharmacokinetic properties in mice and biological activities for H53/L28 and tocilizumab are reviewed, defined using BaF/gp130 (see comparative examples for this method).

The result of measuring the affinity is presented in table 2. The result of the determination of biological activity conducted using BaF/gp130 (the final concentration of IL-6 was 30 ng/ml), shown in Fig. 4. The results showed that compared the Oia with tocilizumab are reviewed by the affinity of H53/L28 was about six times higher and the activity expressed as the concentration that gives 100% inhibition BaF/gp130, was several times higher.

Table 2
ka(1/MS)kd(1/s)KD(M)
Tocilizumab4,9E+052,0E-034,0E-09
H53/L287,6E+055,2E-046,8E-10

The results of determining the value of the isoelectric point known by electrophoresis with isoelectric focusing showed that the isoelectric point of tocilizumab are reviewed and H53/L28 was about to 9.3 and 6.5 and 6.7, respectively. Thus, the isoelectric point of H53/L28 was below the isoelectric point of tocilizumab are reviewed approximately 2.7. In addition, theoretical isoelectric point of the variable regions VH/VL was calculated using GENETYX (GENETYX CORPORATION). The result showed that theoretical isoelectric point of tocilizumab are reviewed and H53/L28 was 9,20 and to 4.52, respectively. Thus, the isoelectric point of H53/L28 was below the isoelectric point is of tocilizumab are reviewed approximately 4.7.

To evaluate the pharmacokinetic properties of the modified antibody H53/L28, which has a lower value of the isoelectric point, we compared the pharmacokinetic properties of tocilizumab are reviewed and H53/L28 in normal mice. To evaluate the dependence of the concentration of tocilizumab are reviewed or H53/L28 in plasma from time to mice (C57BL/6J; Charles River Japan, Inc.) intravenous (i.v.) or subcutaneously (s.c.) was administered one dose of tocilizumab are reviewed or H53/L28 in the amount of 1 mg/kg concentration Dependence of tocilizumab are reviewed and H53/L28 in the plasma of time after intravenous or subcutaneous injection shown in Fig. 5 and 6, respectively. Pharmacokinetic parameters (clearance (CL) and half-life (T1/2))obtained using WinNonlin (Pharsight), are presented in table 3. The half-life (T1/2) H53/L28 in plasma after intravenous injection of approximately 1.3 times the half-life of tocilizumab are reviewed, and clearance was decreased by approximately 1.7 times. The half-life (T1/2) H53/L28 after subcutaneous injection approximately two times the half-life of tocilizumab are reviewed, and clearance was decreased by approximately 2.1 times. Thus, it was found that the pharmacokinetic properties can be significantly improved by reducing the value of the isoelectric point of tocilizumab are reviewed after the introduction of amino acid substitutions.

Table 3
i.v.s.c.
CL, ml/h/kgT1/2dayCL/F, ml/h/kgT1/2day
Tocilizumab0,17718,50,1814,7
N/L28is 0.10223,50,08629,7

[Example 3] the identification of the sites of mutations that reduce the immunogenicity of tocilizumab are reviewed

Identification of mutations that contribute to reducing the risk of immunogenicity of T-cell epitopes present in variable fields

T-cell epitopes present in the sequence of variable regions of tocilizumab are reviewed, analyzed using TEPITOPE (Methods. 2004 Dec; 34(4):468-75). It was suggested that CDR2 L-chain has many T-cell epitopes, which must contact the HLA (that is, this circuit has the sequence responsible for the high risk of immunogenicity). Thus, the analysis was performed TEPITOPE to assess and amino acid substitutions, which will reduce the risk of immunogenicity CDR2 L-chain, but will not reduce the stability, activity of the binding or neutralizing activity.

As described below, the results of the screening showed that the risk of immunogenicity can be reduced without reducing the stability, activity of the binding or neutralizing activity by replacement of threonine at position L51 (numbering according to Kabat; Kabat E.A.et al., (1991) Sequences of Proteins of Immunological Interest, NIH)) CDR2 L-chain (SEQ ID NO: 59) of tocilizumab are reviewed for glycine, and arginine in position L53 to glutamic acid (SEQ ID NO: 60):

CDR2 L-chain of tocilizumab are reviewed (SEQ ID NO: 59)

CDR2 L-chain of tocilizumab are reviewed with remote T-cell epitopes (SEQ ID NO: 60)

[Example 4]. Reducing the risk of immunogenicity by full humanization of frame sequences of the variable regions of tocilizumab are reviewed

In the method of humanization of tocilizumab are reviewed some murine sequences left in the frame sequence in order to preserve the 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 (numbering according to Kabat; Kabat E.A.et al., (1991) Sequences of Proteins of Immunological Interest, NIH)) sequence variable regions of tocilizumab are reviewed. Murine sequences that were saved, can increase the risk of immunogenicity. Thus, the analysis was performed in order on the identify, can fully humanitariannet frame sequence to further reduce the risk of immunogenicity of tocilizumab are reviewed.

The results showed that the entire frame sequence of tocilizumab are reviewed can be completely humanitarian without compromising stability, binding activity or neutralizing activity by replacing FR1 H-chain (SEQ ID NO: 61) of tocilizumab are reviewed on humanitarian FR1-A H-chain (SEQ ID NO: 62), shown below, and replace FR3 H-chain (SEQ ID NO: 63) at humanitarian FR3 H-chain (SEQ ID NO: 64), is presented below:

FR1 H-chain of tocilizumab are reviewed (SEQ ID NO: 61)

Humanitariannet FR1-A H-chain (SEQ ID NO: 62) (derived from the germline of the IMGT hVH_4)

FR3 H-chain of tocilizumab are reviewed (SEQ ID NO: 63)

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

[Example 5]. Identification of mutation sites in order to improve the pharmacokinetic properties carried out on the basis of pH-dependent binding of tocilizumab are reviewed with the receptor of IL-6

One of the ways to improve the pharmacokinetic properties of tocilizumab are reviewed is to improve the properties of the molecule, so that one molecule of tocilizumab are reviewed could several times to contact the receptor of IL-6 and neutralize several molecules of the receptor for IL-6. It has been suggested that after binding to the receptor of IL-6 membrane-type tocilizumab is absorbed NR is triketone endosomes through internalization upon binding with the receptor of IL-6 membrane-type, and then, when binding to a receptor of IL-6 membrane type, it is transferred to a complementary mechanism and decomposes these lysosomes. In particular, one molecule of tocilizumab are reviewed usually associated with one or with two molecules of receptor IL-6 membrane-type (for monovalent or divalent mechanism) and degraded in the lysosomes after internalization. Therefore, one molecule of tocilizumab are reviewed can communicate with only one or two molecules of receptor IL-6 membrane type and neutralize only one or two such molecules.

Thus, the authors of the present invention believe that if you receive tocilizumab are reviewed, which binds depending on pH, where the specified binding of tocilizumab are reviewed supported under neutral conditions, but significantly reduced in acidic conditions, tocilizumab, which is bound depending on the pH, will dissociates of the receptor of IL-6 membrane-type (antigen) in the endosomes and back into the plasma by binding to FcRn present in endosomes, as illustrated in Fig.7. After returning to the plasma tocilizumab, the binding of which depends on the pH, can again be contacted with the receptor of IL-6 membrane type. Obviously, the re-binding in plasma and dissociation in endosomes one molecule of tocilizumab are reviewed several times to contact carried alkemi molecules of the receptor of IL-6/neutralize several molecules of the receptor for IL-6. Thus, it is believed that tocilizumab, the binding of which depends on pH, has improved pharmacokinetic properties compared with standard tocilizumab are reviewed.

In order tocilizumab dissociatively of the receptor of IL-6 in the acid conditions in endosome, tying in acidic conditions should be significantly weaker than in neutral conditions. On the cell surface to neutralize the need of a strong binding with the receptor of IL-6, and therefore, at a pH of 7.4, which is a pH cell surface antibody should bind to the receptor of IL-6 with the same strength as tocilizumab, or more. It was reported that the pH in endosome is usually 5.5 to 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 mitigate its binding to the receptor of IL-6 at pH 5.5 to 6.0, then you can assume that it will dissociates of the receptor of IL-6 in acidic conditions in the endosome. In particular, if tocilizumab, the binding of which depends on pH, has been modified to enhance its binding to the receptor at pH of 7.4, which is the pH of the cell surface, and in order to mitigate its binding to the receptor of IL-6 at pH 5.5 to 6.0, which is endosomal pH, one molecule of tocilizumab are reviewed can communicate with multiple molecules of the receptor of IL-6 and neutralize several molecules of the receptor for IL-6, and so it may have improved pharmacokinetic properties.

A possible method for messages to tocilizumab are reviewed abilities to pH-dependent binding with the receptor of IL-6 is the introduction of a his-tag residues in the variable region of tocilizumab are reviewed, since the pKa of his-tag remnant is approximately 6,0-6,5, and its levels dissociation of protons in a neutral conditions (pH of 7.4) and acidic conditions (pH 5.5 to 6.0) differ. Thus, was screened to identify the sites of injection of histidine in variable areas on the basis of three-dimensional structural model of tocilizumab are reviewed. In addition, the selected sequences of the variable regions of tocilizumab are reviewed were made arbitrary replacement of histidine in order to design libraries for screening. The screening was carried out by the receptor binding of IL-6 at pH 7.4 and dissociation from the receptor, IL-6, or by reducing the affinity at pH 5.5 to 5.8, where these parameters are used as indicators.

As a result, the authors of the present invention were identified sites of mutation, indicating to the tocilizumab are reviewed the ability of pH-dependent binding with the receptor of IL-6 (ability to communicate at a pH of 7.4 and dissociates at pH 5.8). The results are presented in Fig. 8. In Fig. 8 replacement of tyrosine histidine in position H27 is a modification in FR1 H-chain, but not in the CDR. One is to, as described in the publication Eur. J. Immunol. (1992) 22:1719-1728, a sequence with a histidine in position H27 is the human sequence (SEQ ID NO: 65). Thus, this antibody can be completely humanitarian in the following frame sequence in combination with humanization described in example 4, to obtain the humanized FR1-B H-chain (SEQ ID NO: 65).

The combination of mutations, including, for example, H3pI/L73 (H-chain H3pI-IgG1/SEQ ID NO: 66; L-chain L73-Kappa/SEQ ID NO: 67), may inform the tocilizumab are reviewed the ability of pH-dependent binding. H3pI/L73 and tocilizumab compared according to their affinity to the soluble receptor of IL-6 at pH of 7.4, the rate of dissociation from the receptor of IL-6 membrane-type at pH 7.4 and pH 5.8, biological activity, measured using BaF/gp130, and pharmacokinetic properties abacadabra monkeys and mice, transgenic for the human receptor of IL-6 (see comparative examples for this method).

The results of the analysis on the affinity of binding to the soluble receptor of IL-6 at pH of 7.4 are presented in table 4. The results of tests for biological activity conducted using BaF/gp130 (the final concentration of IL-6 is equal to 30 ng/ml), shown in Fig. 9. These results showed that the binding affinity of H3pI/L73 with soluble receptor of IL-6 at pH 7.4 and activity, measured using BaF/gp130 were comparable to the specified parameters for tocilizumab are reviewed.

Table 4
ka(1/MS)kd(1/s)KD(M)
Tocilizumab5,1E+051,0E-032,1E-09
H3pI/L735,4E+057,4E-041,4E-09

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

Table 5
a pH of 7.4
kd(1/s)
pH 5.8
kd(1/s)
Kd(rn,8)/ Kd(rn,4)
the pH-dependence
Tocilizumab2,5E-042,5E-041,00
H3pI/L732,6E-046,7E-042,59

To evaluate the dependence of the concentration of tocilizumab are reviewed or H3pI/L73 plasma from time abacadabra monkeys intravenously was administered one dose of tocilizumab are reviewed or H3pI/L73 in the amount of 1 mg/kg concentration Dependence of tocilizumab are reviewed or H3pI/L73 in the plasma of time after intravenous injection is shown in Fig. 10. The results showed that abacadabra monkeys pharmacokinetic properties H3pI/L73 were significantly better than the pharmacokinetic properties of tocilizumab are reviewed.

To evaluate the dependence of the concentration of tocilizumab are reviewed or H3pI/L73 plasma from time to mice transgenic for the human receptor of IL-6 (hIL-6R-transgenic mice, Proc Natl Acad Sci U S A. 1995 May 23; 92(11):4862-6), intravenously was administered one dose of tocilizumab are reviewed or H3pI/L73, comprising 25 mg/kg concentration Dependence of tocilizumab are reviewed or H3pI/L73 in the plasma of time after intravenous injection is shown in Fig. 11. The results showed that in mice transgenic for the human receptor, IL-6, pharmacokinetic properties H3pI/L73 were significantly better than the pharmacokinetic properties of tocilizumab are reviewed.

H3pI/L73, which represents tocilizumab which is capable of pH-dependent binding was found significantly better pharmacokinetic properties soba is podobnych monkeys and mice, transgenic human receptor, IL-6, compared with tocilizumab are reviewed. This suggests that one molecule can bind to several molecules of IL-6 and neutralize several molecules of IL-6 when the message her ability to bind the antigen at pH 7.4 and to the dissociation of the antigen at pH 5.8. It is also believed that the pharmacokinetic properties can be further improved when the message is even more pronounced dependence of binding on pH than is observed in H3pI/L73.

[Example 6]. Optimization of the constant region of tocilizumab are reviewed

The reduction of the heterogeneity of the C-end of the H-chain of tocilizumab are reviewed

In addition, it was reported that the heterogeneity of the C-terminal sequences of the H-chain antibodies is due to a deletion of the C-terminal lysine amino acid residue and the amidation of the C-terminal carboxyl group in the deletion of two C-terminal amino acids, glycine and lysine (Anal. Biochem. 2007 Jan. 1; 360(1):75-83). In addition, in the tocilizumab are reviewed, the key is the sequence in which the C-terminal amino acid lysine was demeterova in nucleotide sequence in the result of post-translational modifications; however, subcomponents, in which lysine is saved, and subcomponents, in which the C-terminal carboxyl group liderovna as a result of deletion of glycine and lysine, are also recognized by the AMI heterogeneity. The complexity and high cost krupnomasshtabnogo industrial production of pharmaceutical preparations on the basis of such antibodies with maintaining the desired substances/related substances associated with the heterogeneity of different products. If possible, it is desirable to use a single substance, but in the development of antibodies that are used as pharmaceuticals, this substance must be reduced heterogeneity. Thus, the development of antibodies that are used as pharmaceuticals, preferably, C-terminal heterogeneity of the H-chain was missing.

To reduce the heterogeneity of the C-terminal amino acids such C-terminal amino acids have been modified. The obtained data showed that C-terminal heterogeneity can be prevented by pre-deletions made in nucleotide sequence to remove the lysine and glycine residues at the C-end of the constant region of H chain of tocilizumab are reviewed. Tocilizumab, tocilizumab, which lacks the C-terminal lysine residue (TOCILIZUMABΔK: H-chain WT-IgG1ΔK/SEQ ID NO: 68; L-chain WT-Kappa/SEQ ID NO: 54), and tocilizumab, which lacks the C-terminal lysine and glycine residues (TOCILIZUMABΔGK: H-chain WT-IgG1ΔGK/SEQ ID NO: 69; L-chain WT-Kappa/SEQ ID NO: 54), rated for heterogeneity using cation exchange chromatography. Was used Olonka ProPac WCX-10, 4×250 mm (Dionex), where the mobile phase A consisted of 25 mmol/l MES/NaOH (pH 6,1), and mobile phase b consisted of 25 mmol/l MES/NaOH, 250 mmol/l NaCl (pH of 6.1). Chromatography was performed at a corresponding flow rate and the corresponding gradient. The results of analysis by using cation-exchange chromatography is shown in Fig. 12. The results showed that the heterogeneity of the C-terminal amino acids can be reduced by pre-deletions made in nucleotide sequence to remove the lysine and glycine residues at the C-end of the constant region of H chain, but not by pre-deletion only lysine residue at the C-end of the constant region of H chain. All C-terminal sequence of the constant region of a human antibody IgG1, IgG2 and IgG4 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, the method is expected to reduce the heterogeneity of the C-terminal amino acids found in this study may also be applicable to constant regions IgG2 and IgG4 and their options.

Reduce heterogeneity due to disulfide bonds, in the tocilizumab are reviewed isotype IgG2

The isotype of tocilizumab are reviewed is IgG1. Because tocilizumab is neutralization, the binding to Fcγ receptor may be undesirable from the point of view of production immunogenicity and side effects. A possible way to reduce the level of binding to Fcγ receptor is turning isotype IgG antibodies with IgG1 to IgG2 or IgG4 (Ann Hematol. 1998 Jun; 76(6):231-48). From the point of view of the level of binding to Fcγ receptor I and pharmacokinetic it is believed that IgG2 is preferable to IgG4 (Nat. Biotechnol. 2007 Dec; 25(12):1369-72). In addition, physico-chemical properties of proteins, and in particular, the homogeneity and stability are very important in the development of antibodies as pharmaceuticals. It was reported that the antibody isotype IgG2 is 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 on the basis of such antibodies while maintaining the desired substances/related substances associated with the heterogeneity caused by different disulfide bonds in various products. Thus, if possible, it is desirable to use one substance. For example, in the development of antibody isotype IgG2 as pharmaceuticals, it is preferable that the heterogeneity caused by disulfide bonds were reduced, but the button is not led to stability.

To reduce the heterogeneity of the antibodies of the IgG2 isotype was evaluated various options. In the result, it was found that the heterogeneity can be reduced without reducing the stability using the constant region of WT-SKSC (SEQ ID NO: 70), where the sequences of the constant region of IgG2, the cysteine residue at position 131 and the arginine residue at position 133 (in accordance with EU numbering) in the CH1 domain of the H-chain were replaced by serine and lysine, respectively, and the cysteine residue at position 219 (in accordance with EU 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-chain WT-Kappa/SEQ ID NO: 54) and tocilizumab-SKSC (H-chain WT-SKSC/SEQ ID NO: 70; L-chain WT-Kappa/SEQ ID NO: 54) were obtained and analyzed for heterogeneity and stability. Heterogeneity was assessed by using cation-exchange chromatography. Was used column ProPac WCX-10 (Dionex), where the mobile phase A consisted of 20 mm sodium acetate (pH 5.0)as mobile phase consisted of 20 mm sodium acetate, 1M NaCl (pH 5.0). Chromatography was performed at a corresponding flow rate and the corresponding gradient. The results of analysis by using cation-exchange chromatography is shown in Fig. 13. Stability was assessed basedfrom intermediate values of temperature during thermal denaturation (Tm values), defined using differential scanning calorimetry (DSC) (VP-DSC; Microcal). The results of DSC measurement in 20 mm sodium acetate, 150 mm NaCl, pH 6.0 and value Tm Fab-domain is shown in Fig. 14.

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

Identification of the sites of mutations that enhance the pharmacokinetic properties, in the constant region of tocilizumab are reviewed.

As described above, on the basis of IgG1, which is an isotype of tocilizumab are reviewed, can be reduced C-terminal heterogeneity and reducing the heterogeneity of antibody constant regions izote is and IgG2, as well as reducing the binding to Fcγ receptor and high stability. In addition, preferably, the constant region had improved pharmacokinetic properties compared to IgG1, which represents the isotype of tocilizumab are reviewed.

To identify the constant regions, which have a longer half-life in plasma than the constant region of the antibody isotype IgG1, were screened for identification of mutation sites in order to improve the pharmacokinetic properties of tocilizumab are reviewed-SKSC, which has high stability and lower heterogeneity compared to the antibody contains a constant region of IgG2 isotype, as mentioned above. In the result it was found WT-M58 (SEQ ID NO: 72 (amino acid sequence)), compared with WT-SKSC glutamic acid at position 137, according to the European numbering was replaced by glycine, serine at position 138 was replaced by glycine, histidine at position 268 was replaced by glutamine, arginine at position 355 was replaced by glutamine, glutamine at position 419 was replaced with glutamic acid, and glycine at position 446 and the lysine at position 447 were dellarovere in order to reduce the heterogeneity of the C-end of the H-chain. In addition, it was received WT-M44 (SEQ ID NO: 73 (amino acid sequence)), compared with IgG1 arg is NIN in position 434 was replaced by 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 antibodies M44 in order to reduce the heterogeneity of the C-end of the H-chain. In addition, the antibody WT-M73 (SEQ ID NO: 75 (amino acid sequence)) was produced by replacing the asparagine at position 434 to 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-chain WT-Kappa/SEQ ID NO: 54) and tocilizumab-M73 (H-chain WT-M73/SEQ ID NO: 75; L-chain WT-Kappa/SEQ ID NO: 54) were received and evaluated on the binding affinity of human FcRn and pharmacokinetic properties in mice transgenic for human FcRn (see comparative examples for this method).

Binding of tocilizumab are reviewed-IgG1, tocilizumab are reviewed-M44, tocilizumab are reviewed-M58 and tocilizumab are reviewed-M73 with human FcRn were evaluated using Biacore. As shown in table 6, the level of binding of tocilizumab are reviewed-M44, tocilizumab are reviewed-M58 and tocilizumab are reviewed-M73 approximately 2.7 times, 1.4 times and 3.8 times higher than the level of binding of tocilizumab are reviewed-IgG1, respectively.

0,59
Table 6
KD(µm)
tocilizumab-IgG11,62
tocilizumab-M44
tocilizumab-M581,17
tocilizumab-M730,42

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

[Example 7] Get a fully humanized antibody against the receptor of IL-6 with improved PK/PD properties

Options received tocilizumab are reviewed by merging multiple mutations in the variable and constant regions of tocilizumab are reviewed, identified, as described above in point is imarah. Fully humanized antibodies against the receptor for IL-6, identified by different methods of screening are: Fv3-M73 (H-chain VH4-M73/SEQ ID NO: 25; L-chain VL1-Kappa/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 affinity obtained Fv3-M73, Fv4-M73, and Fv5-M83 against the receptor of IL-6 compared with the affinity of tocilizumab are reviewed (see comparative example for this method). The affinity of these antibodies to soluble receptor of IL-6 defined at a pH of 7.4 are presented in table 7. In addition, BaF/gp130 - neutralizing activity compared with the corresponding activities of tocilizumab are reviewed and control (a known high-affinity antibodies against the receptor for IL-6, described in the comparative example, and VQ8F11-21 hIgG1 described in the application U.S. 2007/0280945) (see comparative example for this method). The results obtained in the determination of the biological activity of these antibodies using BaF/gp130 shown in Fig. 16 (tocilizumab, a control antibody, and Fv5-M83 with the final concentration of IL-6, equal 300 ng/ml) and Fig. 17 (tocilizumab, Fv3-M73, and Fv4-M73 with the final concentration of IL-6, equal to 30 ng/ml). As shown in table 7, the affinity of Fv3-M73, and Fv4-M73 about 2-3 times the affinity of tocilizumab are reviewed, and the affinity of Fv5-M83 approximately 100 times greater than the affinity of tocilizumab are reviewed (because of the difficulty of measurement is finesti Fv5-M83, this affinity was determined using Fv5-IgG1 (H-chain VH5-IgG1/SEQ ID NO: 76; L-chain VL5-Kappa/SEQ ID NO: 30), which had constant region type IgG1; and it is usually assumed that the constant region does not have any effect on the 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 is more than 100 times the activity of tocilizumab are reviewed from the point of view of a 50%inhibitory concentration. Fv5-M83 also possessed neutralizing activity, which is about 10 times the neutralizing activity of the control antibody terms of 50%inhibitory concentration (known high-affinity antibodies against the receptor for IL-6).

Table 7
ka(1/MS)kd(1/s)KD(M)
Tocilizumab4,0E+051,1E-032,7E-09
Fv3-M738,5E+058,7E-041,0E-09
Fv4-M73 7,5E+051,E-031,4E-09
Fv5-M831,1E+062,8E-052,5E-11

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

Table 8
a pH of 7.4
kd(1/s)
pH 5.8
kd(1/s)
Kd(rn,8)/ Kd(rn,4)
the pH-dependence
Tocilizumab2,5E-042,5E-041,00
Fv3-M73 4,9E-045,3E-0310,88
Fv4-M735,1E-045,1E-0310,06

Isoelectric point of tocilizumab are reviewed, control, Fv3-M73, Fv4-M73, and Fv5-M83 was determined by electrophoresis with isoelectric focusing, carried out by the method known in the art. The results showed that the isoelectric point was about to 9.3 for tocilizumab are reviewed; about 8.4 and 8.5 for control; approximately 5.7 and 5.8 for Fv3-M73; about 5.6 to 5.7 for Fv4-M73; and 5.4-5.5 for Fv5-M83. Thus, each of these antibodies had significantly lower isoelectric point compared to the value of the isoelectric point for tocilizumab are reviewed and control. In addition, theoretical value of the isoelectric point of the variable regions VH/VL was calculated using GENETYX (GENETYX CORPORATION). The results showed that theoretical value of the isoelectric point was 9,20 for tocilizumab are reviewed; 7,79 to control; 5,49 for Fv3-M73; 5,01 for Fv4-M73; and 4,27 for Fv5-M83. Thus, each antibody had a significantly lower value of isoelectric point compared with the values for tocilizumab are reviewed and control. Since, as shown in example 2 pharmacokinetic properties have been improved by reducing the value of the isoelectric that is CI, it is obvious that the pharmacokinetic properties Fv3-M73, Fv4-M73, and Fv5-M83 were better pharmacokinetic properties of tocilizumab are reviewed and control.

T-cell epitopes in the sequence of variable regions of tocilizumab are reviewed, Fv3-M73, Fv4-M73 or Fv5-M83 were analyzed using TEPITOPE (Methods. 2004 Dec; 34(4):468-75). It was suggested that tocilizumab has a T-cell epitopes, many of which can communicate with the HLA, as described in example 3. In contrast, the number of sequences in Fv3-M73, Fv4-M73, and Fv5-M83, which, as predicted, associated with T-cell epitopes significantly reduced. In addition, the frame region Fv3-M73, Fv4-M73 or Fv5-M83 has no 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 with tocilizumab are reviewed.

[Example 8] analysis of the pharmacokinetic/pharmacodynamic properties (PK/PD) is a fully humanized antibody against the receptor of IL-6 in monkeys

Each of the antibody such as tocilizumab, a control antibody, Fv3-M73, Fv4-M73, and Fv5-M83 intravenously injected abacadabra monkeys in a single dose of 1 mg/kg to evaluate the dependence of the concentration of these antibodies in plasma against time (see comparative example for this method). The concentration of tocilizumab are reviewed, Fv3-M73, Fv4-M73, and Fv5-M83 in p is the AZM depending on time after intravenous presented on Fig. The results showed that each of the antibodies, namely, Fv3-M73, Fv4-M73, and Fv5-M83, had significantly improved pharmacokinetic properties abacadabra monkeys compared with the pharmacokinetic properties of tocilizumab are reviewed and control antibodies. Among these antibodies, Fv3-M73, and Fv4-M73 had a much better pharmacokinetic properties compared with tocilizumab are reviewed.

Was evaluated the effectiveness of each antibody in relation to neutralize the receptor of IL-6 membrane-type in abacadabra monkeys. IL-6 abacadabra monkeys were injected subcutaneously in the lower region of the backrest at a concentration of 5 mg/kg every day from day 6 to day 18 after injection of the antibody (day 3-day 10 for tocilizumab are reviewed), and after 24 hours was determined by the concentration of CRP for each animal (see comparative example for this method). CRP concentrations depending on the time after the introduction of each antibody presented on Fig. To evaluate the effectiveness of each antibody against neutralization of the soluble receptor of IL-6 in abacadabra monkeys was determined by the concentration of free soluble receptor of IL-6 in plasma abacadabra monkeys and calculated the percentage of free soluble receptor of IL-6 (see comparative example for this method). The percent of free soluble receptor of IL-6, depending on the time after the introduction of each and what Titel shown in Fig. 20.

Each of Fv3-M73, Fv4-M73, and Fv5-M83 had a more lasting neutralizing action against the receptor for IL-6 membrane-type in abacadabra monkeys and inhibited the increase of CRP levels over a longer period of time compared to tocilizumab are reviewed and the control (well-known high-affinity antibody against the receptor for IL-6). In addition, each of Fv3-M73, Fv4-M73, and Fv5-M83 had a more lasting neutralizing activity towards soluble receptor of IL-6 in abacadabra monkeys and inhibited the increase in the level of free soluble receptor of IL-6 in abacadabra monkeys over a longer period of time compared to tocilizumab are reviewed and monitored. The data obtained showed that all antibodies, namely, Fv3-M73, Fv4-M73, and Fv5-M83, had a more lasting neutralizing activity against receptor IL-6 membrane-type and soluble receptor of IL-6 compared with tocilizumab are reviewed and monitored. Of these antibodies, especially long-neutralizing action had Fv3-M73, and Fv4-M73. In addition, Fv5-M83 inhibited CRP and free soluble receptor of IL-6 abacadabra monkeys in a higher degree than Fv3-M73, and Fv4-M73. Thus, it is believed that Fv5-M83 has a stronger neutralizing activity against the receptor for IL-6 membrane-type and soluble receptor of IL-6 than antibodies Fv3-73, Fv4-M73, and the control antibody (known high-affinity antibody against the receptor for IL-6). It is believed that the results ofin vivoreceived for abacadabra monkeys showed that Fv5-M83 has a higher affinity to the receptor of IL-6 and stronger biological activity in the test system BaF/gp130 compared to control.

The results suggest that Fv3-M73, and Fv4-M73, compared with tocilizumab are reviewed and a control antibody, are significantly more prolonged activity as antibodies, neutralizing receptor IL-6, and therefore, they can be introduced in a much smaller dose and with less frequency. In addition, it was demonstrated that Fv5-M83 used as antibodies, neutralizing receptor IL-6, has a much higher activity and saves this activity over a longer period of time. Thus, it is expected that antibodies Fv3-M73, Fv4-M73, and Fv5-M83 can be used as pharmaceutical antagonists of IL-6.

[Example 9]

It is known that protein, which is a chemoattractant for monocytes, (MCP)-1, involved in cellular invasion of monocytes, T cells, NK cells and basophils. It was reported that MCP-1 is highly expressed in synovial tissues/synovial fluid of patients with RA (J. Clin. Invest., Sep. 1992, 90(3):772-779), and it is believed that outcasted in the development of pathological conditions of the type RA (Inflamm. Allergy Drug Targets, Mar 2008, 7(1):53-66).

VEGF is a potent angiogenic factor, and it is known that it is produced by, for example, macrophages, fibroblasts and synovial cells in the synovial membrane of patients with RA (J. Rheumatol., Sep 1995, 22(9):1624-1630). In addition, the level of VEGF in the serum of patients with RA correlates with pathological activity and growth parameters on the radiograph (Arthritis Rheum., Jun. 2003, 48(6):1521-1529; Arthritis Rheum., Sep 2001, 44(9):2055-2064), the level of VEGF in the serum is reduced in patients with RA, anti-IL-6R antibody tocilizumab are reviewed, and therefore it is believed that VEGF also plays an important role in the development of pathological conditions in RA (Mod. Rheumatol. 2009, 19(1):12-19; and Mediators Inflamm. 2008, 2008:129873).

Thus, the analysis was performed in order to determine whether tocilizumab and Fv4-M73 to inhibit the production of MCP-1 and VEGF in sinoville human cells with RA, which is the result of sIL-6R and IL-6 stimulation.

Synovial cells with PA (TOYOBO) were sown in 96-well plates in IMDM medium containing 5% FCS at a density of 2×104cells/0.05 ml/well, and were sown within 90 minutes in CO2-incubator (37°C, 5% CO2). Then added 0.05 ml of tocilizumab are reviewed and Fv4-M73, diluted to appropriate concentrations, and the tablets were left for 15 minutes, after which was added 0.05 ml of the soluble receptor of IL-6 (SR344: obtained by the method opican the m in the comparative examples). Then the tablets were left for another 30 minutes, after which was added 0.05 ml IL-6 (TORAY) (final concentration of the soluble receptor of IL-6 and receptor of IL-6 was 50 ng/ml each). After culturing for two days supernatant culture was collected and the concentration of MCP-1 and VEGF in these supernatant were determined using an ELISA kit (Biosource and Pierce Biotechnology). The results are presented in Fig. 21 and 22. Tocilizumab and Fv4-M73 inhibited the production of MCP-1 and VEGF in synovial cells from RA after stimulation of the soluble receptor of IL-6 and its receptor IL-6, depending on the concentration.

In accordance with this duration of effect Fv4-M73 as antibodies, neutralizing the receptor for IL-6 (effect of the receptor binding of IL-6 and blocking signals transmitted by the receptor of IL-6 membrane-type and soluble receptor IL-6)was significantly higher than that of tocilizumab are reviewed, and therefore, the frequency of administration and the dose can be significantly reduced compared with the frequency of administration and dose of tocilizumab are reviewed, and in addition, it was found that Fv4-M73 inhibits the production of MCP-1 and VEGF in synovial cells of a person with RA. Thus, it was shown that Fv4-M73 is the most effective therapeutic tool for the treatment of RA.

Comparative examples

To obtain soluble recombinant human receptor of IL-6

RA is soluble recombinant human receptor, IL-6, derived from the human receptor, IL-6, which is an antigen, obtained as described below. Was derived cell line CHO, constitutively expressing soluble human receptor of IL-6 containing the sequence starting from the first N-terminal amino acids and ending 344-th amino acid, as described in the publication J. Biochem. (1990) 108, 673-676 (Yamasakiet al., Science (1988) 241, 825-828 (GenBank #X12830)). Soluble human receptor of IL-6 was purified from the supernatant of the culture SR344-expressing cells SNO by carrying out column chromatography three types: column chromatography on sepharose Blue Sepharose 6 FF, affinity chromatography on a column with immobilized the antibody specific to SR344, and column chromatography of the type of gel filtration. Faction, erwerbende as the main peak, was used as the final purified sample.

To obtain soluble recombinant receptor of IL-6 abacadabra monkeys (cIL-6R)

Oligo-DNA primers were obtained on the basis of the described sequence of the gene for the receptor of IL-6 rhesus monkeys (Birneyet al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue):D556-61). A DNA fragment having a full-sized gene receptor IL-6 abacadabra monkeys were obtained by PCR using primers, as well as matrix used cDNA isolated from the pancreas with Bampton monkeys. The obtained DNA fragment was embedded in the expression vector mammalian cells, and using this vector was achieved stable expression of the indicated fragment in the cell line CHO (cyno.sIL-6R-producing cell line CHO). Culture medium cyno.sIL-6R-producing CHO cells was purified on a HisTrap column (GE Healthcare Bioscience), and then concentrated using an adsorbent Amicon Ultra-15 Ultracel-10k (Millipore). The final purified sample of the soluble receptor of IL-6 abacadabra monkeys (hereinafter called cIL-6R) were further purified using gel filtration on a column with Superdex200pg16/60 (GE Healthcare Bioscience).

The preparation of recombinant IL-6 abacadabra monkeys (cIL-6)

IL-6 abacadabra monkeys was obtained by the method described below. Was obtained nucleotide sequence encoding a 212 amino acids and deposited in the SWISSPROT reg. No. P79341, and then this sequence cloned in the expression vector in mammalian cells. The resulting vector was introduced into CHO cells to obtain cell lines with stable expression (cyno.IL-6R-producing cell line CHO). Culture medium cyno.IL-6R-producing CHO cells was purified on a column of SP-separate/FF (GE Healthcare Bioscience), and then concentrated using an adsorbent Amicon Ultra-15 Ultracel-5k (Millipore). The final purified sample of IL-6 abacadabra monkeys (called the alley cIL-6R) were further purified using gel filtration on a column with Superdex75pg26/60 (GE Healthcare Bioscience), followed by concentration of the adsorbent Amicon Ultra-15 Ultracel-5k (Millipore).

Getting well-known high-affinity antibodies against the receptor for IL-6

Expression vector mammalian cells designed for expression VQ8F11-21 hIgG1 known high-affinity antibodies against the receptor for IL-6. VQ8F11-21 hIgG1 described in application for U.S. patent 2007/0280945 A1 (US 2007/0280945 A1; amino acid sequence of H-chain and L-chain is presented in SEQ ID NO: 77 and 78, respectively). Variable region antibody was constructed by PCR using a combination of synthetic oligo-DNA (obtained by PCR Assembly), and a constant region used IgG1. Variable and constant region antibody were combined by PCR Assembly, and then embedded in the expression vector mammals in order to construct expression vectors for representing the interest of the H-chain and L-chain. Nucleotide sequence of the obtained expression vectors were determined by a method known in the art. High-affinity antibody against the receptor of IL-6 (hereinafter called "reference antibody") expressed and purified using the constructed expression vectors by the method described in example 1.

Receiving, expression and purification of variants of tocilizumab are reviewed

Options received tocilizumab are reviewed using the kit for site-directed mutagenesis QuikChange (Stratagene) by the method described in the attached is the manual. The obtained plasmid fragments were incorporated into expression vectors in mammalian cells in order to construct expression vectors for representing the interest of H-chains and L-chains. Nucleotide sequence of the obtained expression vectors were determined by a method known in the art. Antibodies expressed by the method described below. Cell lines HEK293H originating from cancer cells of the human embryo kidney (Invitrogen), suspended in DMEM (Invitrogen), which was added 10% fetal bovine serum (Invitrogen). Cells were sown in a volume of 10 ml in a Cup in the Cup for adhesive cells (10 cm in diameter; CORNING) at a density of cells 5-6×105cells/ml and cultured in CO2-incubator (37°C, 5% CO2in one day and one night. Then the medium was removed by suction and was added 6.9 ml of medium CHO-S-SFM II (Invitrogen). The resulting plasmids were introduced into cells by the method of lipofectin. The resulting supernatant culture were collected, centrifuged (approximately 2000×g, 5 minutes at room temperature) to remove cells and sterilized by filtering through a 0,22 µm filter MILLEX(R)-GV (Millipore) to produce supernatants. Antibodies were purified from the obtained supernatant culture known method using recombinant protein a - sepharose™ Fast Flow (Amersham Biosciences). To determine the concentration oceanog the antibody optical density was measured at a wavelength of 280 nm using a spectrophotometer. The antibody concentration was determined on the basis of values calculated using the absorption coefficients, measured by the method of PACE (Protein Science 1995; 4:2411-2423).

Obtaining cell line BaF3 expressing human gp130

Cell line BaF3 expressing human gp130, received in accordance with the procedure described below, the result of which was produced cell line, proliferate depending on IL-6.

Full-size cDNA of human gp130 (Hibiet al., Cell (1990) 63:1149-1157 (GenBank #NM_002184)) amplified by PCR and cloned in the expression vector pCOS2Zeo to construct pCOS2Zeo/gp130. pCOS2Zeo is an expression vector constructed by removing the region of the DHFR gene expression of pCHOI (Hirataet al., FEBS Letter (1994) 356:244-248) and embedding expression region of the gene of resistance to zeocin. Full-size cDNA of human IL-6R amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen) in order to design hIL-6R/pcDNA3.1(+).

10 µg pCOS2Zeo/gp130 was mixed with BaF3 cells (0,8×107cells), suspended in PBS, and then gave impetus to 0.33 kV and 950 μf using a device Gene Pulser (Bio-Rad). The BaF3 cells with a gene that is built by electroporation, were cultured for one day and one night in the medium RPMI 1640 (Invitrogen), which were added to 0.2 ng/ml mouse interleukin-3 (Perotech) and 10% fetal bovine serum (hereinafter referred to FBS, HyClone), and then were selected by adding RPMI medium 1640, to which were added 100 ng/ml human interleukin-6 (R&D systems) and 100 ng/ml of soluble receptor for human interleukin-6 (R&D systems)and 10% FBS, obtaining cell line BaF3 expressing human gp130 (hereinafter called BaF3/gp130"). These cells BaF/gp130 proliferate in the presence of human interleukin-6 (R&D systems) and soluble receptor of human IL-6, and therefore they can be used to estimate the growth-inhibitory activity (or activity, neutralizing receptor IL-6) antibodies against the receptor for IL-6.

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

The activity aimed at neutralizing receptor IL-6, was evaluated using cells BaF3/gp130, which proliferate depending on the IL-6/receptor IL-6. After three washes with RPMI1640 medium, to which was added 10% FBS, the cells BaF3/gp130 at a density of 5×104cells/ml suspended in RPMI1640 medium, to which were added 600 ng/ml or 60 ng/ml human interleukin-6 (TORAY) (final concentration 300 ng/ml or 30 ng/ml), the appropriate amount of soluble human receptor of IL-6 and 10% FBS. Cell suspension was distributed (50 μl/well) in 96-well plates (CORNING). Then purified antibodies were diluted RPMI1640, containing 10% FBS, was added to each well (50 μl/well). Cells were cultured at 37°C in an atmosphere of 5% CO2within three days. Reagent WST-8 (set to count cells - 8; Dojindo Laboratories) was twice diluted with PBS. Then to each well was immediately added to 20 μl of the specified reagent and measured the optical density at a wavelength of 450 nm (reference wavelength: 620 nm) on the device SUNRISE CLASSIC (TECAN). After cultivation for two hours again measured the optical density at a wavelength of 450 nm (reference wavelength: 620 nm). Activity, neutralizing receptor IL-6, was evaluated by the change in optical density for two hours as an indicator.

Analysis Biacore for binding to soluble human receptor of IL-6

The kinetics of the reaction "antigen-antibody" was analyzed using Biacore T100 (GE Healthcare). The interaction of antibodies with soluble human receptor of IL-6 was assessed by immobilization of an appropriate number of protein a or protein A/G or anti-IgG F(ab')2(specific to γ-chain) on the sensor chip by a method of binding an amine, binding interest antibodies on the chip at pH 7.4 and analysis steps soluble receptor of IL-6 in various concentrations at pH of 7.4 on the chip as the analyte. All measurements were carried out at 37°C. the Kinetic parameters, the rate constant of Association ka(1/MS) and to instanty rate of dissociation (k d(1/s) was calculated by sensorama based on the results of the measurements. Then, based on the rate constants of Association and dissociation were determined KD(M). The corresponding parameters were determined using a computer program Biacore T100 Evaluation Software (GE Healthcare).

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

Reaction monitoring "antigen-antibody" with the receptor of IL-6 membrane-type at pH 5.8 and pH of 7.4 was performed using Biacore T100 (GE Healthcare). Binding to the receptor of IL-6 membrane type was analyzed by assessing the binding of soluble human receptor of IL-6 immobilized on the sensor chip. SR344 was subjected to biotinylation method known in the art. Biotinylated soluble human receptor of IL-6 was immobilized on the sensor chip by means of streptavidin on the basis of the affinity of binding of Biotin and streptavidin. All measurements were carried out at 37°C. the Buffer for the mobile phase consisted of 10 mm MES (pH 5.8), 150 mm NaCl and 0.05% tween-20. Clone detecting the pH-dependent binding, was introduced at a pH of 7.4 for binding to soluble human receptor of IL-6 (buffer for injection of the sample consisted of 10 mm MES (pH of 7.4), 150 mm NaCl and 0.05% tween-20). Then monitored for pH-dependent dissociation of each clone at pH 5.8, which corresponds to the pH of the mobile phase. Intercept the NTU rate of dissociation (K d(1/s)) at pH 5.8 was calculated using Biacore T100 Evaluation Software (GE Healthcare) by constructing a curve only for phase dissociation at pH 5.8. The concentration of the sample was 0.25 microgram/ml, the Binding was carried out in 10 mm MES (pH of 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. Similarly, the rate constant of dissociation (Kd(1/s)) at a pH of 7.4 was calculated using Biacore T100 Evaluation Software (GE Healthcare) by constructing a curve only for phase dissociation at pH of 7.4. The concentration of the sample was 0.5 µg/ml of Binding was carried out in 10 mm MES (pH of 7.4), 150 mm NaCl and 0.05% tween-20, and dissociation was also carried out in 10 mm MES (pH of 7.4), 150 mm NaCl and 0.05% tween-20.

Evaluation of the binding to human FcRn

FcRn is a complex FcRn and β2-microglobulin. Oligo-DNA primers were obtained from the described sequence of the human FcRn gene (J. Exp. Med. (1994) 180(6):2377-2381). The DNA fragment corresponding to the full-size gene was obtained by PCR using human cDNA (Human Placenta Marathon-Ready cDNA, Clontech) as the template and using the obtained primers. Using the obtained DNA fragment as a matrix, this DNA fragment encoding the extracellular domain containing the signal region (Met1-Leu290), amplified by PCR and was built in expression vector cells ml is capitalsim (amino acid sequence of human FcRn presented in SEQ ID NO: 79). Similarly, oligo-DNA primers have been described on the basis of the gene sequence of human β2-microglobulin (Proc. Natl. Acad. Sci. USA. (2002) 99(26):16899-16903). The DNA fragment corresponding to the full-size gene was obtained by PCR using human cDNA (Hu-Placenta Marathon-Ready cDNA, CLONTECH) as the template and using the obtained primers. Using the obtained DNA fragment as a matrix, this DNA fragment encoding full-β2-microglobulin, containing the signal region (Met1-Met119), amplified by PCR and was built in expression vector mammalian cells (amino acid sequence of human β2-microglobulin is presented in SEQ ID NO: 80).

Soluble human FcRn expressed in accordance with the following procedure. The plasmids constructed for human FcRn and β2-microglobulin, was introduced by lipofectin cells HEK293H cell line, derived from cancer cells of the human embryo kidney (Invitrogen)with 10% FBS (Invitrogen). The obtained 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 purified on a column of HiTrap Q HP (GE Healthcare).

The concentration of antibodies in the plasma of mice

The antibody concentration in plasma is e mice were determined using ELISA method well-known specialists.

PK/PD test to determine the antibody concentration, the concentration of CRP and freely soluble receptor of IL-6 in plasma of monkeys

The concentration of the antibodies in the plasma abacadabra monkeys were determined using ELISA method.

The concentration of CRP 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 receptor of IL-6 in plasma abacadabra monkeys was determined by the method described below. All antibodies IgG (IgG abacadabra monkeys, the antibody against the human receptor of IL-6 and antibody against the human receptor of IL-6 soluble receptor of IL-6 abacadabra monkeys") in plasma was adsorbing on the protein And by downloading 30 µl of plasma abacadabra monkeys on the appropriate amount of resin, namely recombinant protein a-sepharose Fast Flow (GE Healthcare), drained of 0.22-ál filter with cap (Millipore). Then the solution from the filter with the cap, centrifuged at high speed centrifuge to collect the solution passing through the centrifuge. Missed the solution did not contain complex "associated with protein And antibody against the human receptor of IL-6 soluble receptor of IL-6 abacadabra monkeys". Therefore, the concentration of free soluble re is aptara IL-6 can be determined by measuring the concentration of soluble receptor of IL-6 abacadabra monkeys in solution, passing through protein A. the concentration of the soluble receptor of IL-6 abacadabra monkeys was determined by the known method of measuring the concentrations of soluble human receptor of IL-6. As the standard used soluble receptor of IL-6 abacadabra monkeys (cIL-6R), obtained as described above. The percentage of free soluble receptor of IL-6 was calculated by the following formula:

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

----------------------------------------------------------------------------- ×100

The concentration of the soluble receptor of IL-6 prior to the introduction of antibodies

1. Any antibody to the receptor of IL-6, selected from:
(a) antibodies, which contains a variable region heavy chain comprising CDR1-containing sequence of SEQ ID NO:1 (CDR1 VH4-M73), CDR2-containing sequence of SEQ ID NO:2 (CDR2 VH4-M73), and CDR3-containing sequence of SEQ ID NO:3 (CDR3 VH4-M73), and variable region light chain comprising CDR1-containing sequence of SEQ ID NO:10 (CDR1 VL1), CDR2-containing sequence of SEQ ID NO: 11 (CDR2 VL1), and CDR3, contains the sequence of SEQ ID NO:12 (CDR3 VL1);
(b) antibodies, which contains a variable region heavy chain comprising CDR1-containing sequence of SEQ ID NO:4 (CDR1 VH3-M73), CDR2-containing sequence of SEQ ID NO:5 (CDR2 VH3-M73), and CDR3-containing sequence of SEQ ID NO:6 (DR3 VH3-M73), and variable region light chain comprising CDR1-containing sequence of SEQ ID NO:13 (CDR1 VL3), CDR2-containing sequence of SEQ ID NO:14 (CDR2 VL3), and CDR3-containing sequence of SEQ ID NO:15 (CDR3 VL3); and
(c) antibody, which contains a variable region heavy chain comprising CDR1-containing sequence of SEQ ID NO:7 (CDR1 VH5-M83), CDR2-containing sequence of SEQ ID NO:8 (CDR2 VH5-M83), and CDR3-containing sequence of SEQ ID NO:9 (CDR3 VH5-M83), and variable region light chain,
comprising CDR1-containing sequence of SEQ ID NO:16 (CDR1 VL5), CDR2-containing sequence of SEQ ID NO:17 (CDR2 VL5), and CDR3-containing sequence of SEQ ID NO:18 (CDR3 VL5).

2. Any antibody to the receptor of IL-6, selected from:
(a) antibodies, which contains a variable region heavy chain comprising the sequence of SEQ ID NO:19 (variable region VH4-M73), and variable region light chain comprising the sequence of SEQ ID NO:22 (variable region VL1);
(b) antibodies, which contains a variable region heavy chain comprising the sequence of SEQ ID NO:20 (variable region VH3-M73), and variable region light chain comprising the sequence of SEQ ID NO:23 (variable region VL3); and
(c) antibody, which contains a variable region heavy chain comprising the sequence of SEQ ID NO:21 (variable region VH5-M83), and variable region easily the chain, including the sequence of SEQ ID NO:24 (variable region VL5).

3. Any antibody to the receptor of IL-6, selected from:
(a) an antibody that includes a heavy chain comprising the sequence of SEQ ID NO:25 (VH4-M73), and light chain comprising the sequence of SEQ ID NO:28 (VL1);
(b) an antibody that includes a heavy chain comprising the sequence of SEQ ID NO:26 (VH3-M73), and light chain comprising the sequence of SEQ ID NO:29 (VL3); and
(c) an antibody that includes a heavy chain comprising the sequence of SEQ ID NO:27 (VH5-M83), and light chain comprising the sequence of SEQ ID NO:30 (VL5).

4. The gene encoding the antibody to the receptor of IL-6 according to any one of claims 1 to 3.

5. Vector to obtain antibodies to the receptor of IL-6, carrying the gene according to claim 4.

6. A host cell to obtain antibodies to the receptor of IL-6, the carrier vector according to claim 5, where a host cell is an E.coli cell or a cell of an animal.

7. Method of producing antibodies to the receptor of IL-6 according to any one of claims 1 to 3, which includes the stages of (a) culturing the host cell according to claim 6 and (b) obtaining the antibody from the host cell or outside the cell.

8. Pharmaceutical composition for treatment associated with IL-6 disease containing as an active ingredient an effective amount of the antibody to the receptor of IL-6 according to any one of claims 1 to 3, or antibodies to receptor of IL-6, obtained by the method according to claim 7.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to producing fused proteins. The fused construct consists of an amino acid sequence of glycosyl phosphatidylinositol anchored tissue inhibitor of metalloproteinase.

EFFECT: cictrisation prevention during skin injures treatment when using the fused construct.

10 cl, 33 dwg, 18 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: polypeptide contains extracellular and transmembrane domains of a human tissue factor. A DNA fragment coding this polypeptide, and a plasmid pET28a(+) are used to produce a genetic make-up enabling biosynthesis of the recombinant human tissue factor. Also, the E coli BL21 [DE3]/p6E-tTF strain that is a producer of the recombinant human tissue factor is offered.

EFFECT: high-yield recombinant protein and simplified recovery and purification procedures.

2 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: what is described in a cartridge genetic maker based on GeneClip-Ul-neo vector producing three siRNA - reproduction inhibitors of human immunodeficiency virus type 1 and human CCR5 gene. The maker contains a fragment of the length 27 bp related to a conservative region of a HIV-1 genome reverse transcriptase domain, a fragment of the length 27 bp related to the other conservative region of the HIV-1 genome reverse transcriptase domain, as well as a fragment 19 bp related to CCR5 gene mRNA. The invention can be used in medicine and scientific research.

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3 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: recovered polynucleotide coding aminopeptidase containing a nucleotide sequence presented in the description is presented. Also, polynucleotide hybridised with said polynucleotide in the high stiffness environment is presented. An expression vector containing said polynucleotide, and an applicable host cell are described. Polypeptide related to the sequence presented in the description and being aminopeptidase is presented. A method of producing said polypeptide involving the stages of applicable host cell transformation by said polynucleotide or vector, cell cultivations in the medium adequate for said polynucleotide expression, and optional polypeptide purification from said cell or culture medium is offered. Besides, there has been described diagnostic technique for Aspergillus-infection in an organism involving the stages: a) recovery of a biological sample from said organism which is supposed to be Aspergillus infected, b) recovery of nucleic acid from said sample, c) determination whether said recovered nucleic acid contains polynucleotides hybridised with polynucleotide recovered from Aspergillus niger.

EFFECT: invention allows diagnosing Aspergillus-infection in the organism.

13 cl, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Versions of a human CD37 specific antibody are presented, each containing a variable site of a light and heavy chain. A coding nucleic acid and a based expression vector are described. There are disclosed: a host cell containing a vector, a method of producing the antibody with using the cell, and also a composition for decreasing the B-cell count and a method of treating diseases associated with aberrant B-cell activity, with using the CD37-specific antibodies.

EFFECT: use of the invention provides specific loss of 80% BJAB cells at the antibody concentration 10 mcg/ml, and also increases the survival rate of Daudi mice as compared with Rhithuximab therapy that can find application for treating various tumours.

39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology and deals with conjugates "caliheamicin derivative-carrier". Essence of invention includes methods of obtaining conjugates "monomer cytotoxic medication-caliheamicin/carrier" with considerably higher loading with medication than in earlier described methods, with low aggregation degree and with low content of low-conjugated fraction (LCF), as well as conjugates "cytotoxic medication-caliheamicin derivative/anti-CD22-antibody". Invention also includes compositions, which contain conjugate with CD22 antibody and application of said conjugates.

EFFECT: invention makes it possible to create conjugates with high loading of cytotoxic medication.

181 cl, 11 ex, 13 tbl, 29 dwg

FIELD: medicine.

SUBSTANCE: substance of the invention involves a humanised human osteopontin antibody containing a variable region of a heavy chain consisting of the amino acid sequence SEQ ID NO:1 and a variable region of a light chain consisting of the amino acid sequence SEQ ID NO:3. Furthermore, the invention involves a polynucleotide containing a sequence coding the variable region of the respective light and heavy chains of the humanised antibody, an expression vector containing polynucleotide, a host cell, a medicine, a method of producing the humanised antibody, a medicine for treating an autoimmune disease, a method of treating, and application of the humanised antibody for producing a pharmaceutical agent.

EFFECT: advantage of the invention consists in creation of the humanised antibody exhibiting improved activity or stability, than activity and stability of standard human osteopontin antibodies.

13 cl, 14 ex, 1 tbl, 16 dwg

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

Il2 antibodies // 2425054

FIELD: medicine, pharmaceutics.

SUBSTANCE: humanised monoclonal antibody and its active fragment under the invention neutralises human IL2 activity by binding with said human IL2 prior to, during and/or after binding of said human IL2 with human IL2 receptor. A variable light chain region of said antibody contains an adherent amino acid KAPKA sequence in its second frame region, and in addition, in the CDR1-CDR3 regions, contains the amino acid sequences presented in SEQ ID NO 1-3 disclosed in the description, while a variable heavy chain region contains in the CDR1 - CDR3 regions, amino acid sequences presented in SEQ ID NO 4-6 disclosed in the description. The invention describes a polynecleotide molecule coding the antibody under the invention or its active fragment, a pharmaceutical composition based on said antibody or its active fragment exhibiting human IL2 neutralising action, and also an application of said antibody or its active fragment or the polynecleotide molecule coding it for preparing a drug which optionally contains an auxiliary anti-inflammatory or anticancer agent for treating inflammatory diseases or tumours, respectively.

EFFECT: production of the alternative specific IL2 activity inhibitors which directly bind with human IL2.

24 cl, 18 dwg, 4 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Versions of a human CD37 specific antibody are presented, each containing a variable site of a light and heavy chain. A coding nucleic acid and a based expression vector are described. There are disclosed: a host cell containing a vector, a method of producing the antibody with using the cell, and also a composition for decreasing the B-cell count and a method of treating diseases associated with aberrant B-cell activity, with using the CD37-specific antibodies.

EFFECT: use of the invention provides specific loss of 80% BJAB cells at the antibody concentration 10 mcg/ml, and also increases the survival rate of Daudi mice as compared with Rhithuximab therapy that can find application for treating various tumours.

39 cl, 39 dwg, 7 tbl, 18 ex

FIELD: medicine.

SUBSTANCE: offered are versions of antibodies each of which is specifically bound with IGF-IR, inhibits its activity and is its antagonist, not exhibiting substantially IGF-IR agonist activity. Each of the antibodies is characterised at least by the presence of a variable area of a heavy and easy chain. There are described: antibody conjugates with cytotoxic agents, and also versions of a pharmaceutical composition for cancer diagnosing and therapy, methods of cancer treatment and diagnosing, a cancer diagnosing reagent - on the basis of the antibodies. There are disclosed: a method of producing the antibodies; nucleic acid (NA) coding the antibody; an expression vector containing NA. Offered is a hybridoma EM 164 producing the antibody under the invention, deposited in ATCC, No. PTA-4457, and also the use of the antibody for IGF-IR linkage. The use of the invention presents the antibodies which allow inhibiting MCF cell growth approximately in 12 times that is higher approximately in 5 times than hen using the antibody IR3, and can be used for cancer diagnosing and treatment expressing higher levels of receptor IGF-I, such as breast cancer, colon cancer, lung cancer, prostate cancer, ovarian cancer, synovial carcinoma and pancreatic cancer.

EFFECT: more efficient diagnosing and treatment of said cancers.

58 cl, 28 dwg, 10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: claimed are versions of separated monoclonal antibody, specific to INNAR-1. Described are: bispecific molecule, immunoconjugate and compositions for treatment of IFNAR-1-mediated diseases and disorders based on monoclonal antibody. Also described are methods of inhibiting biological activity of type I interferons, method of treating diseases and disorders, mediated by type I interferon with application of antibody. Claimed are nucleic acid, which codes antibody, vector for antibody expression, cell, transformed by vector, as well as method of obtaining antibodies and antibody-producing hybridoma.

EFFECT: application of invention provides novel IFNAR-1 inhibiting antibodies, which block IFNAR-1 and bind its other epitope, in comparison with known antibody 64G12.

29 cl, 15 dwg, 6 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: protein is capable to inhibit thrombin activity specifically. Besides, what is offered is a nucleic acid coding said chimeric protein, a vector containing this nucleic acid and a host cell carrying the vector. The present invention also refers to a pharmaceutical composition containing the recombinant chimeric protein of neutrophils and girugen inhibition factor. An effect of the given composition consists in thrombocyte aggregation inhibition or peripheral leukocyte activation inhibition.

EFFECT: composition can be used for treating a cardio-cerebrovascular disease or preventing a cerebral ischemic injury or a cerebral hematoma.

13 cl, 11 dwg, 16 tbl, 17 ex

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