Medication for treatment of rheumatoid arthritis

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to the field of immunology. Disclosed is a method of treating rheumatoid arthritis, chronic arthritis in children or Castelman's disease, application of an antibody in the said method, as well as application of an antibody in production of a medication for treatment of rheumatoid arthritis, chronic arthritis in children or Castelman's disease. The antibody by the claimed invention possesses an improved antigen-neutralising ability, pharmacokinetics, immunogenicity, safety and physicochemical properties and can be further applied in therapy of diseases, associated with the activation of the receptor IL-6.

EFFECT: claimed is the medication for treatment of rheumatoid arthritis, chronic arthritis in children or Castelman's disease, representing the antibody against the receptor IL-6, obtained on the basis of the antibody TOCILIZUMAB.

12 cl, 5 dwg, 2 ex

 

Technical area

The present invention relates to medicines for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine that contain antibody to the receptor of IL-6 as an active ingredient.

Background of the invention

IL-6 is a cytokine with diverse functions and is produced by various cell types such as T cells, b cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesangial cells and synovial cells (non-Patent documents 1 and 2). IL-6 binds to IL-6 receptor, and then the signal is transferred into cells after binding of the complex IL-6/receptor IL-6 gp130 (non-Patent document 3). There are two types of receptors IL-6: membrane type and soluble form. A soluble form of the receptor of IL-6 may also form a complex IL-6/receptor of IL-6 and, thus, can provide the transmission of the signal through binding to gp130.

Rheumatoid arthritis (RA) is a systemic inflammatory disease of unknown cause, and its main symptoms are frequent arthritis and progressive destruction of the joints. Extra-articular symptoms include distribution of pathological changes in lungs, kidneys and subcutaneous tissue. The hallmark of RA is the persistent synovitis, who, despite the fact, there are various systemic symptoms. Pathological functional feature of this disease is the destruction of cartilage, synovitis, which causes bone erosion and subsequent functional loss of the joint. When RA is an increase in blood vessels in the joints, and leukocytes, such as lymphocytes and macrophages, migrate from the blood vessels in the synovial joint tissue. In the joint is the local immune response, where the inflammatory response is induced by the action of cytokines produced from lymphocytes and macrophages. As a result, destruction of the cartilage/bone progresses.

When RA is as follows: increases the rate of sedimentation of red blood cells, increasing the level of CRP (C-reactive protein), increases the number of platelets, increased levels of polyclonal immunoglobulins and rheumatoid factor is present. Have any suggestion that IL-6 is involved in these changes. As reported, the level of IL-6 is elevated in serum and synovial fluid of patients with RA, and thus, there is a correlation between the level of IL-6 and degree of disease activity of RA (non-Patent documents 4 to 6). It was also shown that in patients with RA the production of IL-6 in synovial tissue increased (non-Patent document 7). In addition, it was reported that IL-6 is involved in bone resorption by aktivirovani the differentiation of precursor cells of osteoclasts in osteoclasts in the presence of soluble receptor of IL-6 (non-Patent document 8). These discoveries suggest that IL-6 and soluble receptor of IL-6 are involved in destruction of the joint. Indeed, in the joint fluid of patients with RA, the level of soluble receptor of IL-6 increased. The concentration of soluble receptor of IL-6 and IL-6 are quite high and comparable to the levels that give the possibility to induce osteoclastsin vitro(Non-patent document 9). Thus, it is assumed that IL-6 is involved in various processes such as the formation of antibodies, infiltration of lymphocytes, formation of pannus, the destruction of the joints, acute-phase reaction and anemia in the pathogenesis of RA (non-Patent document 10). The results of recent clinical studies of RA have demonstrated excellent effect gumanitarnogo antibodies against IL-6R (TOCILIZUMAB), which can communicate both with the receptor IL-6R membrane type, and soluble receptor of IL-6 and, thus, to inhibit IL-6 signal. This suggests that the inhibition of the receptor for IL-6 is a highly effective therapeutic method for the treatment of RA (non-Patent document 10).

It is also known that, in addition to RA, TOCILIZUMAB is effective in the treatment of chronic arthritis in children and diseases of Castlemaine.

Prototype documents considered area associated with the present invention, is presented below.

Prototype documents races is motivemag region

[Patent documents]

[Non-patent document 1] Kishimoto T. The biology of interleukin-6. Blood 1989; 74: 1-10.

[Non-patent document 2] Guerne PA, Zuraw BL, Vaughan JH, Carson DA, Ltz M. Synovium 20 as a source of interleukin 6 in vitro. Contribution to local and systemic manifestations of arthritis. J Clin Invest 1989; 83: 585-92.

[Non-patent document 3] Nishimoto N, Kishimoto T. Interleukin 6: from bench to bedside. Nature Clinical Practice Rheumatology 2006; 11: 19-26.

[Non-patent document 4] Harada T, Matsuda T, Turner M, Miyasaka N, Buchan G, Tang B, 25 Sato K, Shimizu M, Maini R, Feldmann M, Kishimoto T: Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. European Journal of Immunology 1988; 18: 1797-1801.

[Non-patent document 5] Houssiau FA, Devogelaer JP, Van Damme J, De Deuxchaisnes CN, Van Snick J: Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and 30 other inflammatory arthritides. Arthritis & Rheumatism. 1988; 31: 784-788.

[Non-patent document 6] Madhok R, Crilly A, Watson J, Capell HA. Serum interleukin 6 levels in rheumatoid arthritis: correlations with clinical and laboratory indices of disease activity. Ann Rheum Dis 1993; 52:232-4.

[Non-patent document 7] Sack U, Kinne RW, Marx T, Heppt P, Bender S, Emmrich F: 35 Interleukin-6 in synovial fluid is closely associated with chronic synovitis in rheumatoid arthritis. Rheumatolology International 1993; 13: 45-51.

[Non-patent document 8] Tamura T, Udagawa N, Takahashi N, Miyaura C, Tanaka S, Yamada Y, Koishihara Y, Ohsugi Y, Kumaki K, Taga T, Kishimoto T, Suda T: Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proceedings of the National Academy of Sciences of the United States of America USA 1993; 90: 11924-11928.

[Non-patent document 9] Kotake S, Sato K, Kim KJ, Takahashi N, Udagawa N, Nakamura I, Yamaguchi A, Kishimoto T, Suda T, Kashiwazaki S: Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast - like cell formation.Journal of Bone & Mineral Research 1996; 11: 88-95.

[Non-patent document 10] Inhibiting interleukin-6 in rheumatoid arthritis. Choy E. 10 Curr Rheumatol Rep. 2008 Oct; 10(5): 413-7.

Disclosure of invention

[Problem solved by the invention]

The present invention was made in light of the above circumstances. The purpose of this invention is the provision of medicines for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprise as an active ingredient the antibody to the receptor of IL-6. More specifically, the present invention is a medicinal product for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprise as an active ingredient the antibody against the receptor of IL-6 with improved antigen-neutralizing capacity, pharmacokinetics (save in the plasma), the immunogenicity, safety and physical-chemical properties, due to the replaced amino acids in the Tocilizumab, which was used as a drug for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

[Means for solving these problems]

The authors present invention has created antibodies against the receptor for IL-6 with improved antigen-neutralizing capacity, pharmacokinetics (save in PLA is IU), immunogenicity, safety and physical-chemical properties, due to the replaced amino acids in the Tocilizumab, which was used as a drug for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine. Then the authors present invention showed that the pharmaceutical substance comprising as an active ingredient the above-described antibody against the receptor of IL-6, useful in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

More specifically, the present invention provides the following[1]-[3]:

[1] for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprises as an active ingredient are described below antibody:

(a) an antibody that includes a heavy chain including:

CDR1 (hypervariable site 1), having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73);

(b) an antibody that includes a light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid follower of the awn SEQ ID NO: 6 (CDR3 VL3); or

(c) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),

and light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3);

[2] for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprises as an active ingredient are described below antibody:

(a) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73);

(b) an antibody that includes a light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3); or

(c) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3; and

[3] for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprises as an active ingredient are described below antibody:

(a) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73);

(b) an antibody that includes a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3); or

(c) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3).

The present invention also provides the following:

[4] a method of treating rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which includes a step of introducing the following antibodies:

(a) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73);

(b) an antibody that includes a light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3); Il-Christ.

(c) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),

and light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3);

[5] a method of treating rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which includes a step of introducing the following antibodies:

(a) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73);

(b) an antibody that includes a light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3); or

(c) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3);

[6] the method of treatment of revm toignore arthritis, chronic arthritis in children or illness of Castlemaine, which includes a step of introducing the following antibodies:

(a) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73);

(b) an antibody that includes a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3); or

(c) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3);

[7] using the following antibodies:

(a) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73);

(b) an antibody that includes a light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3); or

(c) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid follower of the awn SEQ ID NO: 3 (CDR3 VH3-M73),

and light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3),

in the production of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine;

[8] using the following antibodies:

(a) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73);

(b) an antibody that includes a light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3); or

(c) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3),

in the production of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine;

[9] using the following antibodies:

(a) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73);

(b) the antibodies is about, which contains a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3); or

(c) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3)

in the production of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine;

[10] described below antibody for use in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine:

(a) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73);

(b) an antibody that includes a light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3); or

(c) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH-M73),

and light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3);

[11] described below antibody for use in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine:

(a) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73);

(b) an antibody that includes a light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3); or

(c) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3); and

[12] described below antibody for use in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine:

(a) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73);

(b) an antibody that includes a light chain having the amine is acid sequence of SEQ ID NO: 10 (VL3); or

(c) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3).

[Effects of the invention]

Tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprise as an active ingredient the antibody to the receptor of IL-6 produced by using the present invention with improved antigen-neutralizing capacity, pharmacokinetics (maintaining plasma), immunogenicity, safety and physico-chemical properties. Therefore, it is possible to reduce the frequency of administration, and they can provide a prolonged therapeutic effect.

Brief description of drawings

Fig. 1 is a graph showing time dependence of the concentration of the antibodies in the plasma after administration of TOCILIZUMAB or Fv4-M73 in the amount of 1 mg/kg Javanese makkam.

Fig. 2 is a graph showing time dependence of the concentration of CRP in plasma after administration of TOCILIZUMAB or Fv4-M73 in the amount of 1 mg/kg Javanese makkam.

Fig. 3 is a graph showing time dependence of the ratio of unbound soluble receptor of IL-6 in plasma after administration of TOCILIZUMAB or Fv4-M73 in the amount of 1 mg/kg Javanese makkam.

Phi is. 4 is a graph showing the inhibiting effect of TOCILIZUMAB and Fv4-M73 on the production of MCP-1 in synovial cells derived from human patients with RA.

Fig. 5 is a graph showing the inhibiting effect of TOCILIZUMAB and Fv4-M73 on the production of VEGF synovial cells derived from human patients with RA.

The method embodiment of the invention

The present invention relates to a means for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprise as an active ingredient the antibody of the following:

(a) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73);

(b) an antibody that includes a light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3); or

(c) an antibody that includes a heavy chain including:

CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),

CDR2 having the amino acid SEQ ID NO: 2 (CDR2 VH3-M73), and

CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),

and light chain, including:

CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),

CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and

CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3).

The present invention also relates to a means for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which comprise as an active ingredient the antibody of the following:

(a) an antibody that includes a heavy chain comprising a variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73);

(b) an antibody that includes a light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3); or

(c) an antibody that includes a heavy chain comprising the variable region of the heavy chain with the amino acid sequence SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of the light chain with the amino acid sequence SEQ ID NO: 8 (variable region VL3).

The present invention also relates to a means for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which shall include as an active ingredient the antibody, listed below:

(a) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73);

(b) an antibody that includes a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3); or

(c) an antibody that includes a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3).

The present invention also provides antibodies containing the amino acid sequence in which one or more amino acids (typically 30 amino acids or less, preferably 10 amino acids or less, more preferably five amino acids or less, and even more preferably by three amino acids or less) is changed, deleted, added, and/or built or modified antibody of the present invention containing the above-described amino acid sequence. Such antibodies containing changes or modifications to the amino acid sequence, preferably have activity (antigen-binding activity of the antigen-neutralizing activity and others), equivalent to that of the original antibody.

In addition, antibodies are used as the antibodies of the present invention, may represent bespecifically antibodies. Bespar the specific antibody refers to an antibody which has a variable region in the same molecule antibodies that recognize different epitopes. Bespecifically antibody of the present invention may be bespecifically antibody, which recognize different epitopes on the receptor molecule IL-6, or bespecifically antibody in which one of the antigen-binding sites recognize the receptor for IL-6, and the other antigen-binding site recognizes another substance. Examples of antigens that are associated with other antigen-binding site especifismo antibodies, which is constructed from a receptor of IL-6-binding antibodies of the present invention include IL-6, TNFα, TNFR1, TNFR2, CD80, CD86, CD28, CD20, CD19, IL-1α, IL-β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxin-α, lymphotoxin-β 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 - α, VEGF, VEGFR, EGF, EGFR, CCR5, APRIL and APRILR.

Frame region FRs used for the antibodies of the present invention, are not particularly limited and can be appropriately selected by the specialists in this field. No special limitation exists, however, it is preferable to use a frame region obtained from people. Frame region FRs can be a frame area having a change of amino acids in natural sequence.

Constant the region, used for antibody according to the present invention are not particularly limited and can be appropriately selected by the specialists in this field. No special limitation exists, however, it is preferable to use a constant region derived from the people. The constant region may be a constant region having a change of amino acids in natural sequence.

The above-described antibodies of the present invention are antibodies against the receptor for IL-6, which are superior to antigen-neutralizing ability, pharmacokinetics (maintaining plasma), immunogenicity, safety, and/or physico-chemical properties. Antibodies are particularly useful as vehicles for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

Antibodies of the present invention can be produced by methods known to experts in this field.

Antibodies of the present invention can be produced, for example, using the following genetically engineered methodology. The gene encoding the antibody of the present invention, design and inserted into an appropriate vector and then the vector is introduced into the host to produce antibodies (see, for example, Borrebaeck, C. A. K. and Larrick J.W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

Thus, the present invention is methods of producing the antibodies of the present invention, which include the stage of culturing the host cell containing the vector, which introduced the gene encoding the antibody of the present invention.

More specifically, the present invention is methods of producing the antibodies of the present invention, which include stages:

(a) culturing the host cell containing the vector, which introduced the gene encoding the antibody of the present invention; and

(b) obtaining the antibody encoded by the genome.

In particular, when the antibody is produced using mammalian cell gene such antibodies can be Express using DNA, which is functionally connected by suitable well-known promoter, a gene expressed antibodies and a polyadenylation signal located downstream of the gene with the 3'-side, or using a vector carrying the DNA. Examples of promoter/enhancer is a fast early promoter/enhancer of the human cytomegalovirus.

In addition, as the promoter/enhancer that can be used for expression of the antibodies of the present invention, it is possible to use viral promoters/enhancers of retroviruses, viruses, p is limy, adenoviruses, simian vacuolating virus 40 (SV-40) or similar, or promoters/enhancers derived from mammalian cells, such as the promoter/enhancer of the elongation factor 1α (HEF1α) person or similar.

The expression of antibodies can be readily implemented using, for example, the method Mulliganet al. (Mulligan, R. C.et al., Nature (1979) 277: p.108-114) using promoter/enhancer SV40, or how Mizushimaet al.(Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18: 5322) using promoter/enhancer HEF1α.

When the antibody is produced usingE. colithe antibody can be Express by means functionally associated common of a suitable promoter, a signal sequence for secretion of antibodies and antibody gene that you want to Express. Examples of promoters include the lacZ promoter and araB promoter. The lacZ promoter can be used in accordance with the method Wardet al.(Ward, E. S.et al., Nature (1989) 341: 544-546; Ward, E. S.et al., FASEB J. (1992) 6: 2422-2427), araB promoter can be used in accordance with way Betteret al.(Better, M.et al., Science (1988) 240: 1041-1043).

When the production of antibodies is carried out in periplasmE. colias a signal sequence for secretion of antibodies can be used signal pelB sequence (Lei, S. P.et al.J. Bacteriol. (1987) 169: 4379-4383). After the Department is of antibodies biogas produced in periplasm, the structure of the antibody is subjected to repeated folding and then use (see, for example, WO 96/30394).

The plot of the beginning of the replication can be obtained from the SV-40, viruses polyoma, adenoviruses, human papilloma virus of cattle (BPV) and the like. In addition, to increase the number of copies of a gene in a cell host expression vector may contain, as a selective marker gene aminoglycoside phosphotransferase (APH), gene timedancing (TK), gene contingenciesliabilities (Ecogpt)E. Coligene digidrofolatreduktazy (dhfr) and the like.

To obtain antibodies of the present invention can use any system of production.

System producingin vitroandin vivosuitable as systems for the production of antibodies. System of production that use eukaryotic cells and prokaryotic cells, are examples of systems producingin vitro.

System of production that use animal cells, plant cells or cells of fungi, are available when using eukaryotic cells. Known animal cells include (1) mammalian cells such as CHO, COS, myeloma, kidney, baby hamster (KSS), HeLa, Vero and the like, (2) cells of amphibians, such as oocytesXenopus laevisand (3) insect cells such as sf, sf21, Tn5 and the like. Known plant cells include cells derived fromNicotiana tabacumand these cells can be grown as calluses. Known cells of fungi include yeast, for example, the genus Saccharomyces, such asSaccharomyces cerevisiaeand filamentous fungi, for example, the genus Aspergillus, such asAspergillus niger.

System of production, which use bacterial cells are available when using prokaryotic cells. Examples of bacterial cells includeE. coliandBacillus subtilis.

Antibodies can be obtained by introducing a gene of interest antibodies in these cells by transformation, and then culturing these transformantsin vitro. Transformants can be grown using known methods. For example, the nutrient medium can be used DMEM, MEM, RPMI 1640, or IMDM, and it can be applied with the addition of serum, such as fetal calf serum (FCS). In addition, antibodies can be produced byin vivoby passing the cells introduced with a gene of the antibody in the abdominal cavity or similar animals.

On the other hand, the system of production ofin vivoinclude a system of production with the use of animals and the production system using plants. Mammals, insects, etc. are used in systems producerof the deposits with animals.

You can use mammals such as goat, pig, sheep, mouse, and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Alternative you can use insects such as silk worm. Plants can be used, for example, tobacco.

Genes of antibodies injected into these animals or plants, then produce antibodies in animals or plants, and then remove. For example, the antibody gene prepared as fused gene by integration of the gene of the antibody in the gene encoding a protein that is specifically produced in milk, such as gene β-casein goats. DNA fragments containing the fused gene, which was incorporated gene antibodies, and then injected into goat embryos, which are then injected into goats. The desired antibody can then be obtained from milk produced by transgenic goats that were born from the goats that received embryos, or their offspring. To increase the production of milk containing an antibody in transgenic goats can properly introduce hormones (Ebert, K.M.et al., Bio/Technology (1994) 12: 699-702).

In the case of silk worm to infect silkworms used baculoviruses carrying the gene of interest antibodies, after which interest is an antibody obtained from body fluids (Maeda, S.et al.,Nature (1985) 315: 592-594). In the case of tobacco, the gene of interest antibodies inserted into the expression vector plants, for example, pMON 530, and the vector can then be introduced into the bacterium, such asAgrobacterium tumefaciens. The bacteria are then used to infect tobacco, such asNicotiana tabacumthat then leaves the tobacco receive the desired antibodies (Julian K.-C. Maet al., Eur. J. Immunol. (1994) 24: 131-138).

When the production of antibodies using the system of production ofin vitroorin vivoas described above, each DNA encoding a heavy chain (H chain) or light chain (L-chain) antibody, can separately be included in the expression vector for simultaneous transformation of the host cell, or alternatively, DNA encoding the H-chain and L-chain can be embedded into a single expression vector for transformation of a host cell (see International patent application No. WO 94/11523).

Antibodies are produced and expressed, as described above, can be distinguished from the internal contents of the cell or cellular environment or from organisms hosts, and then clear to homogeneity. Antibodies used in the present invention, it is possible to select/clear using affinity chromatography. Columns used in affinity chromatography include, for example, a protein-A column and protein G column. The media used for affine who chromatography include, for example, HyperD, POROS, and Sepharose F.F. you can also use other traditional methods of isolation and/or purification of proteins. Such methods are not particularly limited.

For example, antibodies of the present invention can select/clear using appropriately selected/combined chromatography, filter, ultrafiltration, vysalivaniya, dialysis, etc. in addition to the above-described affinity chromatography. Such chromatography include, for example, ion exchange chromatography, hydrophobic chromatography and gel filtration, and can be used for high performance liquid chromatography (HPLC). In addition, you can use reverse-phase HPLC.

The concentration of antibodies, prepared as described above, can be determined by measuring the optical density, enzyme-linked immunosorbent assay (ELISA) or so In particular, in the case of measuring the optical density of the solution of antibodies diluted PBS(-) and measured the optical density at a wavelength of 280 nm. The concentration calculated by the optical density (1,35 OD=1 mg/ml). Alternatively, in the case of ELISA chemical analysis can be carried out by the following method. Namely, 100 μl of the antibody to IgG goat man (TAG) at a dilution of 1 µg/ml in 0.1 M bicarbonate buffer (pH 9,6) are added to 96-well plate (Nunc) and inquiry is over night at 4°C for immobilization of antibodies. After blocking for tablet add appropriately diluted antibody of the present invention, appropriately diluted sample containing the antibody, or IgG (CAPPEL) as the standard (100 μl), then the tablet was incubated for one hour at room temperature.

After washing add 100 ál of diluted 5000 times of antibodies to human IgG labeled with alkaline phosphatase (BIO SOURCE), and incubated for one hour at room temperature. After wash add substrate solution and incubated, and the optical density measured at 405 nm using the microplate reader for Model 3550 (Bio-Rad)to calculate the concentration of interest antibodies.

The activity of the antibodies used in the present invention, the inhibition of IL-6-induced signal can be measured using conventional methods known to experts in this field. For example, IL-6 added to cultures of IL-6-dependent myeloma cell lines of human (S6B45 and KPMM2), T-cell lymphoma line, Lennert person KT3 or IL-6-dependent cell lines MH60.BSF2; and the accumulation of3H-thymidine IL-6-dependent cells to determine the presence of an inhibitor of IL-6. Alternatively, cultivate expressing the receptor of IL-6 cells U266, and to the culture at the same time add125I-labeled IL-6 and the inhibitor I-6, and then define125I-labeled IL-6 associated with the cells expressing the receptor of IL-6. In addition to the group of inhibitors of IL-6, in the above-described test systems include a group of negative control without inhibitor of IL-6. IL-6-inhibitory activity of an inhibitor of IL-6 can be estimated by comparing the results for both groups.

Antibodies used in the present invention may be antibody fragments or modified product thereof, provided that they can be appropriately used in the present invention. Such antibody fragments include Fab, F(ab')2, Fv and single chain Fv (scFv) and sc(Fv)2, which Fvs H - and L-chains are linked together by appropriate linkers.

Antibodies used in the present invention also include modified antibodies, such as antibodies associated with various molecules such as polyethylene glycol (PEG). Here "antibody" includes such modified antibodies. Such modified antibodies can be obtained by chemical modification of the obtained antibodies. Such methods have previously been taken in this field.

Tocilizumab, which is humanitariannet antibody against the receptor of IL-6 person, was approved in Japan as a medicinal product for the treatment of rheumatoid arthritis, chronic what about the arthritis in children (e.g., juvenile idiopathic arthritis, which operates in most joints, and systemic juvenile idiopathic arthritis) and diseases of Castlemaine.

The above-described antibodies of the present invention are antibodies whose antigen-neutralizing capacity, kinetics in the blood, immunogenicity, safety and physico-chemical properties have been improved by changing amino acids in the Tocilizumab. Needless to say, the above-described antibodies of the present invention have the same therapeutic effect as Tocilizumab, and, thus, can be used as drugs for the treatment of rheumatoid arthritis, chronic arthritis in children and diseases of Castlemaine.

Medicines according to the present invention can be introduced in the form of a pharmaceutical product and you can enter systemically or locally via oral or parenteral methods. For example, you can choose intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, the introduction of using suppositories, infusion in the colon or enteric agent for oral administration. The appropriate route of administration can be chosen depending on the patient's age and symptoms. In most cases, the effective dose is chosen within the range from 01 to 100 mg/kg of body weight at each administration. Alternatively, the dose can be selected in the range from 1 to 1000 mg/patient, preferably within the range from 50 to 250 mg/patient. The preferred dose can also find experts in this field.

Drugs of the present invention may contain pharmaceutically acceptable carriers, such as preservatives and stabilizers. "Pharmaceutically acceptable carrier" means a material that can be entered together with the above drugs and who may have or may not have the above-described therapeutic effect. Alternatively, the media can be a material that has no therapeutic effect, but creates an additional or synergistic stabilizing effect when applied in combination with the antibody.

Examples of pharmaceutically acceptable materials include sterile water, saline, stabilizers, fillers, buffers, preservatives, surfactants, complexing means (for example, etc) and a binder, and the like.

In the present invention, examples of surfactants include non-ionic surfactant. Typical examples include sorbitane esters of fatty acids, such as arbitarily ether monocapillary acid, arbitarily ether of monolaurin the th acid or arbitarily ether monopalmitate acid; glycerin fatty acid esters such as glycerol ester monocapillary acid, glycerin ether monodirectional acid or glycerol ether of monostereo acid; polyglyceryl esters of fatty acids, such as decapitaciones ether of monostereo acid, decapitaciones ether of diseasemovie acid or decapitaciones ether monolinoleate acid; polyoxyethylenesorbitan esters of fatty acids, such as polyoxyethylenesorbitan ether of monolauric acid, polyoxyethylenesorbitan ether monolingual acid, polyoxyethylenesorbitan ether of monostereo acid, polyoxyethylenesorbitan ether monoplotting acid, polyoxyethylenesorbitan ether trilingual acid or polyoxyethylenesorbitan ether of tristearin acid; polyoxyethylenesorbitan esters of fatty acids, such as polyoxyethylenesorbitan ether of tetrastearate acid, polyoxyethylenesorbitan ether tetrominoes acid; esters polyoxyethyleneglycol fatty acids, such as polyoxyethyleneglycol ether of monostereo acid; polietilenglikolya esters of fatty acids, such as polietilenglikolya ether of diseasemovie acid; polyoxyethylenesorbitan esters, such as polyoxyethyleneglycol ether; polyoxyethylenesorbitan EPE is, such as polyoxyethyleneglycol, polyoxyethylenesorbitan ether or policyactioninpolicyrule ether; polyoxyethylene alkylphenolic ether, such as polyoxyethylene nonylphenoxy ether; polyoxyethylene hardened castor oils such as polyoxyethylene castor oil, or polyoxyethylene hardened castor oil (polyoxyethylene hydrogenated castor oil); polyoxyethylene derived beeswax, such as polyoxyethylenesorbitan beeswax; polyoxyethyleneglycol derivatives, such as polyoxyethylenated; and polyoxyethylenated fatty acids with hydrophilic-lipophilic balance (HLB) of from 6 to 18, such as polyoxyethylenated.

As surfactants can also list anionic surfactants. Typical examples of anionic surfactants may include alkylsulfate salts, alkyl group having from 10 to 18 carbon atoms, such as cityswift sodium, sodium lauryl sulfate or reinsulate sodium; polyoxyethylenesorbitan salt, whose average mol of ethylene oxide added is from two to four, and the number of carbon atoms in the alkyl group is from 10 to 18, such as polyoxyethyleneglycol sodium salts alkylsulfonate esters with the number of the carbon atoms in the alkyl group is from 8 to 18, as, for example, sodium salt laurylsulphate ether; natural surfactants such as lecithin or glycerophospholipid; sphingophospholipids, such as sphingomyelin; and sucrose esters of fatty acids with the number of carbon atoms in the fatty acid of 12 to 18.

One or a combination of two or more of these surfactants can be added to medicinal products of the present invention. The preferred surfactant for use in the preparations according to the present invention is polyoxyethylenesorbitan ether fatty acids, such as Polysorbate 20, 40, 60, 80 or the like, especially preferred is Polysorbate 20 and 80. Also is preferred polyoxyethyleneglycol presented Poloxamer (e.g., Pluronic F-68®).

The amount of added surfactant varies depending on the type of surfactant, but for Polysorbate 20, Polysorbate 80 or Poloxamer 188 is generally of 0.0001-10% (weight/volume), preferably 0.001 to 5% and more preferably 0.005 to 3%.

The buffers of the present invention include such as buffers on the basis of phosphoric acid, citric acid, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, Caprylic acid, deoxycholic acid, salicylic is islote, triethanolamine, fumaric acid, other organic acids, buffer based on carbonic acid, Tris buffer, his-tag buffer, imidazole buffer, etc.

Dissolved compounds can be prepared by dissolving in aqueous buffers known in the field of dissolved compounds. The concentration of the buffer is usually 1-500 mm, preferably 5-100 mm and even more preferably 10-20 mm.

Drugs of the present invention may optionally include other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, amino acids, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols.

Examples of amino acids according to the present invention include basic amino acids such as arginine, lysine, histidine and ornithine, and inorganic salts of these amino acids (preferably in the form of chloride salts and phosphate salts, or more specifically, the phosphates of amino acids). In the case of free amino acids, the pH is adjusted to a preferred value by adding a suitable physiologically acceptable buffer, such as, for example, inorganic acids, in particular hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, formic acid or their salts. In such cases, the use of a salt of phosphoric acid is about obinna useful since it is possible to obtain particularly stable lyophilized product. This is especially useful when the drug is substantially not contain organic acids such as malic acid, tartaric acid, citric acid, succinic acid or fumaric acid, or when the corresponding anion (ion malate, tartrate ion, citrate ion, succinate ion, ion fumarata or similar) are not present. Preferred amino acids are arginine, lysine, histidine or ornithine. In addition, you can use acidic amino acids such as glutamic acid and aspartic acid and a salt (preferably sodium salt); neutral amino acids such as isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine or alanine; or aromatic amino acids such as phenylalanine, tyrosine, tryptophan or its derivative N-acetyltryptophan.

Examples of sugars and carbohydrates such as polysaccharides and monosaccharides, according to the present invention include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose and raffinose.

Examples of sugar alcohols according to the present invention include mannitol, sorbitol, Inositol, and the like.

When the pharmaceutical agent of the present invention is used in the form of an aqueous solution used for any the capabilities of, the solution can be mixed with, for example, physiological saline and isotonic solutions containing glucose or other additional substances, such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. The solution can also be combined with the appropriate solubilizers substances such as alcohol (e.g. ethanol), polyalcohol (for example, propylene glycol or PEG), or non-ionic surfactants (e.g. Polysorbate 80 or HCO-50).

Optionally, you can include diluents, solubilizing agents, agents to maintain pH, sedatives, sulfur-containing reducing agents and antioxidants.

Examples of sulfur-containing reducing agents of the present invention includeN-acetylcysteine,N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, coorbital, thioglycolic acid and salt of this, sodium thiosulfate, glutathione, and connections, bearing sulfhydryl groups, such as tilkenoidnih acid with the number of carbon atoms and from one to seven.

Examples of the antioxidants of the present invention include erythorbate acid, dibutylaminoethanol, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbicacid, sodium bisulfite, sodium sulfite, tremellales, propylgallate and to plexopathies substances, such disodium salt of ethylenediaminetetraacetic acid (edtc), sodium pyrophosphate and metaphosphate sodium.

If necessary, the pharmaceutical preparations can be enclosed in microcapsules (microcapsules made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate] or similar), or put in colloidal systems drug delivery (as, for example, liposomes, albumen microspheres, microemulsion, nanoparticles and nanocapsules) (see, for example, "Remington''s Pharmaceutical Science 16th edition", Oslo Ed., 1980). Methods of preparation of pharmaceutical products like pharmaceutical controlled release, also well known, and such methods can be prominenet in the present invention (Langeret al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12: 98-105; U.S. Patent No. 3773919; European (EP) patent application No. 58481; Sidmanet al., Biopolymers (1983) 22: 547-556; EP 133988). In addition, the volume of liquid for subcutaneous injection can be increased by adding or mixing hyaluronidase means (for example, see WO 2004/078140).

Used pharmaceutically acceptable carriers properly selected from among the above or combinations thereof, in accordance with the dosage form, but is not limited to this.

The present invention relates to methods for treating rheumatoid arthritis, chronic arthritis in children is or illness of Castlemaine, which include stage introduction to the subject of the medicinal product according to the present invention. Here, "subject" refers to a body or body part of the organism, which introduces the medicinal product according to the present invention. Organisms include animals (e.g., human, varieties of domesticated animals and wild animals), but is not limited to this specific. The above parts of the body are" not particularly limited.

In the present invention, the expression "enter" includes oral or parenteral administration. Oral administration includes the introduction in the form of oral means, and dosage forms for oral means you can choose from granules, powders, tablets, capsules, dissolved funds, emulsions, suspensions and the like.

Parenteral administration includes the introduction of injectable forms, examples of which include intravenous, subcutaneous administration, intramuscular and intraperitoneal administration. In addition, the effects of the methods of the present invention can be achieved by introducing composed of oligonucleotides gene intended for introduction into a living organism using methods of gene therapy. The pharmaceutical agents of the present invention can be applied topically exposed to the treatment site. For example, the means in which the contain by local infusion or catheter during surgery, or by targeted gene delivery DNA that encodes a peptide of the present invention.

Medicines according to the present invention it is possible to introduce the subject after the development of the symptom, or prophylactically to its development.

The present invention also relates to the use of the antibodies of the present invention in the manufacture of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine. In addition, the present invention relates to the antibodies of the present invention for use in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

Amino acids in the amino acid sequences of the present invention, excision can be modified (for example, modification of N-terminal glutamine to pyroglutamic acid by Pyroglutamate well known to specialists in this field). Naturally, such excision of modified amino acids included in the amino acid sequence of the present invention.

Further, chains of sugars that are associated with antibodies of the present invention, can be any structure. Chain sugars at position 297 (EU numbering) can be a chain of sugars of any structure (preferably of fucosylation what I sugar chain), or in this position, you may not be joining any chain of sugars (for example, this can be achieved by producing antibodies inEscherichia colior by making changes so that at position 297, EU numbering, not joined any chain of sugars).

All references cited here prototypes included in this description by reference.

EXAMPLES

Later in this document, the present invention will be specifically described with reference to Examples, but they should not be construed as limitations.

[EXAMPLE 1] RK/PD-test antibodies to the receptor of IL-6 man on monkeys

TOCILIZUMAB (H-chain WT-IgG1/SEQ ID NO: 13; L-chain WT-kappa/SEQ ID NO: 14), and Fv4-M73 (H-chain VH3-M73/SEQ ID NO: 9; L-chain VL3-kappa/SEQ ID NO: 10), which was introduced amino acid replacement or the like, to improve the antigen-neutralizing capacity, kinetics in the blood, immunogenicity, safety and physico-chemical properties of TOCILIZUMAB, expressed and purified using methods known specialists in this field (see reference example for methods). These antibodies were evaluated for their effect as a drug for the treatment of rheumatoid arthritis, according to the following procedure.

Each of TOCILIZUMAB and Fv4-M73 intravenously injected a single dose of 1 mg/kg Javanese makaka to evaluate their temporal curve to the concentrations in plasma (see reference example for the method). Time curves of plasma concentrations for TOCILIZUMAB and Fv4-M73 after intravenous shown in Fig. 1. The result showed that Fv4-M73 found a significant improvement of pharmacokinetics in cynomolgus macaques compared to TOCILIZUMAB.

Estimated efficiency of each of the antibodies to neutralize the receptor of IL-6 membrane-type cynomolgus macaque. IL-6 cynomolgus macaque) was subcutaneously injected in the lumbar region in the amount of 5 mg/kg daily from Day 6 to Day 18 after injection of antibodies (from Day 3 to Day 10 for TOCILIZUMAB), and CRP concentrations for each animal was determined after 24 hours (see reference example for the method). The temporal curve of the concentration of CRP after the introduction of each antibody is shown in figure 2. To evaluate the effectiveness of each of the antibodies to neutralize the soluble receptor of IL-6 cynomolgus macaque, was determined by the concentration of unbound soluble receptor of IL-6 cynomolgus macaque plasma cynomolgus macaques and counted the proportion of unbound soluble receptor of IL-6 (see reference example for the method). The temporal curve of the percentage of unbound soluble receptor of IL-6 after the introduction of each antibody is shown in Fig. 3.

Fv4-M73 neutralized receptor IL-6 membrane-type cynomolgus macaque more sustainable manner and inhibited the increase in CRP over a longer period, when avanyu with TOCILIZUMAB. In addition, Fv4-M73 neutralized soluble receptor of IL-6 cynomolgus macaque more sustainable manner and inhibited the increase in unbound soluble receptor of IL-6 cynomolgus macaque for a longer period compared to TOCILIZUMAB. These results show that Fv4-M73 exceeds TOCILIZUMAB in maintaining neutralization of receptor IL-6 membrane-type and soluble receptor of IL-6.

[EXAMPLE 2]

It is known that chemoatractant protein monocytes (MCP)-1 is involved in cell invasion of monocytes, T-cells, NK-cells and basophils. It was reported that MCP-1 is highly expressed in synovial tissue/synovial fluid in patients with RA (J. Clin. Invest., Sep 1992; 90(3): 772-9) and are considered to be involved in a pathological condition RA (Inflamm Allergy Drug Targets, Mar 2008; 7(1): 53-66).

VEGF is a potent angiogenic factor and, as is well known, is produced by, for example, macrophages, fibroblasts and synovial cells in the synovial membrane of patients with RA (J. Rheumatol., Sep 1995; 22(9): 1624-30). In addition, the level of VEGF in the serum of patients with RA correlates with activation of the disease and radiographic progression of the disease (Arthritis Rheum., Jun 2003; 48(6): 1521-1529; Arthritis Rheum., Sep 2001; 44(9): 2055-64), and the level of VEGF in the serum is reduced when treating patients with RA antibody against IL-6R TOCILIZUMAB; therefore, VEGF is believed, also plays an important role in the pathological condition RA (Mod. Rheumatol.2009; 19(1): 12-9; and Mediators Inflamm. 2008; 2008: 129873).

Thus, it was tested whether TOCILIZUMAB and Fv4-M73 to inhibit the production of MCP-1 and VEGF synovial cells obtained from RA patients of the person, which comes from the stimulation of sIL-6R and IL-6.

Synovial cells from RA patients person (TOYOBO)were seeded in 96-well plates in IMDM medium containing 5% FCS at a concentration of 2×104cells/0.05 ml/well and were placed on 90 minutes in CO2-incubator (37°C, 5% CO2). Added 0.05 ml of TOCILIZUMAB and Fv4-M73, diluted to appropriate concentrations, tablets left for another 15 minutes, then added 0.05 ml of the soluble receptor of IL-6 (SR344: prepared according to the method described in reference example). Tablets left for another 30 minutes and then was added 0.05 ml IL-6 (TORAY) (final concentration of the soluble receptor of IL-6 and IL-6 at 50 ng/ml each). After two days of culturing were collected cultural supernatant, and the concentration of MCP-1 and VEGF in the culture supernatant were determined using an ELISA kit (Biosource and Pierce Biotechnology). The results are shown in Fig. 4 and 5. TOCILIZUMAB and Fv4-M73 inhibited the production of MCP-1 and VEGF synovial cells obtained from RA patient person, after stimulation of the soluble receptor of IL-6 and IL-6-dependent concentration.

Accordingly, the effect lasts for Fv4-M73 ka the antibodies, neutralizing receptor IL-6 (effect of the receptor binding of IL-6 and blocking signals of the receptor of IL-6 membrane-type and soluble receptor of IL-6), significantly greater than any for TOCILIZUMAB, frequency of administration and dosage can be significantly reduced compared to TOCILIZUMAB, and, in addition, Fv4-M73 inhibits the production of MCP-1 and VEGF synovial cells obtained from RA patient person. Thus, it was shown that Fv4-M73 is a very effective drug against RA.

Reference examples

Preparation of soluble recombinant receptor of IL-6 man

Soluble recombinant receptor of IL-6 human receptor of IL-6 person, which is an antigen, obtained as described below. Was established cell line of Cho, stably expressing the soluble receptor of IL-6 person, containing a sequence from the N-terminal 1st to 344-th amino acids, published in J. Biochem. (1990) 108: 673-676 (Yamasakiet al., Science (1988) 241: 825-828 (GenBank #X12830)). Soluble receptor of IL-6 man was purified from the culture supernatant expressing SR344 cells SNO using three chromatography on columns: chromatography on a column of Blue Sepharose 6 FF, affinity chromatography using a column with immobilized antibody specific for SR344, and gel filtration column of chromatogra the AI. Faction elyuirovaniya as the main peak was used as the final purified sample.

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

Oligo-DNA primers were prepared based on the disclosed sequence receptor gene IL-6 macaque-rhesus (Birneyet al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D556-61). The DNA fragment encoding the complete gene for the receptor of IL-6 cynomolgus macaque, prepared by means of PCR using primers and, as a matrix, cDNA, obtained from the pancreas of cynomolgus macaque. The resulting DNA fragment was incorporated into the vector for expression in animal cell, and using the vector prepared line of SNO with stable expression (producing cyno.sIL-6R cell line Cho). Culture medium producing cyno.sIL-6R line SNO was purified using a HisTrap column (GE Healthcare Bioscience), and then concentrated using Amicon Ultra-15 Ultracel-10k (Millipore). Finally the purified sample of the soluble receptor of IL-6 cynomolgus macaque (hereinafter cIL-6R) was obtained by further purification on a gel-filtration column Superde×200pg16/60 (GE Healthcare Bioscience).

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

IL-6 cynomolgus macaque prepared in accordance with the methodology described below. Preparing nucleotide posledovatelno the ü, coding 212 amino acids deposited under SWISSPROT Accession No. P79341, and cloned into a vector for expression in animal cells. The resulting vector was introduced into cells navigational AIDS for training line SNO with stable expression (producing cyno.IL-6R Cho cell line). Culture medium producing cyno.IL-6R line SNO was purified using a column of SP-Sepharose FF (GE Healthcare Bioscience) and then concentrated using Amicon Ultra-15 Ultracel-5k (Millipore). Finally the purified sample IL-6 cynomolgus macaque (hereinafter cIL-6) was obtained by further purification on a gel-filtration column Superde×75pg26/60 (GE Healthcare Bioscience), and then concentrated using Amicon Ultra-15 Ultracel-5k (Millipore).

Preparation, expression and purification options TOCILIZUMAB

Plasmid fragments encoding the desired sequence antibodies, have been built into the vector for expression in animal 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 the method known to specialists in this field. Antibodies expressed using the method described below. Cell line HEK293H (Invitrogen)derived from embryonic kidney cancer man, suspended in DMEM (Invitrogen) supplemented with 10% fetal serum of bovine Invirogen). Cells were seeded into a Cup, in the amount of 10 ml/Cup in the Cup for grown cells (10 cm in diameter; CORNING) with a density of cells from 5 to 6×105cells/ml and cultured in CO2-incubator (37°C, 5% CO2during one full day and night. Then the medium was removed by suction and was added 6.9 ml of medium CHO-S-SFM II (Invitrogen). The prepared plasmids were introduced into cells using the method of lipofectin. The resulting culture supernatant were collected, centrifuged (approximately 2000 g, 5 min, room temperature) to remove cells and sterilized by filtering through a 0,22 µm filter MILLEX(R)-GV (Millipore) to obtain supernatant. Antibodies were purified from the obtained culture supernatants way, known to specialists in this field, using rProtein A SepharoseTMFast Flow (Amersham Biosciences). To determine the concentration of purified antibodies was measured by optical density at 280 nm using a spectrophotometer. The antibody concentration was calculated according to specific values using the absorption coefficient calculated by the method of PACE (Protein Science (1995) 4: 2411-2423).

RK/PD-test to determine the concentration of antibodies in the plasma concentrations of CRP and unbound soluble receptor of IL-6 in Makarov

Plasma concentrations cynomolgus macaques was determined by ELISA using the method, you know what about the professionals. The CRP concentrations were determined using an automated analyzer (TBA-120FR; Toshiba Medical Systems Co.) using Cias R DRR (KANTO CHEMICAL CO., INC.). The plasma concentration of unbound soluble receptor of IL-6 cynomolgus macaque in cynomolgus macaques was determined in accordance with the methodology described below. All antibodies IgG (IgG cynomolgus macaque, the antibody against the receptor of IL-6 on human and complex, antibodies against the receptor for IL-6 person with soluble receptor of IL-6 cynomolgus macaque) in plasma was adsorbing the protein A by loading plasma cynomolgus macaque by the corresponding number of rProtein A Sepharose Fast Flow resin (GE Healthcare), dried of 0.22-μm filter unit (Millipore). Then, the solution in the cell was poured using a high-speed centrifuge to collect the solution that was leaked. The solution, which was leaked, does not contain associated with protein And complex antibodies against the receptor for IL-6 person with soluble receptor of IL-6 cynomolgus macaque. Thus, the concentration of unbound soluble receptor of IL-6 can be determined by measuring the concentration of soluble receptor of IL-6 cynomolgus macaque in the solution, which was filtered through protein A. the concentration of the soluble receptor of IL-6 cynomolgus macaque was determined using the method, known in the art, for measuring the concentration of the receptor of IL-6 person. Rast is orily receptor IL-6 cynomolgus macaque (cIL-6R), prepared as described above was used as standard. The percentage of unbound soluble receptor of IL-6 was calculated by the following formula.

1. Drug for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which is an antibody containing heavy chain, including:
CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),
CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and
CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),
and light chain, including:
CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),
CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and
CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3).

2. Drug for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which is an antibody containing a heavy chain comprising the variable region of the heavy chain having the amino acid sequence of SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of light chain having the amino acid sequence of SEQ ID NO: 8 (variable region VL3).

3. Drug for the treatment of rawmat odnogo arthritis, chronic arthritis in children or illness of Castlemaine, which is an antibody containing a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3).

4. A method of treating rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which includes a step of introducing the antibodies, which contains a heavy chain, including:
CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),
CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and
CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),
and light chain, including:
CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),
CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and
CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3).

5. A method of treating rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, which includes a step of introducing the antibodies, which contains a heavy chain comprising the variable region of the heavy chain having the amino acid sequence of SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of light chain having the amino acid sequence of SEQ ID NO: 8 (variable region VL3).

6. A method for the treatment of PE is matainaho arthritis, chronic arthritis in children or illness of Castlemaine, which includes a step of introducing the antibodies, which contains a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3).

7. The use of antibodies that includes a heavy chain including:
CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),
CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and
CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),
and light chain, including:
CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),
CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and
CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3),
in the production of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

8. The use of antibodies, which contains a heavy chain comprising the variable region of the heavy chain having the amino acid sequence of SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of light chain having the amino acid sequence of SEQ ID NO: 8 (variable region VL3), the production of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

9. the label antibodies, which contains a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3), the production of tools for the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine.

10. The use of antibodies in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, where the specified antibody includes a heavy chain including:
CDR1 having the amino acid sequence of SEQ ID NO: 1 (CDR1 VH3-M73),
CDR2 having the amino acid sequence of SEQ ID NO: 2 (CDR2 VH3-M73), and
CDR3 having the amino acid sequence of SEQ ID NO: 3 (CDR3 VH3-M73),
and light chain, including:
CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 VL3),
CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 VL3), and
CDR3 having the amino acid sequence of SEQ ID NO: 6 (CDR3 VL3).

11. The use of antibodies in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, where the specified antibody contains a heavy chain comprising the variable region of the heavy chain having the amino acid sequence of SEQ ID NO: 7 (variable region VH3-M73), and light chain comprising the variable region of light chain having the amino acid sequence of SEQ ID NO: 8 (variable region VL3).

12. The use of antibodies in the treatment of rheumatoid arthritis, chronic arthritis in children or illness of Castlemaine, where the specified antibody contains a heavy chain having the amino acid sequence of SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence of SEQ ID NO: 10 (VL3).



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: single-domain mini-antibody is proposed, specifically binding the protein-receptor of epidermal growth factor of human HER2/ERBB2/neu obtained by immunisation of two-humped camel (Camelus bactrianus) by the preparation of tumour cells SKBR3, and characterised by the amino acid sequence. Also the method of detecting protein HER2/ERBB2/neu and its expressing cells is considered. The antibody according to the present invention is able to penetrate in the target cell on which surface HER2/ERBB2/neu is exposed, and may find further application in diagnostics and therapy of diseases associated with overexpression of HER2/ERBB2/neu.

EFFECT: improving efficiency of use of the compound.

2 cl, 6 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed are: a pharmaceutical composition and a method, based on an introduction to a patient of a therapeutically efficient amount of an antibody against CD200, which contains G2/G4 constant region, for treatment of the patient with malignant neoplasm, containing CD200 positive malignant cells. The said antibody against CD200 inhibits interaction between CD200 and CD200R.

EFFECT: application of the invention ensures better inhibition of the tumour growth with reduced effector function in comparison with application of the antibody, containing G1constant region, which can be applied in medicine.

24 cl, 29 dwg, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to using an agent reducing B-cell count for treating chronic fatigue syndrome, as well as to a method of treating chronic fatigue syndrome involving the stage of administering the agent reducing B-cell count into an individual suffering from it. The agent reducing B-cell count can represent a preferentially humanised CD20 antibody reducing B-cell count or the fragment of the above CD20 binding antibody.

EFFECT: using the given group of inventions based on B-cell count depletion, is effective in treating chronic fatigue syndrome.

14 cl, 1 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to recovered monoclonal antibodies, particularly CDR-grafted humanised antibodies binding to an epitope of human RAGE molecule, and particularly possess an ability to inhibit RAGE binding to various ligands. The invention also refers to a method for preparing the above antibodies, a recovered nucleic acid coding them, an expression vector, a host cell and a pharmaceutical composition.

EFFECT: invention provides treating the diseases or disorders associated with advanced glycation end product (RAGE) receptor, including Alzheimer's disease effectively.

16 cl, 13 dwg, 10 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to antibodies specifically bound to epidermal growth factor receptors (EGFR), as well as to DNA coding V heavy chain regions of the above antibodies, to DNA coding V light chain regions of the above antibodies. There are disclosed expression vectors containing these DNAs, and animal cell lines for expression of the above antibodies containing these vectors. There are described compositions for treating cancer related to epidermal growth factor receptor (EGFR) containing an effective amount of the above antibodies.

EFFECT: invention enables treating cancer related to epidermal growth factor receptor (EGFR) effectively.

30 cl, 12 dwg, 12 ex, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to biotechnology and provides humanised synthetic analogues (R6313/G2) of variable domains of the anti-angiotensin II type 1 receptor monoclonal scFv antibody 6313/G2, i.e. which specifically bind the molecule which specifically binds with a peptide, having the amino acid sequence EDGIKRIQDD. The invention also discloses a method of treating cancer, use of specifically binding molecules according to the invention when preparing a drug for treating cancer, a combined preparation and pharmaceutical compositions containing a specifically binding molecule according to the invention and angiotensin II.

EFFECT: present invention enables to block the harmful effect of angiotensin II and use said analogues to treat cancer in humans.

9 cl, 15 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology and medicine. What is presented is a method for preventing or treating an ocular condition related to high expression or activity of a complement factor D involving administering an antibody or its antigen-binding fragment into an individual. There are presented an anti-factor D 20D12 antibody and its antigen-binding fragment to be used to prepare a therapeutic agent, as well as containing the above antibody or its antigen-binding fragment, a kit for treating the ocular condition related to high activity or expression of factor D.

EFFECT: invention can find further application in therapy of the complement system related diseases.

14 cl, 6 dwg, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology, more specifically to allergen modifications to reduce an allergenic capacity thereof, and may be used in medicine. A modified allergen is prepared by sequential modification of all primary amino groups or a portion thereof of lysine and arginine residues of an allergen molecule with using potassium cyanate and phenylglyoxal and used as an ingredient of a pharmaceutical composition for treating allergy.

EFFECT: invention provides preparing the modified allergen possessing the reduced allergenic capacity as compared to a respective native allergenic material and allergoids prepared by modification by either cyanate, or phenylglyoxal.

9 cl, 12 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to industrial microbiology, namely to a method of obtaining a composition, intended for prevention of a cow milk protein allergy (CMPA) and a higher sensitivity to allergens in newborn babies and infants. The method includes a stage of milk substrate bioconversion by means of a culture of Bifidobacterium breve BBC50 strain, deposited on May 31 1999 in the National Collection of Cultures of Microorganisms (CNCM) under the number I-2219, by keeping a substrate in contact with the strain culture under conditions unfavourable for production of an acid by the strain.

EFFECT: application of the claimed method makes it possible to obtain the composition, efficient for prevention of the allergy to cow milk proteins and a higher sensitivity to allergens in newborn babies and infants.

10 cl, 5 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to a Fcγ receptor (Fc-gamma receptor) for application in treatment of multiple sclerosis, where multiple sclerosis represents a form of multiple sclerosis, mediated by B cells, and/or a triggered by autoantibodies form of multiple sclerosis. The invention relates to a pharmaceutical composition, containing the Fcγ receptor (Fc-gamma receptor) for application in treatment of multiple sclerosis, where multiple sclerosis represents the form of multiple sclerosis, mediated by B cells, and/or the triggered by autoantibodies form of multiple sclerosis.

EFFECT: obtaining the composition for treatment of multiple sclerosis.

7 cl, 2 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and describes a method for testing a planned or known immunomodulatory agent for T-cell activation which involves the stage of contacting a peripheral blood mononuclear cell (PBMC) culture with the pre-determined amount of the planned or known immunomodulatory agent in vitro and observing the T-cell activation in the PBMC culture using a readout system, when contacting the planned or known immunomodulatory agent, wherein the PBMC culture density at the stage of pre-culture makes at least 2×106/ml, preferentially at least 5×106/ml, more preferentially at least 107/ml, or at least 4×105/cm2, preferentially at least 106/cm2, most preferentially at least 2×106/cm2; the PBMC pre-culture is cultured for at least 12 hours.

EFFECT: invention provides the improved agent for testing the immunomodulatory agents in vitro.

10 cl, 16 dwg, 13 ex

FIELD: chemistry, pharmacology.

SUBSTANCE: group of inventions deals with complex action medication, which as active components contains complex of immunoglbulins of classes G, M, A, interferon alpha-2b human recombinant, zinc gluconate, as well as accessory components: tisolum, buffer solution, bee wax, polysorbate and hard fat in form of homogenised suppository mass, additionally it contains dry extract of aloe with particle size less than 250 mcm in amount from 0.04 g to 0.17 g per one suppository. Method of medication production includes the following stages: fat base, including bee wax in form of solid substance from white to yellow-brown colour, polysorbate and hard fat, is prepared, after that, zinc gluconate is introduced with following mixing, also prepared is mixture of active substances, for which purpose buffer solution is mixed with complex of immunoglobulins of classes G, M, A and interferon, with addition to them of Tisolum mass and mixing until homogeneous solution, after which the latter is introduced into fat base, brought to temperature 38°C, and mixed until homogenous suppository mass of composition given above is formed.

EFFECT: increased pharmacological activity of medication and extension of spectrum of action of medication, obtained by claimed method.

16 cl, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to crystalline forms of (R)-5-[3-chloro-4-(2, 3-dihydroxypropoxy)bez[z]iliden]-2-([z]-propylimino)-3-o-tolylthiazolidin-4-one, methods of their obtaining, pharmaceutical composition, containing said crystalline forms, and application of said forms as compounds, improving vascular function, and as immunomodulatory agents.

EFFECT: obtaining compounds, improving vascular function.

22 cl, 5 dwg, 5 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: compounds can be applied for treatment of oncologic and autoimmune diseases. Invention also characterises method of obtaining conjugates, pharmaceutical composition and medication, which contains modified proteins. In general formulae 1 or 2 , R1 is selected from the group representing (CH3)2N-,

R2 is selected from the group representing where R3 as terminal substituent represents -NH2, or and R4 represents H or C1-C3alkyl.

EFFECT: novel compounds possess affinity for CD16a receptor.

18 cl, 20 dwg, 3 tbl, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical and cosmetic industries and represents a composition for relieving an ultraviolet injury by post-irradiation administration of a composition containing one or more compounds specified in a group consisting of D-glutamic acid and/or its salts, and at least one acceptable additive.

EFFECT: invention provides effective relief of the ultraviolet skin injuries.

11 cl, 34 ex, 1 tbl, 8 dwg

FIELD: medicine.

SUBSTANCE: present invention refers to immunology and biotechnology. What is presented is an IL-1β-binding antibody or its IL-1β-binding fragment containing V heavy and light chain regions. The above antibody binds to human IL-1β with dissociation constant less than 1pM. Versions of the antibody are described. There are disclosed corresponding coding nucleic acids (NA), as well as: a NA passage vector to a host cell, the host cell producing a coded polypeptide. What is described is using the antibody for preparing the other format of the above antibody: "camel-like", VHH antibody, nanobody. What is disclosed is a pharmaceutical composition for treating or preventing an IL-1β-related disease in a mammal on the basis of the antibody, as well as a method of treating or preventing the IL-1β-related disease in a mammal.

EFFECT: using the invention provides the novel IL-1β-specific antibodies with high IL-1β affinity that can find application in medicine for preventing, treating the diseases mediated by IL-1β activity.

39 cl, 20 dwg, 6 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: in formula R1 is H or (1-6C alkyl); R2 represents NRbRc, (1-4C)alkyl, (1-4C)fluoroalkyl, CF3, (1-4C)hydroxyalkyl, -(1-4Calkyl)hetAr1, -(1-4Calkyl)NH2, -(1-4C alkyl)NH(1-4Calkyl), -(1-4Calkyl)N(1-4Calkyl)2, hetAr2, hetCyc1, hetCyc2, phenyl substituted where applicable by NHSO2(1-4Calkyl) or (3-6C)cycloalkyl, substituted where applicable by (1-4C alkyl), CN, OH, OMe, NH2, NHMe, N(CH3)2, F, CF3, CO2(1-4C alkyl), CO2H; C(=O)NReRf or C(=O)ORg; Rb is H or (1-6C alkyl); Rc represents H, (1-4C)alkyl, (1-4C)hydroxyalkyl, hetAr3 or phenyl, wherein the above phenyl is substituted where applicable by one or more substitutes independently from halogen, CN, CF3 and -O(1-4C alkyl); Re represents H or (1-4C)alkyl; Rf represents H, (1-4C)alkyl or (3-6C)cycloalkyl; Rg represents H or (1-6C)alkyl; X is absent or represents -CH2-, -CH2CH2-, -CH2O- or -CH2NRd; Rd represents H or (1-4C alkyl); R3 represents H or (1-4C alkyl); and n is equal to 0-6. The radical values NRbRc, Y, hetAr1, hetAr2, hetAr3, hetCyc1, hetCyc2, NReRf, R4 are specified in the patent claim. The invention also refers to a pharmaceutical composition containing the above compounds, to a method of treating Trk kinase mediated diseases and conditions, such as pain, cancer, inflammation, neurodegenerative disease, Typanosoma cruzi infection, osteolytic disease, and to a method of preparing the above compounds.

EFFECT: invention refers to new derivatives of pyrazolo[1,5-a]pyrimidines possessing an inhibitory activity on tropomyosin-related kinases (Trk).

42 cl, 1 tbl, 105 ex

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