Monoclonal antibodies against il-21 of human being

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

 

BACKGROUND of the INVENTION

[1] the Immune system is the primary defense of the body against diseases caused by pathogens, namely bacteria, viruses, fungi, etc. and also from diseases caused by abnormal growth of cells and tissues of the body (i.e. malignant tumors). Usually, the immune system has the ability to distinguish between normal cells or "their" and alien pathogens or abnormal cells or not, other people". The processes by which the immune system refrains from responding to one's own body, is called tolerance. Sometimes, the immune system loses the ability to recognize "their" as normal and the subsequent response directed against the tissue or cells, results in the loss of tolerance, a state of autoimmunity. Pathology resulting from autoimmunity, often have serious clinical consequences and are one of the main health problems in the world, especially in industrialized countries.

[2] IL-21 is a potent immunomodulatory cytokine type I of the four α-helical strands, which is associated with a heterodimeric receptor consisting of IL-21 R and the common gamma chain (review, Spolski and Leonard, Annu Rev Immunol. Nov 8; 2007). IL-21 is produced by NK-T cells and CD4+T-cells (including Pro-inflammatory Th17 cells and follicular T-to edami-helper TFN, which are important for responses germinal center) and has pleiotropic effects on innate and adaptive immune responses, including enhanced proliferation of b-cells and T-cells, increased cytotoxicity of CD8+T cells and natural (natural killer (NK) cells, the differentiation of b-cells into immunoglobulin-secreting plasma cells, and regulation of Th17 cell lines (see below). IL-21 can inhibit antigen presenting function of dendritic cells and under certain conditions can induce apoptosis in b cells and NK-cells. IL-21 has a strong antitumor activity, but also was associated with the development of various autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and psoriasis (review, Spolski and Leonard, Anna Rev Immunol. Nov. 8. 2007).

[3] it Was shown that IL-21 modulated responses of antibodies direct effect on b cells (Mehta et al., J. Immunol., 170:4111-4118, 2003; Ozaki et al., Science, 298:1630-1634, 2002; Suto et al., Blood. 100:4565-4573, 2002). IL-21 can induce the differentiation of naive b cells In antitelomerase plasma cells (Ozaki et al., J. Immunol. 173:5361, 2004; Ettinger et al., J. Immunol. 175:7867-79, 2005; Ettinger et al., J. Immunol. 128:2872-82, 2007; Kuchen et al., J. Immunol. 179:5886-96, 2007) and to stimulate the production of IgE in human cells (Kobayashi et al., Human Immunol, doi: 1:1016/j.humimm.2008.10.). In animals deprived of IL-21 or IL-21 R, less antidemocratic cells formed in the reaction germinal center and decreases the affinity maturation (Zotos et al., submitted for publication). Extrafollicular antibody productive cells, which are involved in autoimmunity require cognates assistance from the specialized subpopulation of CD4 T cells that secrete IL-21 (Odegard, et al., JEM 205(12): 2873-2886. 2008).

[4] the Formation of antibodies against allogeneic MHC is a major phenomenon in graft rejection. Recipients with graft, have increased titers of anti-MHC antibodies (highly sensitive patients with transplantation), are in temporary risk t for chronic rejection and are poor candidates for new transplants due to likely mediated by antibodies rejection of the new graft (Smith, et al., Am J Transplantation 8: 1-11, 2008). In the model rats of acute rejection of renal allograft IL-21 and IL-21 R were clearly increased in intravascular mononuclear cells of renal allografts, but not sotranslation (Hecker, et al., Immunobiology: doi: 10.1016/i.imbio.2008.04.004. (2008)). In patients with heart transplants, subject to exclusion, the levels of expression of IL-21 and IL-21 R correlate with the degree of rejection according to the ISHLT (trans., ISHLT International Society of transplantati the heart and lungs) and the highest expression is in degrees 1R and 2R (Baan, et al., Transplantation 83 (11): 1485-1492, 2007).

[5] In graft-versus-host (GVHD), the answer is anti-ALLO mediated by uncontrolled activation of T-lymphocytes from the graft, which control the inflammatory response against tissue of the host. Regulatory T cells (Treg) can modulate this response in animal models. It was shown that IL-21 neutralized regulatory function of Treg (Clough et al., J. Immunol. 180: 5395-5401, 2008). In murine models of GVHD transfer of T cells deprived of IL-21 resulted in significantly reduced clinical symptoms and histological indicators and increased survival compared with T-WT cells. A lower frequency of T cells secreting IFN-gamma and increased Tregs was observed in the mucosa of the colon. Blockade of IL-21 with the use of anti-mIL-21 mAb and migration of T cells WT has produced similar results (Bucher et al., Blood (ASH Annual Meeting Abstracts) 2008 112: Abstract #2342).

[6] have Also recently been shown that IL-21 is produced by proinflammatory Th17-cells of the mouse, and is required for differentiation of proinflammatory Th17 cells mouse (Korn et al., Nature, 448:484-487, 2007; Nurieva et al., Nature, 448:480-483, 2007; Zhou et al., Nat. Immunol., 8:967-974, 2007; Wei et al., J. Biol. Chem. 282:34605-34610, 2007). Human Th17 cells also produce IL-21 and studies are continuing to determine whether the effect of IL-21 as an autocrine factor for human Th17 cells as well as IL-21 acts to Th17-the notches of the mouse. Ozaki et al. (J. Immunol. 173:5361, 2004) showed that the expression of IL-21 is increased in predisposed to lupus mice BXSB-Yaa, a model of systemic lupus erythematosus (SLE), the age at which the initial symptoms of autoimmune processes in the first place become pronounced. The treatment of these mice BXSB-Yaa using a soluble receptor of IL-21 mouse (mIL-21R-Fc) partially inhibits various parameters of the disease, including glomerulonephritis (Bubier et al., Ann N Y Acad. Sci. 1110:590-601. 2007). It was also shown that treatment with mIL-21R-Fc was effective in another preclinical murine models of SLE disease, MRL/lpr (Herber et al., J. Immunol. 178: 3822-3830, 2007), as well as in a model of collagen-induced arthritis (CIA) rheumatoid arthritis (Young et al., Arthr. Rheum. 56: 1152-1163, 2007). Preliminary data also suggest dysregulation of IL-21 and IL-21 R in SLE (Mitoma et al., Int. J. Mol. Med. 16: 609-615. 2005; Wang et al., Chinese J. Cell. Mol. Immunol. 23(11):1041-1042. 2007; Sawalha et al., Ann Rheum. Dis. 67: 458-461, 2008). Recently, data Eng et al., obtained from 24 SLE patients and 15 healthy controls (Nguyen et al., ACR/ARHP Scientific Meeting, 1760/482, 2008 Oct 24-29, San Francisco, CA). Eng et al. showed that 1) the expression of mRNA of IL-21 is significantly increased in CD4+T-cells of patients with SLE compared with controls, 2) the levels of IL-21 are significantly elevated in the sera of patients with active SLE compared with inactive SLE or controls, 3) IL-21 increased the AET proliferation of CD4+T-Katok and CD19+b cells in patients and controls dose-dependent manner, 4) IL-21 enhances the differentiation of plasma cells induced by anti-CD40, normal control patients and SLE patients, and 5) elevated levels of IL-21 may contribute to the proliferation of self-reactive CD4+T cells and differentiation of plasma cells in SLE.

[7] Monteleone et al. showed that the expression of RNA and protein IL-21 is increased in inflamed, but not in nevospalennyh tissue of patients with Crohn's disease (CD) (and, to a lesser extent, with ulcerative colitis) and that the production of IL-21 CD3+cells mononeucleosis cells own records of patients with CD is also increased (Monteleone et al. Gastroenterology 128:687-694, 2005; Monteleone et al. Gut 55: 1774-1780, 2006; Peluso et al., J. Immunol. 178:732-739, 2007). These authors suggested that IL-21 regulates experimental colitis by modulating the balance between regulatory T cells (Tregs) and Th17 cells (Fantini et al., Eur. J. Immunol. 37:3155-3163, 2007). Inhibition of IL-21 in vivo using a soluble receptor of IL-21 in mouse and rat models of colitis leads to a significant reduction of clinical symptoms of colitis (Young et al. US 2006/0039902).

[8] the Receptor for IL-21 is expressed in NK cells, and it has been shown that NK cells are responsible for the treatment with IL-21 in vivo and in vitro. In cancer patients treated with recombinant human IL-21, watched the changed distribution of lymphocyte subpopulations, misrepresented the number of NK-cells, and increased expression of markers of activation of NK cells and cytolytic activity of effectors (Frederiksen, et al., Cancer Immunol Immunother 57(10); 1439-1449, 2008). In autoimmune diseases the activity of NK-cells may play a role in the activation of inflammation and associated tissue damage. The homing of NK-cells in the tissue is controlled by chemoattractants released at the site of inflammation (Morris and Ley, Curr Mol Med.; 4(4):431-8. 2004). NK-cells own records of patients with Crohn's disease was released greater amounts of IFN-γ and TNF-α when stimulated in vitro with IL-21 and IgG compared with cells LPNK controls (Liu and Jiu, Chronic Inflammation of Liver and Gut. Falk Symposium abst. No.163. 2008 Mar 14-15). Also reported that NK cells regulate autoimmunity and graft rejection through their interactions with dendritic cells (DC), by destruction of immature or activated DC and by releasing cytokines that disrupt the activation status and function of antigen presentation by DC (Vivier et al., Nat. Immunol. 9(5):503-510. 2008; Laffont et al., Blood 112:661-671. 2008). Comparison of mononuclear cells in the peripheral blood of tolerant and non-tolerant recipients with allograft liver showed changes in the transcriptional program of NK-cells (Martinez-Llordella et al., J. Clin. Invest. 118(8):2845-2857. 2008). Thus, blockade of IL-21 can modulate the activation status of NK-cells, to reduce their contribution to tissue inflammation in touch is immune diseases and change the course of the disease transplant rejection. It is also reported that NK cells regulate autoimmunity and graft rejection by their interactions with dendritic cells (DC)by destruction (trans., killing) immature or activated DC and by releasing cytokines that disrupt the activation status and change the function of antigen presentation DC. Thus, blockade of IL-21 can modulate the activation status of NK cells and reduce their contribution to tissue inflammation in autoimmune diseases.

[9] This invention provides monoclonal antibodies against IL-21 human and methods of using these antibodies, which inhibit the symptoms and the biological activity detected in the form of autoimmune and inflammatory disorders and are associated with interactions IL 21/IL-21 receptor.

BRIEF DESCRIPTION of FIGURES

[10] Figure 1 is an alignment of the amino acid residues containing the variable regions of the heavy chains of the antibodies marked with the numbers of clones 362.78.1.44 (78), 362.597.3 (597), 362.75.1.1 (75), 366.552.11 (552), 366.328.10 (328).

[11] Figure 2 is an alignment of the amino acid residues containing the variable region of light chains of the antibodies described above.

[12] Figure 3 illustrates the mass spectra of MALDI/TOF areas peptide sequences of IL-21 was obtained from IL-21 in pure form and immune complex of IL-21. The peptide is s sequence of IL-21, EKKPPKEF (SEQ ID NO: 2 from residue 129 to 136) (m/z (Tr, mass/charge), 1002.5619 Yes) and LERFKSLL (SEQ ID NO: 2 from residue 137 to 144) (m/z, 1005.6091 Yes), the free state of IL-21 (A). The mass shift of the peptides caused by holding deuteranomaly amides in the presence of IL-21 mAb (B). Another area of the peptide sequence of IL-21, KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2 from residue 141 to 162) (m/z, 2519.2451) the free status of IL-21 (C). A partial shift of the masses of the peptides caused by holding deuteranomaly amides in the presence of IL-21 mAb (D).

[13] Figure 4 shows the selected ion chromatogram acetylated and deacetylating peptides. Selected one ion chromatogram acetylated TCPSCDSYEKKPPKEF (SEQ ID NO: 2 from residue 119 to 136) (m/z, 1986 Da)isolated from IL-21 in its pure form (A) and similar chromatographic curve immune complex of IL-21 (In). Nested mass spectrum represents trehearne the condition of the mass of the peptide (m/z, 662.9). The third curve shows the selected ion chromatogram peptide ion at m/z 1018 Yes, that is an acetylated KSLLQKMI (SEQ ID NO: 2 from residue 141 to 148), isolated from IL-21 in its pure form (S) and similar chromatographic curve immune complex of IL-21 (D). Nested mass spectrum is a double condition of the mass of the peptide (m/z 509.1).

BRIEF description of the INVENTION

[14] In one aspect, the invention provides mono is analnoe antibody against IL-21 containing at least 80% identity with amino acid residues 20-145 SEQ ID NO: 29 and at least 80% identity with amino acid residues 21-126 SEQ ID NO: 37. In some embodiments, antibodies contain changes at least 80% of identity that exist in the variable regions of the heavy chain CDRI SEQ ID NO: 31.

[15] In another aspect, the invention provides a monoclonal antibody against IL-21 human, comprising: (a) region of the heavy chain, containing: (i) variable region of the heavy chain CDR1, containing SEQ ID NO: 31; (ii) the variable region of the heavy chain CDR2, containing SEQ ID NO: 33; and (iii) the variable region of the heavy chain CDR3 containing SEQ ID NO: 35; and (b) a light chain comprising: (i) variable region light chain CDRI, containing SEQ ID NO: 39; (ii) variable region light chain CDR2, containing SEQ ID NO: 41; and (iii) variable region light chain CDR3 containing SEQ ID NO: 43. In specific embodiments, the invention provides a monoclonal antibody against IL-21, containing amino acid residues 20-145 SEQ ID NO: 29 and amino acid residues 21-126 SEQ ID NO: 37. In other embodiments, the antibody is optionally contains amino acid residues 1-145 SEQ ID NO: 29 and amino acid residues 1-126 SEQ ID NO: 37. Another variant of the present invention provides hybridoma marked 362.78.1.44, where hybridoma deposited in the American collection ti the marketing of crops, having a patent deposited designation in ATSC MOUTH-8790, and the invention includes an antibody produced by hybridomas.

[16] Another aspect of the present invention provides a monoclonal antibody against IL-21 human, comprising: (a) region of the heavy chain, containing: (i) variable region of the heavy chain CDR1, containing SEQ ID NO: 47; (ii) the variable region of the heavy chain CDR2, containing SEQ ID NO: 49; and (iii) the variable region of the heavy chain CDR3 containing SEQ ID NO: 51; and (b) a light chain comprising: (i) variable region light chain CDR1, containing SEQ ID NO: 55; (ii) variable region light chain CDR2, containing SEQ ID NO: 57; and (iii) variable region light chain CDR3 containing SEQ ID NO: 59. In some embodiments, the invention provides a monoclonal antibody against IL-21, containing amino acid residues 20-145 SEQ ID NO: 45, amino acid residues 21-126 SEQ ID NO: 53. In other embodiments, the invention is additionally containing amino acid residues 1-145 SEQ ID NO: 45, amino acid residues 21-126 SEQ ID NO: 53. Another variant of the present invention provides hybridoma marked 362.597.3, where hybridoma deposited in the American type culture collection having the patent deposited designation in ATSC MOUTH-8786, and the invention includes an antibody produced by hybridomas.

[17] In another aspect of this izopet the tion provides a monoclonal antibody against IL-21 containing at least 80% identity with amino acid residues 20-141 SEQ ID NO: 13 and at least 80% identity with amino acid residues 21-126 SEQ ID NO: 21. In one embodiment, the invention includes a monoclonal antibody, where amino acid substitutions are conservative amino acid substitutions.

[18] In another aspect, the invention provides a monoclonal antibody against IL-21 human, comprising: (a) region of the heavy chain, containing: (i) variable region of the heavy chain CDR1, containing SEQ ID NO: 15; (ii) the variable region of the heavy chain CDR2, containing SEQ ID NO: 17; and (iii) the variable region of the heavy chain CDR3 containing SEQ ID NO: 19; and (b) a light chain comprising: (i) variable region light chain CDR1, containing SEQ ID NO: 23; (ii) variable region light chain CDR2, containing SEQ ID NO: 25; and (iii) variable region light chain CDR3 containing SEQ ID NO: 27. In some embodiments, the invention includes a monoclonal antibody against IL-21, containing amino acid residues 20-141 SEQ ID NO: 13 and amino acid residues 21-126 SEQ ID NO: 21. In another embodiment, the invention is additionally containing amino acid residues 1-141 SEQ ID NO: 13 and amino acid residues 1 -126 SEQ ID NO: 21. Another variant of the present invention provides hybridoma marked 362.75.1.1, where hybridoma deposited Staffers the second type culture collection, having a patent deposited designation in ATSC MOUTH-8791 cool designer, and the antibody is produced by hybridomas.

[19] In another aspect, the invention provides a monoclonal antibody against IL-21 human, comprising: (a) region of the heavy chain, containing: (i) variable region of the heavy chain CDR1, containing SEQ ID NO: 79; (ii) the variable region of the heavy chain CDR2, containing SEQ ID NO: 81; and (iii) the variable region of the heavy chain CDR3 containing SEQ ID NO: 83; and (b) a light chain comprising: (i) variable region light chain CDR1, containing SEQ ID NO: 87; (ii) variable region light chain CDR2, containing SEQ ID NO: 89; and (iii) variable region light chain CDR3 containing SEQ ID NO: 91. In one embodiment, this invention provides a monoclonal antibody against IL-21, containing amino acid residues 20-136 SEQ ID NO: 77 and amino acid residues 23-129 SEQ ID NO: 85. In another embodiment, the invention is additionally containing amino acid residues 1-136 SEQ ID NO: 77 and amino acid residues 1-129 SEQ ID NO: 85. Another variant of the present invention provides hybridoma marked 366.552.11, where hybridoma deposited in the American type culture collection having the patent deposited designation in ATSC MOUTH-8787, and the antibody is produced by hybridomas.

[20] In another aspect, the invention provides a monoclonal antibody against IL-21 with the holding: (a) region of the heavy chain, containing: (i) variable region of the heavy chain CDR1, containing SEQ ID NO: 63; (ii) the variable region of the heavy chain CDR2, containing SEQ ID NO: 65; and (iii) the variable region of the heavy chain CDR3 containing SEQ ID NO: 67; and (b) a light chain comprising: (i) variable region light chain CDR1, containing SEQ ID NO: 71; (ii) variable region light chain CDR2, containing SEQ ID NO: 73; and (iii) variable region light chain CDR3 containing SEQ ID NO: 75. In some embodiments, the invention provides a monoclonal antibody against IL-21, containing amino acid residues 20-139 SEQ ID NO: 61 and amino acid residues 23-129 SEQ ID NO: 69. In other embodiments, the invention is additionally containing amino acid residues 1-139 SEQ ID NO: 61 and amino acid residues 1-129 SEQ ID NO: 69. Another variant of the present invention provides hybridoma marked 366.328.10, where hybridoma deposited in the American type culture collection having the patent deposited designation in ATSC MOUTH-8789, and the antibody is produced by hybridomas.

[21] In another aspect, the invention provides hybridoma marked 366.345.6.11 where hybridoma is deposited in the American type culture collection having the patent deposited designation in ATSC MOUTH-8788, and includes an antibody produced by hybridomas.

[22] In another aspect of this is subramania this invention provides the selected monoclonal antibody, which is associated with the discontinuous epitope containing at least two regions in the protein IL-21, where the first region comprises at least one amino acid from residue Ile45 to balance Leu56 SEQ ID NO: 2 and a second area consists of at least one amino acid residue Glul29 to balance Leul44 SEQ ID NO: 2. In one embodiment, the invention provides that the first area consists of from 1 to 12 amino acids from residue Ile45 to balance Leu56 SEQ ID NO: 2, and the second area consists of 1 to 16 amino acids from residue Glul29 to balance Leul44 SEQ ID NO: 2.

[23] In each aspect included inventions described above, is a variant in which the monoclonal antibody further comprises an Fc-part, and another variant in which the Fc-part selected from the group consisting of IgG1, IgG2 and IgG4, and another variant in which the Fc portion has reduced effector function.

[24] In another aspect, the invention provides a method of treating diseases mediated by follicular T cells-helper or mediated by b-cells in the subject, by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here, where the disease mediated by follicular T-cells, helper and mediated In cells selected from the group consisting of systemic lupus erythematosus, autoimmune hearing loss, disease, Gr is visa, ordinary water, myasthenia gravis, neuromyelitis optic nerve syndrome?, autoimmune nephritis, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinative polyneuropathy (CIDP), autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (TR).

[25] In another aspect, the invention provides a method of treating diseases mediated T cells or indirect T-cells from the subject, by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here, where the disease mediated by THI cells or indirect T cells selected from the group consisting of psoriasis, spondyloarthropathy, reactive arthritis, enteropathic arthritis, autoimmune myocarditis, Kawasaki disease, gluten enteropathy, uveitis, diseases behceta, coronary artery disease, chronic obstructive pulmonary disease (COPD) and interstitial lung disease.

[26] In another aspect, the invention provides a method of treating inflammatory bowel disease (IBD) in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here, where inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis and irritable bowel syndrome.

[27] In another aspect, the invention provides a method of treating rheumatoid arthritis in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here.

[28] In another aspect, the invention provides a method of treating multiple sclerosis in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here.

[29] In another aspect, the invention provides a method of treating diabetes mellitus type I (IDDM) in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 people.

[30] In another aspect, the invention provides a method of treatment of Sjogren syndrome in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here.

[31] In another aspect, the invention provides a method of treatment of a subject with a transplant by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here, where suppress graft rejection, set the tolerance to the treatment regimen before transplantation or reduce the titers of alloantigen the subject.

[32] is another aspect of this invention provides a method of treating autoimmune disease in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 described here, where the autoimmune disease is selected from the group consisting of pancreatitis, inflammatory myopathies (polymyositis, dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, reactions, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), glomerulonephritis, IgA nephropathy-type vysokomineralizirovannaja patients with transplantation, antiphospholipid syndrome, and asthma and other autoimmune diseases or other diseases, indirect antagonists of IL-21 and its receptor IL-21.

[33] In another aspect, the invention provides a method of treating systemic lupus erythematous (SLE) in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here.

[34] In another aspect, the invention provides a method of treating psoriasis in a subject by introducing a therapeutic amount of the claimed monoclonal antibodies against IL-21 is described here.

Description of the INVENTION

[35] the Following definitions provide for ease of Pont the mania of the invention, are described here.

[36] the Terms "amino-terminal and carboxy-terminal" are used here to denote positions within the polypeptide. Where allows context, these terms are used with reference to a specific sequence or part of a polypeptide to indicate proximity or relative position. For example, a certain sequence, located carboxyl-terminal relative to the reference sequence in the polypeptide that is located proximally relative to carboxyl-end reference sequence, but not necessarily located at carboxyl-end of the full polypeptide.

[37] the Term "antagonist" refers to any compound, including a protein, polypeptide, peptide, antibody, antibody fragment, large molecule, or small molecule (less than 10 kDa), which reduces the activity, activation or function of another molecule. Antagonists of IL-21 to cause at least one of the following: reduced immune function, NK cells, dendritic cells, subpopulations of T cells and subpopulations of b-cells; binding of IL-21, in which the protein interaction of IL-21 with its receptor is blocked, inhibited, reduced or neutralized.

[38] "Antibodies" (Abs) and immunoglobulin" (Igs) are glycoproteins having the same structural characteristics. Then ka the antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody molecules that do not have antigenic specificity. The polypeptides of the latter type, for example, are produced at low levels by the lymphatic system and elevated levels of myeloma. Thus, as used here, the term "antibody" or "peptide (peptide) antibody" refers to intact antibody or its binding fragment that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human and bespecifically antibodies. In some embodiments, binding fragments produced by recombinant DNA technologies. In additional embodiments, binding fragments receive enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but without limitation, Fab, Fab', F(ab')2, Fv and single-chain antibodies, ScFv. As a rule, "natural antibodies and immunoglobulins are heterotetrameric glycoproteins of approximately 150000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while between the heavy chains of different isotypes of immunoglobulins number of disulfide bonds is atlichaetsa. Also each heavy and light chain has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end has a variable domain (VH) followed by some number of constant domains. Each light chain has at one end a variable domain (VL) and a constant domain at the other end; the constant domain of the light chain is oriented to the first constant domain of the heavy chain and the variable domain of the light chain is oriented to the variable domain of the heavy chain. Assume that the boundary surface between the variable domains of light and heavy chains form a specific amino acid residues. (Chothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).

[39] the Term "chimeric antibody" or "chimeric antibodies" refers to antibodies, genes for the light and heavy chains were constructed, typically by genetic engineering, genes constant and variable regions of immunoglobulin belonging to different species. For example, the variable segments of the genes of murine monoclonal antibody may be joined to human constant segments, such as gamma 1 and gamma 3. Thus, a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigennegative domain of mouse antibody and the constant house is a human antibody, although other mammalian species may be used. Specifically, a chimeric antibody receive methods based on recombinant DNA in which all or part of the hinge and constant regions of the light chain, heavy chain or both chains of an antibody have been substituted for the corresponding region of the other light chain or heavy chain immunoglobulin of the animal. Thus, antigennegative part of the parent monoclonal antibody are transplanted into the frame antibodies of another species.

[40] the Term "epitope" refers to any the protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants usually consist of chemically active surface groupings of molecules such as the side chains of amino acids or sugars, and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. More specifically, the term "epitope of IL-21, in the application here, refers to the portion of the polypeptide of IL-21, having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably mouse or human. An epitope having immunogenic activity is a portion of the polypeptide of IL-21, which causes the formation of antibodies in the animal. An epitope having antigenic activity is the second part of the polypeptide of IL-21, which antibody immunospecificity bound that is set by any method well known in this field, such as immunological assays. Antigenic epitopes do not have to be immunogenic. "Discontinuous epitopes are conformational epitopes formed from at least two separate regions of the primary sequence of the protein IL-21. Conformational epitopes lose the ability to specifically bind in the presence of denaturing solvents (for example, Western blot analysis).

[41] This invention provides monoclonal antibodies and antibody fragments that specifically bind to proteins and polypeptides of IL-21. The polypeptides of IL-21 human and mouse proteins and polynucleotides encoding the polypeptides described in Parrish-Novak et al., Nature 408:57 - 63, 2003; U.S. patent No. 6307024 and 6686178; and 7250274. Described here represent the structural and functional characteristics that define regions (epitopes) of the protein IL-21 people who were identified as targets for therapeutic monoclonal antibodies. Are cited as an example of a monoclonal antibody against IL-21 people. Some of these antibodies have the ability to bind natural IL-21 human, recombinant wild-type IL-21 human, recombinant mutant protein IL-21 and/or the peptide region of IL-21 people.

[42] This invention provides anti-IL-21 antibodies, which are applicable for therapeutic treatment of autoimmune and inflammatory diseases. For example, anti-IL-21 antibodies applicable for the treatment of psoriasis, pancreatitis, diabetes mellitus type I (IDDM), graves disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, rheumatoid arthritis, reactive arthritis, enteropathic arthritis, spondyloarthropathy, autoimmune myocarditis, Kawasaki syndrome, celiac disease, uveitis, diseases behceta, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, inflammatory myopathies (polymyositis, dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), Sjogren syndrome, glomerulonephritis, IgA nephropathy, autoimmune nephritis, ordinary water, myasthenia gravis, autoimmune loss of the case is and, of neuromyelitis optic nerve syndrome?, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinative polyneuropathy (CIDP), autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP), transplant rejection, vysokomineralizirovannaja patients with transplantation, antiphospholipid syndrome, allergies and asthma, and other autoimmune diseases, or other diseases mediated by IL-21 and agonists of the receptor for IL-21.

[43] Five classes of immunoglobulins, IgG, IgA, IgM, IgD and IgE, have been identified in higher vertebrates. Proteins IgG, IgD and IgE are typically crosslinked by disulfide bonds by heterotetramer, consisting of two identical heavy chains and two identical light chains. Usually IgM is detected in the form of five tetramers, whereas IgA is found in the form of two tetramers. Modifications can be introduced into the immunoglobulin molecule.

[44] IgG covers the main class, which usually exists in the form of the second most common protein found in the plasma. People have IgG consists of four subclasses, designated IgG1, IgG2, IgG3 and IgG4. Each heavy chain immunoglobulin contains a constant region, which consists of domains of the constant region of the protein (CH1, hinge, CH2 and CH3), which are the same for the top the subclass. The constant region of the heavy chain of the IgG class identify using the Greek symbol γ. For example, the immunoglobulin subclass IgG1 contain a constant region of the heavy chain γI.

[45] Fc-fragment, or Fc-domain consists of crosslinked by disulfide bonds hinged sections of the heavy chain, CH2, and CH3 domains. In fused proteins of the immunoglobulin Fc domains of the IgG1 subclass is often used as part of immunoglobulin molecules, as IgG1 have the longest half-life in serum of any of the whey proteins. Extended half-life in serum may be a desirable characteristic of protein in animal studies and the potential therapeutic use for humans. In addition, the subclass IgG1 has the strong ability to execute effector functions mediated by the antibody. The primary effector function, which can be most suitable in the fused protein of the immunoglobulin, represents the ability for IgG1 antibody to mediate antibody-dependent cellular cytotoxicity. On the other hand, it could be an undesirable feature for a fused protein that functions primarily as an antagonist. It was more of the identified amino acid residues that are important for activity in the subclass IgG1 mediated konstantnoi area antibodies. Thus, the introduction or deletion of these specific amino acids allows you to enable or disable specific activity, mediated constant region of immunoglobulin (see U.S. patent No. 5648260; 5624821).

[46] Modified IgG1 Fc man was designed to create a fused protein with the Fc. For example, mutations Fc4, Fc5 and FC6 has to reduce effector functions mediated by the Fc by reducing the FcγRI binding and binding of complement C1q, described in patent application U.S. 2006-0034852, incorporated by reference in its entirety. Specifically, three amino acid substitutions were introduced to reduce the FcγRI binding. These substitutions are substitutions at positions 234, 235 and 237 index EU. It was shown that replacement of these provisions reduce binding to FcγRI (Duncan et al., Nature 332:563 (1988)). These amino acid substitutions may also reduce binding to FcγRIIa and FcγRIII binding (Sondermann et al., Nature 406:267 (2000); Wines et al., J. Immunol. D:5313 (2000)). These mutations do not change the binding to FcRn, which promotes a long time half-life in serum by recycling of IgG on the endocytic pathway of recyclization.

[47] Several groups have described the importance of the provisions of 330 and 331 index EU binding of complement C1q and subsequent fixation of complement (Canfield and Morrison, J. Exp. Med. 173: 1483 (1991); Tao et al., J. Exp. Med. 128:661 (1993)). Amino acid substitutions in these is alogene were introduced in Fc4 to reduce the fixation of complement. CH3 domain Fc4 is identical to the domain, which is found in the corresponding wild-type polypeptide excluding the stop codon, which was replaced with TGA on TAA for elimination of potential customers of dam methylation in cultivation of cloned DNA in strains of E. coli (dam+). In Fc5 residue arginine at position 218 index EU represents lysine and the remaining sequence Fc5 coincides with the above description for Fc4.

[48] the invention also includes genetically modified antibodies that are functionally equivalent to the above-described antibodies. Preferred are the modified antibodies, providing improved stability and/or therapeutic efficacy. Examples of modified antibodies include antibodies with conservative substitutions of amino acid residues and one or more divisions or insertions of amino acids that do not change essentially destructive antigennegative value. Replacement can be in the range from changing or modifying one or more amino acid residues to complete changes in the design area, while maintaining therapeutic value. Antibodies of this invention can be modified excision (for example, acetylation and phosphorylation) or can be modificirovana synthetic means (e.g., attach labeled group).

[49] the Antibodies of this invention may be described or specified on the basis of epitopes (epitopes) or part (s) of the polypeptide of IL-21 in this invention, which they recognize or specifically bind. The epitope (epitope) or part (s) of the polypeptide can be specified as described herein, for example, using N-terminal and C-terminal positions or by size in a nonessential amino acid residues. Antibodies of the present invention may also be described or specified on the basis of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of the polypeptide of the present invention, is enabled.

[50] Sorting epitopes or epitope-specific sorting, in the original text of "an epitope binning) refers to the use of competitive analyses linking to identify pairs of antibodies that are able, or unable, to communicate with protein IL-21, whereby identify antibodies that bind to the same or overlapping epitopes on the protein. Then the family of antibodies (or bins)that have the same or overlapping binding specificity can be used to facilitate the identification of specific epitopes on the IL-21. Experiments epitope-specific sorting provide evidence that there are antigenic R is zlecenie epitopes. However, by themselves, they do not identify, or "Carteret", epitope in relation to a specific amino acid sequence or the location of a protein molecule IL-21.

[51] Competition binding can be evaluated for any pair of antibodies or fragments. For example, using suitable reagents for detecting the binding specificity of the antibodies or binding fragments of any of the species/source can be compared with the binding specificity of monoclonal antibodies disclosed here. Epitope-specific sorting can be performed using the "isolated antibody" or using supernatant cell culture. Often the sorting is performed by using clonal supernatants the first stage, for conducting the selection of clones, which additionally occur. Antibodies that are compared, should have essentially homogeneous antigennegative domains. In the case of "bespecifically" or "bifunctional" antibody binding specificity of two different binding sites to assess or sort (binned) independently.

[52] This invention describes a ligand-specific antibody. In addition to competitive binding of the antibody can also be used to sort epitopes to identify epitope-specific sorting of the antibody or receptor, or the ligand, which competitive prevent the binding of the ligand with its receptor or activation of its receptor-mediated ligand. Often, the favorable properties of the family (or bin) antibodies may correlate with the binding with a specific epitope specific epitope family (an epitope bin).

[53] Experiments on competitive binding do not measure directly the binding affinity of, but antibodies to be tested should contact is strong enough to act as competitors. Usually, the conditions of the experiments plan to minimize the effects of differences in the affinity of binding.

[54] Anti-IL-21 antibodies can also be used in diagnostic assays for protein IL-21, for example, detecting its expression in specific cells, tissues, or serum. Antibodies are classified in different families (bins) and the ability to communicate with different immunogenic sites of IL-21, or epitopes, can be used as reagents for sandwich assays. In the sandwich-the analysis of the test sample is captured by the first antibody that immobilized on a solid substrate, and then discover by using a secondary antibody that also binds to a target substance, thus forming an insoluble three-part complex. See, for example, U.S. patent No. 4376110. Secondary antibody which itself can be labeled with detectable group (direct sandwich assays) or may be measured using antiimmunoglobulin antibodies labeled detectable group (indirect sandwich assay). For example, one type of sandwich-analysis is an analysis of ELISA, in which the detected group is an enzyme.

[55] the Antibodies of this invention can be analyzed for specific binding using a method known in this field. Many different formats of competitive analysis of binding can be used for epitope-specific sorting. The immunoassays which can be used include, but are not limited to, competitive and non-competitive systems analysis using methods such as Western blots, radioimmunoassay tests (enzyme-linked immunosorbant assay), "sandwich" immunoassays, methods, thus, precipitation reactions, gel-diffusion precipitation reactions, immunodiffusion assays, agglutination reaction, the reaction fixation of complement, Immunoradiometric assays, fluorescent immunoassays, immunoassays using protein And as just a few. Such assays are routine and well known in the art (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology. Vol.1, John Wiley & Sons, Inc., New York). Cited as an example, the immunoassays describe briefly below (but not intended for limiting). In addition, there may be an issue is lnen routine perekrestnotochny analysis, such as described in Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).

[56] BIACORE® (GE Healthcare, Piscataway, NJ) represents only one of various formats analysis of surface plasmon resonance, which is routinely used for panels families epitopes (an epitope bin panels) monoclonal antibodies. Many references (e.g., The an epitope Mapping Protocols, Methods in Molecular Biology. Volume 66 Glenn E.Morris ed. Humana Press, 1996) describe alternative methods that could be used to sort antibodies (antibodies bin) and would be expected to provide comparable information regarding the specificity of antibody binding with protein IL-21. When using the BIACORE® experiments on epitope-specific sorting (an epitope binning experiments) is carried out using soluble, natural or recombinant antigen. The study of epitope-specific sorting (an epitope binning studies can be performed on the BIACORE system 1000® system (GE Healthcare, Piscataway, NJ). Software BIAIogue® v. 1.2 can be used for programming modes methods. An example of using BIACORE® for sorting (to bin) mouse monoclonal antibodies raised against IL-21, polyclonal goat antimurine antibody to IgG Fc (Jackson ImmunoResearch Laboratories, West Grove, PA) can be covalently immobilized to the sensor chip, BIACORE® CM5 and used for binding (capture) Pervin the th monoclonal antibodies series of tests with a chip. Then unoccupied binding sites Fc chip block using polyclonal Fc-fragment of IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Then make protein IL-21 and provide specific contact captured (trans. printed on the chip) primary monoclinal antibody. The instrument BIACORE® measures the mass of protein associated with the sensor chip, and the binding of primary antibodies, and IL-21 antigen can be confirmed for each cycle. After binding of the primary antibody and antigen chip make soluble secondary antibody and allowed to contact with a pre-associated antigen. If a secondary monoclonal antibody able to bind to IL-21 antigen simultaneously with the primary monoclonal antibody, its binding by BIACORE®. However, if the secondary monoclonal antibody is not able to communicate with IL-21 antigen simultaneously with the primary monoclonal antibody, no additional bonding will not detect. Each monoclonal antibody is tested against itself as a negative control to establish the level of background (no binding) signal.

[57] the format of the competition test (LFC-), not carrying a tag can also be used to sort antibodies to antibodies bin). This method is described by Nagata et al., J. Measurement Methods 292: 141-155, 2004. This method epitope-specific sorting (eptope binning) used biotinylated IL-21. As an example, sort (binning) mouse monoclonal antibodies were obtained against IL-21, tablets for micrometrology covered at 100 μl/well using 1 µg/ml specific goat antimelanoma antibodies to Fc-γ IgG (Jackson ImmunoResearch), diluted in PBS, 0.1% tween-20, 1% BSA). After linking the covering antibodies for 3 hours at ambient temperature each air-conditioned environment containing mAb, diluted in for approximate concentration of mAb 0.5 μg/ml and allow them to contact the tablets covered with goat artemisinin IgG overnight at 4°C (mAb#1). In parallel a second set of air-conditioned environments (mAb#2) were diluted in polystyrene test tubes for analysis of up to approximately 0.5 μg/ml mAb in, mixed with 50 ng/ml biotinylated IL-21 antigen and incubated overnight at 4°C. After incubation mAb#1 with covering antibody tablets block using an unrelated antibody to saturate unoccupied binding sites on the tablet. A mixture of mAb#2-Biotin-IL-21 was added to the plate and give contact. As a control for (non-competitive) analysis, 50 ng/ml biotinylated IL-21 was added directly (without pre-incubation with mAb#2) in wells containing immobilized mAb#1. After incubation with biotinylated complex-IL-21-mAb#2, add streptavidin-HRP (Pierce, Rockford, IL) PL is het at 0.5 µg/ml Tablets are the TMB substrate (BioFX Laboratories, Owings Mills, MD) to measure the absorption of the individual wells at 450 nm with a spectrophotometer to read the tablets (tablet reader) (Molecular Devices SPECTRAMAX®340, Sunnyvale, CA). If mAb#1 binds to another epitope mAb#2 complex of Biotin-IL-21-mAb#2 will contact with the tablet, resulting in high indication absorption. If mAb#1 binds to the same epitope as mAb#2 complex of Biotin-IL-21-mAb#2 will not contact with the tablet, which will lead to low indication absorption.

[58] the Ligand-specific antibodies of this invention can easily be contacted with IL-21 or act as antagonists of IL-21. For example, the invention includes antibodies that do not destroy the interaction of the receptor for IL-21 ligand or disrupt the interaction of the receptor for IL-21 ligand or partially, or completely. The invention provides a ligand-specific antibodies that inhibit activation of the receptor. The invention includes neutralizing antibodies that bind the ligand and prevent binding of the ligand to the receptor, and antibodies that bind the ligand, thereby preventing activation of the receptor, but do not prevent the ligand from binding the receptor. Activation of the receptor (i.e. the signal) can be determined by methods described herein or other, Zvezdnyi in this area. For example, activation of the receptor can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by thus with subsequent Western blot or by analysis based on Luminex (for example, as described above). In specific embodiments, the embodiments of the present invention provides antibodies that inhibit the activity of the ligand or receptor of at least 90%, at least 80%, at least 70%, at least 60% or at least 50% activity in the absence of antibodies.

Antibodies against IL-21 (anti-IL-21 antibodies)

[59] Antibodies to IL-21 can be produced, for example, using protein, which is the product of a vector expressing IL-21 or IL-21 isolated from a natural source as an antigen. Anti-IL-21 antibodies of this invention bind specifically" with IL-21. It is believed that antibodies are specifically related if antibodies detect at least one of the following two properties: (1) antibodies bind to IL-21 with the threshold value binding activity, and (2) antibodies do not give significant cross-react with polypeptides related to IL-21. Related polypeptides could include polypeptides other members of the type 1 cytokines, which bind receptors containing the E. common gamma chain (γ), such as IL-2, IL-4, IL-7, IL-9 and IL-15.

[60] with regard to the first characteristic, antibodies specifically bind if they bind to a polypeptide of IL-21, peptide or epitope with a binding affinity of, in the form of apparent affinity constants. To characterize the affinity values of the kinetic rate constants, equilibrium Association constants and equilibrium dissociation constants were calculated for the interaction of antagonists of IL-21 with IL-21 antigen by surface plasma resonance. The rate constant of Association (ka(M-1s-1)) is a value that reflects the rate of formation of complex antigen-antagonist. The rate constant of dissociation (kd(s-1)) is a value that reflects the stability of this complex. The equilibrium binding affinity of usually Express or equilibrium dissociation constants (KD(M)) or the equilibrium Association constants (KA(M-1)). KDthe quotient of the rate constants of dissociation constant rate of Association (kd/ka), while KAthe quotient of the rate constants of Association rate constant dissociation (ka/kd). Antagonists with similar KD(or similar KA) can have significantly different intercept is you speed Association and dissociation. Therefore, the dimension kaand kda kAndor KDthat helps to more clearly describe the affinity of interaction antagonist-antigen. Preferred affinity antibodies reflect in KA (equilibrium constant of Association) of 106M-1or higher, preferably 107M-1or higher, more preferably 108M-1or higher, and most preferably 109M-1or higher. The binding affinity of antibodies can be readily determined by the person of ordinary skill in this field, for example, Katchanovski analysis (Scatchard, Ann. NY Acad. Sci. 5J.: 660, 1949), or by using a commercially available biosensor instrument. Regarding the second characteristic, antibodies do not give significant cross-reaction with a molecule related polypeptide, for example, if they detect IL-21, but not other known polypeptides using a standard Western blot analysis or trap - test. Examples of known related polypeptides include well-known members of the family of IL-2, which belongs to the IL-21 (for example, IL-2, IL-4, IL-7, IL-9 and IL-15).

[61] Monoclonal anti-IL-21 antibodies can be produced using antigenic epitope-bearing peptides and polypeptides of IL-21. Antibodies of this invention bind antigenic epitope-bearing peptides and polypeptides, operasie sequence of at least nine, or from about 15 to about 30 amino acids contained in SEQ ID NO: 2, or another amino acid sequence disclosed here. However, peptides or polypeptides containing the larger part of the amino acid sequence containing from 30 to 50 amino acids, or any length up to the full amino acid sequences, including the complete amino acid sequence of the polypeptide can also be applied to induce antibodies that bind to IL-21. Preferably, the amino acid sequence of an epitope-bearing peptide was selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes a relatively hydrophilic residues, whereas hydrophobic residues are usually correct). In addition, amino acid sequences containing Proline residues may also be desirable for large-scale production of antibodies.

[62] Monoclonal anti-IL-21 antibodies can be produced by methods known to experts in this field. Monoclonal antibodies rodents to specific antigens may be obtained by known methods (see, for example, Kohler et al., Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol.1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan"], Picksley et al., "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems. 2ndEdition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).

[63] the Antibodies of the invention can be obtained by any method known in this field for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by means of recombinant expression. Recombinant expression of the antibodies of the invention, or fragment, derivative or analog, such as heavy or light chain of the antibody of the invention, requires a design expressing vector containing polynucleotide that encodes the antibody. After receiving polynucleotide of the present invention, molecules encoding antibodies or heavy or light chain of the antibody or portion thereof (preferably containing the variable domain of a heavy or light chain), can be obtained, the vector for the production of antibody molecules by recombinant DNA technology using methods well known in the field. Thus, methods of obtaining protein by expression of polynucleotide containing the nucleotide sequence encoding the antibody, described herein. Methods which are well known to specialists in this field can be used to construct expressing vectors containing sequences encoding the antibody, and the appropriate control signals at the level of transcription and translation. These methods include in with the BOJ, for example, recombinant DNA technology in vitro, methods of synthesis and the genetic recombinantly in vivo. Thus, the invention provides vectors can replicate containing the nucleotide sequence encoding the antibody molecule of the invention or a heavy or light chain, or variable domain of a heavy or light chain, functionally attached to the promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecules (see, for example, PCT publication no WO 86/05807; PCT publication no WO 89/01036 and U.S. patent No. 5122464), and the variable domain of the antibody may be cloned into such a vector for the expression of a complete heavy or light chain.

[64] Expressing the vector is transferred into a cell-master traditional ways and then transfetsirovannyh cells are cultivated by traditional methods for obtaining antibodies of the invention. Thus, the invention includes host cells containing polynucleotide encoding the antibody of the invention, or a heavy or light chain, functionally attached to a heterologous promoter. In preferred embodiments for the expression of double-stranded antibodies, vectors encoding both heavy and light chain can be coexpression in the cell-host for expression of the complete molecule of immunoglobu the ina, as described in detail below.

[65] Various vector systems for expression in the cell-master can be used for the expression of antibody molecules of the present invention. Such systems for expression in the cell of the host are the conduits through which interest coding sequence may be produced and subsequently purified, but also represent cells that may, when transforming or transfetsirovannyh with sequences encoding a suitable nucleotide that can Express the antibody molecule of the invention in situ. These systems include, but are not limited to, microorganisms such as bacteria (for example E. coli, B. subtilis)transformed with recombinant DNA bacteriophage expressing vectors based on the plasmid DNA or kosmidou DNA containing the sequence encoding the antibody; yeast (for example Saccharomyces, Pichia)transformed with recombinant yeast expressing vectors containing sequences encoding the antibody; cell system of insects infected with recombinant virus expressing vectors (e.g. baculovirus)containing the sequence encoding the antibody; systems of plant cells, infected with recombin ntih expressing viral vectors (for example, the cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant virus expressing vectors (e.g., Ti plasmid)containing the sequence encoding the antibody; or systems of mammalian cells (such as COS cells, CHO, KSS, 293, T)containing a recombinant expression constructs containing promoters derived from the genome of mammalian cells (for example, metallothionein promoter) or of mammalian viruses (for example, MPSV, CMV, adenovirus late promoter; the promoter C cowpox virus). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the full expression of recombinant antibody molecules used for expression of recombinant antibody molecules. For example, mammalian cells such as cells of the Chinese hamster ovary (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus, CMV enhancer or MPSV promoter, is an effective expression system antibodies (Foecking et al., 1986, Gene 45: 101; Cockett et al., 1990, Bio/Technologv 8:2).

[66] In bacterial systems, a number expressing the vectors may be advantageously selected depending upon the use intended for expressionvisitor antibodies. For example, when there should be obtained a large quantity of such a protein, to create pharmaceutical compositions of antibody molecules can be desirable, vectors that direct the expression of high levels of products fused protein, which is easily cleaned. Such vectors include, but are not limited to expressing vector pUR278 E.coli (Ruther et al., 1983, EMBO J.2: 1791), in which the sequence encoding the antibody can be Legerova separately in vector inside the reading frame with the coding region of the lacZ so that was producyrovtsa protein; vectors pFN (Inouye &Inouye, Nucleic Acids Res. 11:3101 -3109, 1985; Van Heeke & Schuster, J.Biol. Chem. 24:5503-5509, 1989); and the like can Also be applicable pGEX vectors for expression of foreign polypeptides in the form of a fused protein with glutathione-S-transferase. In General, such fused proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose pellet followed by elution in the presence of free glutathione. The pGEX vectors design for inclusion of sites of cleavage by the protease thrombin or factor XA, so that the product of the cloned target gene could be released from the molecule GST.

[67] In the system of insects, the nuclear polyhedrosis virus of Autographa califomica (AcNPV) is used as a vector for e is cpressey alien genes. The virus grows in the cells of Spodoptera frugiperda. The sequence encoding the antibody may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of the AcNPV promoter (for example the polyhedrin promoter).

[68] In the cells of the host mammal a number of expression systems based viruses can be used. When as expressing vector using the adenovirus of interest sequence encoding the antibody can be Legerova with adenovirus complex that controls the transcription/translation, for example, the late promoter and tripartite leader sequence. This chimeric gene could then be built into the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable to Express the antibody molecule in infected hosts (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355 - 359, 1984). Specific initiation signals may also be required for efficient translation of embedded sequences encoding the antibody. These signals include the initiating ATG codon and adjacent sequences. In addition, the initiating codon must be in phase is with the reading frame of the desired coding sequence to ensure translation of the full inserts. Data exogenous control signals broadcast initiator codons can be from a variety of sources, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of suitable enhancer elements, transcription, transcription terminators, etc. (see Bittneret et al., Methods in Enzvmol. 153:51-544, 1987).

[69] moreover, can be chosen line of host cell which modulates the expression of the built-in sequences, or modifies and processes the gene product in a desired way. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host cell can be selected to ensure the correct modification and processing of the expressed foreign protein. For this purpose, can be used in eukaryotic host cells which possess the cellular mechanism for the proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product. Such host cells of mammals include, but are not limited to, Cho, VERO, KSS, Hela, COS, MDCK, 293, 3T3, W138, and in cast the STI, cell lines breast cancer, such as, for example, VT, Hs578T, HTB2, VT20 and T47D, and cell lines of normal mammary gland, such as, for example, CRL7030 and Hs578Bst.

[70] For long-term large-scale production of recombinant proteins preferred stable expression. For example, can be engineered cell lines that stably Express the antibody molecule. Instead of using expressing vectors that contain viral start replication, cell-hosts can be transformed with DNA controlled by appropriate control elements of expression (e.g., promoter and enhancer sequences, transcription terminators, polyadenylation sites, and others), and with the use of breeding marker. After the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched environment, and then transfer to selective medium. Breeding marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid inside their chromosomes and grow with the formation of foci, which in turn can be cloned and expanded into cell lines. This method can be successfully applied to construct cell lines that expressio the t molecule antibodies. Such engineered cell lines may be particularly applicable in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

[71] Can be used a number of selection systems, including, but not limited to, genes timedancing of herpes simplex virus (Wigler et al., Cell C:223, 1977), gipoksantin-guanine-phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202. 1992) and adenine-phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980), which can be employed in tk-, hgprt - or aprt-cells, respectively. Also antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which tells resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:357. 1980: O'hare et al., Proc. Natl. Acad. Sci. USA 78: 1527. 1981); gpt, which confers resistance to mycophenolic acid (Mulligan &Berg, Proc. Natl. Acad. Sci. USA 78:2072. 1981); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95. 1991; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596, 1993; Mulligan, Science 260:926-932, 1993; and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217, 1993; TIB TECH 11 (5): 155-215). May, 1993; and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147, 1984). Traditional methods known in the field of recombinant DNA technologies, which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY; and in chapters Chapters 12 and 13, Dracopoti et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., J. Mol. Biol. 150:1. 1981; which is incorporated herein by reference in their entirety.

[72] the Levels of expression of antibody molecules can be increased by amplification of the vector (for review see, Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). In those cases, when a marker in the vector system expressing antibody is amplificare, raising the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Because amplificatory region is associated with the gene of the antibody, the formation of the antibody will also increase (Grouse et al., Mol. Cell. Biol. 2:257, 1983).

[73] a host Cell can be cotransfection two expressing the vectors of the present invention, the first vector encoding the polypeptide produced from the heavy chain and the second vector encoding the polypeptide produced from the light chain. These two vectors can contain identical selective markers that provide equal expression of heavy and light polypeptide chains. Alternatively, it may be applied only vector that encodes polypeptides in both heavy and light chain. In such cases, the light chain should be on esena before the heavy chain to avoid an excess of toxic heavy chain (Proudfoot, Nature 322:52, 1986; Kohler, Proc. Natl. Acad. Sci. USA 77:2197, 1980). Coding sequences for the heavy and light chains may contain cDNA or genomic DNA.

[74] After the antibody molecule was recombinante expressed, it may be purified by any method known in this field for the purification of immunoglobulin molecules, for example, by chromatography (e.g. ion exchange, affinity, particularly in the affinity for the specific antigen after chromatography with protein a and exclusion column chromatography), centrifugation, differential solubility, or by any other standard method for purification of proteins.

[75] For specific applications it may be desirable to obtain fragments of anti-IL-21 antibodies. Such fragments of antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody. Fragments of antibodies can be obtained by cleavage by pepsin or papain whole antibodies traditional ways. As an illustration, fragments of antibodies can be produced by enzymatic cleavage of antibodies with pepsin to provide 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol-reducing agent to obtain a monovalent 3,5S Fab'-fragments. Arbitrarily, the cleavage reaction can be performed with the use the of a blocking group for the sulfhydryl groups, generated from cleavage of disulfide bonds. Alternatively, an enzymatic cleavage using pepsin produces two directly monovalent Fab fragment and the Fc-fragment. These methods describe, for example, Goldenberg, U.S. patent No. 4331647, Nisonoff et al., Arch Biochem. Biophvs. 89:230, 1960; Porter, Biochem. J.73: 119, 1959; Edelman et al., in Methods in Enzymology Vol.1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

[76] Other ways of splitting antibodies, such as separation of heavy chains with the formation of monovalent fragments of light-heavy chain, additional cleavage of fragments, or other enzymatic, chemical or genetic methods can also be applied, provided that the fragments bind to the antigen that is recognized by the intact antibody.

[77] for Example, Fv fragments contain the Association of the heavy (VH) and light (VL) chains. This Association may be monovalent, as described Inbar et al., Proc. NAT'l Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chains can be linked by intermolecular disulfide bond or cross stitched chemical compounds such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437, 1992).

[78] Fv fragments can contain the VH and VL chains connected by a peptide linker. Data single-stranded antigennegative proteins (scFv) is obtained by constructing p is ktorego gene, containing DNA sequences encoding the VH and VL domains are connected by the oligonucleotide. The structural gene is inserted into the expressing vector, which is subsequently embedded in the cell host, such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide connecting the two V-domain. Methods of obtaining scFvs are described, for example, by Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97 (1991) (see also Bird et al., Science 242:423, 1988, Ladner et al., U.S. patent No. 4946778, Packetal. Bio/Technoloav 11:1271. 1993, and Sandhu, above).

[79] it is Also possible construction of alternative frameworks use collection Monomeric proteins for the formation of Monomeric domain. Data monomer domains can be sufficiently small to penetrate tissue. Monomer domains can be natural or artificial variants, or a combination. Monomer domains can form multimer of two or more domains. Monomer domain binds position, similar to the epitopes described herein, the molecule target. In some cases multimer can be formed of different monomer domains (see, for example, patent application U.S. 2004-0132028 and patent application U.S. 2006-0177831).

[80] the Antibodies of this invention include derivatives that are modified, i.e. covalent attach any type of mole is cooly to the antibody, so that covalent attachment is not protected antibody from binding IL-21 or block activation of the receptor. For example, but without limitation, derivatives of antibodies include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, deriving, using a known defensive/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known methods, including, but not limited to, chemical cleavage, acetylation, formirovanie, metabolic synthesis tunicamycin etc. in Addition, the derivative may contain one or more non-classical amino acids.

[81] Anti-IL-21 antibody can be conjugated with a detectable label with the formation of immunoconjugate anti-IL-21. Suitable label detected include, for example, a radioisotope, a fluorescent label, chemiluminescent label, an enzymatic label, a bioluminescent label, or colloidal gold. How to create and detection of such labeled to detect, immunoconjugates well known to any ordinary person skilled in the art and are described in more detail below. Detected meth is and can be a radioisotope, found autoradiography. Isotopes which are particularly applicable for purposes of the present invention, are3H,125I131I35S and14C.

[82] Immunoconjugate anti-IL-21 can also be too efficient with fluorescent compounds. The presence of a fluorescently-labeled antibody is detected by exposure immunoconjugate in light of a suitable wavelength and detecting the resulting fluorescence. Fluorescently-labeled compounds include fluorescein-isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ortho-phthalaldehyde, Alexa dyes, fluorescent nanoparticles (e.g., Q-dots) and fluorescamine.

[83] it is Also possible that immunoconjugate anti-IL-21 can be too efficient to detect by prishivki component antibody chemiluminescent compound. The presence chemiluminescence-labeled immunoconjugate determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples chemiluminescence-labelled compounds include luminal, isoluminol, aromatic acridine ester, imidazole, acridine salt and oxalate ester.

[84] Similarly, a bioluminescent compound can be used for tagging immunoconjugate anti-IL-21 of the present invention. Biolumina zentia is a type of chemiluminescence, found in biological systems in which a catalytic protein increases the efficiency of the reaction chemiluminescence. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds, which are applicable for tagging include luciferin, luciferase and acorin.

[85] Alternatively, immunoconjugate anti-IL-21 can be too efficient to detect by attaching anti-IL-21 antibodies to the enzyme. When the incubation of conjugate anti-IL-21-enzyme in the presence of a suitable substrate molecule of the enzyme interacts with the substrate for the formation of chemical molecules that can be detected, for example, by spectrophotometric, fluorometrically or visual means. Examples of enzymes that can be used for labeling, detecting the polyspecific immunoconjugates include β-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.

[86] the Professionals in the art will know of other suitable labels that can be used in accordance with this invention. The binding of marker molecules with anti-IL-21 antibodies can be performed using conventional methods known in this field. A typical methodology in this regard is described as follows: Kennedy et al., Clin. Chim.Acta 70:1, 1976; Schurs et al., Clin. Chim. Acta 81:1, 1977; Shih et al., Int'l J.Cancer 46:1101,1990: Stein et al., Cancer Res. 50:1330. 1990; and Coligan, above.

[87] moreover, the convenience and versatility of immunochemical detection can be enhanced by using anti-IL-21 antibodies, which were conjugated with Avidya, streptavidin, and Biotin (see, for example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods In Enzymology, Vol.184 (Academic Press 1990), and Bayer et al., "Immunochemical Applications of Avidin-Biotin Technology," in Methods In Molecular Biology, Vol.10, Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).

[88] How to perform immunologicheskikh analyses are common. See, for example, Cook and Self, "Monoclonal Antibodies in Diagnostic Immunoassays," in Monoclonal Antibodies: Production. Engineering, and Clinical Application. Ritter and Ladyman (eds.), pages 180-208, (Cambridge University Press, 1995), Perry, "The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology", in Monoclonal Antibodies: Principles and Applications. Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (Academic Press, Inc. 1996).

[89] Antibodies or fragments thereof with increased half-life in vivo can be constructed by methods known to experts in this field. For example, antibodies or fragments thereof with increased half-lives in vivo, can be constructed by modification (e.g., substitution, deletion or accession) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, for example, international publication number WO 97/3431 and WO 02/060919, which is incorporated herein by reference in their entirety). Antibodies, or fragments thereof with increased half-life in vivo can be constructed by joining these antibodies or fragments of antibodies of polymer molecules such as high molecular weight polyethyleneglycol (PEG). PEG can be attached to the indicated antibodies or fragments of antibodies together with a multifunctional linker or without a multifunctional linker, or by site-specific conjugation of the PEG, for example, N-end, or the end of the indicated antibodies or fragments of antibodies or via Epsilon-amino groups present on lysine residues. Will use the formation of linear or branched derivatives of polymers, which lead to minimal loss of biological activity. The degree of conjugation will be carefully monitored SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules with antibodies. Unreacted PEG can be separated from the conjugates of the antibody-PEG, for example, gel chromatography or ion exchange chromatography.

The pharmaceutical composition

[90] the invention additionally includes pharmaceutical compositions containing a pharmaceutically acceptable carrier and a polypeptide or antibody described herein. The pharmaceutical composition may include the additional therapeutic agents, including, but not limited to, cytotoxic agents, a cytotoxin, e.g., a cytostatic or a destructive agent, a therapeutic agent or an ion of a radioactive metal. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-tioguanin, cytarabine, 5-fluorouracil with dacarbazine), alkylating agents (e.g., mechlorethamine, thio-TAP chlorambucil, melphalan, carmustine (BCNU) and lomustin (CCNU), cyclophosphamide, busulfan, dibromo-mannitol, streptozotocin, mitomycin C, and CIS-dichlorodiammine platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (for example, dactinomycin (formerly actinomycin), bleomycin, mithramycin and astromicin (AMC)), and antimitoticescoe agents (e.g. vincristine and vinblastine). For example, the pharmaceutical to notice may contain protein or polypeptide, possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-IFN, β-IFN, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g. angiostatin or endostatin; or biological response modifiers such as, for example, lymphokines, interleukin - 1 (IL-1), interleukin - 2 (IL-2), interleukin - 6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), antibodies, designed to counteract the biological response modifiers, other antibodies, other fused with Fc proteins or other growth factors.

[91] For therapeutic purposes molecule anti-IL-21 antibody and a pharmaceutically acceptable carrier is administered to the patient in therapeutically effective amounts. It is believed that the combination of therapeutic molecules of the present invention and a pharmaceutically acceptable carrier is administered in a "therapeutically effective amount"if the quantity entered is physiologically significant. An agent is physiologically significant if its presence leads to a detectable change in the physiology of the patient-recipient. N the example, used to treat inflammation is physiologically significant if its presence soothes inflammatory response.

[92] Degradiruete polymer microspheres were designed to maintain a high systemic levels of therapeutic proteins. The microspheres obtained from degradiruem polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, polychaetes), non-biodegradable polymers ethylvinylacetate, in which proteins are included in the polymer (Gombotz and Pettit, Bioconiugate Chem. 6:332, 1995; Ranade, "Role of Polymers in Drug Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.), pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable Controlled Release Systems Useful for Protein Delivery," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92 (Plenum Press 1997); Bartus et al., Science 281:1161. 1998; Putney and Burke, Nature Biotechnology 16:153. 1998; Putney, Curr. Opin. Chem. Biol. 2:548, 1998). Covered with polyethylene glycol (PEG) nanospheres can also provide carriers for intravenous administration of therapeutic proteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167, 1997).

[93] Other dosage forms can be developed by experts in this field, as shown, for example, Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems. 5th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington''s Pharmaceutical Sciences. 19th Edition (Mack Publishing Company 1995), and Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).

[94] the Pharmaceutical compositions may be supplied as a kit containing a container that contains a neutralizing anti-L-21 antibody. therapeutic polypeptides can be provided in the form of an injectable solution of single or multiple doses, or as a sterile powder that will be resuspendable before the introduction. Alternatively, such a kit may include a dispersant for powder, liquid aerosol generator or nebulizer for administration of therapeutic polypeptide. Such a kit may further comprise written information regarding the indications and use of pharmaceutical compositions.

[95] a Pharmaceutical composition comprising anti-IL-21 antibodies can be prepared in liquid form, in an aerosol or solid form. Liquid forms illustrate injectable solutions, aerosols, eye drops, solutions for local use and suspensions for oral administration. Typical solid forms include capsules, tablets and forms with controlled release. The last form of supply minimations pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239,1997; Ranade, "Implants in Drug Delivery," in Drug Delivery Systems. Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps," in Protein Delivery: Physical Systems. Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of Proteins from a Controlled Release Injectable Implant," in Protein Delivery: Physical Systems. Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)). Other solid forms include creams, pastes, other topologically the applications, etc.

Therapeutic use of anti-IL-21 antibodies

[96] IL-21 is a derived from CD4+T-cells, cytokine that is important for best immunity, mediated by CD8+T-cells, activity of NK-cells and the best humoral responses, such as the formation of antibodies and maturation of b-cells. It was shown that IL-21 induces a number of proinflammatory chemokines and cytokines, such as IL-18, IL-15, IL-5, IL-6, IL-7A, IL-17F, TNFRII, sCD25, and RANTES. IL-21 also induces ostrofsky response in "non-human primates and humans, with the introduction of IV (AC, intravenously) or SC (trans., subcutaneous) injection (Dodds et al., Cancer Immunol Immunother 2008 Oct 17 [e-publication]). In vitro, stimulates the growth of certain populations of neoplastic immune cells, such as cells of multiple myeloma and acute T-cell leukemia (Brenne et al., Blood 99(10):3756-62 (2002), diCarlo E, et al., Cancer Immunol Immunother 56(9): 1323-1324 (2007)). IL-21 also is produced by the cells of the Hodgkin and reed-Sternberg with Hodgkin lymphoma (Lamprecht et al., Blood 112(8):3339-47, 2008). Increased expression of the receptor for IL-21 was shown in the epidermis of patients with systemic sclerosis (Distler et al., Arthritis & Rheumatism 52:865-864, 2004) and in synovial fibroblasts in rheumatoid arthritis (Jungel et al., Arthritis & Rheumatism 50:1468-1476. 2004). In addition, in mice with autoimmune diabetes in NOD increased expression of the receptor for IL-21 (King et al., Cell 117:265-277. 2004). It was shown that exp is Essie IgG and IL-21 is increased in murine models BXSB-Yaa, which develops an autoimmune disease like lupus (Ozaki et al., J. Immunol. 173:5361-5371, 2004); the expression of IL-21 is higher in mice predisposed to lupus sanroque (Vinuesa et al., Nature 435:452, 2005); the expression of IL-21 is higher in inflamed compared to nevospalennyh tissues of the intestine of patients with Crohn's disease (Monteleone, et al., Gastroenterology 128:687-694, 2005). IL-21 is overproduction in the mucosa of patients with coeliac disease (Finn et al. Gut. PMID: 17965065, 2007).

[97] a Therapeutically effective amount of anti-IL-21 antibodies refers to the number of antibodies, which when administered to a subject is effective to prevent, delay, reduction or inhibition of symptom or biological activity associated with the disease or disorder. The introduction may consist of a single dose or multiple doses and may be provided in combination with other pharmaceutical compositions.

[98] This invention provides compositions and methods of using monoclonal anti-IL-21 antibodies in inflammatory and immune diseases or conditions such as psoriasis, pancreatitis, diabetes mellitus type I (IDDM), graves disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, rheumatoid arthritis, reactive arthritis, enteropathies the arthritis, spondyloarthropathy, autoimmune myocarditis, Kawasaki syndrome, celiac disease, uveitis, Behcet's disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, inflammatory muscle disease (polymyositis, dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, systemic lupus erythematosus, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), a syndrome Sjogren's syndrome, glomerulonephritis, IgA nephropathy, autoimmune nephritis, common bladderwort, myasthenia gravis, autoimmune hearing loss, neuromyelitis optic nerve syndrome?, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinizing polyneuropathy (CIDP), autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP), graft rejection, vysokomineralizirovannaja patients with transplantation, antiphospholipid syndrome, allergies and asthma, and other autoimmune diseases. This invention provides compositions and methods using monoclonal antibodies anti-IL-21V treatment of specific cancers of immune system cells, such as multiple myeloma, acute T-cell leukemia or Hodgkin's lymphoma.

Contact dermatit

[99] Allergic contact dermatitis define as mediated T-cell immune response to an antigen that makes contact with the skin. Suggest that CLA+T-cell population is involved in the initiation of dermatitis because allergenicity T-cell responses are largely restricted population of cells CLA+(See Santamaria-Babi, et al., J. Exp. Med. 181:1935. (1995)). Recent data found that only T-memory cells (CD45RO+) CD4+CLA+and not CD8+T memory cells proliferate and produce as type 1 cytokines (IFN-γ)and type 2 cytokines (IL-5) in response to Nickel, standard contact allergen hypersensitivity. In addition, after the Nickel-specific stimulation, the number of cells expressing CLA in combination with CD4, CD45RO (memory) or CD69 increased and the cells Express the chemokine receptors CXCR3, CCR4, CCR10, but not CCR6. See, Moed et al., Br. J. Dermatol. 51:32, (2004).

[100] In animal models it was shown that allergic contact dermatitis is a T-cell-dependent, and that the allergen-reactive T-cells migrate to the site of application of the allergen. See, in General: Engeman, et al., J. Immunol. 164:5207. (2000); Ferguson & Kupper, J. Immunol. 150: 1 172, (1993); and Gorbachev & Fairchild, Crit. Rev. Immunol. 21:451 (2001).

[101] the antibodies against IL-21 in mouse model contact hyperco the reality used to assess the clinical application of anti-IL-21 antibodies to improve symptoms and changes of the disease.

Atopic dermatitis

[102] Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease with a markedly increased frequency of the disease in recent decades. Clinically AD differs greatly itchy, often extorionate plaques and papules, which detect chronic relapsing course of the disease. The diagnosis of AD is often based on major and minor clinical signs. See, Hanifin J.M., Arch Dermatol 135: 1551 (1999). Histopathology detects sponges, hyperparasites and focal parakeratosis in acute lesions, whereas marked epidermal hyperplasia with hyperparasitism and parakeratosis, acanthosis/hypergranulation and perivascular infiltration of the dermis by lymphocytes and excess fat cells, are signs of chronic lesions.

[103] T cells play a Central role in the initiation of local immune responses in tissues, and the data suggest that infiltrating the skin T cells, in particular, can play a key role in initiating and maintaining misaligned immune responses in the skin. Approximately 90% of the infiltrating T cells in cutaneous inflammatory sites Express the cutaneous lymphocyte-associated antigen that binds E-selectin induced molecule adhesion to the endothelium (reviewed in Santamaria-Babi, et al, Eur. J. Dermatol. 14:13, (2004)). It was recorded a significant increase in circulating CLA+T cells in patients with AD compared with control individuals (see Teraki, et al., Br. J. Dermatol. 143: 373 (2000), while others have shown that CLA+T-cell memory in patients with AD should preferably respond to the allergen extract in comparison with the population of CLA (see, Santamaria-Babi, L.F., et al., J. Exp. Med. 181: 1935. (1995)). In humans, the pathogenesis of atopic skin diseases was associated with increased CLA+T cells that Express elevated levels of cytokines Th-2-type, similar to IL-5 and IL-13. Cm. Akdis et al., Eur. J. Immunol. 30: 3533 (2000); and Hamid et al., J. Allergy Clin. Immunol. 98: 225 (1996).

[104] Mouse NC/Nga spontaneously develop AD-like lesions, which in many aspects is similar to AD person, including in the course of illness and signs, histopathology and immunopathology when placing free from nonspecific microflora (non-SPF) conditions, approximately 6-8 weeks of age. On the contrary, mice, NC/Nga contained in SPF conditions, do not develop lesions skin damage. However, the emergence of spontaneous lesions on the skin and pochesyvaya behavior can be synchronized in mice, NC/Nga while providing SPF conditions by weekly subcutaneous injections of crude antigen dust mites. See, Matsuoka et al., Allergy 58: 139 (2003). Thus, the development of AD in NC/Nga is an applicable model for Enki new treatments for AD.

[105] In addition to model NC/Nga spontaneous AD, intradermal sensitization of mice using OVA can also be used as a model to induce antigen-dependent epidermal and dermal thickening using a mononuclear infiltrate in the skin of sensitized mice. This usually coincides with elevated levels of serum total and specific IgE, but no dysfunction of the skin barrier or itching usually does not occur in this model. See, Spergel et al., J. Clin. Invest. 101:1614, (1998). This Protocol can be modified with the intention of inducing dysregulation of the skin barrier and itch sensitization transgenic mice DO11.10 OVA TCR with OVA. The increased number of antigen-specific T cells that could learn sensitizing antigen may increase the level of inflammation of the skin before the appearance of visible pochesyvaya behavior and lichenification/formation of scales on the skin.

[106] the Introduction of anti-IL-21 antibodies murine models of atopic dermatitis is used to assess the clinical application of anti-IL-21 antibodies to improve symptoms and changes of the disease.

Arthritis

[107] Arthritis, including osteoarthritis, rheumatoid arthritis, arthritic joints as a result of injury and the like, are common inflammatory conditions which have received the s benefit from therapeutic use of anti-inflammatory antibodies and binding polypeptides. For example, rheumatoid arthritis (RA) is a systemic disease that affects the whole body and is one of the most common forms of arthritis. Rheumatoid arthritis is inflammation of the membrane lining the joint, which causes pain, stiffness, warmth, redness and swelling. Inflammatory cells release enzymes that can break down bone and cartilage. The result of rheumatoid arthritis inflamed lining of the joint, the synovial layer, can infect and damage bone and joint, which leads to wear of the joint and severe pain, among other physiological effects. Involved joint can lose its shape and the correct combination that leads to pain and loss of mobility.

Rheumatoid arthritis

[108] Rheumatoid arthritis (RA) is immunopositive disease, specifically characterized by inflammation and subsequent tissue damage, leading to severe disability and increased mortality. Various cytokines are produced locally in rheumatoid joints. Numerous studies have shown that IL-1 and TNF-alpha, two prototypical proinflammatory cytokine, plays an important role in the mechanisms involved in synovial inflammation and progressive destruction of the joints. Indeed, the introduction of inhibitors of TNF-alpha and IL-1 patients RA led to a drastic improvement of the biological and clinical signs of inflammation and reduction of bone erosion and destruction of cartilage on radiological indicators. However, despite these encouraging results, a significant percentage of patients do not respond to these substances, giving the opportunity to suggest that other mediators are also involved in the pathophysiology of arthritis (Gabav. Expert. Opin, Biol. Ther. 2(2):135-149. 2002).

[109] There are several known in the field of experimental animal models for rheumatoid arthritis. For example, in a model of collagen-induced arthritis (CIA) mice develop chronic inflammatory arthritis, which has close affinity with rheumatoid arthritis people. Because the CIA has a similar immunological and pathological properties with RA, this makes it an ideal model for screening potential anti-inflammatory compounds for humans. The CIA model is a well-known model in mice, which, for the manifestation of CIA is dependent on the immune response, and inflammatory response. The immune response includes the interaction between b cells and CD4+T cells in response to collagen, which serves as an antigen and produces antibodies against collagen. Phase inflammation is a result of tissue responses to inflammatory mediators, as a result of some of these antibodies, giving a cross-reaction to murine native collagen and activates the path activation of complement. The advantage and the use of models SIA is what are the basic mechanisms of pathogenesis are known. The appropriate T-cell and b-cell epitopes on collagen type II have been identified and various immunological (e.g., delayed-type hypersensitivity and anticollagen antibodies) and inflammatory (e.g., cytokines, chemokines, and matrix-degrading protease) parameters related to immunopositive arthritis were identified and, thus, can be used to assess the effectiveness of the test composition in the model SIA (Wooley, Curr. Opin. Rheum. 3:407-20, 1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life Sci. 61:1861-78, 1997; Wang et al., Immunol. 92:8955-959. 1995).

[110] the Introduction of anti-IL-21 antibodies of this mouse model of CIA used to assess the implementation of anti-IL-21 antibodies to improve symptoms and changes the course of the disease.

Inflammatory bowel disease (IBD)

[111] In the United States approximately 500,000 people suffer from inflammatory bowel disease (IBD)that can affect the colon and direct (ulcerative colitis), or both, thin and thick intestines (Crohn's disease). The pathogenesis of these diseases is unclear, but they are chronic inflammation of the affected tissues. Ulcerative colitis (UC) is an inflammatory disease of the large intestine, commonly called colon rectum characterized by inflammation and ulceration the mucous membrane or located deep within the lining of the colon. This inflammation causes frequent emptying of the colon, resulting in diarrhea. Symptoms include easing chair and associate abdominal spasm, fever and weight loss. Although the exact cause of UC is unknown, a recent study suggests that a natural protective reaction of the body is working against proteins of the body, which the body considers foreign ("autoimmune reaction"). Perhaps because they resemble bacterial proteins in the intestine, these proteins can either provoke or stimulate an inflammatory process that begins to destroy the lining of the colon. Once the lining of the colon is destroyed, an ulcer forms a discharge of mucus, pus and blood. The disease usually begins in the rectal area and can, eventually, to extend through the entire large intestine. Repeated cases of inflammation lead to thinning of the walls of the intestine and rectum on the basis of scar tissue. Death of tissue of the colon or sepsis may occur with severe disease. The symptoms of ulcerative colitis vary depending on the severity, and their origin can be gradual or sudden. Seizures can be provoked by many factors, including respiratory infections or stress.

[112] Although currently there is no treatment UC, treatments focused on the suppression of broken inflammation in the lining of the colon. Methods of treatment, including corticosteroids immunosuppressants (e.g. azathioprine, mercaptopurine and methotrexate) and aminosalicylate are available to treat the disease. However, prolonged use of immunosuppressive drugs such as corticosteroids and azathioprine, may cause serious side effects, including thinning of bones, cataracts, infectious diseases and effects on the liver and bone marrow. In patients for whom current therapy is not successful, the alternative is surgery. However, surgery is to remove the entire colon and rectum.

[113] There are several experimental animal models, which can partially mimic chronic ulcerative colitis. The most widely used model is a model of colitis induced by 2,4,6-trinitrobenzenesulfonic/ethanol (TNBS), which includes chronic inflammation and ulceration in the colon. With the introduction of TNBS into the colon susceptible mice by intrarectal drip, TNBS induces T-cell-mediated immune response in the mucosa of the colon, in this text the tea causing severe inflammation of the mucous membrane, characterized by massive infiltration of T cells and macrophages throughout the walls of the colon. In addition, this histopathological picture is accompanied with a clinical picture of progressive weight loss (wasting), bloody diarrhea, prolapse of the rectum and thickening of the walls of the colon (Neurath et al., Intern. Rev. Immunol. 19:51-62, 2000). Adaptive transfer of naive T cells to mice with mismatching of minor histocompatibility loci or syngeneic mice with a weakened immune system leads to the development of colitis (Leach MW, et al., 1996, Powrie F. et al., 1997), as well as to skin lesions resembling psoriasis (Schon MP, et al., Nat. Med. 2: 183-8, 1997; Davenport CM, et al., Int. Immunopharmacol. 5:653-72, 2002). Transplantation of only 0.2 million CD4+CD25 - T cells from BALB/C mice or in B10.D2 mice with weakened immune systems C. b-17 SCID leads to weight loss, positive hemocult-test Kala and development of skin lesions. The symptoms in these mice vary from colony to colony. This model of colitis/psoriasis has some features in common with Crohn's disease and psoriasis person and has been widely used to test the effectiveness of therapy of these diseases in humans.

[114] Another experimental model of colitis uses dextran sulfate sodium (DSS), which induces acute colitis manifested by bloody diarrhea, weight loss, shortening of the colon and Izyaslav the receiving mucosal neutrophil infiltration. DSS-induced colitis differs histologically by infiltration of inflammatory cells in own plate mucosa, lymphoid hyperplasia, focal (focal) defeat crypts and epithelial ulceration. It is believed that such changes occur because of the toxic effect of DSS on the epithelial and phagocytic cells own plates mucosa and formation of TNF-alpha and IFN-gamma. Despite its common use, several issues regarding the mechanisms DSS about the appropriateness in relation to human disease remain unresolved. DSS considered as a T-cell-independent model, because it is found in animals deficient in T-cells, such as the SCID mouse.

[115] the Introduction of anti-IL-21 antibodies records TNBS, DSS or CD4+T-cell transfer models can be used to assess the use of antagonists of IL-21 to improve symptoms and changes the course of gastrointestinal disease. IL-21 may play a role in the inflammatory response in colitis, and the neutralization activity of IL-21 by introducing antagonists of IL-21 is a potential therapeutic approach for IBD.

Psoriasis

[116] Psoriasis is a chronic skin disease that affects more than seven million Americans. Psoriasis type plaque is the most widely distr the common form, different inflammatory foci skin ("foci"), covered with white silvery scales. Psoriasis can be limited to a few plaques or affect moderate to large areas of the skin appearing more often on the scalp, knees, elbows and torso. Although he is strong, psoriasis is not contagious disease. The pathogenesis of the disease is the activation of T cells, the modified antigen presentation and the formation of inflammatory cytokines by dendritic cells and chronic inflammation of the affected tissues. Anti-IL-21 antibodies of the present invention could serve as a valuable therapeutic agent to reduce inflammation and pathological effects in psoriasis, other inflammatory skin diseases, allergic diseases of the skin and mucous membranes and related diseases.

[117] Psoriasis is a mediated T-cell inflammatory violation of the skin, which can cause significant discomfort. Psoriasis is a disease for which there is no treatment and that affects people of all ages. Psoriasis affects approximately two percent of the population of Europe and North America. Although people with psoriasis mild severity can often control their disease with the help methodistic funds, over the eat one million patients worldwide in need of treatment with ultraviolet light or immunosuppressive therapy. Unfortunately, the inconvenience and risk of UV exposure and the toxicity of many therapies limit their prolonged use. In addition, patients usually occurs recurrence of symptoms of psoriasis and in some cases the return of symptoms shortly after cessation of immunosuppressive therapy. Anti-IL-21 antibodies can be tested using a recently developed model of psoriasis on the basis of CD4+CD45RB transport models (Davenport et al., Internat. Immunopharmacol., 2:653-672, 2002).

[118] In addition to other disease models described here, the activity of anti-IL-21 antibodies to inflamed tissues obtained from psoriatic plaques person, can be measured in vivo using a murine model of severe combined immunodeficiency (SCID). We developed several mouse models, in which human cells or tissue graft is implanted mice with the human immunodeficiency virus (all together referred to as model xenografts); see, for example, Cattan and Douglas, Leuk. Res. 8:513-22, 1994 and Flavell, Hematological Oncology. 14:67-82, 1996. As in vivo xenograft models of psoriasis psoriatic skin tissue of a human implanted in SCID murine model and subsequently critically evaluate with an appropriate antagonist. In addition, other experimental models of psoriasis on animals in this area can be used to estimate antagonists L-21, such as skin grafts psoriatic human tissue implanted in a mouse model AGR129 and critically evaluated with an appropriate antagonist (see, for example, Boyman et al., J. Exp. Med. Online publication #20031482, 2004). Similarly, tissue or cells derived from foci of diseases, colitis, IBD, IBS, arthritis or other inflammatory foci may be used in the SCID model to assess the anti-inflammatory properties, anti-IL-21 antibodies described here.

[119] the Efficacy of treatment is measured and statistically evaluated in the form of increased anti-inflammatory effect within the treated population over a period of time using methods that are well known in this field. Some typical methods include, without limitation, for example, the dimension in the model of psoriasis thickness of the epidermis, the number of inflammatory cells in the papillary layer of the dermis and the severity of parakeratosis. Such methods are known in this area, described here. See, for example, Zeigler et al., Lab Invest 81:1253, 2001; Zollner et al., J. Clin. Invest. 109:671, 2002; Yamanaka et al., Environ. Immunol. 45:507, 2001; Raychaudhuri et al., Br. J. Dermatol. 144:931, 2001; Boehncke et al., Arch. Dermatol. Res. 291:104. 1999; Boehncke et al., J. Invest. Dermatol. C:596, 2001; Nickoloff et al., Am. J. Pathol. 146:580, 1995; Boehncke et al., J. Cutan. Pathol. 24:1, 1997; Sugai et al., J. Dermatol. Sci. 11:85, 1998; and Villadsen et al., J. Clin. Invest. 112: 1571, 2003. Inflammation can also be controlled during the belts using well-known methods, such as flow cytometry (or PCR) to quantify the number of inflammatory or damaged cells present in the sample, using estimates (weight loss, diarrhea, rectal bleeding, length of the colon) for IBD, the scale of the disease in the legs and scale of inflammation model SIA RA.

[120] the Introduction of anti-IL-21 antibodies in this murine model of psoriasis used to assess the implementation of anti-IL-21 antibodies to improve symptoms and changes the course of the disease.

Systemic lupus erythematosus

[121] Systemic lupus erythematosus (SLE) is a disturbance-mediated immune complex, characterized by chronic production of IgG antibodies directed against universal autoantigens (e.g., anti-dsDNA). The effects of SLE are more systemic than localized to a specific organ, although in some cases can lead to glomerulonephritis (i.e. the lupus nephritis). Multiple chromosomal loci have been associated with the disease and may contribute to various aspects of the disease, for example, anti-dsDNA antibodies and glomerulonephritis. It was shown that CD4+T cells are actively involved in murine models of SLE (Horwitz, Lupus 10:319-320, 2001; Yellin and Thienel, Curr. Rheumatol. Rep., 2:24-37, 2000). The role of CD8+T cells is not clearly established, but there is evidence before alagat, "suppressor function of CD8+T cells is impaired in patients with lupus erythematosus (Filaci et al., J. Immunol. 166:6452-6457. 2001; Sakane et al., J. Immunol., 137:3809-3813, 1986).

[122] it Was demonstrated that IL-21 induces the differentiation of naive B-cells of human antitelomerase plasma cells (Ozaki et al., J. Immunol. 173:5361, 2004; Ettinger et al., J. Immunol. 175:7867-79, 2005; Ettinger et al., J. Immunol. 178:2872-82, 2007; Kuchen et al., J. Immunol. 179:5886-96, 2007). Ozaki et al., (J. Immunol. 173:5361, 2004) also showed that the expression of IL-21 increased in predisposed to lupus mice BXSB-Xaa model for SLE, the age at which first become apparent initial characteristic of autoimmune processes. The treatment of these mice BXSB-Kao using the mouse antagonist of IL-21 partially inhibits various parameters of the disease, including glomerulonephritis (Bubier et al., Ann N Y Acad. Sci. 1110:590-601. 2007). It was shown that the same antagonist IL-21 is effective in different preclinical models of the disease SLE, mouse, MRL/lpr (Herber et al., J. Immunol. 178: 3822-3830, 2007). Furthermore, since IL-21 limits the development of Treg cells, the introduction of anti-IL-21 antibodies could provide more reliable suppressor function of T cells in patients with lupus, in which the feature is broken (Lamprecht et al., Blood. 112 (8):3339-47. 2008).

[123] the Data obtained from 24 patients with SLE and 15 healthy controls, showed that 1) the expression of mRNA of IL-21 is increased in CD4+T-cells p of the patients with SLE compared with controls, 2) the levels of IL-21 are significantly elevated in the sera of patients with active compared to inactive SLE or controls that are installed using a commercial kit IL-21 kit (Invitrogen, Carlsbad, CA), 3) IL-21 enhances the proliferation of CD4+T cells and CD 19+b cells in patients and controls, depending on the dose, 4) IL-21 increases induced by anti-CD40 differentiation of plasma cells from normal controls and patients with SLE, and 5) elevated levels of IL-21 may contribute in the proliferation of self-reactive CD4+T cells and differentiation of plasma cells in SLE (Rus, V., ACR Presentation #1760. 2008 American College of Rheumatology meeting, October 24-29, 2008).

[124] Anti-IL-21 antibodies can be introduced in combination with other agents already in use in autoimmunity, including immunomodulators, such as IFNγ, NOVANTRONE®, ENBREL®, BETAFERON®, REM1CADE®, LEUKINE® and PROLEUKIN®. Anti-IL-21 antibodies can be introduced in combination with other agents already used in cancer therapy for multiple myeloma, Hodgkin's lymphoma or acute T-cell leukemia, such as THALOMID® or with steroids, such as dexamethasone or prednisone. The determination of the optimum dose level and patterns of use of anti-IL-21 antibodies do in a variety of ways, including a study of the pharmacokinetics and pharmacodynamics of anti-IL-21 antibodies; the determination of effective doses in animal models, ocenco the toxicity of anti-IL-21 antibodies. Direct pharmacokinetic measurements were carried out on primates and then clinical trials can be used to predict theoretical doses in patients who have anti-IL-21 antibodies in the plasma reach levels that are of sufficient magnitude and duration to achieve a biological response in patients.

Graft rejection

[125] the Recipients of transplanted solid organs may develop acute or chronic allograft rejection due to a mismatch of histocompatibility. The formation of antibodies directed against HLA molecules (alloantigen) in these patients is due to the presentation of foreign antigen to T cells. Allintitle can mediate tissue damage in the graft through the formation of immune complexes, complement fixation and antibody-dependent cellular cytotoxicity, managed related allintitle. Path activation of complement also frees up local factors that activate endothelial cells and cause disorder of vessels in the graft. C4d-product of complement is an early marker of acute and chronic graft rejection and can be detected in preclinical cases to clear pathological changes (Racusen and Haas, Clin. J. Am. Soc. Nephrol. 1:415-420. 2006; Moll and Pascual, Am. J. Transplantation 5:2611-268. 2005; Tinkam and Chandraker, J. Clin Am. Soc. Nephrol. 1:404-414, 2006). Patients performed the screening for anti-HLA, allintitle (paninirvana antibody) before transplantation. Patients may be vysokomineralizirovannaja due to previous allograft rejection, blood transfusions or pregnancies. The presence of alloantigen at vysokomineralizirovannaja patients with transplantation complicates the care of them, as may be necessary reinforced immunosuppressive treatment and the chance of acute rejection is high (Baid et al., Curr. Opin. Immunol. 13:577-581, 2001). In some cases the use of agents aimed at In-cell (mikofenolna acid or rituximab), although this therapeutic strategy is not directly aimed at antitelomerase plasma cells. Also used plasmapheresis to reduce circulating immunoglobulin. In all cases, recipients with graft treated with immunosuppressive agents, focused on T cells to reduce the risk of rejection, and recipients may be slowly "weaned" from these modes, as set tolerance to the graft (Seyfort-Margolis and Turka, J. Clin. Invest. 118(8):2684-2685. 2008; Taylor et al., Crit. Rev. Oncol. Hematol. 56:23-46, 2005; Amante and Ejercito, Transplant. Proc. 40:2274-2280, 2008).

[126] the Development of plasma cells secreting antibodies requires cognates help from CD4 T cells in addition is a group of specialized microenvironment which supports the survival of plasma cells (Tarlington et al., Curr. Opin. Immunol. 20:162-169, 2008). Secretion of cytokines by activated T cells is required for differentiation and survival of plasma cells and is known to affect the nature of antibodies and isotype Ig. There are models for monitoring T-cell-dependent or antibody-based test responses in the murine lines and non-human primates". These methods are well understood specialists in this field. The kinetics and amplitude of the primary or secondary or antibody-based test model responses against peptide antigens such as ovalbumin, tetanus toxoid, sheep erythrocytes, or modified trinitrophenyl hemocyanin lymph snails monitorium using assays that detect total or antigen-specific antibodies, including subtypes of IgG, IgM, IgE, or IgA in the serum of treated animals. In some models, the affinity maturation of antibodies can also be controlled. These models can be used to test the effects of drugs that alter b-cell help T-cells that inhibit cytokines, which are important for the differentiation and survival of plasma cells.

[127] the Study of allograft rejection is performed on many kinds of animals. For example, a model of renal what about the transplant in cynomolgus monkeys may represent the effects of chronic rejection of renal allograft, mediated allintitle people. Tolerogen for allograft regimes perform in some cases prior to transplantation. The presence of donor-specific alloantigen monitorium running cytometrical analysis of the serum of the recipient that are not of peripheral blood leukocytes and deposits of C4d-component of the complement to be detected in the biopsies of renal allograft (Smith et al., Am. J. Transplant. 6:1790-1798, 2006; Smith et al., Am. J. Transplant. 8:1-11, 2008). Acute and chronic models of transplantation can be performed on murine kinds of specialists in this field.

[128] the Introduction of anti-IL-21 antibodies in the model of T-cell-dependent or antibody-based test response or model of allograft rejection is used to assess the clinical application of anti-IL-21 antibodies to reduce responses alloantigen and improve symptoms of allograft rejection, or as part of a treatment regimen before transplantation or tolerogenic mode for recipients with graft, including vysokomineralizirovannaja patients with transplantation.

[129] the Invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1. Getting proteins and antibodies to IL-21

1A. Immunization and hybridoma

[130] the Protein IL-21 was obtained as described in U.S. patent No. 7250274 included here in full. Soluble cocktail recipes. the major proteins of IL-21 was received, as described in patent application U.S. 2007-0122413 and U.S. patent No. 6777539, both included here in full. Monoclonal antibodies against IL-21 were obtained in wild-type mice, generating mouse antibodies, and in transgenic mice, generating a fully human antibody (Medarex, Princeton, NJ). Mice were immunized with a protein IL-21 people. Briefly, the original mice were immunized by subcutaneous injection of 30 μg of purified recombinant IL-21 (obtained in E. coli in ZymoGenetics), conjugated to BSA (Imject Pharmalink Immunogen Kit, Pierce)and used in combination with CpG (oligonucleotide ligand murine TLR9 and GM-CSF (granulocyte-macrophage colony-stimulating factor, R&D, Minneapolis, MN) and adjuvant Emulsigen® - R (MVP Laboratories, INC., Omaha, NE) according to the manufacturer's instructions. After the initial immunization, each mouse was given three additional 30 μg IL-21 in Freund Emulsigen® - P using the subcutaneous route of administration with weekly intervals. Seven days after the fourth immunization in mice the blood was collected through retroorbital plexus and separated serum for analysis of the ability of serum to contact with IL-21.

[131] was Collected and pooled splenocytes of two BALB/c mice with high titers or transgenic mice and was merged with cells of the mouse myeloma P3-H-Ag8:653 using PEG 1450 in one procedure merge (the ratio at the confluence of 2:1, splenocytes to alumnim cells", Antibodies: A Laboratory Manual", E.Harlow and D.Lane, Cold Spring Harbor Press). After 9 days of growth after the merger identified pools hybridomas producing specific antibody, direct way for ELISA and trapping ELISA using recombinant protein IL-21, unlabeled and labeled human IgG Fc, in the form of targets for specific antibodies. Positive pools hybridomas were additionally analyzed for their ability to block binding of ligand to the receptor, which is measured as the level of STAT3 phosphorylation after ligand-receptor interaction (reaction inhibiting STAT3 phosphorylation") of purified recombinant protein of IL-21 in BaF3 cells expressing the sequence of the receptor for IL-21. Monoclonal antibodies are purified from the medium for tissue culture, were distinguished by their ability to block ligand-receptor interaction (reaction inhibiting STAT3 phosphorylation") of purified recombinant protein of IL-21 in BaF3 cells expressing the sequence of the receptor. "Neutralization" of monoclonal antibodies identified in this way.

[132] the hybrid Pools, giving positive results in the neutralization of STAT3 phosphorylation and ELISA formats, cloned at least twice by the serial dilution method. In these tests the samples were titrated using standard razvedeni the low density (less than one cell per well), to see what the clone will maintain the highest reading of the optical density OD. Using the results as the neutralization and titration analyses, two specific clone from each source uterine wells were selected for additional analyses. Clones are subjected to an additional round of cloning to ensure the homogeneity of culture and subjected to screening using the direct method of conducting ELISA. After one additional analysis titration method was selected two end of clone IL-21. Hybridoma clones were maintained in culture medium containing 90% of the modified environment Iscove''s Modified Dulbecco's (Tr, environment Dulbecco, modified by the method of Claims) with 2 mm L-glutamine, 100 µg/ml penicillin and 100 μg/ml streptomycin sulfate and 10% Fetal Clone I Serum (Hyclone Laboratories). The clones were propagated by sowing crops with 2×105cells/ml and the maintenance of between 1×105and 5×105cells/ml at 37°C and 5-6% CO2. Cells adapted to serum-free conditions in sequential transfers. Cells frozen in 90% serum, 10% DMSO and stored in the gas phase apparatus for freezing by immersion in liquid nitrogen.

[133] Purified monoclonal antibodies produced by hybridoma clones, characterized in various ways, including sorting (binning) (i.e. by definition, could ka is the antibody to inhibit the binding of any antibodies) epitope mapping using peptides, the relative affinity and neutralization.

[134] the Methods of obtaining heterologous antibodies from transgenic mice are known, see, e.g., Lonberg, Nat. Biotech. 23(9): 1117-25. 2005; Tomizuka et al., PNAS 97(2):722-727. 2000; and U.S. patent No. 5625126.

[135] the Following hybridoma producing murine monoclonal antibodies (mAb)directed against IL-21 were deposited in the American type culture collection, Manassas, VA. clone 338.5.4 ATS No. (PTA-8317), clone 338.11.5 ATS No. (PTA-8314), clone 338.14.3 ATS No. (PTA-8313), clone 338.15.5 ATS No. (PTA-8315), clone 338.17.3 ATS No. (PTA-8316), clone 338.24.5 ATS No. (PTA-8430), clone 338.25.6 ATS No. (PTA-8431), clone 338.39.5 ATS No. (PTA-8432), clone 338.29.2 ATS No. (PTA-8433), clone 338.28.6 ATS No. (PTA-8434).

[136] the Following hybridoma producing human monoclonal antibodies directed against IL-21 were deposited in the American type culture collection, Manassas, VA. Table 1 provides a complete amino acid sequences of variable heavy (VH) and variable light (VL) chains of antibodies. Also included are sequences for CDRI, CDR2 and CDR3 regions of the VH and VL for each antibody. The corresponding nucleotide sequence indicated in the list of sequences.

Included in the Bank, but not in table 1, is hybridoma marked 366.345.6.11, ATS No. PTA-8788.

1B: Expression clone 362.78.1.44 genes of heavy and light chain immunoglobulin in mammalian cell lines to obtain 362.78-Cho

[137] Human monoclonal antibody against IL-21 (produced from hybridoma clone 362.78.1.44) expressed in cells SNO using two expression cassettes. VH-chain poured with a modified constant region of human IgG1. Modified IgG1, IgG1.1, contains five amino acid substitutions to reduce effector functions. Heavy chain of human antibodies against IL-21 person connected with breeding marker dihydrotetrazolo (DHFR) sequence internal website landing ribosomes (IRES). The expression of the heavy chain of human antibodies against IL-21 human and breeding marker DHFR was sent constitutive synthetic promoter consisting of the merger of cytomegalovirus (CMV) enhancer and enhancer/promoter myeloproliferative sarcoma virus (MPSV). The polyadenylation signal of simian virus 40 (SV40) used for termination of transcription at the end of breeding marker DHFR. VL-chain was merged with the constant region of the Kappa chain of human immunoglobulin. Light chain of human antibodies against IL-21 was associated with a selective marker of resistance to puromycin (puroR) with the IRES sequence. The expression of the light chain of the human PR is against IL-21 human and breeding marker puroR sent constitutive synthetic promoter, consisting of a merger of the CMV enhancer person and enhancer/MPSV promoter. The SV40 polyadenylation signal used for termination of transcription at the end of breeding marker. Expression cassettes of the heavy and light chain human against IL-21 people were cotranslationally in host cell Cho DXB-11. Selection on puromycin accompanied by a selection on methorexate to get high, stable expression of human monoclonal antibodies against IL-21 people. Expressed in Cho variant mAb IL-21 clone 362.78.1.44 will be called in the following examples as "362.78-Cho."

Example 2. Monoclonal antibodies anti-IL-21 is associated proteins and peptides of IL-21

2A. Binding and neutralizing peptides

[138] the Ability of binding and neutralizing monoclonal antibodies against IL-21 human contact with IL-21 human mutant protein IL-21 human and synthetic peptides produced from IL-21 showed a direct format ELISA.

[139] Used the following peptides:

[140] peptide #1 ((SEQ ID NO:3) pyroGlu GQDRHMIRMRQLIDIVDQLKC;

[141] peptide #2 ((SEQ ID NO: 4) NDLVPEFLPAPEDVETNC,

[142] peptide #3 ((SEQ ID NO: 5) NVSIKKLKRKPPSTNAGRRQKHRLTC,

[143] peptide #4 ((SEQ ID NO:6) CDSYEKKPPKEFLERFKSLLQKMIHQHLS and

[144] Recombinant IL-21 (SEQ ID NO: 2), synthetic peptides produced from the sequence of IL-21 and recombinant mutant IL-21 is the ne (SEQ ID NO: 7) separately was immobilized on the surface of 96-well polystyrene plates to ELISA in the amount of 100 μl/well at a concentration of 1 μg/ml in covering buffer (0.1 M Na 2CO3pH 9,6). The plates were incubated overnight at 4°C, after which the unbound protein was aspirated and the tablets were washed twice with 300 μl/well of buffer for washing (PBS-Tween defined as 0,M NaCl, 0,0022M KCl, 0,0067M Na2HPO4, 0,0020 M KH2PO4, 0,05% vol./mass. Polysorbate 20, pH of 7.2). The wells were blocked with 200 µl /well blocking buffer (PBS-Tween plus 1% wt./about. bovine serum albumin (BSA)for 1 hour, after which the tablets were washed twice with wash buffer. Breeding antibodies were prepared in 5% fetal bovine serum (FBS)/environment Dulbecco modified by the method of Claims (IMDM) and brought up to 1 mcg/ml. Then two samples of each dilution of antibody transferred in tablets for analysis, 100 μl/well, for binding proteins anti-IL-21 people. After 1 hour incubation at ambient temperature the material from the wells was aspirated, and the tablets were washed twice as described above. Then labeled with horseradish peroxidase (HRP) Fc-specific goat antibody against mouse IgG or Fc-specific goat antibody against rat IgG or Fc-specific goat antibody against human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:5000 with 5% medium FBS/IMDM was added to each well, 100 μl/well, and the plates were incubated at ambient temperature for 1 hour. After removal of the bore is provided antibodies conjugated with HRP, the tablets were washed five times, 100 μl/well of tetramethylbenzidine (TMB) (BioFX Laboratories, Owings Mills, MD) was added to each well and the plates were incubated for 3 minutes at ambient temperature. The color development was stopped by adding 100 µl/well reagent 450 nm TMB Stop Reagent (BioFX Laboratories, Owings Mills, MD) and was calculated values of absorption content of the holes on the instrument Molecular Devices Spectra MAX 340 to 450 nm.

2B. Measurement of the affinity of binding of the monoclonal antibody against IL-21 human 362.78-Cho with IL-21 and IL-21 cynomolgus macaques by means of surface plasma resonance (Biacore)

[145] anti-IL-21 monoclonal antibody 362.78-Cho was evaluated for its binding affinity of with recombinant IL-21 and recombinant human IL-21 monkeys using surface plasmon resonance.

[146] Determination of affinity. Kineticist rate constants and equilibrium dissociation constants were measured for the interaction of monoclonal antibodies against IL-21 human 362.78-Cho with IL-21 and IL-21 monkeys by surface plasmon resonance. The rate constant of Association (ka(M-1s-1)) is a value that reflects the rate of formation of the complex antigen-antibody. The rate constant of dissociation (kd (s-1)) is a value that reflects the stability of this complex. By dividing the rate constant dissociation constant rate of Association (kd/ka) get the equilibrium dissociation constant (KD(M)). This value describes the binding affinity of the interaction. Antibodies with similar KDcan have different rate constants of Association and dissociation. Therefore, the dimension kaand kdantibodies helps to more clearly describe the affinity of interaction of the antibody with the antigen.

[147] the Materials and methods. The study of binding kinetics and affinity performed on the system Biacore T100™ (GE Healthcare, Piscataway, NJ). Methods for Biacore T100™ was programmed using the software BIACORE KZT100™ Control Software, v 1.1.1. For these experiments, the antibody 362.78-Cho or recorded on the sensor chip CM4 using Fc-gamma-specific goat antibodies against human IgG (Jackson immunoResearch, West Grove, PA), or antibody 362.78-Cho had minimum biotinilated using Sulfo-NHS-LC-Biotin, a mass ratio of 1:100 (Pierce, Rockford, IL) in PBS buffer pH of 7.4, and then recorded on the chip with streptavidin (SA). All experiments on the binding was performed at 25°C in a buffer of 10 mm HEPES, 300 mm NaCl, 5 mm CaCl2, of 0.05% Surfactant P20 (Biacore), 1 mg/ml bovine serum albumin, pH 8.0.

[148] For Dan who's experiments with Fc-gamma specific goat antibody against human IgG, exciting antibody was diluted to a concentration of 50 μg/ml in 10 mm sodium acetate pH 5.0, and then was covalently immobilized on all four flow cells of sensor chip CM4 using chemistry stitching amine (EDC:NHS). After immobilization of the antibody remaining active sites on the flow cell were blocked with 1 M ethanolamine. Received the density of the exciting antibodies approximately 3500 RU. anti-IL-21 antibody 362.78-Cho was fixed on flow cell 2, 3 and 4 of the chip CM4 in three different densities (varying from 25 to 150 RU). The capture antibodies 362.78-Cho immobilized on the surface was performed with a flow rate of 10 ál/min Biacore System measures the mass of protein bound to the surface of sensor chip, and thus, capture the test antibodies were tested for each cycle. Serial dilution of recombinant human IL-21 or recombinant simian IL-21 (ZymoGenetics) was prepared from 40 nm to 0.003 nm (serial dilution 1:5). Serial dilution were injected with the surface and allowed to specifically bind with the antibody 362.78-Cho, captured on the sensor chip. Double injections of each concentration of the antigen IL-21 was performed at the time of the Association or 6.5 or 7 minutes and time of dissociation or 10, 15, or 60 minutes. The study of the kinetics of binding was performed with a flow rate of 50 ál/min Between cycles flow-through cuvette p is washed 20 mm hydrochloric acid for regeneration of the surface. This stage wash was removed as captured by the test antibody, and any associated antigen from the surface of the immobilized antibodies. Then the antibody 362.78-Cho was again captured in the next cycle.

[149] For experiments with minimally biotinylated 362.78-Cho, biotinylated antibody captured (translated, recorded) on flow cell 2, 3, and 4 chip SA in three different densities (varying from 150 to 1200 RU). The capture of the biotinylated antibodies 362.78-Cho surface was performed at flow rate of 10 ál/min Serial dilution of recombinant human IL-21 or recombinant simian IL-21 (ZymoGenetics) prepared or from 50 nm to 0.001 nm (serial dilution 1:4) or from 40 nm to 0.003 nm (serial dilution 1:5). Data serial dilution were injected with the surface and allowed to specifically bind with the antibody 362.78-Cho, captured on the sensor chip. Double injections of each concentration of the antigen IL-21 was performed at the time of the Association or 6.5 or 7 minutes and time of dissociation or 10, 15 or 60 minutes. The study of the kinetics of binding was performed with a flow rate of 50 ál/min Between cycles flow-through cuvettes were washed in 20 mm hydrochloric acid for regeneration of the surface. This stage leaching has removed any associated antigen from the surface of the immobilized antibodies. The rinse cycle does not delete biotinylated antitle-SNO from the surface of the sensor and the antibody was subsequently available for binding of the next sample antigen.

[150] the Data were collected using software BIACORE KZT100™ Evaluation software (version 1.1.1). The data were processed by subtracting the reference flow cell and a control injection. Base line stability was evaluated to ensure that the stage of regeneration of providing a solid surface for binding throughout the sequence of injections. Identical curves injection was examined in terms of reproducibility. Based on the monovalent binding of IL-21 with bivalent antibody was determined binding model is 1:1, which is appropriate. Curves binding minus three reference flow-through cuvettes (FC2-1, FC3-1, FC4-1) were generally consistent with model binding 1:1 with multiple Rmax and set RI with respect to zero. The data agree well with the model binding 1: 1 with good agreement between experimental and theoretical binding curves. Chi-square (chi2and the average RMS errors associated with the smoothing, were low. Deviations in the residuals did not exist.

[151] Results. To interact 362.78-Cho IL-21, data were collected from four separate experiments, kaseveral experiments were in the range from ZE+07 to 5+07 (M-1s-1), while kdranged from ZE-06 to SEE-05 (s-1). Calculated KDwas in the limits of from E-13 to 8E-13 (M).

[152] For interaction 362.78-Cho IL-21 monkey data were collected from three separate experiments, ka for each experiment was ZE+07 (M-1s-1), while kdranged from 2E-04 to 5E-04 (s-1). Calculated KDranged from 0,9F-11-2E-11 (M).

Example 3. Experiments on cross-reactivity between species

[153] Determining the ability of antibodies against IL-21 people to give cross-react and bind IL-21 mice or cynomolgus macaque IL-21 or synthetic peptides produced from IL-21

[154] the Study of cross-reactivity between species may be important to demonstrate specificity for therapeutic strategies for the development of antagonists. To determine whether or not described here binding and neutralizing form against IL-21 people to give cross-react and bind with the mouse or obezyany IL-21 (and thus to equalize the Javanese macaco or mouse as a suitable species for testing), it was necessary to demonstrate competitive binding of the antibody with recombinant IL-21 human, murine and cynomolgus macaques in a variety of formats analysis. One way to test the binding of monoclonal antibodies is their behavior in the immunoblot-analysis (Western blot). Recombinant human IL-21 (SEQ ID NO:2), recombinant the first murine IL-21 (SEQ ID NO:11), recombinant simian IL-21 (SEQ ID NO:9), synthetic peptides produced from the sequence of IL-21 human: peptide #1 pyr30-K50 (Seq ID 3), peptide #2 N54-C71 (SEQ ID NO:4), peptide #3 N97-C122 (SeqID N0:5), peptide #4 C125-S153 (SEQ ID NO:6), conjugated with ovalbumin, or irrelevant control cytokine recombinant human IFN-λ (ZymoGenetics) were subjected to gel-electrophoresis in polyacrylamide the gel with sodium dodecyl sulfate (SDS-PAGE using 4-12% BisTris poliakrilamidnykh gels (Invitrogen, Inc.) and transferred to nitrocellulose membranes using standard methods and buffer containing 25 mm Tris, 186 mm glycine and 40% methanol.

[155] For Western blots nonspecific sites on the membrane were blocked with buffer containing 20 mm Tris, pH 7.4, 0.5 mm EDTA, and 0.5% IGEPAL CA-630, 150 mm NaCl, and 0.25% gelatin and 1% blocking buffer casein hydrolysate (Western Blocking Reagent, Roche Diagnostics, Inc., Basel Switerzerland) (blocking buffer). Then membranes were incubated for 2 hours at room temperature with purified monoclonal antibody (10 ng/ml or 100 ng/ml) in blocking buffer followed by incubation for 2 hours with conjugated with horseradish peroxidase donkey antibody against human IgG (Jackson Laboratories, Bar Harbor, ME). Membranes were washed 5 times with Tris/EDTA/IGEPAL/NaCI/blocking buffer with gelatin without casein hydrolysate, and showed with a solution SUPERSIGNAL™ DuraWest Luminol/Enhancer/ Proxidase Solution (Pierce, Rockford, IL) to detect chemiluminescence. The blots were shown using standard methods based on x-ray film.

Example 4. Evaluation of the ability of antibodies against IL-21 human cross-reactivity and binding γ-family cytokines IL-2, IL-4, IL-7, IL-9, IL-15 human

[156] Another important property of specific antibodies is the ability of the antibodies to contact with the protein target (protein-targets) and to counteract the protein binding target (protein-targets), but not to link related proteins (not targeted) to a significant extent. The ability of antibodies against IL-21 human contact related cytokines tested in the format of a Western blot. Received samples of all members γ-family cytokines and dispersed in SDS-PAGE and transferred to nitrocellulose membranes for blotting. Recombinant human IL-2 (202-IL/CF), human IL-4 (204-IL/CF), human IL-7 (207-IL/CF), human IL-9 (209-IL/CF), human IL-15 (247-IL/CF) were all obtained from R&D Systems, Minneapolis MN), human IL-21 (ZymoGenetics) and human IFN-λ (U.S. patent No. 6927040; 7252969) were used to evaluate specificity the antibodies. All of the tested antibodies did not show significant binding above background with γ-family of cytokines, with the exception of human IL-21, which observed a clear link, coz asusena with previous Western blots using antibodies (see Example 3).

Example 5. The study of competitive epitope-specific sorting (binning epitopes)

[157] Experiments on epitope-specific sorting (binning) was performed to determine which anti-IL-21 monoclonal antibodies are able to bind simultaneously with human IL-21. Resuspendable both human and mouse antibodies. Monoclonal antibodies anti-IL-21, which compete for the same, or overlapping the binding site (epitope) on the antigen, does not have the ability to simultaneously connect and functionally unite in one family or "bin epitopes" (store, bunker epitopes). Anti-IL-21 monoclonal antibodies that do not compete for the same binding site on the antigen, have the ability to connect simultaneously, and are grouped in a separate collection, or "bins epitopes". The experiments were performed using BIACORE T100™. Experiments on binning (sorting epitopes, sorting, an epitope-specific sorting, epitope-specific sorting) was performed with soluble human IL-21 (ZymoGenetics) as antigen.

[158] the Study of epitope-specific sorting (an epitope binning) was performed on the system BIACORE T100™ (GE Healthcare, Piscataway, NJ). Methods were programmed using software BIACORE T100™ Control Sofware, v 1.1.1. Individual monoclonal antibodies anti-IL-21 was covalently immobilized to separate flow cells of sensory BIACORE chip CM4. Then IL-21 antigen were injected with and gave specifically bind to a monoclonal antibody immobilized on the sensor chip. The BIACORE system measures the mass of protein bound to the surface of sensor chip, and thus, the immobilization of the primary antibody pairs analyzed and specific binding of IL-21 antigen primary antibody was tested for each test cycle. After binding of IL-21 antigen were injected with anti-IL-21 monoclonal antibody and allowed to contact. If a secondary anti-IL-21 monoclonal antibody was capable of binding the antigen simultaneously with the primary monoclonal antibody, we detected the increase of mass on the surface of the chip, or binding. However, if a secondary anti-IL-21 monoclonal antibody was not capable of binding the antigen simultaneously with the primary monoclonal antibody, the additional mass, or binding, was not found. Each anti-IL-21 monoclonal antibody, and analyzed against itself, used as a negative control to establish the level of background signal (no binding).

[159] a Series of experiments was performed to analyze the binding properties of purified monoclonal and the antibodies anti-IL-21, obtained from hybridoma m spleens of mice immune to human IL-21. The first anti-IL-21 monoclonal antibody pairs analyzed was immobilized covalently with the use of EDC:NHS to a density of approximately 1000 RU (trans., units resonance). IL-21 antigen was diluted to 100 nm and allowed to spread over the surface of the immobilized antibodies. Then the secondary antibody pairs analyzed was diluted to 5 μg/ml (approximately 32,2 nm) and allowed to contact with the captured IL-21 antigen. A subpopulation of anti-IL-21 monoclonal antibodies were analyzed as primary antibodies in combination with a full panel secondary anti-IL-21 monoclonal antibodies. Experiments on binding was performed with a flow rate of 30 μl/min at 25°C. the Buffer for these studies consisted of 10 mm Hepes, 0.3 M NaCg, of 0.05% surfactant P20, 5 mm CaCl21 mg/ml bovine serum albumin, pH 8.0. Between cycles the antibody on the chip was regenerated with 20 mm hydrochloric acid. Data was collected using a BIACORE KZT100™ Evaluation software (version 1.1.1), then transferred to EXCEL ™ for further data processing.

[160] Received characteristics of the purified monoclonal antibodies anti-IL-21 and defined in the family epitopes (an epitope bins). Signal (RU, resonance unit)represented by BIACORE, directly correlated with the mass on the surface of sensor chip. After the establishment level, the background signal (EN), associated with the negative control (the same anti-IL-21 monoclonal antibody used for the primary and secondary antibodies), sort results (binning results were presented in the form of positive and negative binding. Positive binding indicates that two different anti-IL-21 monoclonal antibodies capable of binding the antigen simultaneously. Negative binding indicates that two different anti-IL-21 monoclonal antibodies capable of binding the antigen simultaneously. The difference between the values of positive and negative response in this experiment was significant and provided a clear distribution of monoclonal anti-IL-21 antibodies to six separate families, or family epitopes (an epitope bins). The first family epitopes (an epitope bin) was presented anti-IL-21 monoclonal antibodies, for example, from clones 338.5.4; 362.78.1 and 362.597.3. The second family epitopes (an epitope bin) was presented anti-IL-21 monoclonal antibodies, for example, from clones 338.14.3; 362.75.1.1 and 366.328.10. It was found that additional third family (third bin) overlaps the binding epitopes of antibodies bin #1 bin and #2. The third family (third bin) were represented by a monoclonal antibody, for example, from clone 366.552.11. Antibodies that neutralize IL-21, is detected in each of these three families (ins).

[161] have Identified three additional family epitopes (an epitope bins). Each of these families (bins) were represented by a monoclonal antibody, for example, from hybridoma clones 366.345.6.11 (bin #4), 338.28.6 (bin#5) and 338.39.5 (bin#6). Antibodies identified in these three families (bins), does not neutralize the biological activity of human IL-21.

Example 6. The study of competition soluble receptor

[162] Experiments on competition performed to determine which monoclonal antibodies against IL-21 people have the ability to bind IL-21 simultaneously with the soluble receptor of IL-21. Monoclonal antibodies against IL-21, which compete with soluble receptor for the same or overlapping binding site (epitope) on the antigen does not have the ability to communicate at the same time. Monoclonal anti-IL-21 antibodies that do not compete with soluble receptor for the same binding site on the antigen, are not able to connect simultaneously. Experiments on competition performed with soluble recombinant human IL-21 in the form of the antigen. IL-21 antigen was allowed to bind monoclonal antibody to compete with a soluble receptor of IL-21. Two variants of the soluble receptor of IL-21 (both produced ZymoGenetics) used for the analysis of monoclonal antibodies in the data the research: the First variant of the receptor consists of homodimeric receptor (IL-21 R-MS), consisting of the extracellular domain of the receptor, IL-21, fused with the Fc molecule produced from a human immunoglobulin. Second soluble form of the receptor was a heterodimeric receptor (IL-21 R/γc Fc), consisting of one subunit, containing the extracellular domain of the receptor, IL-21, fused with the Fc molecule produced from a human immunoglobulin, and a second subunit, containing the extracellular domain of the common γ-chain, fused with the Fc molecule produced from human immunoglobulin, as described in the joint U.S. patent No. 6777539 included here by reference in its entirety.

[163] Research the competition was carried out on a BIACORE system KZT100™ (GE Healthcare, Piscataway, NJ). Methods were programmed using software BIACORE KZT100™ Control Software, v 1.1.1. Individual anti-IL-21 monoclonal antibody was immobilized covalently to a separate flow cells of sensory BIACORE chip CM4. Then were injected with IL-21 antigen (SEQ ID NO: 2), gave specifically bind to a monoclonal antibody immobilized on the sensor chip. The Biacore system measures the mass of protein bound to the surface of sensor chip, and thus, the immobilization of the first antibodies and specific binding of IL-21 antigen with a first antibody was tested for each test cycle. After binding of IL-21 antigen were injected with soluble the th receptor and gave them to communicate. If soluble receptor was capable of binding the antigen simultaneously with the first monoclonal antibody on the chip surface was found to increase in mass, or binding. However, if the soluble receptor was not capable of binding the antigen simultaneously with the first monoclonal antibody, no additional mass, or binding, was not found. Each monoclonal anti-IL-21, are analyzed against himself, was used as negative control to determine the level of background signal (no binding). As a positive control, each anti-IL-21 monoclonal antibody were analyzed by anti-IL-21 antibodies from another family epitopes (an epitope bin) to determine the level of the positive signal (binding).

[164] a Series of experiments was performed to analyze the binding properties of 5 purified monoclonal anti-IL-21 antibodies from hybridoma clones 362.78.1, 366.75.1.1, 366.328.10, 366.552.11.31 and 366.345.6.11)that bind human IL-21. The first monoclonal anti-IL-21 antibody pairs analyzed was covalently immobilized using a mixture of 0.4 M EDC [N-ethyl-N'-(3-diethylaminopropyl)carbodiimide] and 0.1 M NHS (N-hydroxysuccinimide) to a density of approximately 1000 RU. After immobilization of the antibody active sites on the flow cell were blocked with 1 M ethanolamine. IL-21 antigen Rabaul is whether to 100 nm and allowed to spread over the surface of the immobilized antibodies. Then the soluble receptor was diluted to 10 μg/ml and allowed to contact with the captured IL-21 antigen. Experiments on binding was performed with a flow rate of 30 μl/min at 25°C. the Buffer for these studies consisted of 10 mm Hepes, 0.3 M NaCl, 0,05% surfactant P20, 5 mm CaCl21 mg/ml bovine serum albumin, pH 8.0. Between cycles the flow cell was washed with 20 mm hydrochloric acid for regeneration of the surface. This stage leaching has removed the IL-21 antigen and any associated soluble receptor from the surface of the immobilized antibodies and provided the subsequent binding of the next test sample. Data were collected using software BIACORE T 100™ Evaluation software (version 1.1.1).

[165] Purified monoclonal anti-IL-21 antibodies differed in their ability to compete with human soluble receptor of IL-21 for binding to IL-21 antigen. Signal (RU, response unit), presents BIACORE, directly correlated with the mass on the surface of sensor chip. After establishing the level of background signal (EN)associated with negative controls (the same anti-IL-21 monoclonal antibody used both as primary and as secondary antibody), the results of the competition were presented in the form of positive and negative binding. Positive link points to is about, that anti-IL-21 monoclonal antibody and soluble receptor of IL-21 is capable of binding the antigen simultaneously. Negative binding indicates that anti-IL-21 monoclonal antibody and soluble receptor of IL-21 is not capable of binding the antigen simultaneously. The difference between the values of positive and negative response in this experiment was significant and provided unambiguous definition of competition between anti-IL-21 monoclonal antibodies and soluble receptor of IL-21.

[166] Monoclonal antibodies from hybridoma clones 362.78.1 and 366.552.11.31 competed with both versions of the soluble receptor of IL-21 (homodimer IL-21 r-fc and heterodimeric IL-21 R/γc-Fc) for binding to human IL-21 antigen. Monoclonal antibodies from hybridoma clones 366.328.10 and 366.345.6.11 not compete with any of the variants of the soluble receptor for binding to the antigen. Monoclonal antibody from hybridoma clone 362.75.1.1 showed partial competition for binding to both soluble forms of the receptor. These studies were performed with IL-21 antigen, previously associated with a monoclonal antibody. It was shown that three of these antibodies (362.78.1, 366.328.10, 366.552.11.31) neutralize human IL-21, whereas monoclonal antibodies from hybridoma clones 362.75.1.1 and 366.345.6.11 depending on the analysis are very the labs or not neutralize the biological activity of human IL-21.

Example 7. STAT3 analysis of the biological activity of IL-21 Baf/hulL-21 R

[167] the Following analysis of the biological activity of phosphorylated STAT3 was used as primary screening for measuring neutralizing titers of anti-IL-21 in mouse serum, as well as the relative levels of neutralizing IL-21 monoclonal-supernatants and purified anti-IL-21 antibodies. The activity of IL-21 was determined by measuring the level of phosphorylation of STAT3 (STAT3 protein is a signal transmitter and activator of transcription 3) after ligand-receptor interaction in cells BaO/KZ134/hulL-21 R (see, Spolski and Leonard, Annu Rev Immunol. Nov 8: 2007). Relative neutralizing activity was determined based on the reduced levels of phosphorylated STAT3 using EU50concentration (EC50the concentration of drug causing 50% inhibition of enzyme) IL-21 and titration antagonist.

[168] Cells BaO/KZ134/hulL-21 R washed twice with the medium for analysis (RPMI 1640 with 5% fetal bovine serum, 1 × Glutamax, 1% sodium pyruvate and 2 mm β-mercaptoethanol; all from Invitrogen, Carlsbad, CA) before seeding at 40,000 cells/well in 96-well, round-bottom plates to tissue culture (Becton Dickinson, Franklin Lakes, NJ). Cells were placed in a thermostat for tissue culture at 37°C. at the time of preparation of solution for analysis. To determine EU50EU90the concentration of IL-21 in genomanalyse was prepared by serial dilution of recombinant human IL-21 in the environment for analysis and were sown in separate 96-well plate with a U-shaped bottom. Alternatively, for the analysis of neutralizing IL-21 EC50the concentration of IL-21 (some equal 33 PM) plaincourault with serial dilutions immunized IL-21 mouse serum, depleted environment for hybrid, purified soluble human IL-21 R/γc-Fc or purified monoclonal anti-IL-21 antibodies. Then as tablets with cells and tablets solutions were incubated in a humid chamber for tissue culture for equilibration for 30 minutes at 37°C and 5% CO2. After 30 minutes the interaction initiated by the transfer of solutions of IL-21 in the tablets to the cells and incubation for 10 minutes at 37°C and 5% CO2.

[169] After 10 minutes of incubation the reaction was stopped by placing the tablets on the bath with ice and adding 125 ál of ice-cold buffer Cell Wash Buffer (BIO-PLEX Cell Lysis Kit, BIO-RAD Laboratories, Hercules, CA) to each well. The cells are then precipitated with centrifugation at 1500 rpm at 4°C for 5 minutes and the medium was aspirated. For lysis of the cells in each well was added 50 μl/well lyse buffer (prepared in accordance with the instructions of the manufacturer, BIO-RAD Labs). Then cell lysates were pietravalle up and down five times, all the time in the bath with ice, and mixed on a rocking platform for the microplate for 20 minutes at 600 rpm at 4°C. Then the plates were centrifuged at 3000 rpm at 4°C for 20 min is so Supernatant collected and transferred into a new tablet for micrometrology and mixed 1:1 with buffer for analysis of Assay Buffer (BIO-RAD) for storage at -20°C.

[170] the Granules to capture Capture beads (BIO-PLEX Phospho-S3 Assay, BIO-RAD Laboratories) was diluted and added into 96-well plates with filters (Millipore Corporation, Ireland) according to the manufacturer's recommendations. The tablets were washed twice with the buffer for washing Wash Buffer (BIO-RAD) and 50 μl of a mixture of cell lysate were made in each well. Then each plate was wrapped in aluminium foil and shaken over night at room temperature at 300 rpm On the following day the tablet was placed in a vacuum apparatus for micrometrology and washed twice with the buffer for washing the Wash Buffer. After adding 25 μl/well detection antibody (BIO-RAD) closed foil plates were incubated at room temperature for 30 minutes with shaking at 300 rpm Tablet was subjected to filtration and washed twice with the buffer for washing. Added streptavidin-PE (BIO-RAD; 50 µl/well) and closed foil plates were incubated at room temperature for 15 minutes with shaking at 300 rpm Tablet was subjected to filtration and washed two times and resuspendable in 125 μl/well of buffer resuspendable granules Bead Resuspension Buffer (BIO-RAD). Then the level of phosphorylated STAT3 was analyzed using R the Dera for matrices (BIO-PLEX, BIO-RAD Laboratories) according to manufacturer's instructions. Data were analyzed using analytical software (BIO-PLEX MANAGER 3.0, BIO-RAD Laboratories). Increased levels of phosphorylated transcription factor STAT3 present in the lysates, testified about the interaction of IL-21 receptor with the ligand. For analysis of neutralization, reduced levels of phosphorylated transcription factor STAT3 present in the lysates, testified to neutralize the interaction of IL-21 receptor with the ligand. The values of the IC50(the concentration of the antagonist which gives 50% inhibition of the activity of the ligand) was calculated using the software GraphPad Prism®4 software (GraphPad Software, Inc., San Diego, CA) and were expressed as molar concentrations for each reagent in the analysis of neutralization.

[171] the Human IL-21 induced the phosphorylation of STAT3, depending on the dose at the prescribed EU50concentration, which is approximately 33 PM. Table 7 summarizes the values of the IC50for the positive control (human soluble protein IL-21 R/γc Fc) and for items that neutralizing IL-21, are described here. These data indicate that IL-21 neutralizing antibody was active and was equal to the positive control or better than the positive control by reducing the phosphorylation of STAT3, induserve the aqueous IL-21.

Table 7
The values of the IC50in the analysis of STAT3 phosphorylation
IC50(PM) Experiment #1IC50(PM) Experiment #2IC50(PM) Experiment #3*
soluble hIL-21 R/γc-Fc25102140,2
Clone#mAbIL-21:
362.75.1No neutralization
362.78.11460
362.78.1.444166,7
362.78-CHO (A2162F)42,0
366.328.10.63210
366.552.1.31 59
366.617.769
366.345.6.11Low

* Experiment 3 was performed using a 96 PM IL-21

Example 8. STAT-luciferase analysis of the biological activity of IL-21 Baf3/huIL-21 R

[172] This 24-hour analysis measures the activity of STAT-luciferase induced by IL-21 in transfected cells Baf3/KZ134/huIL-21 R. Transfetsirovannyh cells Baβ/KZ134/huIL-21 R washed twice with the medium for analysis (RPMI medium 1640 without phenol red with 5% fetal bovine serum, 1 × Glutamax, 1% sodium pyruvate and 2 mm β-mercaptoethanol; all from Invitrogen, Carlsbad, CA) before seeding at 40,000 cells/well in 96-well, flat-bottomed opaque white cultural tablet (Corning/Costar, Lowell, MA). Then the cells were placed in a thermostat for tissue culture at the time of preparation of solutions for analysis. In a separate tablet mixed human IL-21 or with the environment or with a number of antagonists of IL-21 (or monoclonal antibodies, or soluble receptor of IL-21 person/γc-Fc). After mixing, this tablet is also placed in a humid chamber thermostat for tissue culture at 37°C. H is cut to 30 minutes, the solutions for analysis were made in the tablet to the cells and mixed. Then the tablet was placed back in the incubator for 24 hours. After 24 hours, cells were removed from thermostat and allowed to cool to room temperature. Then each well was diluted 1:1 by volume 100 μl of luciferase reagent Steady-Glo Luciferase reagent (Promega, Madison, Wl) and thoroughly mixed. The tablets were covered and shaken at room temperature for 10 minutes and measured the relative luciferase units (RLU) in a luminometer.

[173] To determine EU50EU90concentrations of IL-21 in this analysis were analyzed by serial dilution of recombinant human IL-21 in the range from 0 to 100 ng/ml EU90the concentration of IL-21, ~15 ng/ml (961 PM)was used in the subsequent experiments neutralization. In these experiments, clone 362.78.1 (and its subclone 362.78.1.44 and expressed in Cho variant 362.78-Cho; see example 1) and clone 362.328.10.63 showed maximum anti-IL-21 activity, with IC50concentrations in the range of 300-850 PM, whereas the values IC50for instant control of receptor IL-21 person/γc-Fc ranged from 650-1830 PM. The relative activity of neutralizing elements described here summarize in table 8.

Table 8
IC50 values in 24-hour STAT-luciferase analysis
IC50(PM) Experiment #1IC50(PM) Experiment #2IC50(PM) Experiment #3*
Soluble hIL-21R/γc-Fc650-8501830140,2
Clone#mAbIL-21:
362.75.1No neutralization
362.78.1400775760
362.78.1.44850
362.78-CHO (A2162F)533
366.328.10.63300-500
366.552.11.312400
366.617.76360
366.345.6.113184

Example 9. Cross-reaction with IL-21 activity cynomolgus macaques, mice or rats

[174] the Study of species cross-reactions (especially cross-reaction to "non-human primates") is important to complete prior preclinical pharmacological/Toxicological studies when creating a therapeutic antagonist. To determine whether form, neutralizing anti-IL-21 man, described here, to give cross-react and neutralize the activity induced monkey IL-21, murine IL-21 or mouse IL-21 (and, thus, to recognize or cynomolgus macaques, mice and/or rats as a viable species for analysis), it was necessary to demonstrate the biological activity of recombinant monkey, mouse or rat IL-21. Methods of analysis STAT-luciferase activity towards IL-21 as described in example 8, was used to determine the values of the EU50EU90for recombinant IL-21 human, monkey IL-21, murine IL-21 and rat IL-21 (all produced in ZymoGenetics). The results shows the Ute, what levels of STAT-luciferase activity induced by human, monkey and murine IL-21 in this analysis vary considerably. Determine the values of the EU90used in the following experiments, which are the following: 961 PM for IL-21; 102 PM for IL-21 cynomolgus macaques; 6,41 nm for murine IL-21 and 1.08 nm for rat IL-21. Soluble receptor of IL-21 (hIL-21 R/γc Fc) neutralized the effects of the monkey, mouse and rat IL-21.

[175] IL-21. The addition of purified monoclonal antibodies against IL-21 are shown in table 9, in varying degrees neutralized monkey IL-21, but not neutralized mouse or rat IL-21 (note, only 362.78-CHO and 366.552.11 analyzed against rat IL-21). The values of the IC50to neutralize the monkey IL-21 neutralizing forms described here were in the range of ~ 100 PM to 431 PM and are summarized in table 9. It should be noted that the best neutralizing antibodies to IL-21 people were all able to effectively neutralize monkey IL-21, but not mouse IL-21, or rat IL-21. In addition, analyzed produced SNO-cell IL-21 mAb (362.78-CHO) in a separate experiment using 800 PM concentration of IL-21 cynomolgus macaques.

Example 10. Assessment of potential cross-reaction to IL-4 in the analysis based on the cellular system is IU

[176] In the development of therapeutic cytokine antagonist is important to know whether this antagonist to give cross-react with structurally related cytokines and neutralizing of structurally related cytokines. Analysis of proliferation of primary b-cells were developed for the analysis of forms, neutralizing IL-21, is described here in relation to cross-react and neutralize human IL-4.

[177] the Allocation of primary b cells. To obtain primary b cells 200 ml of peripheral blood was collected from healthy human volunteers (ZymoGenetics). The blood was diluted to 400 ml with PBS buffer (lane, phosphate buffered saline) at room temperature and was doing 35-ml aliquots in 50-ml conical tubes. Was pagkawala fourteen milliliters of ficoll-vipaka Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) at room temperature and the tubes were centrifuged for 20 minutes at 2000 rpm/min Layer RVMS (trans., mononuclear cells of peripheral blood) at the interface was aspirated and washed twice with MACS buffer (PBS, 20 mm HEPES and 1% BSA; Invitrogen, Carlsbad, CA). Cells were counted and cells were isolated by negative selection using a dial for selecting In-cell Isolation Kit from Miltenyi Biotec (Auburn, CA)following the Protocol provided by the manufacturer. A small sample of purified b cells were analyzed for purity FACS analysis and is obnaruzhili, in all experiments, CD19+B-cells are >97% purity.

[178] Analysis of cell proliferation. B cells proliferate in response to the joint cultivation of IL-4 with immobilized anti-lgM. To determine the potential cross-reaction to IL-4 and neutralization of IL-4 monoclonal antibodies (mAbs) anti-IL-21, pre-selected b cells were first sown at 40 000-50000 cells/well in 96-well plate for tissue culture with a U-shaped bottom (Becton Dickinson, Franklin Lakes, NJ), which was pre-coated with the use of 1.0 μg/ml anti-lgM (Southern Biotech, Birmingham, AL). Then cells were treated with 10 ng/ml recombinant IL-4 (R&D Systems; Minneapolis, MN) and m-series IL-21 antagonist (analyzed antibodies or controls). Then cells were incubated for 3 days at 37°C and 5% CO2in a humid chamber thermostat for tissue culture. Three days later cells were irradiated with 1 µci/well of [3H]-thymidine (Amersham Biosciences, Piscataway, NJ). After 16 hours the cells were collected on glass-fiber filters and produced quantitative analysis of [3H]-enable using a beta-counter (Topcount NXT, Packard). None of the three analyzed monoclonal anti-IL-21 antibodies (362.78.1, 366.328.10.6 and 366.552.11.31) did not show any neutralization of proliferation induced by IL-4, up to 250-fold molar excess.

Example 1.

11A. Assessment of potential cross-reaction to IL-2 and IL-15 in the analysis based on the cellular system

[179] When developing a therapeutic cytokine antagonist is important to know whether this antagonist to give cross-react with structurally related cytokines and neutralizing of structurally related cytokines. Murine T-cell line, CTLL-2, can be induced to proliferation in response to IL-2 or IL-15 people. Thus, this analysis selected for analysis forms, neutralizing IL-21, described here, in a cross-reaction to IL-2 and IL-15 human and neutralization of IL-2 and IL-15 human.

[180] Cells CTLL-2 were washed three times in medium for biological analysis of proliferation (RPM1 1640, 2×Glutamax, 10% FBS, 2 x NaPyr (trans., sodium pyruvate), 1×β-mercaptoethanol and 20 mm Hepes; Invitrogen, Carlsbad, CA), and were sown at 50,000 cells per well in 96-well round-bottom plates to tissue culture (Becton Dickinson, Franklin Falls, NJ). These cells were added to a predefined dose of the EU90or IL-2 (3.0 ng/ml) or IL-15 (0.5 ng/ml) in combination with a serial dilution of forms, neutralizing IL-21. The ratio of the cytokine to the antibody varied from 250-fold molar excess to the ratio of 1:1. Anti-IL-2 or anti-IL-15 neutralizing antibodies (both from R&D Systems, Minneapolis, MN) were used as positive controls. Then cells were incubated for 24 hours at 37°C and 5% CO2what about the wet chamber of thermostat for tissue culture. After 24 hours, cells were irradiated with 1 µci/well of [3H]-thymidine (Amersham Biosciences, Piscataway, NJ). After 16 hours the cells were collected on glass-fiber filters and produced quantitative analysis of the number of [3H]-enable using a beta-counter (Topcount NXT, Packard).

[181] Results. None of the three analyzed monoclonal anti-IL-21 antibodies (362.78.1, 366.328.10.6 and 366.552.11.31) did not show any neutralization of proliferation induced by IL-2 or IL-15.

11. Confirmation using surface plasma resonance (Biacore) that IL-21 mAb 362.78-Cho does not bind the cytokines IL-2. IL-4, IL-7, IL-9 or IL-15, related to IL-21 people.

[182] anti-IL-21 mAb 362.78-Cho was evaluated by surface plasmon resonance with respect to potential cross-reactivity cross-reactivity with human IL-2 human IL-4 human IL-7, human IL-9 human IL-15.

[183] the Materials and methods. The experiments were performed for analysis of cross-reactivity of anti-IL-21 monoclonal antibodies 362.78-Cho with human IL-2 human IL-4 human IL-7, human IL-9 human IL-15. The study of binding were performed on BIACORE KZT100™ (GE Healthcare, Piscataway, NJ). Methods were programmed using software BIACORE T100™ Control Software, v 2.0. Fc-gamma specific goat antibody against human is IgG (Jackson ImmunoResearch, West Grove, PA) was covalently immobilized on flow cells 1 and 2 of sensor chip CM4 using chemistry stitching amine (EDC:NHS). Then purified anti-IL-21 monoclonal antibody 362.78-Cho were applied to the flow cell of sensor chip 2 at a density of approximately 240 EN. Flow cell 1 was used as the reference surface.

[184] IL-2, IL-4, IL-7, IL-9 and IL-15 (all purchased from R&D Systems, Minneapolis, MN) was made by injecting on the surface of the captured antibodies (flow cell 2) and the reference flow cell (flow cell 1) at concentrations of 100, 20 and 4 nm. As a positive control for this series of experiments, IL-21 (manufactured ZymoGenetics) was also made by injecting in the same concentrations. Study linking was performed with a flow rate of 25 μl/min, time of the Association 2 minutes and time of dissociation 3 minutes. All experiments on the binding was performed at 25°C in buffer: 10 mm HEPES, 300 mm NaCl, 5 mm CaCl2, of 0.05% Surfactant P20 (Biacore), 1 mg/ml bovine serum albumin, pH 8.0. Between cycles the flow cell was washed with 20 mm hydrochloric acid for regeneration of the surface. This stage leaching has removed the captured antibody 362.78-Cho, and any associated antigen from the surface of the chip. Data were collected using software BIACORE T100™ Evaluation software (version 2.0). Data obrabatyvaniya subtracting the injection reference flow cells and control. Base line stability was evaluated to ensure that the stage of regeneration provided the appropriate surface for binding throughout the sequence of injection.

[185] Results. Did not observe binding of IL-2, IL-4, IL-7, IL-9 or IL-15 antibody 362.78-Cho. On the contrary, the positive control IL-21 showed a dose-dependent binding, which is consistent with previous research.

[186] This absence of cross-reactivity subsequently explained by the study of epitope mapping of clone 362.78 (see example 17). Amino acid residues located near the D-helix of IL-21, which, as shown, are linked clone 362.78 (EKKPPKEFLERFKSLL; SEQ ID NO: 2 from residue 129 to 144)), are not the most conservative among related cytokines person containing gamma chain, also of IL-21 mouse, as shown in table 10.

Table 10
IL21_ - TCPSCDSYEKK-PPKEFLERFKSLLQKMIHQHLSSTHGSEDS
IL21_ - KCPSCDSYEKR-TPKEFLERLKWLLQKMIHQHLS
IL15_ - CKECEELEEK-NIKEFLQSFVHIVQMFINTS
IL2_ - TTFMCEYADET-ATIVEFLNRWITFCQSIISTLT
IL4_ - GLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS
IL7_ - SLEENKSLKEQKK-LNDLCFLKRLLQEIKTCWNKILMGTKEH
IL9_ - CEQPCNQTTAG-NALTFLKSLLEIFQKEKMRGMRGKI

[187] IL-21 is shown as SEQ ID NO: 2; IL-21 mouse are shown as SEQ ID NO: 11; IL-15 person shown as SEQ ID NO: 92; IL-2 shown in SEQ ID NO: 93; IL-4 person shown as SEQ ID NO: 94; IL-7 person shown as SEQ ID NO: 95; IL-9 person shown as SEQ ID NO: 96.

Example 12. Analyses of b-cell proliferation

Analyses of primary b-cells

[188] For further analysis activity forms, neutralizing IL-21, developed two analysis of primary b cells. Analysis of b-cell proliferation was used to demonstrate neutralization of proliferation induced by IL-21, in 4 days, and the analysis of the differentiation of b-cells for 8 days showed neutralization differentiation into plasma cells induced by IL-21. These experiments showed neutralization of IL-21 in biologically significant long analysis.

[189] the Allocation of primary b-cells. To obtain primary b cells 200 ml of peripheral blood was collected from healthy human volunteers (ZymoGenetics). The blood was diluted with 200 ml of PBS buffer at room temperature the tours and did a 35-ml aliquots in 50-ml conical tubes. Was pagkawala fourteen milliliters of ficoll-vipaka Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) at room temperature and the tubes were centrifuged for 20 minutes at 2000 rpm/min Layer RVMS (mononuclear cells of peripheral blood) at the interface was aspirated and washed twice with MACS buffer (PBS, 20 mm HEPES and 1% BSA; Invitrogen, Carlsbad, CA). Cells were counted and cells were isolated by negative selection using a dial for selecting In-cell Isolation Kit from Miltenyi Biotec (Auburn, CA)following the Protocol provided by the manufacturer. A small sample of purified b cells were analyzed for purity FACS-analysis and found that in all experiments, b cells are >97% purity.

[190] Analysis of cell proliferation. B cells proliferate in response to joint cultivation with anti-CD40 and IL-21. To determine the neutralizing activity of monoclonal antibodies anti-IL-21, b-cells were sown at 40000-50000 cells/well in the treated 96-well plate for tissue culture with a U-shaped bottom (Becton Dickinson, Franklin Lakes, NJ). Then cells were treated with 0.1 µg/ml anti-CD40 (goat polyclonal antibody against human CD40; R&D Systems, Minneapolis, MN), 50 ng/ml (3,21 nm) of recombinant IL-21 (ZymoGenetics, A 1207F) and titration antagonist of IL-21 (mAbs analyzed or controls). Then the tablets with cells were incubated for 3 days at 37°C and 5% CO2in humid chambers thermostat for tissue culture. Three days later cells were irradiated with 1 µci/well of [3H]-thymidine (Amersham Biosciences, Piscataway, NJ). Then after 16 hours the cells were collected on glass-fiber filters and produced quantitative analysis of [3N]-enable using a beta-counter (Topcount NXT, Packard). Curves IC50measuring effective neutralization of proliferation induced by IL-21 was calculated and expressed as molar concentration. The values of the IC50for best neutralizing mAbs described here were in the range from 0.71 nm to 6,55 nm and are summarized in table 11.

Table 11
Neutralization of IL-21 in the analysis of b-cell proliferation
The antagonist of IL-21IC50 (nm)
Soluble hIL-21R/γc-Fc3,5
362.75.1No neutralization
362.78.11.17
366.328.100,71
366.552.114,75
366.617.76,55

[191] the Analysis of b-cell differentiation. Diff is retireve naive b cells In antitelomerase plasma cells greatly facilitated in vitro with a combination of IL-21 with anti-CD40 and IL-4 (Ettinger et al., J Immunol. 175:7867-79, 2005; Ettinger et al, J Immunol. 178:2872-82, 2007; Kuchen et al. J Immunol. 179:5886-96. 2007). To demonstrate activity in the analysis, longer than these two screening analysis based on Baf3 described in examples 7 and 8, used to neutralize the form described here, for the neutralization of IL-21 and inhibiting differentiation of plasma cells. To accomplish this primary b-cells were sown at 150,000 cells/well in the treated 96-well flat-bottomed tablet for tissue culture (Becton Dickinson, Franklin Lakes, NJ). Then cells were treated with 0.1 µg/ml anti-CD40 (goat polyclonal antibody against human CD40; R&D Systems, Minneapolis, MN), 10 ng/ml recombinant human IL-4 (R&D Systems) and 25 ng/ml (1.6 nm) recombinant human IL-21 (ZymoGenetics). Then added antagonists of IL-21 (analyzed mAbs or control) and cells were incubated at 37°C and 5% CO2for eight days in a humid chamber thermostat. At the end of eight days, conditioned medium was collected for antibody titers), and the cells were besieged for subsequent flow cytometrics tests.

[192] the Analysis of the b-cells using flow cytometry: Cells resuspendable in the buffer for FACS person (HBSS, 20 mm HEPES, 1% BSA (all from Invitrogen) and 2% human serum antibodies to Human Ab Serum (Gemini Bio-Products, Woodland, CA)for five minutes to block Fc-receptors. Then the entrances were centrifuged (5 minutes at 1200 rpm) and was sucked out. The dyes were prepared by dilution of the antibody 1:100 in buffer for FACS person and they dosaged 100 μl per sample. Mix with one dye (to adjust the compensation settings cytometer) and several dyes were prepared using the following antibodies: anti-CD 138-FITC, anti-lgD-PE, anti-CD38-PE-A and anti-CD 19-ARS. Plasma cells were defined as large (estimated direct scattering) cells CD19+, IgD10, CD38+and CD138+. The percentage of plasma cells relative to total cells was used to determine the effectiveness of neutralizing forms described here.

[193] Result: With the combination of IL-21 with IL-4 and anti-CD40, approximately 50% of living, a large In-cells on day 8 was a CD138+plasma cells IgDlow. In the absence of IL-21 proportion of plasma cells was ~ 8% of large cell b-cells. The addition of various antagonists of IL-21 to cultures containing IL-21, reduced the proportion of plasma cells depending on the dose. Clone 362.78.1 was the most effective neutralizer and almost completely neutralized the activity of IL-21 at the ratios of the antagonist ligand 10:1 and 2.5:1. Other analyzed antibodies, clones 366.328.10.63 and 366.552.11.31 were almost as effective in neutralizing differentiation-stimulated IL-21. These Yes the data are summarized in table 12.

Table 12
The inhibition of differentiation of plasma cells by neutralizing monoclonal anti-hlL-21 antibodies
The ratio of the antagonist ligand
10:12,5:10,6:10,16:1
The antagonist of IL-21% plasma cells CD138+IgDlow
362.78.113,715,534,142,8
366.328.10.6315,420,241,452,1
366.552.11.31*15,426,444,850,3
The receptor for IL-2123,441,754,.266,7
* Note that the actual ratio of the antagonist ligand for clone 366.552.11.31 was 14.4, 3.6, 0.9, and 0,22:1

Example 13. Murine model PTH (trans., hypersensitivity of the delayed type)

[194] DTH responses are classical immune responses that are triggered by CD4+T cells and mediated by T-cells, neutrophils and macrophages. DTH response is a good indicator of CD4+T-cell-mediated response. Mice subjected to immunization subcutaneously using egg protein ovalbumin (OVA) in one of two adjuvant, complete Freund's adjuvant (CFA; Sigma) or Ribi (Sigma; also known as adjuvant MPL+TDM+CWS) (trans. consisting of monophosphorylated A (MPL), dimycolate trehalose (TDM) and cell wall skeleton (CWS)). This phase is called the phase of sensitization (days 0-6). Measurement of the ear produce seven days. Then mice were injected with in ear control PBS (left ear) or OVA (right ear). This phase is called phase provocation (days 7-8). Immune responses generated by OVA, induce inflammation of the ear, which leads to an increase in the thickness of the ear within 24 hours, the ear treated with OVA, but not ear-treated PBS. The thickness is measured with the help of kalibaru.

[195] C57BL/6 Mice (n=8/group) are subjected to immunization in the back with 100 μg of egg ovalbumin (OVA)emulsified in CFA in a total volume of 200 μl. When using Ribi instead CFA, add 0.5 mg/ml ovalbumin in one vial, RIBI and strongly stirred for 2 minutes to education em is lsii, which is used for the injection of mice. Seven days after immunization of mice injected with 10 μl of PBS in the left ear (control) using 10 μl of OVA in PBS in the left ear in the volume of 10 µl. The thickness of the ear of all mice was measured before the injection of the mouse ear (0 dimension). The ear thickness was measured 24 hours after provocation. Calculate the difference in ear thickness between 0 measurement and 24-hour measurement it reflects inflammation of the ear. Groups of mice injected with PBS or anti-IL-21 antibodies with different concentrations intraperitoneally or days 0-6 (sensitization phase), or 7-8 days (phase provocation). Injecting 7 and 8 provide day 2 hours before measurement of ear thickness in the 0 and 24 hour time points. At the end of the 24-hour period, after measuring the thickness of the ear, ears cut off and placed in formalin for histological analysis.

Example 14. Murine model of multiple sclerosis

[196] For the analysis does anti-IL-21 whatever the effects on multiple sclerosis, analyzed the ability of anti-IL-21 antibodies to inhibit experimental autoimmune encephalomyelitis (EAE-MS), a murine model of MS. Used the well-studied T-cell-dependent immunization with myelin peptide oligodendrotsitarnym glycoprotein (MOG) (35-55) used in the mouse model, C57BL/6. The experiment is carried out to determine that the anti-IL-21 antibody could b the delay and/or inhibit the scale of the disease EAE or by inhibition mediated DC (AC, dendritic cell presentation of antigen, or by enhancing CD8 T-cell responses. The lack of effective CD8 T-cell responses in this model exacerbates EAE (Malipiero et. al., Eur. J. Immunol. 27:3151-3160, 1997). Late manifestation of the disease in the EAE model, depending on the dose suggests that the use of anti-IL-21 antibodies can be useful in MS.

[197] EAE is a murine model of MS. In one such model of C57BL/6 mice subjected to immunization with 100 μg of MOG peptide (MOG 35-55) or 100 µg of recombinant MOG protein, emulsified in Freund CFA. Two milliliters of 0.5 mg/ml MOG 35-55 in PBS was added into the vial with CFA and was vigorously shaken on a vortex for turning into an emulsion, solution or get in the ratio of 1:1 recombinant MOG in CFA. The backs of mice shave and injected subcutaneously with 100 μg of MOG/CFA in the backs of mice. The readings of the mass of the mice take two days before and every day after immunization. Then mice on day 2 is injected intravenously (i.v.) using 200 μl pertussis toxin (PT), the final concentration of 200 ng/mouse. Mice exposed daily monitoring of clinical indicators. Groups of mice injected intraperitoneally (I.P. Pavlova.) using 200 μl PBS, 100 μg BSA, 10 μg - 200 μg anti-IL-21 antibody in a volume of 200 μl on days 0-20, or 3 x per week for 3 weeks. Mass mice, clinical indicators and the incidence of disease estimate is depicted graphically.

Example 15. Anti-mIL-21 antibody decreases disease incidence and progression in mouse models of adaptive T-cell transfer colitis and psoriasis

[198] the Adaptive transfer of naive T cells to mice with mismatching of minor histocompatibility loci or syngeneic mice with a weakened immune system leads to the development of colitis (Leach MW, et al., 1996, Powrie F. et al., 1997), as well as skin lesions similar to psoriasis (Schon MP, et al., Nat Med. 2: 183-8, 1997; Davenport CM, et al., Int. Immunopharmacol. 5:653-72, 2002). Transplantation of only 0.2 million CD4+CD25-T cells from BALB/C mice or B10.D2 mice with weakened immune systems C. b-17 SCID leads to weight loss, positive hemocult-test and the development of skin lesions. The symptoms in these mice differ from colony to colony.

[199] This model of colitis/psoriasis, which has some similarities to Crohn's disease and psoriasis person was actively used to analyze the effectiveness of therapy for these diseases in humans. For this experiment, mice (8 female mice donors B10.D2; 50 females recipients C. b-17 SCID) were obtained from Jackson Laboratories or Charles River Laboratories, respectively. Collected spleen 8 mice [10]. D2. CD4+CD25 - T cells were isolated from the United spleens using conventional methodology known in this field. The purity of the T-cell population was assessed by flow cytometry.

[200] Naive mice C. b-17 SCID received the 5×10 5CD4+CD25-T-cells isolated from spleens of mice B10.D2) by intravenous injection on day 0. All mice were weighed at least five times a week and watched weight loss, which may be associated with colitis. In addition, a clinical indicator of colitis [stool consistency and blood in the stool] evaluated at least one day a week. Mice were also subjected to careful monitoring, at least five days a week and identified indicators of psoriasis symptoms (hair loss, scratching, alopecia etc).

[201] Rat antibody against mouse IL-21 (mIL-21), a control antibody, rat isotype, or filler (PBS) was administered to groups of mice beginning on day 0. The treatments were carried out in the form of intraperitoneal injection, twice a week, using antibodies, injected at 0.2 or 0.8 mg per mouse per dose. The treatment could also be produced using a similar dosing regimen or other way of introduction. In groups of anti-IL-21 antibodies were 9-10 mice per group, 6-7 mice per group in groups izotopicheskogo control antibodies and 10 mice per group in the PBS group. This dosage regimen is called "preventive dose".

[202] In a separate experiment, groups of mice given a dose of the same antibodies, and doses described above (10 mice per group)started their ways Le is to be placed on day 12 after transfer of cells, which is the approximate day on which the mice began the signs of psoriasis and/or colitis. These dosing schedules referred to as "therapeutic dosage".

[203] At the end of the study (45 day) tissue of the colon was on histological confirmation of colitis and serum for analysis of the levels of cytokines and chemokines.

[204] the results of the prophylactic dosage. Mice receiving anti-IL-21 antibody as in doses of 0.2 mg and 0.8 mg differed significant (at least p<0.05 or below) the reduction of body weight reduction and a significant reduction in psoriatic skin and symptom of colitis during the whole period of the experiment compared with mice that were administered PBS or 0.2 mg of isotype control monoclonal antibody. At the end of the study (45 day) mouse treated with any dose of antibodies mIL-21, had approximately 100% of their initial body weight, whereas mice treated with PBS, was found lost on average 16% of their initial body weight, and mice treated with izotopicheskogo control antibodies had a 10-15% loss of initial body weight. 45 day mouse treated with any dose of antibodies mIL-21, had approximately 6.5-7 times lower average clinical indicators colitis and approximately 5-7 times lower average psoriatic skin. Only 20% of mice, cennych using 0.2 mg of anti-IL-21 antibody, developed psoriasis, which was weak, whereas none of the mice treated with a dose of 0.8 mg, did not develop any symptoms of psoriatic skin. On the other hand, 100% of mice treated with PBS, developed psoriasis, together with approximately 50% of these mice develop severe symptoms. At the end of the study was also a significant reduction of histological indices of colitis (assessed according to the scale for cellulitis bowel lesions and patterns) in mice treated with anti-IL-21 antibody, compared with mice treated with PBS and 0.2 mg isotyping control antibodies.

[205] Mice treated with anti-IL-21 antibodies had significantly lower levels of serum IL-6, RANTES (trans. RANTES is a chemokine expressed and indirect T-cell activation), TNF-α, - MIP-I β compared with treated with PBS, additionally providing anti-inflammatory role of anti-IL-21 antibodies.

[206] the Results of therapeutic dosage. Mice receiving anti-IL-21 antibody as in doses of 0.2 mg and 0.8 mg, ranging from 12 days after transfer of T cells, characterized by a reduction in the loss of body weight and significant reductions in symptoms of colitis during the period of the experiment compared with mice that were injected isotopique control monoclonal antibody. 45 day mouse treated with any dose of antibodies mIL-21, had rough is about 3.5-4 times lower average clinical indicators of colitis compared with mice treated with isotyping control monoclonal antibodies. Mice treated with a dose of 0.8 mg of anti-IL-21 antibodies had lower clinical characteristics of psoriasis than mice treated with isotyping control antibody or PBS.

[207]. Taken together, these results indicate that the introduction of in vivo anti-IL-21 antibody was effective in reducing the occurrence and severity of colitis and psoriasis in a murine model of T-cell migration, and suggest that anti-IL-21 antibodies can be effective in the treatment of inflammatory bowel disease and/or psoriasis in people.

Example 16. Murine model of contact hypersensitivity

[208] Contact hypersensitivity can be induced in mice using different contact allergens, including dinitrofluorobenzene (DNFB) and oxazolone. Mice sensibiliser locally using allergen in the carrier of acetone and olive oil and then subjected to immunization with allergen in the ear in olive oil in its purest form. The change in ear thickness is a measure of the immune response to an antigen. anti-IL-21 antibodies are introduced, or in the sensitization phase (days 0-5), or during the phase of provocation (days 5-6). Inhibition of ear thickness antagonists of IL-21 indicates the role of IL-21 in the induction of contact hypersensitivity.

[209] Mice S B1/6 paint the back with p the power of 0.5% DNFB in a mixture of acetone: olive oil (4:1) or a mixture of acetone and olive oil in its pure form at 0 day. On day 5 the ear thickness of mice was measured using calipered and mice infect the ears of olive oil in its pure form (control) or 0.25% DNFB in olive oil by applying drops of a solution of 25 µI ear. The change in ear thickness was measured on day 6 and inflammation is calculated as the difference in ear thickness between 5 and day 6 day. Groups of mice were injected with intraperitoneal (I.P. Pavlova.) PBS or 10-100 μg anti-IL-21 antibodies or 0-5 days, or on days 5-6.

[210] the Inhibition of ear thickness anti-1b21 antibodies shows that anti-IL-21 antibodies can be applied for inhibition of contact hypersensitivity.

Example 17. Mapping of epitopes A. Experiment hydrogen-deuterium exchange (HPX)

[211] for the purpose of identification of epitope regions of IL-21 that are recognized by neutralizing anti-IL-21 mAbs 362.78.1.44, 362.597.3, 366.328.10, 366.552.11 and soluble hIL-21 R/γc-Fc, used hydrogen-deuterium exchange (HDx) on the basis of immunoaffinity with subsequent mass spectrometric analysis. In particular, purified mAbs were immobilized on the granules CNBr-activated sepharose and translated in deuterium buffer. Denatured IL-21 was associated with immunoaffinity granules during incubation and the pellets were washed in deuterated buffer to remove unbound proteins. Then the complex antigen-antibody were subjected to solution of PBS to initiate reverse interaction between the amide hydrogen on unbound in the areas of IL-21. Then hydrogen-deuterium exchange extinguished and IL-21 were suirable in a buffer with low pH, IL-21 was then subjected to proteolytic destruction of immobilized pepsin. Then created a mass spectrometric peptide maps mass spectrometry by MALDI-TOF and compared with peptide mass spectrometry cards control sample produced in the free state of IL-21, which was exchanged back to the amide hydrogen of deuterated IL-21 by dilution with a solution of PBS. Figure 3 shows the extended mass spectra of products of proteolytic destruction by pepsin peptides of IL-21 in the free state (figa and 3C), and is associated with the antibody IL-21 (figv and 3D). As indicated on figa, overlapping peptide isotope attributed to the corresponding peptides EKKPPKEF (SEQ ID NO: 2 from residue 129 to 136) (mass/charge, m/z, 1002.5619 Yes) and LERFKSLL (SEQ ID NO: 2 from residue 137 to 144) (m/z, 1005.6091 Yes) IL-21 in the free state with theoretical peptide masses with a precision determination of the mass of 10 ppm (parts per million h/m). Observed a small amount of residual deuterated peptide in the region of m/z, 1002.5619 Yes, due to incomplete exchange of the amide hydrogen.

[212] FIGU is a spectrum of the same mass range figa showing two overlapping zones peptide isotopes with monoisotopic ions when 1014.49 m/z and 1015.00 m/z. As the two peptidnaya were grouped in clusters around the same mass range, it was difficult to establish the identity of each peptide between the two ions. However, these tandem mass spectrometry peptide ions figa and showed that they were identical fragmentation pattern of the peptide (data not shown). Although there was a small percentage Mediterraneo peptide detected in the sample obtained from the complex antigen-antibody, the majority of peptide ions retained deuterium and shifted to higher masses due to the limited accessibility of solvent to the binding regions of the antibody/antigen, indicating that the binding site mAb, probably contains the EKKPPKEFLERFKSLL (SEQ ID NO: 2 from residue 129 to 144).

[213] Another peptide, a proteolytic product of the destruction of pepsin as IL-21 in the free state and in complex antigen-antibody watched as shown in figs and D and identified as KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2 from residue 141 to 162) (m/z, 2519.2451) on the basis of theoretical mass of product a proteolytic destruction by pepsin peptide fragment of IL-21. As shown in fig.3D, comparison of the shear mass of this peptide of 22 amino acid residues (Δ=9,0 Yes) shift the weight of these two upstream residues (figure 2, EKKPPKEF (SEQ ID NO: 2 from residue 129 to 136) and LERFKSLL (SEQ ID NO: 2 from residue 137-144), this area is only slightly defended upon the binding of mAb, indicating that only the part of this peptide may be involved in binding to mAb. In fact, this peptide sequence is a C-terminal tail, and it contains four amino acid residue overlapping with the peptide (LERFKSLL (SEQ ID NO: 2 from residue 137 to 144), which, apparently, are substantially protected mAb. Based on mass spectrometric measurements of retention deuterated derivative, the inventors have found that the epitope of IL-21 to bind mAb EKKPPKEFLERFKSLL (SEQ ID NO: 2 from residue 129 to 144) and upstream sequence KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2 from residue 141 to 162).

C. Experiment protection of lysine from tagging

[214] using HDx analysis determined the epitope region of IL-21 binding mAb, and noticed that the five lysine residues (out of the total 12 lysine residues in IL-21) are installed in the area of the binding epitope. Since it is most likely that the lysine residues are present in the areas of protein solvent-accessible, due to their property of charge, lysine, apparently, is the ideal option for selective chemical modification for parallel detection region epitope of the antigen. The idea after strategy for chemical modification is that the protection of the lysine in the antigen from the modification in the presence of antibody or in the absence of antibodies is due to its binding epitope (Scholten et al., J. Amer. Soc. Mass Spectr. 17: 983-994, 2006). T is thus, to further characterize epitope region and to determine the lysine residues involved in the binding of mAb was performed selective acetylation of lysine residues in affine-bound and free States of IL-21. Website modification/protection against modification of lysine was determined by analysis of the mapping of the total mass and peptides using MALDI-TOF mass spectrometry with ionization by sputtering in an electric field (ESI).

[215] Scholten et al. (Scholten et al., J. Amer. Soc. Mass Spectr. 17: 983-994, 2006) investigated the molar ratio of reagent for acetylation (NHS-acetate) and the antigen to complete the acetylation of lysine residues, solvent-accessible, and found that the tagging was effective at 250-fold molar excess of the reagent with 3-minute reaction. To determine the binding epitope same reaction conditions were used as for the free state of IL-21 and affine-associated IL-21 with several neutralizing IL-21 mAbs, as well as heterodimeric receptor protein IL-21 (IL-21 R/γc-Fc). In this condition the reaction tagging various accessibility to solvent lysine residues of IL-21 in pure form and affine-associated IL-21 has caused the distribution of acetylation of lysine (degree of filling of the acetyl) molecule IL-21. The number of protected lysine residues on affinity-bound IL-21 was comparable with L-21 in its pure form is based on the most intense ion. Spectral alignment on the basis of the most intense ions showed clearly that the number of lysine azetilirovaniu on the antigens isolated from a variety of immune complexes is different. It was obvious that the labeling reagent lysine was less available in affinity-bound IL-21. Therefore, acetylation was reduced by binding of the antigen with the antibody.

[216] Lisini protected by acetylation, which may be involved in the binding of mAb, were further probed by cleavage by proteases and subsequent analysis mapping of peptide masses using liquid chromatography-mass spectrometer. As covalently modified lysine residues are resistant to cleavage by trypsin, used proteolytic enzyme pepsin to generate peptides, to a greater extent detectable mass spectrometry, for a more detailed study of the modification of lysine. Modification of individual peptides was investigated using ion-exchange chromatography of a single peptide. As shown in figure 4, the selected ion chromatogram was obtained from both the control (IL-21 in its pure form)and test samples (affine-associated molecules IL-21) to determine acetylated and deacetylating lysine residues. Figa is a selected ion-exchange chromatogram obtained is about as a result of proteolysis of the peptide, lirovannomu on 56,22 min under these chromatographic conditions, and the monoisotopic mass of the peptide was about 662,9 Yes, which, apparently, is trehserijnom state (Δm=0,3 Yes), as shown in the inserted mass spectrum. Made the identification of this peptide in trehserijnom state in the form containing acetylated lysine peptide fragment formed by cleavage by pepsin, TCPSCDSYEKK.PPKEF (SEQ ID NO: 2 from residue 119 to 136) (m/z, 1986 Yes), taking into account that the mass deacetylating peptide is 1860 Da (m/z). The difference of the masses (acetylated peptide / deacetylating peptide) was 126 Yes, indicating that all three lysine residue in this peptide were azetilirovanny in the free state of IL-21. However, the selected ion chromatogram of affinity-bound IL-21 showed no traces of the peptide (pigv), indicating that the three lysine residue were fully protected by binding IL-21 antibody.

[217] it Was found that additional peptide after the destruction of pepsin (KSLLQKMI (SEQ ID NO: 2 from residue 141 to 148) is secured upon the binding of IL-21 mAb (figure 4C and 4D). Monoisotopic peptide ion was approximately 509 Da (m/z) in the form of double-ion (Δm=0,5 Da) and the mass difference (acetylated peptide / deacetylating peptide) was 42 Yes, indicating that only one of the two lysine residues in the peptide was protected. Earlier experiments H/D exchange was observed that the left lysine (upstream) C-terminal tail of the peptide sequence, KSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 2 from residue 141 to 162), was most likely protected by the binding of an antibody. Thus, the most likely is that K113, not C involved in the binding of an antibody.

[218] Four lysine residue (K, K, K and ZCTU) were protected from acetylation binding clones 362.78.1.44 and 362.597.3, and lysine residues were located in the proposed epitope region, linking mAb IL-21, as shown in the analysis of the HDx. In aggregate, the analysis of the protection acetylation ensured the involvement of specific lysine residues in the interaction of antigen-antibody and additionally confirmed epitope sequence, estimated from the analysis of HDx.

17C. Comparison of amino acid sequences of IL-21 different types

[219] For a better understanding of cross-reactivity between species, the results in examples 3 and 9, in the light of established epitopes on IL-21, the bound mAbs IL-21 (example 17A and b), obtained amino acid sequences and compared the IL-21 with many types crosswise (table 13). The complete human sequence was more than 96% identical to the sequences of cynomolgus macaques and rhesus whereas only 61-65% identical to the sequence is lnasty IL-21 rodents. Namely, discontinuous epitope linked clones 362.78.1.44 and 362.597.3 as described in example 17 (shown in table 13)is identical to IL-21 human, cynomolgus macaques and rhesus whereas rat IL-21 sequence differs from the human IL-21 within 6 residues in these areas, and the mouse IL-21 sequence differs 7 residues.

[220] table 13
Alignment of the amino acid sequence of IL-21 to IL-21 human, cynomolgus macaques, rhesus, rat, and mouse.
Neither IL-21MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLV
CynolL-21MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLD
RhesuslL-21MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLD
RatIL-21MERTLVCLILIFLGTVAHKSSPQRPDHLLIRLRHLMDIVEQLKIYENDLD
MulL-21MERTLVCLWIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLD
HuPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPP
CynoPEFLPAPEDVETNCEWSAISCFQKAQLKSANTGNNERIINLSIKKLKRKSP
RhPEFLPAPEDVETNCEWSAISCFQKAQLKSANTGNNERIINLSIKKLKRKSP
RatPELLTAPQDVKGQCEHEAFACFQKAKLKPSNTGNNKTFINDLLAQLRRRLP
MuPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLP
HuSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
CynoSTGAERRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
RhSTGAERRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
RatAKRTGNKQRHMAKCPSCDLYEKKTPKEFLERLKWLLQKMIHQHLS
MuARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS

[221] IL-21 is shown as SEQ ID NO:2; IL-21; mouse are shown as SEQ ID NO: 11; IL-21 cynomolgus macaques are shown as SEQ ID NO:9; IL-21 rhesus shown as SEQ ID NO:9; IL-21 rats are shown as SEQ ID NO: 97.

[222] Discontinuous epitope set for two highly related anti-hIL-21 mAbs 362.78.1 and 362.597.3 underlined above.

Example 18. Antiidiotypic monoclonal antibodies to 362.78-Cho for use in preclinical and clinical immunological assays.

[223] Antiidiotypic mAbs produced specificity the ski to 362.78-Cho for use in preclinical and clinical immunoassays, so the potential responses to anti-36278-SNO antibody in individuals treated with this therapeutic anti-IL-21 mAb, can be specifically measured.

[224] To differentiate between the immunogen (362.78-Cho), which itself is an antibody, and antiadiotipiceskih antibodies in this example, the immunogen will be denoted in the form Ab1, and antiidiotypic antibody will be denoted as Ab2. Antiidiotypic antibody should inhibit (neutralize) the binding of Ab1 with its antigen (IL-21). However, it should be noted that this method will be made anti-Abl antibodies that are not antiadiotipiceskih. By definition, these antibodies will bind anti-362.78-Cho, no neutralizing antibodies and can also be applied in preclinical and clinical immunoassays.

[225] Way. For immunization of mice with 362.78-Cho, five BALB/c mice 6 to 8 weeks of age (Charles River Laboratories, Wilmington, MA) were immunized with 362.78-Cho. The original mice were immunized by subcutaneous injection with ~50 µg purified 362.78-Cho (Lot# A2125F) in combination with adjuvant Emulsigen® - R (MVP Laboratories INC, Omaha, NE) according to the manufacturer's instructions. After the initial immunization, each mouse received an additional 50 µg 362.78-Cho in Freund Emulsigen® - P using the subcutaneous route of administration each is two weeks over a six-week period. Seven days later the mice of the third and fourth immunization was taken away by the blood through retroorbital plexus and the serum separated from the blood for analysis of the ability of serum to contact 362.78-Cho.

[226] the Selection of animals with a merger with the help of trap options analysis analysis of neutralization.

[227] Trap analysis. The ability of mouse anti-362.78-Cho (Ab2, antiidiotypic) antibodies in anticigarette contact 362.78-Cho (Ab1 produced in the cells of Cho, lot # E10569) was assessed using trap option ELISA. In this analysis wells 96-well polystyrene tablets for ELISA were first coated with 100 µl/well Fc-specific goat antibodies against human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a concentration of 1 μg/ml in covering buffer (0.1 M Na2CO3pH 9,6). The plates were incubated overnight at 4°C, after which the unbound antibody was aspirated and the tablets were washed twice with 300 μl/well of buffer for washing (PBS-Tween installed as M NaCI, 0.0022M KCl, M Na2HPO4, 0,0020M KH2PO4, 0,05% vol./wt. Polysorbate 20 (Tween 20), pH of 7.2). The wells were blocked with 200 µl/well blocking buffer (PBS-Tween plus 1% wt./about. bovine serum albumin (BSA) for 60 minutes at room temperature (RT), was aspirated and the tablets were washed twice with 300 μl/well PBS-Tween. The plan is Yety incubated with 362.78-Cho (Ab1, ZGI produced in the cells of Cho, lot # E10569) at a concentration of 1 mg/ml (1% BSA in PBS-Tween). After 1 hour incubation at RT, the contents of the wells of the tablets was aspirated and the tablets were washed twice as described above. Preparing serial 10-fold dilution (in 1% BSA in PBS-Tween) of antisera (Ab2), starting with an initial dilution of 1:1000 to 1:1000000. Then double samples of each dilution was transferred into a tablet for analysis, 100 µl/well. Normal mouse serum served as negative control. After 1 hour incubation at RT the contents of the wells of the tablets was aspirated and tablets twice washed as described above. Then Fc-specific goat antibody against mouse IgG conjugated with HRP (horseradish peroxidase) (Jackson ImmunoResearch Laboratories)at a dilution of 1:5000 was added to the wells, 100 μl/well. After 1 hour incubation at RT unbound detection antibody was aspirated from the wells and tablets twice washed. After extraction with 100 μl/well of tetramethylbenzidine (TMB) (BioFX Laboratories, Owings Mills, MD) was added to each well and the plates were incubated for 1 minute at RT. The color development was stopped by adding 100 µl/well of stop reagent (BioFX Laboratories, Owings Mills, MD) and the absorption values of the content of the wells was read on the instrument Molecular Devices Spectra MAX 340 to 450 nm.

[228] the Reaction of neutralization. The ability of mouse anti-362.78-Cho antiidiotypic antibodies (Ab2) in antises orode to inhibit (neutralize) binding activity 362.78-Cho (Ab1) was estimated using the neutralization on tablets. In this assay, wells of 96-well polystyrene tablets for ELISA were first coated with 100 μl/well of ligand IL-21 (lot # A 1207F) at a concentration of 1 μg/ml in covering buffer (0.1 M Na2CO3pH 9,6). The plates were incubated overnight at 4°C, after which the unbound ligand was aspirated and the tablets were washed twice with 300 μl/well of buffer for washing (PBS-Tween set as 0,M NaCl, M KCl, 0,M Na2HPO4, 0,M KH2PO4, 0,05% vol./wt. Polysorbate 20 (Tween 20), pH of 7.2). The wells were blocked with 200 µl/well blocking buffer (PBS-Tween plus 1% wt./about. bovine serum albumin (BSA) for 1 hour, after which the tablets were washed twice with the buffer for washing. Preparing serial 10-fold dilution (in 1% BSA in PBS-Tween) of antisera (Ab2), starting with an initial dilution of 1:100 to 1:1000000. Normal mouse serum served as a negative control. Then double samples of each dilution was transferred into a 96-well plate for cultivation, 100 µl/well. Ab1 was added in the form of a 2 × solution, 100 µl/well. After 45 minutes incubation at RT, 100 μl/well was transferred into a tablet for analysis after the extraction of the blocking buffer. After 1 hour incubation at RT the contents of the wells was aspirated and tablets twice washed as described above. Then Fc-specific goat antibody against human IgG labeled with horseradish peroxidase, conjugated with HRP (horseradish peroxidase) (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:5000 was added to each well, 100 μl/well, and the plates were incubated at RT for 1 hour. After removing unbound detecting antibody tablets twice washed, 100 μl/well of tetramethylbenzidine (TMB) (BioFX Laboratories, Owings Mills, MD) was added to each well and the plates were incubated for 2 minutes at RT. The color development was stopped by adding 100 µl/well of stop reagent (BioFX Laboratories, Owings Mills, MD) and the absorption values of the content of the wells was read on the instrument Molecular Devices Spectra MAX 340 to 450 nm.

[229] the Merger. Two mice with the highest neutralizing titers of anti-362.78-Cho were immunized at the time of ending with approximately 50 μg 362.78-Cho (Ab1) in PBS without adjuvant by subcutaneous injection. Four days later he collected the spleen and lymph nodes of this mouse. Electroline performed using conventional methods known in this field to merge lymphocytes with mouse myeloma cells P3-H-Ag8.653 (American type culture collection, CRL 1580) with a ratio of 1:1 lymphocyte:myeloma device Cyto-pulse CEEF-50 (Cyto Pulse Sciences, Inc., Glen Burnie, MD). The mixture to merge distributed in 96-well flat-bottomed plates. Wells tablets to merge filled three times with 70% replacement of hybridoma culture medium (IMDM with 1 x L-glut the min (100x), 1 × penicillin-streptomycin (100x), all Gibco Invitrogen, Carlsbad, CA, 10% serum Fetalclonel not inactivated by heating (HyClone, Logan, UT)with 10% factor to clone hybrid Hybridoma Cloning Factor (BM Condimed HI Roche Diagnostic, Indianapolis, IN), 1 × additive HAT (50 ×, Gibco Invitrogen). (translated content) holes was evaluated in ten days after application on the tablets of the merge.

[230] the Selection of main (master) holes. Organized the screening of 96-well plates to confluence in the presence of mouse anti-362.78-Cho idiotypical antibodies using trap option ELISA, as described above, except that the hybridoma supernatant analyzed undiluted from the culture plates. Hybridoma cells positive holes has successfully increased in 24-hole culture plates. When reaching a density cultures in 24-hole tablets approximately 4-6×105cells/ml of the supernatant (approximately 1.5 ml) were collected from each well separately and kept, and the cells from each well were kryokonservierung. Environment for freezing consisted of 90% serum Fetalclone 1 and 10% DMSO. Each of the 24-hole of supernatants analyzed again, as in trap variant ELISA and neutralizing variant ELISA plates as described above. The results showed that after the expansion of all supernatant core holes have retained their ability to learn 362.78-Cho antibody (Ab1) in races of the thief. Seven out of supernatants major holes retained its ability to neutralize the binding of Ab1 to human IL-21 ligand.

[231] the Cloning. Cells from 5 core holes were chosen according to their neutralizing activity and cloned in the growth environment for hybrid, equipped with 1×HT (100x, Gibco Invitrogen) for selection cloned hybridoma producing interest neutralizing mAb. The cells were cloned in 96-well culture plates to micrometrology using traditional breeding approach low density (less than 1 cell per well) and monoclonality was assessed by microscopic examination (content) holes for one source of growth before analysis. Six days after seeding in tablets all tablets were subjected to screening neutralizing ELISA for inhibiting anti-362.78-Cho antiidiotypic antibodies. Hybridoma cells from positive wells were successfully propagated in 24-hole tablets.

[232] the Selection of clones from the first round. Supernatant of approximately 6 holes each cloned hybridoma lines that were positive for specific mAb and came from a hole of only one colony growth hybridoma, were collected from each set clone and they were re-screened at various dilutions in neutralizing ELISA for identification of l is chego clone producing neutralizing mAb. When the density of the best clone 24-hole cultures approximately 4-6×105cells/ml, the supernatant was collected separately and kept the contents from each well and the cells from each well with careful cryopreservation.

[233] Conclusions. Produced mouse monoclonal antibodies (b2) reactive against recombinante expressed 362.78-Cho antibody (Ab1), and antibodies showed neutralizing activity, is able to block binding 362.78-Cho antibody with IL-21 people. These antibodies can be used as reagents in pre-clinical and clinical immunological assays.

Example 19. Linking native intracellular IL-21 and IL-21 cynomolgus macaques (but not mouse or rat IL-21) (a monoclonal antibody) IL-21 mAb 362.78.1.44

[234] Neutralizing anti-1b-21 monoclonal antibodies described herein are produced from transgenic mice expressing the genes of human immunoglobulin, and immunized with recombinant human IL-21 (see example 1). It was important to confirm that IL-21 mAb clone 362.78.1.44 can bind and neutralize native IL-21 in addition to the recombinant form of IL-21. In addition, to support pre-clinical Toxicological studies it is useful to understand the binding ability of IL-21 mAb clone 362.78.1.44 with native IL-21 different species. For this anal is one of the approaches is the tagging of IL-21 mAb clone 362.78.1.44 with a fluorescent dye and the use of labelled antibodies for the detection of intracellular IL-21 in activated T-cells flow cytometry. In this study, freshly isolated leukocytes of peripheral blood and cynomolgus macaques, and splenocytes rats and mice were activated in vitro with PMA (lane, phorbol-12-myristate-1-3-acetate (PMA)) and ionomycin for 24 hours for stimulation of the formation of IL-21. Cells were collected, recorded, was permeabilities and stained to detect the expression of CD3 or CD4 (to determine populations of helper T-cells), and IL-21 with IL-21 mAb clone 362.78.1.44 labeled with ALEXA FLUOR-647 (AF-647), and compared with the intensity of the coloration induced izotopicheskii similar control antibody. A positive signal in this analysis, higher signal observed for izotopicheskogo control mAb, demonstrating specific binding of IL-21 mAb clone 362.78.1.44 with endogenous IL-21 test species, although it is not an indicator of neutralizing the activity of IL-21. Additional research is needed to demonstrate neutralization of IL-21, which gives a positive result in relation to the binding of IL-21 mAb against human IL-21 clone 362.78.1.44 (see example 20).

[235] the Selection RVMS person (trans., mononuclear cells of peripheral blood): 100 ml of peripheral blood was collected from healthy men volunteers (ZymoGenetics) in heparin vacuum tubes Vacutainer (Vacutaine) with green lids (Becton Dickinson, San Jose, CA). The blood of resbala and with 100 ml of PBS at room temperature and 35 ml aliquots were distributed into 50 ml conical tubes. Was pagkawala 14 ml picola Ficoll/Paque PLUS (Pharmacia, Uppsala, Sweden) at room temperature and the tubes were centrifuged for 20 minutes at 2000 rpm Extracted layer RUMS on the interface and washed twice with the medium for analysis (RPMI 1640, equipped with penicillin/streptomycin, 10% fetal bovine serum, sodium pyruvate, 2 mm β-mercaptoethanol, all from Invitrogen, Carlsbad, CA). Viable cells were counted in trypanosome blue using traditional methods.

[236] the Selection RVMS cynomolgus macaques: 40 ml of peripheral blood was collected in heparin vacuum tubes Vacutainer (Vacutaine) with green lids (BD Biosciences) in cynomolgus macaques contained in the University of Washington in Seattle. The blood was diluted with 40 ml of PBS at room temperature and 35 ml aliquots were distributed into 50 ml conical tubes. Was pagkawala fourteen ml of ficoll Ficoll/Paque PLUS (Pharmacia, Uppsala, Sweden) at room temperature and the tubes were centrifuged for 20 minutes at 2000 rpm Extracted layer RUMS on the interface and washed twice with the medium for analysis (RPMI 1640, equipped with penicillin/streptomycin, 10% fetal bovine serum, sodium pyruvate, 2 mm β-mercaptoethanol, all from Invitrogen, Carlsbad, CA). Viable cells were counted in trypanosome blue using traditional methods.

[237] the Selection of mouse and rat splenocytes. As the rat, the AK and mouse splenocytes were prepared in accordance with the following Protocol. Viewsat spleen was carefully destroyed until a homogeneous suspension of cells with the ends of two frosted glass slides. Then the cells were passed through a nylon filter with holes 70 μm to remove lumps. Erythrocytes were literally by resuspendable sediment cells in 2 ml of lyse buffer ASC for 10 at room temperature. This interaction was stopped by adding an environment for analysis, then the cells were centrifuged (1200 rpm for 5 minutes), resuspendable and was passed through nylon with other holes to remove debris. Viable cells were counted in trypanosome blue using traditional methods.

[238] the Activation of the cells overnight with PMA and ionomycin. Cells of all types resuspendable at 2.0×106cells / ml and Then one ml of cells were sown with or without added 20 ng/ml PMA and 200 ng/ml ionomycin in the 24-well plate and incubated at 37°C for 20 hours in a humid chamber thermostat for tissue culture with 5% CO2. After 20 hours in each well was added with 1.0 ál of GolgiPlug (BD Pharmingen) and cells were incubated for an additional four hours.

[239] the Collection of cells and staining the surface. After 24 hours of incubation, as described above, cells were collected, washed with cold FACS buffer and were sown at 2.0×105-5,0×105cells is as well in 96-well plate for tissue culture (Becton Dickinson and Co., Franklin Lakes, NJ). The cells are then, if necessary, were stained with 1 μg/ml of one or more of the following antibodies: antimachine CD4-PE, anticrimine CD3-PE, anticrimine 220V-PE, or anti-monkey CD4-PE, or anti-human CD4-PE for 20 minutes in a bath with ice. Then cells were washed twice in PBS in preparation for the commit.

[240] Fixation and permeability of cells. For fixation of cells each sediment cells resuspendable in 200 μl of 2% paraformaldehyde and incubated at room temperature for 5 minutes. The cells are then centrifuged (5 minutes at 1200 rpm) and supernatant was aspirated and cells resuspendable in the buffer for permeabilization [PBS supplied with 0.1% saponin (Calbiochem) and 0.5% BSA (Sigma)] for 10 minutes at room temperature.

[241] Intracellular staining. After fixation and permeabilization cells were stained with ~1 mg/ml of one of the following labeled antibodies: antimisting IL-21-AF647, against human IL-21 clone 362.78.1.44-AF647 (both produced ZymoGenetics) or antibody comparison against human IL-21-AF647 from BD Pharmingen. The cells are then incubated at room temperature in the dark for 40 minutes. After 40 minutes the cells were washed twice with FACS buffer (HBSS (trans., balanced salt solution Hanks)with 1% BSA, 2% serum AB man and 0.05% HEPES).

[242] the retrieval and analysis of data is H. Upon completion of the dyeing and washing cells resuspendable 400 μl FACS buffer and the data were obtained using a run through a FACS Calibur (BD Pharmingen) software CellQuest software. Data were analyzed using the software FCS Express data analysis software (De Novo Software, Los Angeles, CA).

[243] Results. Detection of human IL-21 in cells of the human T-cells induced by PMA+ionomycin: while only 0,015% CD3+T cells were positively stained using izotopicheskogo control, approximately 9% of CD4+T cells positively stained for IL-21 using the IL-21 mAb clone 362.78.1.44 labeled with AF-647. The same fraction of IL-21+cells were detected using a commercially available IL-21 mAb from eBiosciences. This shows that IL-21 mAb can bind IL-21, endogenously produced in CD4+T-cells.

[244] the Detection of IL-21 cynomolgus macaques in mononuclear peripheral blood cells induced by PMA+ionomycin: Approximately 3.6% monkey CD3+T cells positively stained for IL-21 using the IL-21 mAb clone 362.78.1.44 labeled with AF-647, compared with the 0.1% positive using izotopicheskogo control. This number is higher than the number detected by using commercially available mAb against human IL-21 from eBiosciences. This discrepancy may be due to a weaker affinity is wyzwania antibodies eBiosciences in respect of monkey IL-21. These results indicate that clone 362.78.1.44 mAb against human IL-21 can bind IL-21, endogenously produced in CD4+T-cells in cynomolgus macaques.

[245] Detection of mouse IL-21 in splenocytes induced by PMA+ionomycin: using as a positive control, rat anti-mouse IL-21 monoclonal antibodies produced ZymoGenetics, approximately 13.5% of activated murine CD4+T cells were positive for IL-21. However, as was predicted from the Western blot and neutralizing activity assays showing that mAb against IL-21 human clone 362.78.1.44 not bind or neutralize murine IL-21 (see examples 3 and 9), mAb against IL-21 human clone 362.78.1.44 labeled with AF647, did not detect any IL-21-positive cells. This further demonstrates that mAb against IL-21 human clone 362.78.1.44 is not associated with murine IL-21.

[246] Detection of rat IL-21 in splenocytes induced by PMA + ionomycin. The rat splenocytes activated during the night in the presence of PMA and indolizine. These activation conditions were sufficient for the formation of IL-21-positive T-cells of human, monkey and murine T cells. In this experiment, neither antimachine-IL-21 mAb or mAb against IL-21 human clone 362.78.1.44, did not detect any cells that were positive for IL-21. However, since exp is Rimante no positive control, this negative result does not completely exclude the possibility that mAb against IL-21 human clone 362.78.1.44 can connect with mouse IL-21. However, these data, considered together with the lack of neutralizing the biological activity of rat IL-21 using mAb against IL-21 human clone 362.78.1.44, in other analyses (see example 9), convincingly prove that this mAb, probably does not bind rat IL-21.

[247]. Described here mAb IL-21 clone 362.78.1.44 undoubtedly associated with the native forms of the protein IL-21 human and cynomolgus macaques, but not with the mouse or mouse IL-21.

Table 14
TypesIzotopicheskii controlmAb against IL-21 human (clone 78)The positive control for anti-IL-21
People0,015% CD3+T cells positively stained with the control hIgG4-AF6479% CD3+T cells were IL-21+10% CD3+T cells were IL-21+(eBiosciences aIL-21 mAb*)
Cynomolgus macaque0.1% of CD3+T-cells positively stained with the control hIgG4-AF6473.6% of CD3+T cells were IL-21+,2% CD3+T cells were IL-21+*
MouseNo izotopicheskii control not usedNothing found13.5% of CD4+T cells were IL-21+
RatNo izotopicheskii control not usedNothing foundNot available
*(this mAb may not closely associated with monkey IL-21)

Example 20. Binding of native IL-21 human and neutralization of biological activity of native IL-21 human clone 362.78.1.44

[248] Neutralizing monoclonal antibodies anti-IL-21 (IL-21 mAb, described here, was produced in transgenic mice expressing the genes of the human immunoglobulin and immunized with recombinant IL-21 (see example 1). It was important to confirm that mAb IL-21 clone 362.78.1.44 can bind and neutralize native IL-21 in addition to the recombinant form of IL-21. To demonstrate neutralization of native IL-21 used the previously described analysis on the basis of cells Baf3/IL-21 R pSTAT (see example 7) and samples of conditioned medium from activated CD4+T cells were used as a source of native IL-21. In this experiment, samples of air-conditioned T-cells with the food pre-incubated with different amounts of mAb IL-21 clone 362.78.1.44 and then measured induced IL-21 levels of phosphorylation of STAT3 (pSTAT3) in Baf3 transfectants/hIL-21R. Neutralization of native IL-21 demonstrated through samples of air-conditioned T-cells environment from four separate people healthy donors.

[249] the Selection RVMS people and creating samples of air-conditioned T-cell medium: 100 ml of peripheral blood from four healthy men volunteers (ZymoGenetics) were collected in heparin vacuum tubes Vacutainer (Vacutaine) with green lids (Becton Dickinson, San Jose, CA). Then the blood was diluted with 100 ml PBS at room temperature and distributed in 35-ml aliquots in 50-ml conical tubes. Was pagkawala 14 ml ficoll Ficoll/Paque PLUS (Pharmacia, Uppsala, Sweden) at room temperature and the tubes were centrifuged for 20 minutes at 2000 rpm Extracted layer RUMS on the interface and washed twice with the medium for analysis (RPMI 1640, equipped with penicillin/streptomycin, 10% fetal bovine serum, sodium pyruvate, 2 mm β-mercaptoethanol, all from Invitrogen, Carlsbad, CA). Viable cells were counted in trypanosome blue using traditional methods. T-cells were selected by negative selection using a set of selection for CD4+T-cells of Human CD4+T Cell Selection Kit from Miltenyi Biotec (Auburn, CA)following the Protocol from the manufacturer. Using the traditional method of immunophenotyping was subsequently identified flow cytometry that CD4+T cells are >95% purity. Then T-cells and incubated for three days at 5×e cells per well in 24-hole tablet, pre-coated using a 5.0 μg/ml anti-CD3 antibodies in the environment for the bevel Th1, containing 5.0 μg/ml anti-IFNγ, 1.0 microgram/ml anti-CD28 (all from Becton Dickinson) and 10 ng/ml recombinant IL-12 (R&D Systems, Minneapolis, MN). After three days cells were washed, perseval in the medium containing 25 ng/ml PMA and 500 ng/ml ionomycin and incubated for five hours at 37°C. After five hours, samples of conditioned medium was collected and frozen and stored at -80°C until the day of the experiment.

[250] To determine the approximate concentration of IL-21 in samples of air-conditioned T-cell medium was prepared by serial dilution of 1:4 and analyzed the induction of STAT3 phosphorylation in Baf3 transfectants/hIL-21 R. Following the 10-minute Protocol analysis of biological activity of pSTAT3 set forth in example 7, the relative concentration of IL-21 in each sample of conditioned medium was determined by comparison of the level of pSTAT3 level obtained during the titration of recombinant IL-21. Using these data, a certain concentration of IL-21 in each of the four samples of conditioned medium were between 5.0 and 10.0 ng/ml

[251] To demonstrate neutralization of Feofilova STAT3 induced native IL-21, 1:10 dilution (final concentration of IL-21 between 0.5 and 1.0 ng/ml) of four samples of air-conditioned T-cell environment has plaincourault for 30 at 37°C With 1:4 serial again in the institutions of IL-21 mAb clone 362.78.1.44. The concentration of clone 362.78.1.44 ranged from 0.4 to 400 ng/ml After 30 minutes, samples of conditioned medium + IL-21 mAb was transferred into a tablet for cells Baf3/hIL-21 R, and incubated for an additional 10 minutes at 37°C. After 10 minutes of interaction was stopped with cold buffer for washing, cells were literally and measured the number of pSTAT3 using the method described in example 7.

[252] In all four samples, air-conditioned environment clone 362.78.1.44 IL-21 mAb effectively neutralized the activity of IL-21 (data summarized in table 15). The data presented clearly demonstrate effective linking with native IL-21 human and neutralization of native IL-21 human clone 362.78.1.44.

[253] table 15
Neutralization of native IL-21 clone 362.78.1.44 IL-21 mAb
Induction of pSTAT3 (times above background)
IL-21 mAb Concentration (ng/ml)Donor 1Donor 2Donor 3Donor 4
0,469,49 56,3261,1162,73
1,670,8461,7368,2465,41
6,2570,7610,467,5160,65
252,161,621,702,49
1001681,431,511,49
4001,591,301,141,51

Example 21.

21A. Pilot toxicity study using IL-21 mAb 362.78-Cho in cynomolgus macaques

[254] the Epitope specificity of the IL-21 mAb 362.78-Cho is common for humans, rhesus monkeys and cynomolgus macaques (see examples 17 and 17b)thus, tolerability and toxicity of IL-21 mAb were analyzed in cynomolgus monkeys that are suitable for this analysis type testing without the danger.

[255] Cynomolgus macaques were treated with a single injection of IL-21 mAb 362.78-Cho and monitored for clinical signs between 4 and 8 week post-treatment. IL-21 mAb gave subcutaneous or intravenous injection at doses of 5 or 100 mg/kg No clinical signs were observed. No significant changes in body weight or coagulation was observed. No changes in the biochemical analysis of blood or General analysis. blood-related drug toxicity were observed. A single treatment with IL-21 mAb 362.78-CHO at 5 or 100 mg/kg was well tolerated by all animals.

[256] an Autopsy was performed in animals at the high dose (100 mg/kg). No significant anatomical changes were observed. Histopathological analysis of animals at the high dose showed minimal lymphoid hyperplasia in lymphoid tissue. Immunohistochemical analysis of lymphoid tissues showed a moderate increase of the size of the follicle and cell types associated with follicles. These changes could be related to the pharmacological activity of IL-21 mAb 362.78-CHO, since it is known that IL-21 directly affect the development and release of b-cells of the lymphoid follicles and keep switching classes, affinity maturation and the development of plasma cells.

[257] the Pharmacokinetic behavior and bioavailability of IL-21 mAb362.78-CHO monitorrole in the study at a single dose in cynomolgus monkeys. Eight male cynomolgus macaques were treated with IL-21 mAb 362.78-CHO. Three were treated with subcutaneous injection of 5 mg/kg and three were treated by intravenous injection of 5 mg/kg Two were treated by intravenous injection of 100 mg/kg serum Samples were taken for analysis of the levels of IL-21 mAb 362.78-CHO for four weeks after injection to groups of 100 mg/kg, and eight weeks after injection for two groups of 5 mg/kg Analysis without compartmentalization pharmacokinetic profiles showed that the effect was increased proportionally to the dose intravenous 5 or 100 mg/kg IL-21 mAb. The bioavailability of IL-21 mAb 362.78-CHO was approximately 50% after subcutaneous injection. Calculated final half for IL-21 mAb was 10-14 days. Calculated final half for IL-21 mAb *insert: 362.78-CHO in cynomolgus macaques was 10-14 days.

21B. Pilot pharmacological research 362.78-CHO in cynomolgus macaques

[258] the Epitope specificity of the IL-21 mAb 362.78-CHO is common for humans, rhesus monkeys and cynomolgus macaques (see examples 17 and 17b)thus, in vivo pharmacology of IL-21 mAb were analyzed in cynomolgus macaques, in suitable forms for pharmacodynamic evaluation.

[259] Cynomolgus macaques were treated with a single injection of IL-21 mAb 362.78-CHO and monitorrole clinical indicators within 4-8 weeks after treatment. Delivery of IL-21 mAb 362.78 - Cho produced subcutaneous, nutrion the th injection in doses of 5 or 100 mg/kg All animals were monitoring changes in the composition of human peripheral blood flow cytometry. No treatment-related changes in the concentration of monocytes and b cells, and no changes in T-cell subpopulations CD4 and CD8, nor in the ratio of CD4 cells to CD8 was observed. The decrease in the concentration of NK-cells was observed after administration of IL-21 mAb 362.78-CHO. In all groups, treatment of NK cells from peripheral blood were increased at 24 h after treatment compared to background values. Five of the eight animals the levels of NK-cells remained at 60% below background values in the last two weeks. Confirming the study by using appropriate controls to manage stress and other sources of instability concentration of NK-cells in peripheral blood will be needed to confirm this observation.

Example 22. Anti-mIL-21 antibody reduces the incidence and progression of collagen-induced arthritis (CIA) in the mouse model

Murine model of collagen-induced arthritis (CIA).

[260] There are several animal models of rheumatoid arthritis, known in this area. For example, in a model of collagen-induced arthritis (CIA) mice develop a chronic inflammatory arthritis, which is very similar to human rheumatoid arthritis. Because the CIA has a total SAS are the major immunological and pathological properties with RA, this makes it an ideal model for screening potential anti-inflammatory compounds for humans. The CIA model is a well-known model in mice, the occurrence of which depends on the immune response, and inflammatory response. The immune response involves the interaction of b cells and CD4+T cells in response to collagen, which is given as antigen, and leads to the formation of anticollagen antibodies. The inflammatory phase is the result of tissue responses to inflammatory mediators, as a result, some of these antibodies to cross-react with murine native collagen and activate the path of complement. The advantage of using the CIA model is that the basic mechanisms of pathogenesis are known. Identified relevant T-cell and b-cell epitopes on collagen II and identified various immunological (e.g., delayed-type hypersensitivity and anticollagen antibody) and inflammatory (e.g., cytokines, chemokines, and matrix-degrading enzymes) parameters related to immunopositive arthritis, and, thus, can be used to assess the effectiveness of the tested compounds in the CIA model (Wooley, Curr. Qpin. Rheum. 3:407-20, 1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life Sci. 61: 1861-78, 1997; Wang et al., Immunol. 92:8955-959, 1995). Sweat is dzielna efficiency rat antimisting mAb to IL-21, produced ZymoGenetics, was analyzed in the CIA model, as described below.

[261] Male mice DBA/1J (Jackson Labs) ten weeks of age were used for therapeutic treatment of a drug (i.e. because the mice developed established disease). On day 21, all animals were given an intradermal injection in the tail 100 microlitres 1 mg/ml collagen type II chicken in complete Freund's adjuvant (prepared Chondrex, Redmond, WA), and three weeks later, on day 0, they were given the same injection except that prepared in incomplete Freund's adjuvant. anti-IL-21 antibody or vehicle (PBS) was administered as intraperitoneal injections every other day, for a total of 6 doses, as soon as the mice developed an established disease. Mice (n=7 per treatment) received or 0.15 mg anti-IL-21 antibodies to animal per dose, or media control, PBS (Life Technologies, Rockville, MD). The animals began to show symptoms of arthritis after the second injection of collagen, along with most animals that develop inflammation in 1-2 weeks. The spread of the disease was assessed in each foot with the help of caliper for measuring the thickness of the legs and assigning a clinical score (0-3) for each foot (see below).

[262] the Monitoring of the disease.

[263] Animals may begin to show signs of inflammation of the paws soon after the second injection of collagen, and some of the animals which can even begin to show signs of inflammation of the fingers before the second injection of collagen. Most animals develop arthritis within 1-2 weeks booster injections, but some may require a longer period of time. The frequency of the disease in this model is usually 90-100%, and typically 0-5 do not respond to treatment (defined after 6 weeks of observation), seen in the experiment using 60 animals. Because this study only induced mice with established disease, the mice that did not develop arthritis, are not used. It is noteworthy that, once the inflammation begins, it may be common transient appearance of various mild inflammation of feet or toes. For this reason, it is not considered that the animal has an established disease before it develops expressed, sustainable swollen feet.

[264] All animals were monitored daily to assess the status of the disease in their legs, which was done by assigning a clinical quality indicator for each of the legs. Every day, each animal was evaluated on a scale of clinical symptoms 4 feet in accordance with their state of clinical disease. To determine the clinical indicator presser foot can be considered as having 3 zones, the fingers, the actual foot bone or paw), and the wrist or ankle joint. The distribution and severity of the inflammation related to these areas, take into account, including: observation of each finger against swelling; her claws or redness of the fingers; taking into account any signs of swelling or redness in any of the tabs; taking into account any loss of clear anatomical demarcation tendons or bones; evaluation of the wrist or ankle, with respect to any swelling or redness; and considering whether the inflammation proximally up the leg. The scale of legs 1, 2 or 3, first, based on the General impression of severity, and secondly, on how many zones were involved. The scale used for the evaluation of the clinical indicators, shown below.

[265] Clinical indicator:

[266] 0 = norm

[267] 0,5 = Involved one or more fingers, but the fingers are inflamed

[268] 1 = secondary inflammation involving the foot (1 zone), and may include a finger or fingers

[269] 2 = moderate inflammation in the foot, and may include some of the fingers and/or wrist/ankle (2 zones)

[270] 3 = severe inflammation in the foot, wrist/ankle and some or all of the fingers (3 zones)

[271] Established disease was defined as a qualitative assessment of the scale of inflammation of the legs, with a ranking of 1 or more, which was maintained for two days in a row. Once established, the disease appeared, the date of the recordings is Ali and was designated as the first day of the animal with established disease."

[272] Mice receiving anti-mIL-21 antibody was characterized by a decrease swelling feet during the entire period of the experiment and had approximately 25% lower, on average, arthritis compared to mice receiving PBS. These results indicate that anti-IL-21 antibody reduced the swelling of the legs and the progression of the disease associated with this model of arthritis and suggest that anti-IL-21 antibody may be effective in the treatment of arthritis in humans.

Example 23. The expression of IL-21 R in samples of psoriatic human skin

[273] the Expression of IL-21 R is normally restricted to cells of hematopoietic origin. However, when establishing inflammatory diseases of the expression of IL-21 R on nonhematopoietic cells can provide a direct incentive for cell types that mediate functional changes in the affected tissues. In psoriasis, the growth of keratinocytes misaligned, together with epidermal hyperplasia with psoriatic rash, aberrant terminal (Tr, end) differentiation and incomplete development of the stratum corneum. IL-21 produced by infiltrating in psoriatic skin T-cells (Th1 and Th17, can activate functional changes in keratinocytes, including cell proliferation, formation of chemokines and altered differentiation. For this reason, the e investigated IL-21 R keratinocytes in psoriatic lesions of the skin.

[274] Methods. Immunohistochemical analysis of 18 samples of skin taken by the method of biopsies from 4 normal people donors and 9 patients with psoriasis was performed using a murine IgG1 antibody against human IL-21 R, produced in ZymoGenetics. For patients with psoriasis subpopulation (5) provided samples as affected and unaffected skin. A high degree of epidermal hyperplasia was noted in the histopathological examination of the affected skin all donors. Immunoreactivity (staining) IL-21 R on specific types of cells was assessed based on the frequency of positive cells and intensity of staining.

[275] Results. In normal skin and unaffected skin of patients with psoriasis, staining for IL-21 R was positive on a random vnutrepenialnyh monocularly cells (MNC), scattered macrophages and fibroblast-like cells. Positive staining for IL-21 R was present on a large number of MNC in all samples of the affected skin of patients with psoriasis. Samples dyed using control antibodies murine isotype, were negative. Minimal staining or no staining for IL-21 R were present in epidermal keratinocytes of normal skin. In samples of unaffected skin, taken by the method of biopsy, patients were observed weak staining in epidermal keratinocytes in 4 Ave is Bach, and moderate staining was observed in the spinous layer in the fifth test. Staining medium to heavy existed in the focal areas of keratinocytes in 8 samples from 9 patients with psoriasis.

[276]. In psoriatic skin lesions, the expression of IL-21 R is not limited by leukocytes infiltrating, but is also positive (up-regulated) in epidermal keratinocytes. The increased staining for IL-21 R on epidermal keratinocytes in unaffected skin of patients with psoriasis compared with normal controls suggests that even in unaffected skin keratinocytes can respond aberrante on the activation of IL-21. In the presence of inflammation was noted increased levels of IL-21 R infiltrating MNC and in hyperplastic epidermal layer. Thus, the treatment of psoriasis using antibodies that block IL-21 can inhibit inflammation by blocking the signals of IL-21 as in inflammatory cells, and epidermal keratinocytes.

1. The selected antibody against IL-21, containing the variable region of the heavy chain, containing SEQ ID NO: 31, 33 and 35, and a variable region light chain containing SEQ ID NO: 39, 41 and 43.

2. The antibody of claim 1, wherein the heavy chain comprises amino acid residues 20-145 SEQ ID NO: 29, or a sequence having at IU the e 80% identity to that, and in which light chain comprises amino acid residues 21-126 SEQ ID NO: 37, or a sequence having an identity of at least 80%.

3. The selected antibody against IL-21, containing the variable region of the heavy chain, containing SEQ ID NO: 47, 49 and 51, and the variable region of the light chain containing SEQ ID NO: 55, 57 and 59.

4. The antibody according to claim 3, in which the heavy chain comprises amino acid residues 20-145 SEQ ID NO: 45 or a sequence having at least 80% identity to it, and in which light chain comprises amino acid residues 21-126 SEQ ID NO: 53, or a sequence having an identity of at least 80%.

5. The antibody according to claim 1 or 2, where Fc part of the antibody has reduced effector function.

6. The antibody according to claim 3 or 4, where the Fc part of the antibody has reduced effector function.

7. The antibody according to claim 1, where Fc part of the antibodies selected from the group consisting of IgG1, IgG2 and IgG4.

8. The antibody according to claim 1 or 2 for use in the treatment of rheumatoid arthritis, for the treatment of inflammatory bowel disease (IBD), where inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis and irritable bowel syndrome.

9. The antibody according to claim 1 or 2 for use in the treatment of rheumatoid arthritis.

10. The antibody according to claim 1 or 2 for use is in the treatment of multiple sclerosis.

11. The antibody according to claim 1 or 2 for use in the treatment of diabetes mellitus type I (IDDM).

12. The antibody according to claim 1 or 2 for use in the treatment of Sjogren syndrome.

13. The antibody according to claim 1 or 2 for use in treating an autoimmune disease selected from the group consisting of pancreatitis, inflammatory myopathies (polymyositis, dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), glomerulonephritis, IgA nephropathy-type vysokomineralizirovannaja patients with transplantation, antiphospholipid syndrome, autoimmune uveitis and asthma.

14. The antibody according to claim 1 or 2 for use in the treatment of systemic lupus erythematosus (SLE) or psoriasis.

15. The antibody according to claim 1 or 2 for use in the treatment of diseases mediated by follicular T cells-helper, or diseases mediated by b-cells selected from the group consisting of systemic lupus erythematosus, autoimmune hearing loss, graves disease, ordinary water, myasthenia gravis, narom is elite optic nerve, syndrome?, autoimmune nephritis, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinative polyneuropathy (CIDP), autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (ITP).

16. The antibody according to claim 3 or 4, where the Fc part of the antibodies selected from the group consisting of IgG1, IgG2 and IgG4.

17. The antibody according to claim 3 or 4 for use in the treatment of rheumatoid arthritis, for the treatment of inflammatory bowel disease (IBD), where inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis and irritable bowel syndrome.

18. The antibody according to claim 3 or 4 for use in the treatment of rheumatoid arthritis.

19. The antibody according to claim 3 or 4 for use in the treatment of multiple sclerosis.

20. The antibody according to claim 3 or 4 for use in the treatment of diabetes mellitus type I (IDDM).

21. The antibody according to claim 3 or 4 for use in the treatment of Sjogren syndrome.

22. The antibody according to claim 3 or 4 for use in the treatment of autoimmune diseases selected from the group consisting of pancreatitis, inflammatory myopathies (polymyositis, dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, ankylosing is its spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), glomerulonephritis, IgA nephropathy-type vysokomineralizirovannaja patients with transplantation, antiphospholipid syndrome, autoimmune uveitis and asthma.

23. The antibody according to claim 3 or 4 for use in the treatment of systemic lupus erythematosus (SLE) or psoriasis.

24. The antibody according to claim 3 or 4 for use in the treatment of diseases mediated by follicular T cells-helper, or diseases mediated by b-cells selected from the group consisting of systemic lupus erythematosus, autoimmune hearing loss, graves disease, ordinary water, myasthenia gravis, neuromyelitis optic nerve syndrome?, autoimmune nephritis, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinative polyneuropathy (CIDP), autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (ITP).

25. Hybridoma, which produces the antibody containing the variable region of the heavy chain, containing SEQ ID NO: 31, 33 and 35, and a variable region light chain containing SEQ ID NO: 39, 41 and 43, and which is deposited in the collection of cultures American Type Culture Collection having the room ATSC Patent Deposit Desgnation MOUTH-8790".

26. Anti-IL-21 antibody produced by hybridomas on A.25.

27. Hybridoma, which produces the antibody containing the variable region of the heavy chain, containing SEQ ID NO: 47, 49 and 51, and the variable region of the light chain containing SEQ ID NO: 55, 57 and 59, and which is deposited in the collection of cultures American Type Culture Collection having the room ATSC Patent Deposit Designation MOUTH-8786".

28. Anti-IL-21 antibody produced by hybridomas on item 27.

29. The antibody according p, where Fc part of the antibody has reduced effector function.

30. The antibody according p, where Fc part of the antibodies selected from the group consisting of IgG1, IgG2 and IgG4.

31. The antibody according p for use in the treatment of inflammatory bowel disease (IBD), where inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis and irritable bowel syndrome.

32. The antibody according p for use in the treatment of rheumatoid arthritis.

33. The antibody according p for use in the treatment of multiple sclerosis.

34. The antibody according p for use in the treatment of diabetes mellitus type I (IDDM).

35. The antibody according p for use in the treatment of Sjogren syndrome.

36. The antibody according p for use in the treatment of autoimmune diseases selected from the group consisting of pancreatitis, inflammatory myopathies (polemize is a, of dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), glomerulonephritis, IgA nephropathy-type vysokomineralizirovannaja patients with transplantation, antiphospholipid syndrome, autoimmune uveitis and asthma.

37. The antibody according p for use in the treatment of systemic lupus erythematosus (SLE) or psoriasis.

38. The antibody according p for use in the treatment of diseases mediated by follicular T cells-helper, or diseases mediated by b-cells selected from the group consisting of systemic lupus erythematosus, autoimmune hearing loss, graves disease, ordinary water, myasthenia gravis, neuromyelitis optic nerve syndrome?, autoimmune nephritis, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinative polyneuropathy (CIDP), autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (ITP).

39. The antibody according p, where Fc part of the antibody has reduced effector the th function.

40. The antibody according p, where Fc part of the antibodies selected from the group consisting of IgG1, IgG2 and IgG4.

41. The antibody according p for use in the treatment of rheumatoid arthritis, for the treatment of inflammatory bowel disease (IBD), where inflammatory bowel disease selected from the group consisting of Crohn's disease, ulcerative colitis and irritable bowel syndrome.

42. The antibody according p for use in the treatment of rheumatoid arthritis.

43. The antibody according p for use in the treatment of multiple sclerosis.

44. The antibody according p for use in the treatment of diabetes mellitus type I (IDDM).

45. The antibody according p for use in the treatment of Sjogren syndrome.

46. The antibody according p for use in the treatment of autoimmune diseases selected from the group consisting of pancreatitis, inflammatory myopathies (polymyositis, dermatomyositis), microscopic polyangiitis, autoimmune aplastic anemia, autoimmune thyroiditis, autoimmune hepatitis, syndrome Wegener, diverticulosis, ankylosing spondylitis, scleroderma, systemic sclerosis, psoriatic arthritis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host (GVHD), cutaneous T-cell lymphoma (CTCL), glomerulonephritis, IgA nephropathy-type vysokomineralizirovannaja PAC the patients with transplantation, antiphospholipid syndrome, autoimmune uveitis and asthma.

47. The antibody according p for use in the treatment of systemic lupus erythematosus (SLE) or psoriasis.

48. The antibody according p for use in the treatment of diseases mediated by follicular T cells-helper, or diseases mediated by b-cells selected from the group consisting of systemic lupus erythematosus, autoimmune hearing loss, graves disease, ordinary water, myasthenia gravis, neuromyelitis optic nerve syndrome?, autoimmune nephritis, cryoglobulinemia, Guillain - Barre syndrome, chronic inflammatory demyelinative polyneuropathy (CIDP), autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura (ITP).



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to a molecule of nucleic acid, which is a cyclic or a linear vector fit for expression, of at least one target polypeptide in cells of mammals, including (a) at least one expressing cassette (POI) for expression of the target polypeptide; (b) an expressing cassette (MSM), including a gene of a selective marker of mammals; (c) an expressing cassette (MASM), including an amplificated gene of a selective marker of mammals; besides, the expressing cassette (POI) is flanked in direction 5' by the expression cassette (MASM), the expression cassette (MSM) is localised in direction 3' from the expression cassette (POI) and in which the expression cassettes (MASM), (POI) and (MSM) are arranged in the same orientation from 5' to 3'. Also the method is disclosed to produce the specified molecule of nucleic acid of the vector, as well as a cell of a host mammal, containing the specified molecule of nucleic acid of the vector, the method to produce a host cell containing the specified molecule of nucleic acid of the vector, and also the method to produce the target polypeptide, using the specified host cell.

EFFECT: invention makes it possible to efficiently produce a target polypeptide in mammal cells.

24 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented: a method for tumour cell growth inhibition in an individual and a method for immune response enhancement in an individual involving the introduction of a PD-1 monoclonal antibody and a CTLA-4 10D1 monoclonal antibody to the individual. The PD-1 monoclonal antibody has the following properties: it binds to human PD-1 having the value KD equal to 1×10-8 M or less; however it binds neither to human CD28, nor to CTLA-4, nor to ICOS; it is able to enhance the T-cell proliferation in the mixed lymphocyte reaction (MLR) analysis; it is able to enhance the gamma interferon production in the MLR analysis; it is able to enhance the interleukine-2 (IL-2) secretion in the MLR analysis.

EFFECT: invention provides a synergic effect when using the above antibodies in a combination.

4 cl, 54 dwg, 7 tbl, 25 ex

Novel antibodies // 2490277

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed is an anti-α5β1 antibody, which is described through amino acid sequences of six hypervariable regions and an antigen-binding moiety thereof. Described are conjugates of the disclosed antibodies with a medicinal agent or a label, a pharmaceutical composition, use of the disclosed antibodies to prepare a medicinal agent, methods and an industrial product for inhibiting angiogenesis and/or vascular permeability in a subject, and for treating cancer, an ophthalmic disease and an autoimmune disease in a subject. The invention describes an isolated nucleic acid, an expression vector, a cell and a method of producing an antibody or an antigen-binding moiety thereof, as well as a method of detecting α5β1 protein in a sample.

EFFECT: present invention can find further use in therapy and diagnosis of α5β1-mediated diseases.

52 cl, 11 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: disclosed are versions of human IL13 specific antibodies and a producing hybridoma cell line deposited in ATCC under number PTA-5657. Described are: versions of encoding polynucleotides; an antibody expression vector; a host cell for antibody expression, as well as versions of a method of producing an antibody using a vector, polynucleotide, hybridoma or host cell. The invention discloses a pharmaceutical composition for treating IL13-mediated diseases and methods of treating allergic, inflammatory and other diseases, which employ an anti-IL13 antibody.

EFFECT: providing antibodies which do not bind with target IL13 and neutralise activity of human IL13.

39 cl, 29 dwg, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 2B8 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-20.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to obtain monoclonal antibodies against the Yersinia pestis V antigen. The strain of hybrid animal cells Mus musculus 5G6 is obtained by immunising BALB/c mice. The mice are immunised by four-time administration of a recombinant V antigen in a dose of 100 mcg/mouse. On the third day after the last immunication, splenocytes of immune mice (1×108 cells) are hybridised with mouse myeloma cells RZ-X63 Ag/8-653 (1×107 cells). The fusion agent used is polyethylene glycol (Sigma, USA). Hybridisation is followed by selection, screening, cloning and cryopreservation of the hybridoma. The strain is deposited in the state collection of pathogenic microorganisms and cell cultures (GKPM-Obolensk) under number N-19.

EFFECT: strain of hybrid cultured cells, which produces monoclonal antibodies which are specific to the Y pestis V antigen, is suitable for constructing test systems for detecting plague pathogens.

8 dwg, 2 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention presents a method for characterising a light chain type in a composition of recombinant polyclonal antibodies which may be used for assessment of various antibodies produced by a polyclonal cell line in the process of production, as well as batch consistency of the antibodies found in the polyclonal products. A method for structural characterisation is based on heavy chain removal and residual light chain separation by chromatographic separation and mass spectrometry of intact light chain types. What is also disclosed is a method for detecting the presence of versions in intact light chain populations in two or more compositions of the recombinant polyclonal antibodies prepared of one polyclonal cell culture in different moments of time in the process of cultivation, or of various polyclonal cell cultures in a specific moment of time.

EFFECT: use of the invention enables making the specific drug batch complying with the pre-set production specification.

19 cl, 8 dwg, 2 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: this invention relates to biotechnology and immunology. One proposes: JAM-A protein antibody or functional fragment thereof, hybridoma secreting such antibody, nucleic acid, expression vector and host cell as well as a method for the antibody and composition production. One considers application of the JAM-A protein antibody or functional fragment thereof.

EFFECT: invention usage ensures creation of new JAM-A protein antibodies which may be further applied in treatment or prevention of diseases related to proliferation of tumour cells extracting JAM-A protein.

34 cl, 31 dwg, 5 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention presents cultivated hybrid cell strains of Mus. musculus animals Sp2/0-BC/rhPC-4F10, Sp2/0-BC/rhPC-1C6, Sp2/0-BC/rhPC-3H6, producers of monoclonal antibodies specific to human protein C. The strains are deposited in the Russian National Collection of Industrial Microorganisms of Federal Unitary Enterprise State Research Institute 'Genetics', No. (VKPM H-111), (VKPM H-112), respectively. The antibodies belong to hPROC-specific murine immunoglobulin G possessing cross-responsiveness, are selectively bound with human protein C and form a stable complex.

EFFECT: antibodies under the invention may be used for purposes of pharmaceutical and biomedical analytical studies, particularly for quantitative detection of the human recombinant factor C.

1 dwg, 4 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, namely to infectious diseases, and can be used for treating whooping cough and/or for Bordetella infection protection. Polypeptide under the invention represents a fragment of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain from an amino acid sequence extended from position 1166 to position 1281 SEQ ID NO:1. Said invention also concerns the specific fragments of adenylate cyclase Bordetella containing CD11b/CD18 interaction domain, and to their application, particularly for targeting of molecules of interest to CD11b expressing cells.

EFFECT: use of the inventions allows extending the range of products for treatment and prevention of the Bordetella infection.

26 cl, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: in invention described is hybridoma, producing monoclonal antibodies, which bind with protein 161P2F10B in a specific way. Invention describes versions of monoclonal antibodies, one of which includes amino acid sequence of heavy and light chain of antibody, produced by said hybridoma, and the other includes amono acid sequence of variable part of heavy and light chain of antibody, produced by said hybridoma, or amino acid sequence Fab, F(ab')2, Fv or Sfv of antibody fragment, which binds with protein 161P2F10B, 161P2F10B demonstrates tissue-specific expression in healthy, mature tissue and is abnormally expressed in case of oncologic diseases.

EFFECT: antibodies can be used as anti-cancer medication and in composition of pharmaceutical composition for treating cancer, in particular, kidney cancer, reducing tumour growth, as well as for detection of protein 161P2F10B in biological sample.

11 cl, 212 dwg, 8 tbl, 20 ex

FIELD: medicine.

SUBSTANCE: essence of invention includes contact of liquid sample, taken from mammal organism, with one or several monoclonal antibodies to Lawsonia intracellularis antigen, secreted by cell lines of ECACC hybridomes, which have registration numbers. Invention also includes diagnostic test-kit, containing antibodies specific for Lawsonia intracellularis.

EFFECT: increased specificity of antigen detection.

11 cl, 5 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of genetic engineering and cloning. Transgenic cow is disclosed, having low activity of prion protein as a result of one or several genetically designed mutations. Specified transgenic cows are also genetically modified for expression of xenoantibodies.

EFFECT: as a result of their resistance to prion diseases, such as trembling disease of cattle, such cows are a safe source of human antibodies for use in pharmaceutics and a safe source of agricultural products.

12 cl, 112 dwg, 13 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.

EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.

16 cl, 34 dwg, 7 tbl, 20 ex

FIELD: chemistry.

SUBSTANCE: proposed is a chimeric or humanised monoclonal antibody against hepatocyte growth factor, produced from L2G7 antibody. Invented is a mouse antibody L2G7, produced by hybridoma ATCC PTA-5162, and the said hydbridoma. Described is a cell line, producing a chimeric or humanised monoclonal antibody against hepatocyte growth factor. Proposed is a pharmaceutical composition and a method of treating tumours based on the said antibody.

EFFECT: use of the invention provides for a neutralising antibody against hepatocyte growth factor, which can be used in treating human cancer.

7 cl, 12 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns the therapeutic agent for treatment of diseases related to children's chronic arthritic diseases, for example, actually children's chronic arthritic diseases, Still's disease and similar to it, including an antagonist of the interleukin-6 IL-6 receptor as an active ingredient, humanised antibody PM-1. Advantage of the invention consists in efficiency increase.

EFFECT: efficiency increase.

5 cl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

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