CN101052419A - Antagonizing interleukin-21 receptor activity - Google Patents

Abstract

Methods and compositions for inhibiting interleukin-21 (IL-21)/IL-21 receptor (MU-1) activity using antagonists of IL-21 or IL-21 receptor (''IL-21R'' or ''MU-1''), are disclosed. IL-21/IL-21R antagonists can be used to induce immune suppression in vivo,e.g., for treating, ameliorating or preventing autoimmune or inflammatory disorders, including, e.g., inflammatory bowel disease (IBD), rheumatoid arthritis (RA), transplant/graft rejection, psoriasis, asthma, fibrosis, and systemic lupus erythematosus (SLE).

Description

Antagonizing interleukin-21 receptor activity

[0001] the application requires in the U.S. Provisional Patent Application serial number 60/599 of application on August 5th, 2004, the U.S. Provisional Patent Application serial number 60/639 of 086 and 2004 on December application in 23,, 176 rights and interests, whole disclosures of these two parts of provisional application all are attached to herein by reference.

Background of invention

Invention field

[0002] the present invention relates to the IL-21 receptor antagonist is used for antagonism, reduction and/or suppresses interleukin-21 (IL-21)/active method and composition of IL-21 receptor (MU-1).Method and composition disclosed herein can be used as the immunization therapy medicine.

Relevant background technology

[0003] human IL-2 1 is a kind of cytokine, its sequence and IL-2, IL-4 and IL-15 homology (Parrish-Novak etc. (2000) Nature 408:57-63).Although sequence homology is low between the interleukin cytokine, cytokine all is folded into " four-helix bundle (four-helix-bundle) " structure representative in this family.Most cytokines can with I class or II type cytokines receptors bind.II type cytokines receptor comprises IL-10 receptor and interferon receptors, and I type cytokines receptor comprises the receptor of IL-2 to IL-7, IL-9, IL-11, IL-12, IL-13 and IL-15, and hemopoietic growth factor receptor, leptin receptor and growth hormone receptor (Cosman (1993) Cytokine 5:95-106).

[0004] human IL-2's 1 receptor (IL-21R) is the expressed I type cytokines receptor of lymphoid tissue, especially NK, B and T cell (Parrish-Novak etc. (2000), ibid).The nucleotide sequence and the aminoacid sequence of coding human interleukin-2 1 (IL-21) and receptor (IL-21R) thereof are described in WO 00/53761; WO 01/85792; Parrish-Novak etc. (2000), ibid; With (2000) Proc.Natl.Acad.Sci.U.S.A.97:11439-44 such as Ozaki.IL-21R and IL-2 receptor β chain and IL-4 receptor alpha chain have highest homology sequence (Ozaki etc. (2000), ibid).In case the part combination, IL-21R and common gamma cells factor acceptor chain (γ c) associate, the receptor of IL-2, IL-3, IL-4, IL-7, IL-9, IL-13 and IL-15 all can share this receptor chain (Ozaki etc. (2000), ibid; Asao etc. (2001) J Immunol.167:1-5).IL-21R extensively distributes in lymph, and prompting IL-21 may have effect in immunomodulating.In fact, in vitro study knows that IL-21 obviously can regulate B cell, CD4 ⁺And CD8 ⁺The function of T cell and NK cell (Parrish-Novak etc. (2000), ibid; Kasaian etc. (2002) Immunity.16:559-69).Yet, support that the evidence of regulating action in the IL-21 body is but very limited.

Summary of the invention

[0005] herein disclosed is the active and/or interactional method and composition of interference interleukin-21 (IL-21) and IL-21 receptor (also claiming " IL-21R " or " MU-1 " in this article), for example use IL-21 antagonist or IL-21R antagonist (also claiming " IL-21/IL-21R antagonist " or " antagonist " or " IL-21/IL-21R pathway antagonists " in this article).

[0006] for example, the applicant knows, by using IL-21 antagonist (fusion rotein that for example comprises the IL-21R extracellular domain that merges with the Fc immunoglobulin domain), reduce the IL-21R activity, in some animal models, improved inflammatory symptoms, described model can rational prediction inflammatory diseases and/or autoimmune disease, for example inflammatory bowel (EBD), rheumatoid arthritis (RA), transplanting/transplant rejection, graft versus host disease, asthma, systemic lupus erythematosus (sle) (SLE) (comprising the glomerulonephritis form) and psoriasis (embodiment 7-14).In collagen-induced arthritis (CIA) mice claw, (embodiment 8) are raised in the expression of IL-21R mRNA.In addition, in asthmatic model, the IL-21R deficient mice shows sx (embodiment 12).Therefore, the activity of IL-21/IL-21R antagonist can be used in vivo induction of immunity and suppresses, and for example is used for the treatment of or prevents inflammatory diseases or autoimmune disease.These antagonisies also can be used for treatment or epidemic prevention cell related diseases, for example with one or more mature T cells (for example ripe CD8 ⁺Or ripe CD4 ⁺The T cell), ripe NK cell, B cell, disease that macrophage is relevant with the megalokaryocyte abnormal activity.

[0007] therefore, on the one hand, the invention is characterized in the method for treatment (for example cure, suppress, postpone), the inflammatory diseases of improving (for example alleviate, alleviate, reduce, reduce) patient or autoimmune disease and/or prevention (for example prevent its outbreak or prevent its recurrence or deterioration).Described method comprises: give patient IL-21/IL-21R antagonist, for example its consumption is enough to treatment, improves or prevents this disease, and perhaps its consumption is enough to suppress or reduce immunologic cellular activity and/or cell number.

[0008] the IL-21/IL-21R antagonist can give the patient separately, perhaps with the coupling of IL-21/IL-21R antagonist, perhaps with other therapeutic form coupling as herein described.Preferred patient suffers from inflammatory diseases or autoimmune disease or is in mammal among its danger, for example the people.For example, described method can be used for treating or preventing patient's inflammatory diseases or autoimmune disease.The example of such disease includes but not limited to: transplant rejection; Diabetes (for example I type); Multiple sclerosis; Arthritis (for example rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis, psoriasis arthropathica or ankylosing spondylitis (preferred RA)); Myasthenia gravis; Vasculitis; Systemic lupus erythematosus (sle) (SLE); Glomerulonephritis; Autoimmune thyroiditis; Inflammatory disease of the skin (for example dermatitis (comprising atopic dermatitis and eczematoid dermatitis), scleroderma or psoriasis); Lupus erythematosus; Fibrosis or fibre modification disease (for example pulmonary fibrosis or hepatic fibrosis); Respiratory disease (for example asthma or COPD); Atopic diseases (for example comprising allergy); Or inflammatory bowel disease (IBD for example, for example Crohn disease (Crohn ' s disease) or ulcerative colitis).

[0009] preferably uses IL-21 of the present invention or the following disease of IL-21R antagonist for treating: lupus erythematosus, inflammatory disease of the skin (for example psoriasis), inflammatory bowel disease (for example IBD, Crohn disease, ulcerative colitis), transplant rejection, asthma, atopic diseases or rheumatoid arthritis.

[0010] in one embodiment, the IL-21/IL-21R antagonist influences IL-21 or IL-21R (for example combining with it), the IL-21 of preferred mammal (for example people) or IL-21R (being called " IL-21 antagonist " and " IL-21R antagonist " in this article), and reduce or suppress one or more IL-21 and/or IL-21R activity.In conjunction with IL-21 or IL-21R, for example affinity costant is at least about 10 with high-affinity for preferred antagonist ⁷M ^-1, preferred about 10 ⁸M ^-1, more preferably from about 10 ⁹M ^-1To 10 ¹⁰M ^-1Or it is stronger.

[0011] for example, during the IL-21/TL-21R antagonist can pass through and IL-21 reduce and/or suppress the IL-21R activity.In one embodiment, antagonist can be a fusion rotein, and it comprises IL-21R fragment and non-IL-21R fragment (for example immunoglobulin fc region).In other embodiments, antagonist is IL-21 receptor, peptide or the micromolecular inhibitor of anti-IL-21R antibody or anti-IL-21 antibody or its Fab, soluble form.

[0012] in one embodiment, the IL-21/IL-21R antagonist is anti-IL-21R antibody or anti-IL-21 antibody or its Fab; For example antibody is monoclonal antibody or monospecific antibody, and described antibody capable is in conjunction with IL-21 (for example the human IL-2 1) or IL-21 receptor (human IL-2's 1 receptor polypeptides or its Fab (for example Fab, F (ab ') for example ₂, Fv or strand Fv fragment)).Preferred antibody is the antibody at people's antibody, humanized antibody, chimeric antibody or the external generation of human IL-2 1 or human IL-2's 1 receptor polypeptides.Preferred antibody is neutralizing antibody.

[0013] in other embodiments, the IL-21/IL-21R antagonist comprises total length IL-21 polypeptide or its fragment, the inhibition IL-21 receptor binding domains of IL-21 polypeptide for example, for example human IL-2's 1 polypeptide (human IL-2 as herein described 1 polypeptide that for example has the aminoacid sequence that is shown in SEQ ID NO:19) or have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them; Perhaps by the corresponding nucleotide sequence that is shown in SEQ ID NO:18 or with they have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity coded.Perhaps, antagonist comprises total length (for example about amino acid/11-538 or the 20-538 of SEQ ID NO:2; Perhaps about amino acid/11-529 or the 20-529 of SEQ ID NO:10), perhaps IL-21 receptor polypeptides fragment, for example the IL-21 of IL-21 receptor polypeptides is in conjunction with the territory, for example the IL-21R soluble fragments (IL-21R fragment for example, it comprises Mus or human IL-2 1R extracellular domain; For example about amino acid/11-235,1-236,20-235, the 20-236 of SEQ ID NO:2 (people), perhaps about amino acid/11-236, the 20-236 of SEQ ID NO:10 (Mus), perhaps by the corresponding nucleotide of SEQ ID NO:1 or SEQ ID NO:9 or with they have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity coded.

[0014] in one embodiment, antagonist is a fusion rotein, it comprises aforementioned IL-21 or IL-21 receptor polypeptides or its fragment, and for example with the second portion of its fusion, for example polypeptide (for example immunoglobulin chain, GST, Lex-A or MBP peptide sequence).In a preferred embodiment, fusion rotein comprises at least can be in conjunction with the IL-21R polypeptide fragment of IL-21, the soluble fragments of IL-21R (IL-21R fragment for example for example, it comprises Mus or human IL-2 1R extracellular domain, about amino acid/11-235 of SEQ ID NO:2 (people) for example, 1-236,20-235,20-236, perhaps about amino acid/11-236 of SEQ ID NO:10 (Mus), 20-236, or have at least 85% by the corresponding nucleotide of SEQ ID NO:1 or SEQ ID NO:9 or with them, 90%, 95%, 98% or the sequence of higher homogeneity coded), and for example with the second portion of its fusion, polypeptide (the immunoglobulin chain of different isotypes for example for example, the Fc fragment, CH comprises: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE).For example, fusion rotein can comprise human IL-2 1R extracellular domain, for example about amino acid/11-235,1-236,20-235, the 20-236 of SEQ ID NO:2, and with the human normal immunoglobulin Fc chain of its fusion (human IgG for example, human IgG1 for example, the human IgG1 of for example naturally occurring human IgG1 or mutant form).In one embodiment, people Fc sequence has sudden change on one or more aminoacid, and for example sudden change on the residue 254 and 257 of naturally occurring SEQ ID NO:28 sequence is to reduce the Fc receptors bind.In other embodiments, fusion rotein can comprise Mus IL-21R extracellular domain (for example about amino acid/11-236, the 20-235 of SEQ ID NO:10 (Mus)), and for example with the rat immune globulin Fc chain (for example Mus IgG, for example Mus IgG2a or Mus IgG2a mutant form) of its fusion.

[0015] fusion rotein also can comprise the joint sequence that first's (for example IL-21R fragment) is connected with second portion (for example immunoglobulin fragment).In other embodiments, other aminoacid sequence can add the N end or the C end of fusion rotein to, so that expression, spatial flexible, detection and/or isolated or purified.

[0016] example that can be used for the antagonism fusion rotein of the inventive method is seen Fig. 7-15.In one embodiment, fusion rotein comprises and is selected from for example following aminoacid sequence: SEQID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39 perhaps have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.In other embodiments, fusion rotein comprises and is selected from for example following coded aminoacid sequence of nucleotide sequence: SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38 perhaps have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.Preferred fusion protein has the aminoacid sequence (seeing Fig. 8 A-8C and Figure 10 A-10C respectively) that is shown in SEQ ID NO:25 or SEQ ID NO:29, perhaps has at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.In other embodiments, fusion rotein comprises and is selected from for example following coded aminoacid sequence of nucleotide sequence: SEQ ID NO:24 or SEQ ID NO:28 (seeing Fig. 8 A-8C and Figure 10 A-10C respectively), or have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.Most preferred fusion rotein has the aminoacid sequence that is shown in SEQ ID NO:29 or has the coded aminoacid sequence (Figure 10 A-10C) of nucleotide sequence that is shown in SEQ ID NO:28.

[0017] IL-21/IL-21R antagonist as herein described, fusion rotein for example as herein described can be derived or (for example another kind of peptide or albumen (for example Fab ' fragment) are connected with another functional molecular.For example, fusion rotein or antibody or antigen-binding portion thereof can functionally connect (for example by chemical coupling, gene fusion, non-covalent combination or alternate manner) one or more other molecular entities, for example antibody (for example bi-specific antibody or multi-specificity antibody), toxin, radiosiotope, cytotoxic agent or cytostatic agent etc.

[0018] in one embodiment, IL-21/IL-21R antagonist as herein described (for example its pharmaceutical composition) in conjoint therapy with other medicines for example curative unite and give, described curative can be used for treating and for example is selected from following one or more inflammatory diseasess or autoimmune disease: arthritis (comprising RA, juvenile rheumatoid arthritis, osteoarthritis, psoriasis arthropathica or ankylosing spondylitis); SLE; Glomerulonephritis; Inflammatory disease of the skin (for example psoriasis); Respiratory disease (for example asthma, COPD); Atopic diseases; Fibre modification disease (for example pulmonary fibrosis or hepatic fibrosis); Inflammatory bowel disease (for example IBD, for example Crohn disease or ulcerative colitis); Or transplant rejection.For example, conjoint therapy can comprise one or more IL-21/IL-21R antagonisies (for example anti-IL-21 antibody or anti-IL-21R antibody or its Fab; The IL-21R fusion rotein; Solubility IL-21R receptor; Inhibitor peptides or micromolecular inhibitor) prepare and/or give described other curative one or more cytokines for example as herein described and growth factor receptor inhibitors, immunosuppressant, anti-inflammatory agent, metabolic poison, enzyme inhibitor and/or cytotoxic agent or cytostatic agent together with one or more other curatives.

[0019] can give and/or the example of preferred other curative of preparation together with one or more IL-21/IL-21R antagonisies, include but not limited to one or more following medicines: the TNF antagonist (for example antibody of chimeric antibody, humanized antibody, people's antibody or external generation or its Fab, they can be in conjunction with TNF; The soluble fragments of TNF receptor, for example p55 or p75 people TNF receptor or derivatives thereof, for example 75kdTNFR-IgG (75kDa TNF receptor-IgG fusion rotein, ENBREL ^TM), p55kDa TNF receptor-IgG fusion rotein; TNF enzyme antagonist, for example TNF α invertase (TACE) inhibitor); The antagonist of IL-6, IL-12, IL-15, IL-17, IL-18, IL-22; T cell and B cell consumption agent (depleting agent) (for example anti-CD 4 antibodies or anti-CD22 antibody); Micromolecular inhibitor, for example methotrexate and leflunomide; Sirolimus (rapamycin) and analog, for example CCI-779; The inhibitor of Cox-2 and cPLA2; NSAID; The inhibitor of p38 inhibitor, TPL-2, Mk-2 and NF κ b; RAGE or solubility RAGE; P-selects the inhibitor (for example micromolecular inhibitor, its antibody, for example anti-P-selects proteic antibody) of albumen or PSGL-1; Erss (ERB) agonist or ERB-NF κ b antagonist.Can with one or more IL-21/IL-21R antagonisies give and/or together the preparation most preferred other curative comprise one or more following medicines: TNF receptor soluble fragments, for example p55 or p75 people TNF receptor or derivatives thereof, 75kdTNFR-IgG (75kDa TNF receptor-IgG fusion rotein, ENBREL for example ^TM); Methotrexate, leflunomide or sirolimus (rapamycin) or its analog, for example CCI-779.

[0020] on the other hand, provide the method that for example is used to reduce following one or more activity of immune cells: mature T cells (ripe CD8 ⁺, CD4 ⁺, lymph node T cell, memory T cell), ripe NK cell, B cell, antigen-presenting cell (APC) (for example dendritic cell, macrophage or megalokaryocyte) or cell colony, for example mix or the immune cell population of basic purification.This method comprises the IL-21/IL-21R antagonist that makes immunocyte contact q.s, and antagonist for example as herein described is to reduce immunologic cellular activity.

[0021] on the other hand, the invention is characterized in fusion rotein, it comprises at least can be in conjunction with the IL-21R polypeptide fragment of IL-21 polypeptide, for example IL-21R soluble fragments (the IL-21R fragment that for example comprises Mus or human IL-2 1R extracellular domain; About amino acid/11-235 of SEQ ID NO:2 (people) for example, 1-236,20-235,20-236, perhaps about amino acid/11-236 of SEQ ID NO:10 (Mus), 20-236, perhaps have at least 85% by the corresponding nucleotide of SEQ ID NO:1 or SEQ ID NO:9 or with them, 90%, 95%, 98% or the sequence of higher homogeneity coded), and for example with the second portion of its fusion, polypeptide (the immunoglobulin chain of different isotypes for example for example, the Fc fragment, CH comprises: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE).For example, fusion rotein can comprise human IL-2 1R extracellular domain (for example about amino acid/11-235,1-236,20-235, the 20-236 of SEQ ID NO:2), and for example with the human normal immunoglobulin Fc chain (for example human IgG, for example human IgG1 or human IgG1's mutant form) of its fusion.In one embodiment, people Fc sequence has sudden change on one or more aminoacid, for example has sudden change on the residue 254 and 257 of wild-type sequence SEQ IDNO:28, to reduce the Fc receptors bind.In other embodiments, fusion rotein can comprise Mus IL-21R extracellular domain (for example about amino acid/11-236, the 20-236 of SEQ ID NO:10 (Mus)) and for example with the rat immune globulin Fc chain (for example Mus IgG, for example mutant form of Mus IgG2a or Mus IgG2a) of its fusion.Fusion rotein also can comprise the joint sequence that IL-21R fragment and second portion are linked together.In other embodiments, other aminoacid sequence can be added on the N end or the C end of fusion rotein, so that expression, detection and/or isolated or purified.

[0022] the feature of the present invention nucleotide sequence of fusion rotein described herein that also is to encode.

[0023] on the other hand, the invention is characterized in host cell and the carrier that contains nucleic acid of the present invention.Preferred host cell is eukaryotic cell (for example mammalian cell, insect cell or yeast cells) or prokaryotic cell (for example escherichia coli (E.coli)).For example, mammalian cell can be cultured cells or cell line.Exemplary mammalian cell comprises lymphocyte series (for example NSO), Chinese hamster ovary cell (CHO), COS cell, oocyte and from the cells of transgenic animal, for example galactophore epithelial cell.For example, the encode nucleic acid of fusion rotein as herein described can be expressed in transgenic animal.In one embodiment, nucleic acid is under the control of tissue-specific promoter's (for example mammary gland-specific promoter), and antibody produces in transgenic animal.For example, fusion rotein is secreted in the milk of transgenic animal, for example in transgenic cow, pig, horse, sheep, goat or the rodentine milk.

[0024] on the other hand, the invention provides for example preparation method of fusion rotein as herein described of fusion rotein.This method comprises: (a) cultivate host cell of the present invention and (b) purified fusion protein from culture in suitable culture medium.The protein that according to said method produces also is provided.

[0025] on the other hand, the invention provides for example pharmaceutical composition of compositions, it comprises pharmaceutically acceptable carrier and at least a IL-21/IL-21R antagonist as herein described (fusion rotein for example as herein described).In one embodiment, compositions for example pharmaceutical composition comprise the combination of two or more IL-21/IL-21R antagonisies.(for example the combination of curative (for example inhibitor, immunosuppressant, anti-inflammatory agent, metabolic poison, enzyme inhibitor and/or cytotoxic agent or the cytostatic agent of one or more cytokines as herein described and somatomedin) or antigen (for example antigenic peptide) and/or antigen-presenting cell is also included within the scope of the present invention for IL-21/IL-21R antagonist and medicine.

[0026] in one embodiment, pharmaceutical composition comprises IL-21/IL-21R antagonist and at least a other curative and pharmaceutically acceptable carrier.Can with one or more IL-21/IL-21R antagonisies be formulated in compositions for example the example of preferred other curative in the pharmaceutical composition include but not limited to one or more following medicines: the TNF antagonist (for example antibody of chimeric antibody, humanized antibody, people's antibody or external generation or its Fab, they can combine with TNF; TNF receptor soluble fragments, for example p55 or p75 people TNF receptor or derivatives thereof, for example 75kdTNFR-IgG (75kDa TNF receptor-IgG fusion rotein, ENBREL ^TM), p55kDa TNF receptor-IgG fusion rotein; TNF enzyme antagonist, for example TNF α invertase (TACE) inhibitor); The antagonist of IL-6, IL-12, IL-15, IL-17, IL-18, IL-22; The depleting agents of T cell and B cell (for example anti-CD 4 antibodies or anti-CD22 antibody); Micromolecular inhibitor, for example methotrexate and leflunomide; Sirolimus (rapamycin) and analog, for example CCI-779; Cox-2 and cPLA2 inhibitor; NSAID; The inhibitor of p38 inhibitor, TPL-2, Mk-2 and NF κ b; RAGE or solubility RAGE; P-selects the inhibitor (for example anti-P-selects proteic antibody for micromolecular inhibitor for example, its antibody) of albumen or PSGL-1; Erss (ERB) agonist or ERB-NF κ b antagonist.Can give jointly and/or most preferred other curative of preparing comprises one or more following medicines: TNF receptor soluble fragments with one or more IL-21/IL-21R antagonisies, for example p55 or p75 people TNF receptor or derivatives thereof, 75kdTNFR-IgG (75kDa TNF receptor-IgG fusion rotein, ENBREL for example ^TM); Methotrexate, leflunomide or sirolimus (rapamycin) or its analog, for example CCI-779.

[0027] on the other hand, the invention is characterized in for example method of the atopic diseases of mammal (for example people) of treatment, improvement or prevention patient.This method comprises and gives patient IL-21/IL-21R antagonist, and for example its consumption is enough to treatment, improves or prevents this disease, and perhaps its consumption is enough to suppress or reduce immunologic cellular activity and/or cell number.In one embodiment, atopic diseases is an allergic asthma.In another embodiment, atopic diseases is atopic dermatitis, urticaria, eczema, allergic rhinitis or allergia gastroenteritis.In one embodiment, the IL-21/IL-21R antagonist can be united with other curative and given, and described curative is cytokine inhibitor, immunosuppressant, anti-inflammatory agent, enzyme inhibitor, leukotriene antagonist, bronchodilator, beta-2-adrenoreceptor agonists, antimuscarinic drug or mast cell stabilizers for example.Can give with the IL-21/IL-21R antagonist combination and treated, improve or prevent the example of the preferred therapeutic medicine of atopic diseases to comprise for example TNF antagonist, IL-6 antagonist, IL-12 antagonist, IL-15 antagonist, IL-17 antagonist, IL-18 antagonist, IL-22 antagonist, t cell depletion agent, the agent of B cell consumption, methotrexate, leflunomide, sirolimus (rapamycin) or its analog, Cox-2 inhibitor, cPLA2 inhibitor, NSAID and p38 inhibitor.

[0028] on the other hand, the invention is characterized in treatment, the method for improving or preventing patient's autoimmune disease.This method comprises and gives patient IL-21/IL-21R antagonist, and for example its consumption is enough to treatment, improves or prevents this disease, and perhaps its consumption is enough to suppress or reduce immunologic cellular activity and/or cell number.In one embodiment, autoimmune disease is a lupus, for example SLE.In one embodiment, the IL-21/IL-21R antagonist can be united with other curative and given, and described curative is cytokine inhibitor, growth factor receptor inhibitors, immunosuppressant, anti-inflammatory agent, metabolic poison, enzyme inhibitor, cytotoxic agent or cytostatic agent for example.Can be treated with the IL-21/IL-21R antagonist combination, the example of the preferred therapeutic medicine of improvement or prevention autoimmune disease comprises for example TNF antagonist, the IL-6 antagonist, the IL-12 antagonist, the IL-15 antagonist, the IL-17 antagonist, the IL-18 antagonist, the IL-22 antagonist, the t cell depletion agent, the agent of B cell consumption, chloroquine, oxychloroquine, methotrexate, leflunomide, sirolimus (rapamycin) or its analog, the Cox-2 inhibitor, the cPLA2 inhibitor, NSAID and p38 inhibitor.

[0029] on the other hand, the invention is characterized in the method for treatment, improvement or prevention patient's fibre modification disease.This method comprises and gives patient IL-21/IL-21R antagonist, and for example its consumption is enough to treatment, improves or prevents this disease, and perhaps its consumption is enough to suppress or reduce immunologic cellular activity and/or cell number.For example, the patient may suffer from visceral nerve fiberization (for example hepatic fibrosis, renal fibrosis or pulmonary fibrosis), fibrosis of skin disease or eye fibrosis, perhaps is among its danger.

[0030] on the other hand, the invention is characterized in the method for organ, tissue or cell transplantation being given the patient.This method comprises and gives patient IL-21/IL-21R antagonist, for example before transplanting, during or afterwards.Transplanted organ and tissue can include but not limited to for example heart, kidney, liver, lung, pancreas, bone marrow, cartilage, cornea, neuronal tissue and cell thereof.In one embodiment, IL-21/IL-21R antagonist and other curative are united and are given, and described curative is cytokine inhibitor, growth factor receptor inhibitors, immunosuppressant, anti-inflammatory agent, metabolic poison, enzyme inhibitor, cytotoxic agent and cytostatic agent for example.Can comprise for example rapamycin, cyclosporin, anti-CTLA-4 antibody, anti-CD 40 antibodies, anti-CD40L antibodies and anti-CD154 antibody with the example of the preferred therapeutic medicine of IL-21/IL-21R antagonist coupling.

[0031] on the other hand, the invention is characterized in the transplant rejection symptom of transplant recipient or the evaluation and the Therapeutic Method of transplant rejection relevant disease (for example fibrosis or graft versus host disease (GVHD)).This method comprises the transplant rejection symptom of differentiating the patient and gives IL-21/IL-21R antagonist that for example its consumption is enough to treatment or improves the transplant rejection symptom.The transplant rejection symptom comprises that for example inflammation, organ dysfunction go down, unusual biopsy and fibrosis.In another embodiment, the invention provides the method for preventing (for example reducing its danger) transplant rejection or transplant rejection relevant disease by giving the IL-21/IL-21R antagonist.

[0032] on the other hand, the invention is characterized in treatment, improvement or prevention patient's the transplant rejection or the method for transplant rejection relevant disease.The method is characterized in that to give patient IL-21/IL-21R antagonist, its consumption is enough to treatment or improves or prevention (for example reducing its danger) repulsion, and perhaps its consumption is enough to suppress or reduce immunologic cellular activity and/or cell number.Transplant organ or tissue can comprise for example heart, kidney, liver, lung, pancreas and bone marrow.In one embodiment, the IL-21/IL-21R antagonist can with other curative coupling, described curative is cytokine inhibitor, growth factor receptor inhibitors, immunosuppressant, anti-inflammatory agent, metabolic poison, enzyme inhibitor, cytotoxic agent or cytostatic agent for example.Can with the coupling of IL-21/IL-21R antagonist with treatment, improve or the example of the preferred therapeutic medicine of prevention transplant rejection comprises for example rapamycin, cyclosporin, anti-CTLA-4 antibody, anti-CD 40 antibodies, anti-CD40L antibodies and anti-CD154 antibody.

[0033] " MU-1 " and " IL-21R " and terms such as peptide, polypeptide and protein are used interchangeably in this article.

[0034] except as otherwise noted, otherwise all scientific and technical terminologies used herein all have one skilled in the art's art-recognized meanings of the present invention.Although method and material all are similar to or are equal to used method and material in the present invention's practice and the experiment, suitable method and material are described below.All publications, patent application, patent and other list of references mentioned in this article all are attached to herein by reference.Under contradictory situation, this description comprises definition, will adjust.In addition, material, method and embodiment only are illustrative, rather than restrictive.

[0035] according to following detailed Description Of The Invention and appending claims, further feature of the present invention and advantage will be conspicuous.

The accompanying drawing summary

[0036] Fig. 1 represents the full length cDNA sequence of Mus IL-21R/MU-1.This nucleotide sequence is corresponding to the nucleotide 1-2628 of SEQ ID NO:9.

[0037] Fig. 2 A-2B represents the aminoacid sequence of Mus and human IL-2 1R/MU-1.Fig. 2 A represents the aminoacid sequence (corresponding to the amino acid/11-529 of SEQ ID NO:10) of Mus IL-21R/MU-1.Have the targeting sequencing (through SPScan prediction) of prediction at amino acid/11-19, it must be divided into 10.1 (boldface letters).The aminoacid 237-253 of SEQ ID NO:10 is the membrane-spanning domain (underscore) of prediction.The prediction signal motif comprises following each district: box 1: aminoacid 265-274, box 2: aminoacid 310-324 (runic+underscore); Six tyrosine are positioned at the amino acid position 281,319,361,368,397 and 510 of SEQ ID NO:10.WSXWS motif (SEQ IDNO:8) is positioned at amino acid residue 214 to amino acid residue 218 (large size word+runic).Potential STAT berth (docking site) comprises aminoacid 393-398 and the aminoacid 510-513 of SEQ ID NO:10.Fig. 2 B represents the aminoacid sequence (corresponding to SEQ ID NO:2) of people MU-1.Indicated the signal sequence (about amino acid/11-19 of SEQ ID NO:2) of prediction; WSXWS motif (about aminoacid 213-217 of SEQ ID NO:2); And membrane-spanning domain (about aminoacid 236-252 of SEQ ID NO:2,236-253,236-254 (underscore).Potential JAK binding site, signal motif and STAT berth have also been indicated.The approximate location in these sites has all added square frame.

[0038] Fig. 3 represents that the GAP of people and Mus MU-1 cDNA sequence (corresponding respectively to the nucleic acid 1-2665 of SEQ ID NO:1 and the nucleic acid 1-2628 of SEQ ID NO:9) compares.HuMU-1=people MU-1, murMU-1=Mus MU-1.Room parameter: room weight=50, average coupling=10.000, length weight=3, average mispairing=0.000, % homogeneity=66.116.

[0039] Fig. 4 represents that the people MU-1 albumen (corresponding to the aminoacid of SEQ ID NO:2) and the GAP of Mus MU-1 albumen (corresponding to the aminoacid of SEQ ID NO:10) compare.Produce comparison (Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.U.S.A.89:10915-19) by BLOSUM62 aminoacid replacement matrix.Room parameter=room weight: 8, average coupling=2.912, length weight=2, average mispairing=-2.003; % homogeneity=65.267.

[0040] Fig. 5 represents people MU-1 aminoacid (corresponding to SEQ ID NO:2), Mus MU-1 aminoacid (corresponding to SEQ ID NO:10) and people IL2 β chain amino acid (GENBANK ^Searching number M26062) multisequencing comparison.Targeting sequencing and stride the film district and have underscore.Conservative cytokine receptor module motif is represented with boldface letter.Potential signal conducting region is represented with underscore+boldface letter.

[0041] Fig. 6 represents the signal conduction by MU-1.In Clone E7 EPO-MU-1 chimera, MU-1 makes STAT 5 phosphorylations.Under the specified conditions of embodiment 3, the signal by MU-1 causes all making STAT 5 phosphorylations at all testing time points.Handle contrast or chimeric BAF-3 cell with IL-3, cause the phosphorylation of STAT 3 rather than STAT 1 or 5.

[0042] Fig. 7 A-7B represents that human IL-2 1R monomer (corresponding to the aminoacid 20-235 of SEQ ID NO:2) is at its aminoterminal and Apis targeting sequencing and His ₆Nucleotide sequence that labelling (amino acid/11-44 of SEQ ID NO:23) merges and aminoacid sequence comparison.This nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:22 and SEQ ID NO:23 respectively.

[0043] Fig. 8 A-8C represents nucleotide sequence and the aminoacid sequence comparison that human IL-2 1R extracellular domain (corresponding to the amino acid/11-235 of SEQ ID NO:2) merges by joint (corresponding to the aminoacid 236-243 of SEQ ID NO:25) and immunoglobulin G while 1 (IgG1) Fc sequence (corresponding to the aminoacid 244-467 of SEQ ID NO:25) at its C-end.This nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:24 and SEQ ID NO:25 respectively.

[0044] Fig. 9 A-9C represents that human IL-2 1R extracellular domain (corresponding to the amino acid/11-235 of SEQ ID NO:2) passes through joint (corresponding to the aminoacid 236-243 of SEQ ID NO:27) and immunoglobulin G while 1 (IgG1) Fc sequence (corresponding to the aminoacid 244-467 of SEQ ID NO:27) and His at its C-end ₆Nucleotide sequence that sequence mark (corresponding to the aminoacid 468-492 of SEQ ID NO:27) merges and aminoacid sequence comparison.This nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:26 and SEQ ID NO:27 respectively.

[0045] Figure 10 A-10C represents nucleotide sequence and the aminoacid sequence comparison that human IL-2 1R extracellular domain (corresponding to the amino acid/11-235 of SEQ ID NO:2) merges by joint (corresponding to the aminoacid 236-243 of SEQ ID NO:29) and immunoglobulin G while 1 (IgG1) Fc mutant nucleotide sequence (corresponding to the aminoacid 244-467 of SEQ ID NO:29) at its C-end.People Fc sequence has sudden change at the residue 254 and 257 of wild-type sequence, to reduce the Fc receptors bind.This nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:28 and SEQ ID NO:29 respectively.

[0046] Figure 11 A-11B represents nucleotide sequence and the aminoacid sequence comparison that human IL-2 1R extracellular domain (corresponding to the amino acid/11-235 of SEQ ID NO:2) merges at its C-end and rhodopsin sequence mark.This nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:30 and SEQ ID NO:31 respectively.

[0047] Figure 12 A-12C represents that human IL-2 1R extracellular domain (corresponding to the amino acid/11-235 of SEQ ID NO:2) is in nucleotide sequence and the aminoacid sequence comparison of its C-end with EK cleavage site and sudden change IgG1 Fc district (corresponding to the aminoacid 236-470 of SEQ ID NO:33) fusion.This nucleotide sequence and aminoacid sequence are shown in SEQ ID NO:32 and SEQ IDNO:33 respectively.

[0048] Figure 13 A-13B represents that Mus IL-21R extracellular domain is in nucleotide sequence and the aminoacid sequence comparison of its C-end with mouse immuning ball protein G2a (IgG2a) fusion.This nucleotide (genome) sequence and aminoacid sequence are shown in SEQ ID NO:34 and SEQ ID NO:35 respectively.

[0049] Figure 14 A-14B represents that Mus IL-21R extracellular domain is at its C-end and Flag and His ₆Nucleotide sequence that sequence mark merges and aminoacid sequence comparison.This nucleotide (genome) sequence and aminoacid sequence are shown in SEQ ID NO:36 and SEQ ID NO:37 respectively.

[0050] Figure 15 A-15B represents that (Apis targeting sequencing) Mus IL-21R extracellular domain is at its C-end and Flag and His ₆Nucleotide sequence that sequence mark merges and aminoacid sequence comparison.This nucleotide (genome) sequence and aminoacid sequence are shown in SEQ ID NO:38 and SEQ IDNO:39 respectively.

[0051] Figure 16 is preventative, the therapeutic that experimentizes with collagen-induced arthritis (CIA) mouse model and the General Schedule of half therapeutic treatment scheme.

[0052] Figure 17 is a curve chart, and the effect of double therapeutic CIA mice of expression MuIL-21RFc (200 μ g/ mices, 3 times/week) is the function of treatment back natural law.With mice Ig (200 μ g/ mices, 3 times/week) in contrast.

[0053] Figure 18 A-18B is a photo, and expression is compared with negative control (figure B), and the CIA mice suffers from IL-21R mRNA expression increase (figure A) in the arthritic claw.

[0054] Figure 19 and Figure 20 are line graphs, and expression is compared with IgG contrast, and the clinical score of the IBD sample symptom of the rat for the treatment of with muIL-21RFc and mEnbrel significantly descends.Left side figure among Figure 19 is a photo, the in situ hybridization of MU-1mRNA in expression normal person's intestinal lymphocyte and the lymph node.

[0055] Figure 21 is for after giving MuIL-21RFc, the statistical table that the histological score of the disease severity of rat IBD model descends.

[0056] Figure 22 is a line graph, be illustrated in and injected in the mice of T cell, muIL-21RFc or the contrast (GFP) of the expression IL-21 of retrovirus transduction, graft survival percentage ratio with adoptive transfer after the variation of natural law of (post-adoptive transfer).

[0057] Figure 23 is a line graph, be illustrated in give MuIL-21RFc after, the adopt improvement of clinical score of psoriasis sexually transmitted disease (STD) kitchen range of metastasis model of CD45RB height.Left figure among Figure 23 represents with the mice photo before and after the MuIL-21RFc treatment.

[0058] Figure 24 is a broken line graph, air flue overreaction (AHR) level of expression ovalbumin (OVA) sensitized mice after phosphate buffered saline(PBS) (PBS) or OVA attack.The sequential methacholine that gives the cumulative amount of mice.It is the sign of AHR that Penh (pausing increases) changes.

[0059] Figure 25 A-25D is a block diagram, and expression OVA sensitized mice is the cell number in its bronchoalveolar lavage fluid (BALF) after PBS or OVA attack.Figure 25 A represents the total cellular score among the BALF.Figure 25 B represents the eosinophilic granulocyte's number among the BALF.Figure 25 C represents the lymphocyte number among the BALF.Figure 25 D represents the neutrophil cell number among the BALF.Open tubular column is represented the WT mice through the PBS attack; Solid post is represented the WT mice through the OVA attack; The Lycoperdon polymorphum Vitt post represent the IL-21R-that attacks through PBS/-mice; The twill post represent the IL-21R-that attacks through OVA/-mice. ^*Expression p＜0.05 is measured by Mann-Whitney U check.

[0060] Figure 26 and Figure 27 block diagram of cytokine levels that is the OVA sensitized mice after OVA attacks among its BALF.Figure 26 represents TNF α and IL-5 level.Figure 27 represents the IL-13 level.Open tubular column is represented the WT mice through the PBS attack; Solid post is represented the WT mice through the OVA attack; The Lycoperdon polymorphum Vitt post represent the IL-21R-that attacks through PBS/-mice; The twill post represent the IL-21R-that attacks through OVA/-mice. ^*Expression p＜0.05 is measured by Mann-Whitney U check.

[0061] Figure 28 A-28B is a block diagram, and expression OVA sensitized mice is attacked its serum IgE level of back with OVA or PBS.Figure 28 A represents serum total IgE level.Figure 28 B represents anti-OVA specific IgE level.Open tubular column is represented the WT mice through the PBS attack; Solid post is represented the WT mice through the OVA attack; The Lycoperdon polymorphum Vitt post represent the IL-21R-that attacks through PBS/-mice; The twill post represent the IL-21R-that attacks through OVA/-mice. ^*Expression p＜0.05 is measured by Mann-Whitney U check.

[0062] Figure 29 A-29D is expression MRL-Fas ^LprThe figure of the circulation dsDNA autoantibody level of mice after MuIL-21RFc or contrast treatment.Figure 29 A represents the IgG1 level.Figure 29 B represents the IgG2a level.Figure 29 C represents the IgG2b level.Figure 29 D represents the IgG3 level. ^*Expression p＜0.05 is measured by Mann-Whitney U check.

[0063] Figure 30 A-30D is expression MRL-Fas ^LprThe figure of the circulation total IgG level of mice after MuIL-21RFc or contrast treatment.Figure 30 A represents the IgG1 level.Figure 30 B represents the IgG2a level.Figure 30 C represents the IgG2b level.Figure 30 D represents the IgG3 level. ^*Expression p＜0.05 is measured by Mann-Whitney U check.

[0064] Figure 31 is to be the figure of the fluorescence level of the mice kidney segment of expression after goat anti-mouse IgG-FITC dyeing.

[0065] Figure 32 is a sketch map, and expression IL-21 is to the example effect of immunne response.

[0066] Figure 33 is a sketch map, and expression suppresses the exemplary policy of IL-21/IL-21R approach.

[0067] Figure 34 is a sketch map, represents exemplary solubility IL-21RFc receptor fusion protein.

[0068] Figure 35 is a curve chart, and expression MuIL-21RFc treatment group and contrast treatment group mice stimulate the average psoriasis scoring in back with Eimeria tenella (E.tenella) (" Etenella ").

[0069] Figure 36 is a statistical table, and expression is compared with contrast treatment mice, stimulates the outbreak of the psoriasis symptom of mice to postpone and alleviates with the Eimeria tenella of MuIL-21RFc treatment.

[0070] Figure 37 is a curve chart, and expression is compared with contrast treatment mice, and the Eimeria tenella for the treatment of with MuIL-21RFc stimulates mice to reduce to some extent aspect losing weight.Body Mass Index is defined as the ratio of measuring body weight and initial body weight.

[0071] Figure 38 A is a line graph, and expression is compared with contrast treatment mice, and the Eimeria tenella for the treatment of with MuIL-21RFc stimulates the average stools scored of mice to descend.

[0072] Figure 38 B is the figure of every Eimeria tenella stimulation mice of each treatment group of expression in the stools scored of transfer in the time of back 77 days.

[0073] Figure 39 is the data statistic of Figure 38 A.

[0074] Figure 40 A is that expression is compared with contrast treatment mice, and the Eimeria tenella for the treatment of with MuIL-21RFc stimulates the figure of the IFN-γ serum levels of mice.

[0075] Figure 40 B is that expression is compared with contrast treatment mice, and the Eimeria tenella for the treatment of with MuIL-21RFc stimulates the figure of the stools scored of mice.

[0076] Figure 41 is a curve chart, after expression is treated with IL-21, ³The H-thymidine is incorporated into activatory CD45RB ^HiAnd CD45RB ^LoIn the cell.

[0077] Figure 42 A-B is a block diagram, after expression is treated with IL-21, and activatory CD45RB ^HiThe cytokine secretion of cell increases.

[0078] Figure 43 is a block diagram, after expression is treated with MuIL-21RFc, and activatory CD45RB ^HiThe cytokine secretion of cell reduces.

[0079] Figure 44 A-B is block diagram (A) and scatter diagram (B), be illustrated in the GVHD model of SLE, the IL-21R that has transplanted B6 bm12 splenocyte pounds out the autoantibody (A) that mice does not produce anti-dsDNA, and does not also observe IgG deposition (B) in these little Ren Mus.

Detailed Description Of The Invention

[0080] herein disclosed is the antagonist of use IL-21 or IL-21 acceptor (" IL-21R " or " MU-1 ") to suppress the active method and composition of interleukin-21 (IL-21)/IL-21 acceptor (MU-1). The IL-21/IL-21R antagonist can be used in vivo induction of immunity and suppresses, and for example is used for the treatment of or prevents inflammatory disease or autoimmune disease (for example with one or more mature T cells (ripe CD8⁺T cell, ripe CD4⁺The T cell), ripe NK cell, B cell, disease that macrophage is relevant with the megacaryocyte abnormal activity, comprise graft rejection, psoriasis and autoimmune disease, for example rheumatoid arthritis and IBD).

[0081] in one embodiment, the applicant proposes, by in using and fusion (it comprises the IL-21R extracellular domain that merges with the Fc immunoglobulin domain) reduce IL-21R activity, with the inflammatory symptom (embodiment 7) that improves collagen-induced arthritis (CIA) animal model, and IBD (embodiment 9 and 11), graft rejection (embodiment 10), psoriasis (embodiment 11) and the lupus (embodiment 13) of improving animal model. In CIA mouse claw, (embodiment 8) are raised in the expression of IL-21R mRNA. The airway inflammation that the IL-21R deficient mice demonstrates antigen induced alleviates (embodiment 12). Therefore, can antagonism the IL-21R bond of IL-21/IL-21R activity can be used in vivo that induction of immunity suppresses, for example be used for the treatment of or prevent inflammatory disease or autoimmune disease (for example glomerulonephritis, graft rejection, psoriasis, atopic diseases, asthma, autoimmune disease (for example rheumatoid arthritis and SLE) and IBD (for example regional ileitis, ulcerative colitis)).

[0082] for the ease of understanding the present invention, at first defines some terms. Other definition will provide in detailed Description Of The Invention.

[0083] term used herein " MU-1 ", " MU-1 albumen ", " interleukin-21 acceptor " or " IL-21R " refer to I type cytokines family receptors, and (WO 01/85792 to be also referred to as NILR; Parrish-Novak etc. (2000) Nature 408:57-63; Ozaki etc. (2000) Proc.Natl.Acad.Sci.U.S.A.97:11439-444). The β chain homology (Ozaki etc. (2000), ibid) that MU-1 and IL-2 acceptor and IL-15 acceptor and IL-4 α share. In case ligand binding, IL-21R/MU-1 can affect common gamma cells factor acceptor chain (γ c) (Asao etc. (2001) J.Immunol.167:1-5), and induces STAT1 and STAT3 phosphorylation (Asao etc. (2001)) or STAT5 phosphorylation (Ozaki etc. (2000)). MU-1 extensively distributes in lymphoid tissue. Term " MU-1 " refer to can with IL-21 (preferably from mammal, for example mouse or human IL-2 1) acceptor (the preferred mammal acceptor of interact (for example in conjunction with), for example from mouse or people), and has one of following characteristics: (i) amino acid sequence of naturally occurring mammal MU-1 polypeptide IL-21R/MU-1 or its fragment, for example amino acid sequence or its fragment shown in SEQ ID NO:2 (people) or the SEQ ID NO:10 (mouse); (ii) basically has for example amino acid sequence of at least 85%, 90%, 95%, 98% or 99% homology with amino acid sequence shown in SEQ ID NO:2 (people) or the SEQ ID NO:10 (mouse) or its fragment; (iii) by naturally occurring mammal IL-21R/MU-1 nucleotide sequence or the coded amino acid sequence of its fragment (for example SEQ ID NO:1 (people) or SEQ ID NO:9 (mouse) or its fragment); (iV) by having the coded amino acid sequence of the nucleotide sequence of at least 85%, 90%, 95%, 98%, 99% homology for example with nucleotide sequence shown in SEQ ID NO:1 (people) or the SEQID NO:9 (mouse) or its fragment; (v) by the amino acid sequence coded with the nucleotide sequence (for example SEQ ID NO.1 (people) or SEQ ID NO:9 (mouse) or its fragment) of naturally occurring IL-21R/MU-1 nucleotide sequence or its fragment degeneracy; Or (vi) stringency for example under the height stringency with the nucleotide sequence of one of foregoing nucleotide sequence hybridization.

[0084] IL-21R/MU-1 derives from mammal, preferred people. The nucleotide sequence of human IL-2 1R/MU-1 and predicted amino acid sequence are shown in SEQ ID NO:1 and SEQ ID NO:2. Analyst IL-21R/MU-1 amino acid sequence (SEQ ID NO:2; Fig. 2 B), disclose following architectural feature: targeting sequencing (about amino acid/11-19 of SEQ ID NO:2 (Fig. 2 B)); WSXWS motif (about amino acid 213-217 of SEQ ID NO:2); Membrane-spanning domain (about amino acid 236-252 (Fig. 2 B) of SEQ ID NO:2); The extracellular domain of about amino acid/11-235 of SEQ ID NO:2; Born of the same parents' internal area with about 253-538 of SEQ ID NO:2. It is believed that ripe human IL-2 1R/MU-1 has the sequence of the amino acid 20-538 of SEQ ID NO:2.

[0085] IL-21R/MU-1 cDNA was preserved in American type culture collection (American Type Culture Collection), preserving number ATCC 98687 on March 10th, 1998.

[0086] any form that is shorter than the IL-21R/MU-1 albumen of total length all can be used for method and composition of the present invention, as long as its keeps the ability in conjunction with the IL-21 polypeptide. Be shorter than the IL-21R/MU-1 albumen of total length, solubility IL-21R for example can be expressed in host cell and produced by the polynucleotides respective segments of coding total length MU-1 albumen. These corresponding polynucleotide passages also are parts of the present invention. Aforesaid modification polynucleotides can prepare by standard molecular biological technique, and described technology comprises structure, the site-directed mutagenesis method of suitable required deletion mutant or the PCR of passing through to use suitable Oligonucleolide primers.

[0087] " solubility IL-21R/MU-1 polypeptide " used herein is the IL-21R/MU-1 polypeptide that self can not be anchored on the film. This class soluble polypeptide comprises for example MU-1 or IL-21R polypeptide, they lack enough cross-film district parts of the described polypeptide of grappling, perhaps they are modified so that cross-film district nonfunctional, the soluble fragments of IL-21R (IL-21R fragment for example for example, it comprises the extracellular domain of mouse or human IL-2 1R, comprise about amino acid/11-235,1-236, the 20-235 of SEQ ID NO:2 (people), the amino acid sequence of 20-236, perhaps about amino acid/11-236 of SEQ ID NO:10 (mouse), the amino acid sequence of 20-236. Solubility IL-21R/MU-1 polypeptide can also comprise second portion (for example with its fusion), and described second portion is polypeptide (for example immunoglobulin chain, GST, Lex-A or MBP peptide sequence) for example. For example, fusion can comprise with second portion merge at least can be in conjunction with the IL-21R polypeptide fragment of IL-21, IL-21R soluble fragments (the IL-21R fragment that for example comprises mouse or human IL-2 1R extracellular domain for example; For example about amino acid/11-235,1-236,20-235, the 20-236 of SEQ ID NO:2 (people), perhaps about amino acid/11-236, the 20-236 of SEQ ID NO:10 (mouse); Described second portion is polypeptide (for example the immunoglobulin chain of different isotypes, Fc fragment, CH comprise IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE) for example.

[0088] term " interleukin-21 " or " IL-21 " refer to have with IL-2, IL-4 and IL-15 cell factor (Parrish-Novak etc. (2000) Nature 408:57-63) of sequence homology. Although sequence homology is low between the interleukin cell factor, cell factor all is folded into " four-helix bundle " structure representative in this family. It is mainly at activation CD4⁺The T cells, it is reported it can affect NK, B and T cell (Parrish-Novak etc. (2000), ibid; Kasaian etc. (2002), ibid). IL-21 is in conjunction with IL-21R (being also referred to as in this article MU-1 and NILR). In case IL-21 combination, the activation of IL-21R will cause STAT5 or STAT3 signal conduction (Ozaki etc. (2000), ibid). Term " IL-21 " or " IL-21 polypeptide " refer to can with IL-21R (preferably from mammal, for example mouse or human IL-2 1) protein of interact (for example in conjunction with) is (preferably from mammal, for example mouse or people), and has one of following characteristics: (i) amino acid sequence of naturally occurring mammal IL-21 or its fragment, for example amino acid sequence shown in the SEQ ID NO:19 (people) or its fragment; (ii) basically has for example amino acid sequence of at least 85%, 90%, 95%, 98%, 99% homology with amino acid sequence shown in the SEQ ID NO:19 (people) or its fragment; (iii) by naturally occurring mammal IL-21 nucleotide sequence or the coded amino acid sequence of its fragment (for example SEQ ID NO:18 (people) or its fragment); (iv) by having the coded amino acid sequence of the nucleotide sequence of at least 85%, 90%, 95%, 98%, 99% homology for example with nucleotide sequence shown in the SEQ ID NO:18 (people) or its fragment; (v) by the amino acid sequence coded with the nucleotide sequence (for example SEQ ID NO:19 (people) or its fragment) of naturally occurring IL-21 nucleotide sequence or its fragment degeneracy; Or (vi) stringency for example under the height stringency with the nucleotide sequence of one of foregoing nucleotide sequence hybridization.

[0089] " biologically active " of phrase MU-1 or IL-21R polypeptide refers to one or more biologically actives of corresponding ripe MU-1 albumen, includes but not limited to (1) and IL-21 polypeptide (for example human IL-2's 1 polypeptide) interaction (for example being combined); (2) associate with signal transduction molecules such as γ c, JAK1; (3) stimulate stat albumen (for example STAT5 and/or STAT3) phosphorylation and/or activation; And/or (4) adjusting (for example stimulating or reduction), propagation, differentiation, effector cell function, cell lysis activity, cytokine secretion and/or immunocyte survival, described immunocyte is T cell (CD8 for example⁺、CD4 ⁺The T cell), NK cell, B cell, macrophage and megacaryocyte).

[0090] " the IL-21/IL-21R antagonist " that can be used for the inventive method used herein refers to can reduce, suppress or eliminate one or more bioactive medicines of IL-21R/MU-1 polypeptide. In a preferred embodiment, antagonist and IL-21R/MU-1 polypeptide interaction (for example being combined). In another preferred embodiment, antagonist and IL-21 polypeptide interaction (for example being combined). Antagonist with IL-21/IL-21R carries out antagonism, not necessarily shows IL-21R/MU-1 polypeptide and/or the bioactive fully elimination of IL-21 polypeptide.

[0091] " the treatment effective dose " of IL-21/IL-21R antagonist used herein refers to give the patient the medicinal effective dose of (for example people patient) with single dose or multiple dose, with cure, improve prevent one or more symptoms of this disease or life cycle of reducing its order of severity or making the patient longer than the life cycle without the patient of this treatment.

[0092] " the prevention effective dose " of IL-21/IL-21R antagonist used herein refers to give with single dose or multiple dose patient's (for example people patient) IL-21/IL-21R antagonist effective dose, with prevent disease, disease described herein or postpone its outbreak or recurrence for example.

[0093] term " induce ", " bringing out ", " inhibition ", " enhancing ", " raising ", " increase ", " reduction " etc., when for example being used for the quantity variance between two states, refer to quantitatively have at least statistically significant difference between two states.

[0094] term " coupling " in this article refers to basically simultaneously (together or sequential) and gives several drugs. If sequential administration, when giving the second compound, preferably still there is detectable valid density in the first in these two kinds of compounds in therapentic part or patient body.

[0095] " fusion " used herein part protein of protein portion for example of referring to contain two or more effective associations (for example connecting). Covalency associates between preferred each several part. Each several part can directly associate or connect by spacer region or joint.

[0096] term used herein " antibody " refer to comprise at least one, the protein of preferred two weights (H) chain variable region (being abbreviated as in this article VH) and at least one, preferred two light (L) chain variable regions (being abbreviated as in this article VL). VH district and VL district also can be further divided into hypervariable region and framework region (FR), and the former is called " complementary determining region " (" CDR "), is wherein interleaving more conservative framework region. Framework region and CDR have accurately been defined (referring to such as (1991) Sequences of Proteins of Immunological Interest such as Kabat, the 5th edition, U.S. HHS (U.S.Department of Health and Human Services), NIH publication No. 91-3242, with (1987) J.Mol.Biol. 196:901-17 such as Chothia, described document is all incorporated herein by reference). Each VH and VL are comprised of 3 CDR and 4 FR, arrange from the aminoterminal to the c-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0097] antibody also can comprise CH and constant region of light chain, thereby forms respectively heavy chain immunoglobulin and light chain immunoglobulin. In one embodiment, antibody is the tetramer of two heavy chain immunoglobulins and two light chain immunoglobulins, and wherein heavy chain immunoglobulin is connected with light chain immunoglobulin by for example disulfide bond connection. CH is comprised of following three domains: CH1, CH2 and CH3. Constant region of light chain is comprised of a domain C L. Variable region of heavy chain and variable region of light chain contain the binding structural domain with AI. The combination of the common mediate antibody of the constant region of antibody and host tissue or the factor, the described factor comprise first component (C1q) of immune various cell (for example effector cell) and classical complement system.

[0098] term used herein " immunoglobulin (Ig) " refers to the protein that is comprised of one or more coded polypeptide of immunoglobulin gene by basically. The human immunoglobulin gene who has identified comprises κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ constant region gene, and countless immune globulin variable region gene. Total length immunoglobulin (Ig) " light chain " (about 25kD or 214 amino acid) is by NH₂The κ of the variable region gene (about 110 amino acid) of-end and COOH-end or λ constant region coded by said gene. Equally, total length immunoglobulin (Ig) " heavy chain " (about 50kD or 446 amino acid) is coded by one of variable region gene (about 116 amino acid) and other above-mentioned constant region gene (for example γ (about 330 amino acid of encoding)).

[0099] " isotype " used herein refers to by the coded antibody isotype of weight chain constant area gene (for example IgM or IgG1).

[0100] " Fab " of term antibody used herein (or being called for short " antibody moiety " or " fragment "), refer to one or more fragments of full length antibody, and it keeps the ability with antigen (for example CD3) specific binding. The example of binding fragment is included in term antibody " Fab " scope, and it comprises (i) Fab fragment, the unit price fragment that namely is comprised of VL district, VH district, CL district and CH1 district; (ii) F (ab ')₂Fragment namely is included in hinge area with the divalence fragment of two continuous Fab fragments of disulfide bond; (iii) the Fd fragment that is formed by VH district and CH1 district; (iv) the Fv fragment that is formed by VL district and the VH district of antibody single armed; (v) dAb fragment (Ward etc. (1989) Nature 341:544-546), it is comprised of the VH district; The complementary determining region (CDR) that (vi) separates. In addition, although two districts of Fv fragment are VL and VH, by separately coded by said gene, but can use recombination method, by synthetic joint they are connected to each other, described joint can make them become an albumen strand, and wherein VL district and VH district pairing formation monovalent molecule (is called scFv (scFv); Referring to such as (1988) Science 242:423-426 such as Bird; With (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883 such as Huston). This class single-chain antibody is also included within " Fab " of term antibody. Available routine techniques well known by persons skilled in the art obtains these antibody fragments, and these fragments can be used as complete antibody in an identical manner through screening.

[0101] also be the application's part with sequence disclosed herein sequence similar or homology (for example having at least about 85% sequence homogeneity). In some embodiments, sequence homogeneity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Perhaps, when lower in selective cross condition (for example height stringency hybridization condition) complementary strand with this chain when nucleic acid segment is hybridized, namely has basic homogeneity. Nucleic acid can be present in intact cell, the cell lysate, or exists with partial purification or basic purified form.

The calculating of " homology " or " sequence homogeneity " (these two terms are used interchangeably in this article) between [0102] two sequence is carried out as follows. For the best compares purpose, sequence is compared (for example in order to carry out the best comparison, can in the first and second amino acid sequences or nucleotide sequence or among both, introduce the room, and for purpose relatively, non-homogeneous sequence can be ignored). In a preferred embodiment, the length of the reference sequences of comparing in order to compare is at least 30%, preferably at least 40%, more preferably at least 50% even more preferably at least 60% of reference sequences length, even more preferably at least 70%, 80%, 90%, 100%. Then, amino acid residue or the nucleotides on more corresponding amino acid position or the nucleotide position. When the position in the First ray was occupied by the amino acid residue identical with relevant position in the second sequence or nucleotides, so described molecule was identical (" homogeneity " of amino acid used herein or nucleic acid is equal to amino acid or nucleic acid " homology ") in this position. % homogeneity is the function of the shared same position number of two sequences between two sequences, has wherein considered the length in room number and each room, and they are introduced for the purpose of the best comparison of two sequences.

Sequence comparison between [0103] two sequence and determine % homogeneity can be adopted mathematical algorithm and finishes. In a preferred embodiment, % homogeneity between two amino acid sequences adopts Needleman and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm and draws, described algorithm is attached in the GAP program of GCG software kit (can derive from http://www.gcg.com), with BLOSUM 62 matrixes or PAM250 matrix, the room weight is 16,14,12,10,8,6 or 4, and the length weight is 1,2,3,4,5 or 6. In another preferred embodiment, % homogeneity between two nucleotide sequences is used the GAP program of GCG software kit (can derive from http://www.gcg.com) and is drawn, use the NWSgapdna.CMP matrix, its room weight is 40,50,60,70 or 80, and the length weight is 1,2,3,4,5 or 6. One group of particularly preferred parameter is (with the parameter that should adopt, if the technical staff does not know what parameter to determine that certain molecule is whether in sequence homogeneity of the present invention or homology limited field with) be BLOSUM62 marking matrix, its gap penalty is 12, it is 4 that point penalty is extended in the room, and the frameshit gap penalty is 5. % homogeneity between two amino acid or nucleotide sequence is available E.Meyers and W.Miller ((1989) CABIOS also, algorithm 4:11-17) and drawing, this algorithm is attached in the ALIGN program (2.0 editions), with PAM120 weight residue table, room length point penalty is 12, and gap penalty is 4.

[0104] term used herein " under stringency, hybridize " refer to hybridization and wash conditions. Stringency is well known by persons skilled in the art, can find in Publication about Document: Current Protocols in Molecular Biology, John Wiley Sons, N.Y. (1989), 6.3.1-6.3.6. Have water method and anhydrous process to be described in this list of references, two kinds of methods all can adopt. A preferred embodiment of stringency hybridization condition is in 6X sodium chloride/sodium citrate (SSC), hybridizes at about 45 ℃, again in 0.2X SSC, 0.1%SDS, in 50 ℃ of washing one or many. Another example of stringency hybridization condition is in 6X SSC, hybridizes at about 45 ℃, again in 0.2X SSC, 0.1%SDS, in 55 ℃ of washing one or many. Another example of stringency hybridization condition is in 6X SSC, hybridizes at about 45 ℃, again in 0.2X SSC, 0.1%SDS, in 60 ℃ of washing one or many. Preferred stringency hybridization condition is in 6X SSC, hybridizes at about 45 ℃, again in 0.2X SSC, 0.1%SDS, in 65 ℃ of washing one or many. Particularly preferred stringency hybridization condition is (with the condition that should adopt, if the technical staff does not know what condition to determine that certain molecule is whether in hybridization limited field of the present invention with) be in 0.5M sodium phosphate, 7%SDS, in 65 ℃ of hybridization, again in 0.2X SSC, 0.1%SDS, in 65 ℃ of washing one or many. The polynucleotides that the present invention separates can be used as hybridization probe and primer, to identify and to separate the nucleic acid that has sequence homogeneity or similitude with coding polynucleotides disclosed by the invention. Comprise PCR (PCR), DNA hybridization, in situ hybridization and RNA hybridization for the identification of the hybridizing method with isolating nucleic acid, these methods all are that those skilled in the art are well-known. This paper provides more disclosures of relevant hybridization conditions and reaction.

[0105] can under different stringency, carry out hybridization reaction. The stringency of hybridization reaction comprises the difficulty that any two nucleic acid molecules are hybridized each other. The polynucleotides of preferred each hybridization are its corresponding multi-nucleotide hybrid under the stringency that reduces, more preferably stringency, most preferably height stringency. The example of stringency sees the following form 1: the height stringency is the same with for example condition A-F at least strict condition; Stringency is the same with for example condition G-L at least strict condition; And the stringency that reduces is the same with for example condition M-R at least strict condition.

Table 1

Stringency	Multi-nucleotide hybrid	Hybridization length (bp)1	Hybridization temperature and buffer solution2	Wash temperature and buffer solution2
					A	DNA:DNA	＞50	65 ℃; 1X SSC or 42 ℃; 1X SSC, 50% formamide	65℃；0.3X SSC
B	DNA:DNA	＜50	T B *；1X SSC	T B *；1X SSC
					C	DNA:RNA	＞50	67 ℃; 1X SSC or 45 ℃; 1X SSC, 50% formamide	67℃；0.3X SSC
D	DNA:RNA	＜50	T D *；1X SSC	T D *；1X SSC
					E	RNA:RNA	＞50	70 ℃; 1X SSC or 50 ℃; 1X SSC, 50% formamide	70℃；0.3X SSC
F	RNA:RNA	＜50	T F *；1X SSC	T F *；1X SSC
					G	DNA:DNA	＞50	65 ℃; 4X SSC or	65℃；1X SSC

			42 ℃; 4X SSC, 50% formamide
					H	DNA:DNA	＜50	T H *；4X SSC	T H *；4X SSC
I	DNA:RNA	＞50	67 ℃; 4X SSC or 42 ℃; 4X SSC, 50% formamide	67℃；0.3X SSC
					J	DNA:RNA	＜50	T J *；4X SSC	T J *；4X SSC
K	RNA:RNA	＞50	70 ℃; 4X SSC or 50 ℃; 4X SSC, 50% formamide	67℃；1X SSC
					L	RNA:RNA	＜50	T L *；2X SSC	T L *；2X SSC
M	DNA:DNA	＞50	50 ℃; 4X SSC or 40 ℃; 6X SSC, 50% formamide	50℃；2X SSC
					N	DNA:DNA	＜50	T N *；6X SSC	T N *；6X SSC
O	DNA:RNA	＞50	55 ℃; 4X SSC or 42 ℃; 6X SSC, 50% formamide	55℃；2X SSC
					P	DNA:RNA	＜50	T P *；6X SSC	T P *；6X SSC
Q	RNA:RNA	＞50	60 ℃; 4X SSC or 45 ℃; 6X SSC, 50% formamide	60℃；2X SSC
					R	RNA:RNA	＜50	T R *；4X SSC	T R *；4X SSC

¹Hybridization length is the hybridization region of hybridization polynucleotides. When the target polynucleotide of polynucleotides and unknown nucleotide sequence was hybridized, hybridization length was assumed to the length of hybridization polynucleotides. When the multi-nucleotide hybrid of known array, can and identify that by the polynucleotide sequence comparison the complementary district of optimal sequence determine to hybridize length.

²(1xSSPE is 0.15M NaCl to SSPE, 10mM NaH₂PO ₄With 1.25mM EDTA, pH 7.4) alternative SSC (1xSSC is 0.15M NaCl and 15mM natrium citricum) in hybridization and lavation buffer solution, can wash after hybridization is finished in 15 minutes.

T _B ^*-T _R ^*: should be than the melting temperature (T of heterozygote less than the hybridization temperature of the heterozygote of 50 base-pairs for length_m) low 5-10 ℃, wherein obtain T according to following formula_m For the heterozygote of length less than 18 base-pairs, Tm (℃)=2 (A+T base number)+4 (G+C base number). For the heterozygote of length between 18-49 base-pair, Tm (℃)=81.5+16.6 (log₁₀Na ⁺)+0.41 (%G+C)-(600/N), wherein N is heterozygote base number, Na⁺The Na ion concentration (Na of 1X SSC in the hybridization buffer⁺＝0.165M)。

Other example of the stringency of multi-nucleotide hybrid is referring to Sambrook etc., Molecular Cloning A Laboratory Manual, the 9th and 11 chapters, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), with (writing) such as Ausubel, Current Protocols in Molecular Biology, the 2.10﹠6.3-6.4 joint, John Wiley ﹠ Sons, Inc (1995), described document is incorporated herein by reference.

[0106] the isolating polynucleotide of the present invention can be used as hybridization probe and primer, to identify and the DNA that separates the sequence with coding polynucleotide allelic variant disclosed herein.Allelic variant is the naturally occurring alternative form of polynucleotide disclosed herein, and its encoded polypeptide and polynucleotide encoded polypeptide disclosed herein have homogeneity or significant similarity.Preferred allelic variant and polynucleotide disclosed herein have at least 90% sequence homogeneity (more preferably at least 95% homogeneity; At least 99% homogeneity most preferably).

[0107] the isolating polynucleotide of the present invention also can be used as hybridization probe and primer, to identify and to separate the DNA that has with the sequence of the homologous coded polypeptide of polynucleotide disclosed herein.These homologous sequences be from the polypeptide disclosed herein species different with polynucleotide separate polynucleotide and the polypeptide that obtains, although perhaps be from same species, to separate, have and polynucleotide disclosed herein and polypeptide obvious sequence similarity.Preferred polynucleotide homologous sequence and polynucleotide disclosed herein have at least 50% sequence homogeneity (more preferably at least 75% homogeneity; And homologous peptide sequence and polypeptide disclosed herein have at least 30% sequence homogeneity (more preferably at least 45% homogeneity at least 90% homogeneity most preferably); At least 60% homogeneity most preferably).The homologous sequence of preferred polynucleotide disclosed herein and polypeptide is isolating from mammal.

[0108] the isolating polynucleotide of the present invention also can be used as hybridization probe and primer, express the cell of polypeptide of the present invention and the condition of tissue and their expression with evaluation.

[0109] people know, IL-21/IL-21R antagonist of the present invention can have other conservative or non essential amino acid replacement, and these replace the not influence basically of its function." conserved amino acid replacement " is such replacement: wherein amino acid residue is had the amino acid residue replacement of similar side chain.The amino acid residue family of similar side chain has been determined to have in this area.These families comprise the aminoacid that has basic side chain (lysine for example, arginine, histidine), aminoacid (the aspartic acid for example that has acid side-chain, glutamic acid), uncharged polar side chain aminoacid (glycine for example, agedoite, glutamine, serine, threonine, tyrosine, cysteine), aminoacid (the alanine for example of band non-polar sidechain, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), the aminoacid of band β side chain side chain (threonine for example, valine, isoleucine) and the aminoacid (tyrosine for example of band aromatic side chains, phenylalanine, tryptophan, histidine).

[0110] term used herein " recombinant host cell " (or abbreviation " host cell ") is meant the cell that wherein imports recombinant expression carrier.Be appreciated that this term not only is meant concrete subject cell, and be meant the filial generation of described cell.Because sudden change or environmental effect, some modification can take place among the offspring afterwards, so such offspring is in fact also inequality with parental cell, but such cell still is included within the scope of term used herein " host cell ".

The IL-21/IL-21R antagonist

[0111] in one embodiment, IL-21R/MU-1 polypeptide or its active fragment can for example immunoglobulin or its fragment (for example its Fc binding fragment) merge with second portion.For example, the soluble form of IL-21R/MU-1 can be by the Fc partial fusion of " joint " sequence and immunoglobulin.Also can use other fusion rotein, for example the albumen that merges with GST, Lex-A or MBP.

[0112] fusion rotein also can comprise the joint sequence that IL-21 or IL-21R fragment are connected with second portion.For example, fusion rotein can comprise peptide linker, for example the about 4-20 of length amino acid whose peptide linker, more preferably 5-10 amino acid whose peptide linker; In one embodiment, peptide linker length is 8 aminoacid.Each aminoacid in the peptide linker all is selected from Gly, Ser, Asn, Thr and Ala; In one embodiment, peptide linker comprises the Gly-Ser element.In other embodiments, fusion rotein comprises peptide linker, and peptide linker comprises and has formula (Ser-Gly-Gly-Gly-Gly) _ySequence, wherein y is 1,2,3,4,5,6,7 or 8.

[0113] in other embodiments, the N of fusion rotein end or C end also can add other aminoacid sequence, so that expression, detection and/or isolated or purified.For example, the IL-21/IL-21R fusion rotein can be connected with one or more extra sections, and described extra section is GST, His for example ₆Labelling, FLAG labelling.For example, fusion rotein can also be connected with gst fusion protein, and wherein the C-of fusion rotein sequence and GST (being glutathione S-transferase) sequence end merges.Such fusion rotein helps the purification of MU-1 fusion rotein.

[0114] in another embodiment, fusion rotein comprises allos signal sequence (promptly on MU-1 nucleic acid encoded polypeptide and non-existent peptide sequence) at its N-end.For example, natural MU-1 signal sequence can be removed, and is replaced by another kind of proteinic signal sequence.In some host cell (for example mammalian host cell), can use the allos signal sequence to increase expression and/or the secretion of MU-1.

[0115] can produce chimeric protein of the present invention or fusion rotein by the recombinant DNA technology of standard.For example, for example use flush end or cohesive end to connect according to routine techniques, the dna fragmentation of the different peptide sequences of coding is linked together with meeting frame, digest through restriction endonuclease, appropriate end is provided, if required, mends flat cohesive end,, carry out enzymatic again and connect to avoid undesired connection with alkaline phosphatase treatment.In another embodiment, can be by the synthetic fusion gene of routine techniquess such as automatic dna synthesizer.Perhaps, use anchor primer can carry out the pcr amplification of genetic fragment, this can produce the complementary jag (overhang) between two consecutive gene fragments, it can be annealed subsequently and increase again to produce chimeric gene sequence (referring to (writing) Current Protocols in MolecularBiology such as for example Ausubel, John Wiley; Sons, 1992).In addition, many expression vectors are commercially available, and their codifieds merge part (for example heavy chain immunoglobulin Fc district).The MU-1 code nucleic acid can be cloned in such expression vector, makes to merge partly to link together with the immune globulin albumin with meeting frame.In certain embodiments, for example dimer or trimeric form exist the MU-1 fused polypeptide with oligomer.Can use means known in the art, make up first polypeptide and/or the nucleic acid of first polypeptide of encoding.

[0116] in certain embodiments, be provided at the MU-1 polypeptide portion of the anomaly MU-1 polypeptide that has sudden change in the naturally occurring MU-1 sequence (wild type), it can produce MU-1 polypeptide and the bonded higher affinity of IL-21 (with respect to the sequence of not suddenling change).

[0117] in certain embodiments, be provided at the MU-1 polypeptide portion of the anomaly MU-1 polypeptide that has sudden change in the naturally occurring MU-1 sequence (wild type), it can make the MU-1 sequence that Proteolytic enzyme is had higher resistance (with respect to the sequence of not sudden change).In certain embodiments, first polypeptide comprises total length MU-1 polypeptide.Perhaps, first polypeptide comprises the polypeptide less than total length MU-1.

[0118] signal peptide that can comprise in the fusion rotein is MPLLLLLLLLPSPLHP (SEQ ID NO:21).If required, also can be with one or more aminoacid insertion between first polypeptide portion and second polypeptide portion that contain the MU-1 part.

[0119] second polypeptide is preferably soluble.In certain embodiments, second polypeptide protracting connect the half life (for example serum half life) of polypeptide.In certain embodiments, second polypeptide includes and is beneficial to fused polypeptide and the associating sequence of the 2nd MU-1 polypeptide.In preferred embodiments, second polypeptide comprises the immunoglobulin polypeptides district at least.The immunoglobulin fused polypeptide is known in the art, referring to for example U.S. Patent number 5,516,964,5,225,538,5,428,130,5,514,582,5,714,147 and 5,455,165.

[0120] in some embodiments, second polypeptide comprises the total length immunoglobulin polypeptides.Perhaps, second polypeptide comprises less than the total length immunoglobulin polypeptides, for example heavy chain, light chain, Fab, Fab ₂, Fv or Fc.Preferred second polypeptide comprises the immunoglobulin polypeptides heavy chain.Preferred second polypeptide comprises immunoglobulin polypeptides Fc district.

[0121] in another aspect of this invention, second polypeptide has the effector function still less than wild type heavy chain immunoglobulin Fc district.The Fc effector function comprises for example Fc receptors bind, complement combination and t cell depletion activity (referring to No. the 6th, 136,310, United States Patent (USP) for example).The assay method of t cell depletion activity, Fc effector function and antibody stability is known in the art.In one embodiment, second polypeptide only has low affinity or does not have affinity the Fc receptor.In an alternate embodiment, second polypeptide only has low affinity or does not have affinity complement protein C1q.

[0122] preferred second peptide sequence comprises the aminoacid sequence of SEQ ID NO:17.This sequence comprises the Fc district.Aminoacid with underscore is different from the aminoacid on the wild type immunoglobulin sequences relevant position:

HTCPPCPAPE ALG APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPP

SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ

QGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：17)

[0123] example that can be used for the antagonism fusion rotein of the inventive method is seen Fig. 7-15.In one embodiment, fusion rotein comprises and is selected from for example following aminoacid sequence: SEQID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37 or SEQ ID NO:39 or have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.In other embodiments, fusion rotein comprises and is selected from for example following coded aminoacid sequence of nucleotide sequence: SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38, or have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.Preferred fusion protein has aminoacid sequence and is shown in SEQ ID NO:25 or SEQ ID NO:29 (seeing Fig. 8 A-8C and Figure 10 A-10C respectively), or has at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.In other embodiments, fusion rotein comprises and is selected from for example following coded aminoacid sequence of nucleotide sequence: SEQ ID NO:24 or SEQ ID NO:28 (seeing Fig. 8 A-8C and Figure 10 A-10C respectively), or have at least 85%, 90%, 95%, 98% or the sequence of higher homogeneity with them.Most preferred fusion rotein has the aminoacid sequence shown in the SEQ ID NO:29, perhaps has the coded aminoacid sequence (Figure 10 A-10C) of nucleotide sequence shown in the SEQ ID NO:28.

[0124] in other embodiments, the IL-21/IL-21R antagonist is antibody or its Fab, and they can be in conjunction with IL-21 or IL-21R, preferably can be in conjunction with mammal (for example people or Mus) IL-21 or IL-21R.

[0125] MU-1 albumen of the present invention also can be used for immune animal, obtaining polyclonal antibody and monoclonal antibody, such antibody can with the reaction of MU-1 protein-specific, can suppress combining of part and receptor.Can use complete MU-1 as immunogen, perhaps, obtain such antibody by using the MU-1 fragment.Less segmental MU-1 also can be used for immune animal.Peptide based immunogens also can contain cysteine residues at c-terminus, and puts together with hapten, and described hapten is keyhole  hemocyanin (KLH) for example.Can prepare other peptide based immunogens by substituting tyrosine residue with the sulphation tyrosine residue.The synthetic method of these peptides is well-known in the art.

[0126] can also can be used for treating above-mentioned disease in conjunction with proteic neutralizing antibody of MU-1 or nonneutralizing antibody (preferred monoclonal antibody).These neutralizing monoclonal antibodies energy block ligand combine with the MU-1 receptor chain.

[0127] the present invention also provides compositions, and it comprises the antibody with IL-21 or IL-21R specific reaction.

[0128] with the transgenic mice of carrier's immunoglobulin gene (rather than mice system), can produce human monoclonal antibodies (mAb) at IL-21 or IL-21R.The splenocyte of the transgenic mice of these target antigen immunity of hanging oneself, be used to produce hybridoma, the secretion of this hybridoma has the people mAb of specificity affinity (referring to for example Wood etc. to people's albumen epi-position, international publication number WO91/00906, Kucherlapati etc., international publication number WO 91/10741; Lonberg etc., international publication number WO92/03918; Kay etc., international publication number 92/03917; Lonberg, 1994 Nature 368:856-859 such as N.; Green, L.L. etc. (1994) NatureGenet.7:13-21; Morrison, S.L. etc. (1994) Proc.Natl.Acad.Sci.U.S.A.81:6851-6855; Bruggeman etc. (1993) Year Immunol 7:33-40; Tuaillon etc. (1993) Proc.Natl.Acad.Sci.U.S.A.90:3720-3724; Bruggeman etc. (1991) Eur J Immunol 21:1323-1326).

[0129] also can produce monoclonal antibody by known other method of recombinant DNA technology those skilled in the art.Developed an alternative method, be called " combinatorial antibody is showed (combinatorial antibody display) " method, identified and separate to have the specific antibody fragment of specific antigen, this method can be used for producing monoclonal antibody; This method is well-known in the art.Behind the immunogen immune animal, all antibody of clone's gained Blymphocyte repertoire.By using oligomerization primer mixture and PCR method, normally known with the DNA sequence of obtaining each colony variable region of immunoglobulin molecules.For example, mixing oligonucleotide primers corresponding to 5 ' leading (signal peptide) sequence and/or framework 1 (FR1) sequence, and at the primer of conservative 3 ' constant region primer, can be used for pcr amplification, so that from various murine antibody amplification variable region of heavy chaines and variable region of light chain (Larrick etc. (1991), Biotechniques 11:152-156).Similarly strategy also can be used for from people's antibody amplification people's variable region of heavy chain and variable region of light chain (Larrick etc. (1991), Methods:Companion to Methods in Enzymology 2:106-110).

[0130] chimeric antibody comprises the gomphosis immunoglobulin chain, can prepare by recombinant DNA technology known in the art.For example, gene with restriction endonuclease digestion coding Mus (or other species) monoclonal antibody molecule Fc constant region, removing Mus Fc coding region, and personnel selection Fc constant region encoding gene be equal to part replace (referring to Robinson etc., international patent application no PCT/US86/02269; Akira etc., european patent application 184,187; Taniguchi, M., european patent application 171,496; Morrison etc., european patent application 173,494; Neuberger etc., international publication number WO 86/01533; Cabilly etc., United States Patent (USP) the 4th, 816, No. 567; Cabilly etc., european patent application 125,023; Better etc. (1988) Science 240:1041-1043); Liu etc. (1987) Proc.Natl.Acad.Sci.U.S.A.84:3439-3443; Liu etc. (1987) J.Immunol.139:3521-3526; Sun etc. (1987) Proc.Natl.Acad.Sci.U.S.A.84:214-218; Nishimura etc. (1987) Canc.Res.47:999-1005; Wood etc. (1985) Nature 314:446-449; With (1988) J.Natl CancerInst.80:1553-1559 such as Shaw).

[0131] means known in the art be can pass through, antibody or immunoglobulin chain humanization made.Can be equal to sequence by personnel selection Fv variable region and replace the sequence of not participating in the bonded Fv of antigen variable region directly, and produce humanized antibody (comprising the Humanized immunoglobulin chain).The universal method that produces humanized antibody is provided by following document: Morrison, S.L. (1985) Science229:1202-1207, Oi etc. (1986) BioTechniques 4:214 and Queen etc., United States Patent (USP) the 5th, 585,089,5,693,761 and 5,693, No. 762, the content of described document all is attached to herein by reference.These methods comprise separation, manipulation and the expression of nucleotide sequence of all or part of IgF v variable region of coding at least one heavy chain or light chain.To those skilled in the art, the source of described nucleic acid is well-known, for example can derive from the hybridoma of generation at the antibody of predetermined target.Then, coding humanized antibody or its segmental recombinant DNA can be cloned in the suitable expression vector.

[0132] can transplant or CDR replaces by CDR, preparation humanization or CDR grafted antibody molecule or immunoglobulin, one of them, the CDR of two or all immunoglobulin chains can be substituted (referring to No. the 5th, 225,539, United States Patent (USP) for example; Jones etc. (1986) Nature 321:552-525; Verhoeyan etc. (1988) Science 239:1534; Beidler etc. (1988) J.Immunol.141:4053-4060; Winter, United States Patent (USP) the 5th, 225, No. 539, the content of all these documents all is attached to herein by reference.Winter has described the CDR implantation method, and described method can be used for preparing humanized antibody of the present invention (the UK Patent Application GB 2188638A of application on March 26th, 1987; Winter, United States Patent (USP) the 5th, 225, No. 539, the content of described document is attached to herein by reference).All CDR of specific people's antibody can be replaced by at least a portion non-human CDR, perhaps only have portion C DR to be replaced by non-human CDR.Only be necessary to replace humanized antibody combines required CDR with predetermined antigen quantity.

[0133] monoclonal antibody, chimeric antibody and the humanized antibody of modifying by disappearance, interpolation or replacement antibody other parts (for example constant region) is also included within the scope of the invention.For example, antibody can be modified by the following method: (i) disappearance constant region; (ii) replace original constant region with another constant region, described another constant region for example can increase the constant region of antibody half life, stability or affinity, and perhaps described constant region is from another species or antibody classification; Or (iii) modify one or more aminoacid in the constant region, with the number of Change Example such as glycosylation site, effector cell function, Fc receptor (FcR) combination, complement fixation etc.

[0134] method of change antibody constant region is known in the art.Can be by replacing at least one amino acid residue with a different residue at the antibody constant portion, generation has the antibody of changing function (for example the affinity of effect part such as FcR on the pair cell or complement C1 component changes) (referring to for example E.P.388,151A1, U.S.5,624,821 and U.S.5,648,260, the content of described document all is attached to herein by reference).When being used for the immunoglobulin of Mus or other species, similarly changing form and to reduce or to eliminate these functions.

[0135] for example, can specify residue by replacing with the residue that has appropriate functional group on the side chain, perhaps by introducing charged functional group (for example glutamic acid or aspartic acid, perhaps aromatics non-polar residue, for example phenylalanine, tyrosine, tryptophan or alanine), the Fc district that changes antibody (for example IgG, for example human IgG) to FcR (for example Fc γ R1) or to the bonded affinity of C1q (referring to for example U.S.5,624,821).

[0136] the IL-21 amino acid sequence of polypeptide is known.For example, human IL-2 1 nucleotide sequence and aminoacid sequence can derive from GENBANK ^Searching number X_011082.Disclosed human IL-2's 1 nucleotide sequence is as follows:

1 gctgaagtga aaacgagacc aaggtctagc tctactgttg gtacttatga gatccagtcc

61 tggcaacatg gagaggattg tcatctgtct gatggtcatc ttcttgggga cactggtcca

121 caaatcaagc tcccaaggtc aagatcgcca catgattaga atgcgtcaac ttatagatat

181 tgttgatcag ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga

241 agatgtagag acaaactgtg agtggtcagc tttttcctgc tttcagaagg cccaactaaa

301 gtcagcaaat acaggaaaca atgaaaggat aatcaatgta tcaattaaaa agctgaagag

361 gaaaccacct tccacaaatg cagggagaag acagaaacac agactaacat gcccttcatg

421 tgattcttat gagaaaaaac cacccaaaga attcctagaa agattcaaat cacttctcca

481 aaagatgatt catcagcatc tgtcctctag aacacacgga agtgaagatt cctgaggatc

541 taacttgcag ttggacacta tgttacatac tctaatatag tagtgaaagt catttctttg

601 tattccaagt ggaggag(SEQ ID NO：18)

[0137] disclosed human IL-2's 1 amino acid sequence of polypeptide is as follows:

MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPED

VETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKP

PKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO：19)

[0138] the present invention also comprises such nucleic acid: described nucleic acid is hybridized with the nucleotide shown in SEQ ID NO:1, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or the SEQ ID NO:38 down in height stringency (for example 0.1X SSC, 65 ℃).The present invention also comprises the isolating polynucleotide of coding MU-1 albumen or fusion rotein, but described polynucleotide are different from the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or the SEQ ID NO:38 because of the genetic code degeneracy.The present invention also comprises because of point mutation or introduces and modify the variation in the nucleotide sequence shown in the following sequence that is caused: SEQ ID NO:1, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 or SEQ ID NO:38.

[0139] the isolating polynucleotide of the present invention can with expression control sequenc ((1991) Nucleic Acids Res.19 such as Kaufman for example, disclosed pMT2 expression vector or pED expression vector among the 4485-4490) operability connects, so that generation reorganization MU-1 albumen.Many suitable expression control sequencs are known in the art.The universal method of express recombinant protein also is known, for example referring to R.Kaufman (1990) Methods in Enzymology 185,537-566." operability connection " defined herein is meant that the isolating polynucleotide of the present invention and expression control sequenc are connected or chemistry is connected to form covalent bond through enzymatic, its mode makes MU-1 albumen by host cell expression, and described cell has used the polynucleotide/expression control sequenc that is connected to transform (transfection).

[0140] term used herein " carrier " is meant the nucleic acid molecules that can transport connected another nucleic acid molecules.One class carrier is " plasmid ", and it is the circular double stranded DNA ring, wherein can connect other DNA section.Another kind of carrier is a viral vector, and wherein other DNA section can be connected in the viral genome.Some carrier can self-replicating (bacteria carrier and the additive type mammal carrier that for example have the antibacterial origin of replication) behind the host cell that imports them.Other carrier (for example non-add type mammal carrier) can be incorporated in the host cell gene group after importing host cell, thereby can duplicate and duplicate with host genome.In addition, some carrier can instruct the expression of gene that is connected with its operability.This class carrier is referred to herein as " recombinant expression carrier " (or abbreviation " expression vector ").Generally speaking, the expression vector that is used for recombinant DNA technology is the plasmid form usually.In this manual, " plasmid " and " carrier " is used interchangeably, because plasmid is the most frequently used carrier format.Yet, the present invention includes the expression vector of other form, viral vector (for example replication defect type retrovirus, adenovirus and adeno associated virus) for example, they exercise identical functions.

[0141] term " adjusting sequence " comprises promoter, enhancer and other expression control element (for example polyadenylation signal), its control antibody chain gene transcription or translation.This class is regulated sequence for example referring to Goeddel (1990) Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, CA.It will be understood to those of skill in the art that the design of expression vector, comprise the selection of regulating sequence, all depend on the selection of host cell to be transformed, the factors such as expression of desirable proteins.Expression for mammalian host cell, the preferred sequence of regulating is included in the viral element that instructs the albumen high level expression in the mammalian cell, for example from following promoter and/or enhancer: FF-1a promoter and BGH poly A, cytomegalovirus (CMV) (for example CMV promoter/enhancer), simian virus 40 (SV40) (for example SV40 promoter/enhancer), adenovirus (for example adenovirus major late promoter (AdMLP)) and polyoma virus.Further describing of relevant viral regulating element and sequence thereof can be referring to No. the 4th, 968,615, the United States Patent (USP) of No. the 4th, 510,245, the United States Patent (USP) of No. the 5th, 168,062, the United States Patent (USP) of for example Stinski, Bell etc. and Schaffner etc.

[0142] recombinant expression carrier portability additional sequences of the present invention is for example regulated sequence (for example origin of replication) and the selected marker that carrier duplicates in host cell.The selected marker helps selecting wherein to import the host cell (referring to the United States Patent (USP) the 4th, 399,216,4,634,665 and 5,179 of for example Axel etc., No. 017) of carrier.For example, the selected marker in the host cell that imports carrier has Drug resistance usually, and described medicine is G418, hygromycin or methotrexate for example.Preferred selected marker comprises dihydrofolate reductase (DHFR) gene and (is used for dhfr ^-Host cell is with methotrexate selection/amplification) and neo gene (being used for G418 selects).

[0143] cell of many types can be used as proper host cell, is used to express MU-1 albumen or its fusion rotein.Can all can use by the proteic any cell type of expressive function MU-1.Suitable mammalian host cell comprises for example monkey COS cell, Chinese hamster ovary (CHO) cell, people's kidney 293 cell, people's epidermis A431 cell, people Colo205 cell, the 3T3 cell, the CV-1 cell, the primate cell system of other conversion, normal diploid cell, cell strain from former generation vitro tissue culture, former generation explant, the HeLa cell, mouse Lcell, BHK, HL-60, U937, HaK, Rat2, BaF3,32D, FDCP-1, PC12, M1x or C2C12 cell.

[0144] also can be by in one or more insecticide expression vectors, the isolating polynucleotide of the present invention are connected use insect expression system, generation MU-1 albumen or its fusion rotein with suitable control sequence operability.The materials and methods that is used for baculovirus/insect cell expression system is commercially available with kit form, derives from for example Invitrogen, San Diego, Calif.U.S.A. (MAXBAC ^Test kit), described method is well-known in the art, is described in Summers and Smith, Texas Agricultural Experiment StationBulletin No.1555 (1987), and described document is attached to herein by reference.The also available aforesaid suitable isolating polynucleotide of the proteic soluble form of MU-1 produce in insect cell.

[0145] or, MU-1 albumen or its fusion rotein can produce in prokaryotes such as lower eukaryotes such as yeast or antibacterial.Suitable yeast strain comprises saccharomyces cerevisiae (Saccharomyces cerevisiae), schizosaccharomyces pombe (Schizosaccharomycespombe), Crewe Vickers Saccharomyces (Kluyveromyces) bacterial strain, mycocandida (Candida) or the proteic any yeast strain of energy expressing heterologous.Suitable bacterial isolates comprises escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonella typhimurium) or the proteic any bacterial isolates of energy expressing heterologous.

[0146] in antibacterial, expresses, cause forming the inclusion body that contains recombiant protein.Therefore, need carry out refolding, activity or active higher material be arranged so that produce to recombiant protein.The certain methods of correctly obtaining folding heterologous protein from the antibacterial inclusion body is known in the art.These methods generally include the described albumen of stripping from inclusion body, make the complete degeneration of albumen with chaotropic agent then.When having cysteine residues in the proteinic one-level aminoacid sequence, it need finish refolding usually in the environment (oxidation-reduction system) that allows correct formation disulfide bond.The universal method of refolding is disclosed in Kohno (1990) Meth.Enzym., 185:187-195; E.P.0433225 and United States Patent (USP) have been described other suitable method the 5th, 399, No. 677.

[0147] MU-1 albumen or its fusion rotein can also be expressed as the product of transgenic animal, for example be expressed as the milk composition of immunocow, goat, pig or sheep, the feature of described transgenic animal is the polynucleotide sequence that contains coding MU-1 albumen or its fusion rotein in somatic cell or the sexual cell.

[0148] can be by under the essential condition of culture of expression desirable proteins, cultivating transformed host cells, preparation MU-1 albumen or its fusion rotein.The expressing protein of gained can come out by purification from culture medium or cell extract then.The soluble form of MU-1 albumen or its fusion rotein can come out by purification from conditioned medium.Can be by the total film component of preparation from express cell and with nonionic detergent (TRITON for example ^X-100) extract film, the proteic film combining form of purification MU-1 of the present invention.

[0149] can adopt method known to those skilled in the art, purification MU-1 albumen or fusion rotein.For example, MU-1 albumen of the present invention can concentrate filter (AMICON for example with commercially available albumen ^Or PELLICON ^Ultra filtration unit, Millipore, Billerica MA) concentrates.After the concentration step, concentrated solution can be added on the purification substrate example gel filter medium.Perhaps, can use anion exchange resin, for example, have diethyllaminoethyl (DEAE) or polyethylene imine based (polyetheyleneimine, PEI) substrate of side group or substrate.These substrate can be acrylamide, agarose, glucosan, cellulose or other kind that is generally used for protein purification.Perhaps, can adopt cation-exchange step.Suitable cationite comprises the various insoluble matrixs that contain sulfo group propyl group or carboxyl methyl.Preferred sulfo group propyl group (S-SEPHAROSE for example ^Post).Purification MU-1 albumen or fusion rotein from culture supernatant also can comprise one or more post step, for example concanavalin A-agarose, heparin-TOYOPEARL of crossing ^Or Cibacron blue 3GA SEPHAROSE ^Etc. affine resin; Or by hydrophobic interaction chromatography, with resins such as phenyl ether, butyl ether or propyl ethers; Or pass through immunoaffinity chromatography.At last, can use one or more RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC) step,, for example have the silica gel of methyl or other aliphatic side group, be further purified MU-1 albumen with hydrophobic RP-HPLC medium.The affinity column that comprises anti-MU-1 protein antibodies also can be used for carrying out purification according to known method.The part steps of above-mentioned purification step or Overall Steps make up with various combination or with other known method, also can be used for providing the isolating recombiant protein of purification basically.The MU-1 albumen of preferable separate is purification, makes it be substantially free of other mammalian proteins.

[0150] MU-1 albumen of the present invention or fusion rotein also can be used for the screening can with the bonded medicine of MU-1.With required conjugated protein (no matter whether immobilization) be well-known in the art in conjunction with measuring, can be used for using proteic this purpose of MU-1 of the present invention.Can be used for identifying described medicine based on purifying cells or based on the screening test of albumen (acellular).For example, MU-1 albumen can be fixed on the carrier by purified form, can measure proteic binding partner of purification MU-1 or potential part.

Pharmaceutical composition

[0151] the IL-21/IL-21R-antagonist is when with after the medicine acceptable carrier mixes, the useful as drug compositions.Except IL-21/IL-21R-antagonist and carrier, described compositions also can contain various diluent, filler, salt, buffer agent, stabilizing agent, solubilizing agent and other material well-known in the art.Term " pharmaceutically acceptable " is meant non-toxic material, and it is the effect of the biologic activity of interferon activity composition not.The characteristic of carrier depends on route of administration.

[0152] pharmaceutical composition of the present invention can be the liposome form, wherein the IL-21/IL-21R-antagonist except with other pharmaceutically acceptable carrier coupling, also with amphiphilic species (for example have lipid, the liquid crystal that becomes micelle, insoluble monolayer or in aqueous solution, be sheet form) coupling with aggregated forms.The suitable lipid that is used for Liposomal formulation includes but not limited to monoglyceride, diglyceride, sulfur cerebroside ester, LYSOLECITHIN SUNLECITHIN A, phospholipid, saponin, bile acid etc.The preparation of this lipoid plastid preparation is in those skilled in the art's horizontal extent, and is and open in following document: for example United States Patent (USP) the 4th, 235, and 871,4,501,728,4,837,028 and 4,737, No. 323, all these documents all are attached to herein by reference.

[0153] term used herein " treatment effective dose " is meant the total effective dose that is enough to bring each active component of the pharmaceutical composition of useful benefit or method to the patient, and described benefit is for example improved symptom, cures described disease or improved its cure rate.When being used for the single-activity composition and giving separately, this term is meant the consumption of the one-component that produces curative effect.When being used for coupling, this term is meant the total amount of the active component that produces curative effect, no matter is associating, sequential or give simultaneously.

[0154] in the practice of Therapeutic Method of the present invention or purposes, gives the patient with the IL-21/IL-21R-antagonist for the treatment of effective dose, for example mammal (for example people).The IL-21/IL-21R-antagonist can according to method of the present invention single with or unite with the other therapies of following detailed description and to give.When with one or more medication combined giving, IL-21-and/or IL-21R-antagonist can with second medicine simultaneously or sequential giving.If sequential administration, the attending doctor will determine to give the proper order of IL-21/IL-21R-antagonist and other medicines.

[0155] can be used for pharmaceutical composition or implement the used IL-21/IL-21R-antagonist of method of the present invention, for example oral absorption of described approach, suction or skin, subcutaneous or intravenous injection by various conventional route.Preferred intravenous gives the patient.

[0156] when the treatment IL-21/IL-21R-agonist of effective dose or antagonist are the oral administration administration, described bonding agent will be the form of tablet, capsule, powder, solution or elixir.When with the tablet administration, pharmaceutical composition of the present invention also can contain solid carrier, for example gelatin or adjuvant.Tablet, capsule and powder contain the bonding agent of the 5-95% that has an appointment, preferably contain the bonding agent of the 25-90% that has an appointment.When with the liquid form administration, can add liquid-carrier, for example water, oil, animal oil or vegetable oil, for example Oleum Arachidis hypogaeae semen, mineral oil, soybean oil or Oleum sesami, or artificial oil.The liquid form of pharmaceutical composition also can contain normal saline, glucose or other sugar juice, or glycol, for example ethylene glycol, propylene glycol or Polyethylene Glycol.When with the liquid form administration, pharmaceutical composition contains the bonding agent of the 0.5-90% that has an appointment (weight), preferably the bonding agent of about 1-50%.

[0157] the IL-21/IL-21R-antagonist when the treatment effective dose is when intravenous, skin or subcutaneous injection give, and described bonding agent will be no thermal source, the acceptable aqueous solution form of parenteral.The preparation of the acceptable protein solution agent of described parenteral with suitable pH, isotonicity, stability etc. is in the art technology scope.Except bonding agent, the preferred pharmaceutical compositions of using for intravenous, skin or subcutaneous injection such as also should contain at vadose solution matchmaker, for example sodium chloride injection, ringer's injection, glucose injection, dextrose ﹠ sodium chloride injection, lactated Ringer's injection or other solvent known in the art.Pharmaceutical composition of the present invention also can contain stabilizing agent, antiseptic, buffer agent, antioxidant or other additive well known by persons skilled in the art.

[0158] consumption of IL-21/IL-21R-antagonist will depend on the character and the order of severity of the disease for the treatment of in the pharmaceutical composition of the present invention, and the character of the treatment once accepted in the past of this patient.Finally, the attending doctor will determine to be used for the treatment of the consumption of each patient's bonding agent.Beginning, the attending doctor will give the bonding agent of low dosage, and observe reaction.Can give more heavy dose of bonding agent, reach optimum therapeuticing effect, and no longer continue to increase dosage usually at this point up to described patient.The different pharmaceutical compositions that is used for the practice of the inventive method should contain the 0.1 μ g that has an appointment to about 100mg IL-21/IL-21R-antagonist/kg body weight.

[0159] carrying out persistent period of intravenous therapy with pharmaceutical composition of the present invention will be different, depends on the order of severity of the disease for the treatment of and each patient's situation and potential specific reaction.Each persistent period scope of using the IL-21/IL-21R-antagonist will be at 12-24 hour continuous intravenous administration.At last, the attending doctor will determine the suitable persistent period of the intravenous therapy of pharmaceutical composition of the present invention.

[0160] polynucleotide of the present invention show one or more purposes or the biological activity (comprising relevant those purposes or the biological activity of quoting with this paper of test) that this paper identifies with protein.By giving or use described protein or giving or use the polynucleotide (for example in gene therapy or be suitable for importing in the carrier of DNA) of code for said proteins, can provide proteinic purposes of the present invention or activity.

Use the IL-21/IL-21R antagonist to reduce immunologic cellular activity

[0161] aspect another, the invention is characterized in the method that suppresses immunologic cellular activity, described immunocyte is mature T cells (ripe CD8 for example ⁺T cell, ripe CD4 ⁺The T cell), ripe NK cell, B cell, macrophage and megalokaryocyte or its colony, described method comprises: make the IL-21/IL-21R antagonist of T cell colony contact q.s, to suppress the activity of immunocyte or colony.Also the antagonist (fusion rotein for example as herein described or neutralizing antibody) of IL-21 and/or IL-21R can be wished to suppress the patient of immunne response.These diseases or disease comprise for example autoimmune disease (for example arthritis, RA, IBD), SLE, asthma, glomerulonephritis, psoriasis or graft/organ transplantation (and relevant repulsion).

[0162] applicant knows, by in using and fusion rotein (fusion rotein that for example comprises the IL-21R extracellular domain that merges with the Fc immunoglobulin domain) reduction IL-21R activity, in some animal models, improved inflammatory symptoms, described model is collagen-induced arthritis (CIA) animal model (embodiment 7) for example, and the animal model of Crohn disease, ulcerative colitis and IBD (embodiment 9 and 11), the animal model (embodiment 13) of transplant rejection (embodiment 10), psoriasis (embodiment 11) and lupus.In CIA mice claw, (embodiment 8) are raised in the expression of IL-21R mRNA.In the airway inflammation of antigen induced, the IL-21R deficient mice shows symptom light (embodiment 12).Therefore, the active IL-21R bonding agent of antagonism IL-21/IL-21R can be used in vivo, and induction of immunity suppresses, for example be used for the treatment of or pathology that the epidemic prevention cell is relevant, comprise autoimmune disease (for example arthritis, RA, IBD), SLE, glomerulonephritis, asthma, psoriasis or graft/organ transplantation.

[0163] also IL-21R DNA is mapped on the chromogene seat of Crohn disease, therefore, provide extra support for using the IL-21/IL-21R antagonist to treat Crohn disease and other inflammatory bowel.

[0164] described method also can be used for regulating (for example suppressing) immunologic cellular activity (for example breed, break up, survive), therefore can be used for treatment or prevents various immune diseases.The limiting examples of the disease that can treat or prevent includes but not limited to transplant rejection, autoimmune disease (comprises for example diabetes, arthritis (comprises RA, teenager RA, osteoarthritis (OA), psoriasis arthropathica), multiple sclerosis, encephalomyelitis, myasthenia gravis, SLE, glomerulonephritis, autoimmune thyroiditis, dermatitis (comprising atopic dermatitis and eczematoid dermatitis), (for example UV damages associated conditions for psoriasis and relevant dermatosis, for example light decay is old, atopic dermatitis, cutaneous T cell lymphoma is mycosis fungoides for example, anaphylaxis and irritant contact dermatitis, lichen planus, alopecia areata, vitiligo, eye cicatricial pemphigoid and urticaria), xerodermosteosis (Sj  gren ' s syndrome), Crohn disease, aphthous ulcer, iritis, ulcerative colitis, the spondylosis arthrosis, ankylosing spondylitis, intrinsic asthma, allergic asthma, chronic obstructive pulmonary disease (COPD), interstitial pulmonary fibrosis, the skin lupus erythematosus, scleroderma, drug eruption, the autoimmunity uveitis, allergic encephalitis, Wegner granulomatosis (Wegener ' s granulomatosis), hepatitis, Stevens-Johnson syndrome (Stevens-Johnson syndrome), the special property sent out sprue, Graves disease (Graves ' disease), sarcoidosis, hepatic fibrosis, primary biliary cirrhosis, posterior uveitis (uveitis posterior), graft versus host disease and allergy, for example atopic allergy.The disease that available IL-21/IL-21R antagonist is treated preferably includes arthritis (for example RA, teenager RA, OA, psoriasis arthropathica and ankylosing spondylitis (preferred class rheumatic arthritis)), multiple sclerosis, type i diabetes, lupus (SLE), IBD (Crohn disease, ulcerative colitis), asthma, vasculitis, allergy, scleroderma, glomerulonephritis and psoriasis.

[0165] in another embodiment, IL-21/IL-21R antagonist list with or with other curative as herein described (for example TNF antagonist) coupling, can be used for treating multiple myeloma and relevant bone-marrow-derived lymphocyte malignant tumor (Brenne etc. (2002) Blood 99 (10): 3756-3762).

[0166] use the IL-21/IL-21R antagonist can regulate immunne response in many ways.Downward modulation can be to suppress or blocking-up has begun the mode of the immunne response of carrying out, and perhaps can participate in preventing inducing immunne response.Can reply or, suppress the function of activating T cell by suppressor T cell by luring T cell-specific toleration or the two.The immunosuppressant of t cell response is normally active, the process of non-antigenic specificity, and this process need makes the T cell continue to be exposed to inhibitor.Toleration (being inducing T cell no response or anergy) and immunosuppressant difference are: toleration is antigenic specificity normally, and still can exist lastingly after stopping to be exposed to the tolerance agent.From operation, after the tolerance agent is exposed to specific antigen when not existing once more, still lack t cell response, provable thus toleration.

[0167] downward modulation or prevention immunologic function are for example used the IL-21/IL-21R antagonist, will be used under the situation of tissue, skin and organ transplantation and graft versus host disease (GVHD).For example, the suppressor T cell function can reduce disorganization in tissue transplantation.Usually in tissue transplantation, transplant rejection begins because of the T cell is identified as the xenobiotics with graft, is the immunoreation that destroys graft with that.Give IL-21/TL-21R antagonist, no matter be before transplanting, during or use separately afterwards or with suppress or block the interactional molecule coupling of other immunoeffectors, all can be used for reducing immunne response.

[0168] effect of available animal model evaluation IL-21/IL-21R antagonist in prevention of organ transplant rejection or GVHD, such animal model can be used for prediction at intravital effect of people and dosage.The example of spendable appropriate system comprises allogeneic heart transplantation of rat and the xenogenic islets transplanting of mice, the two all is used for detecting in vivo the immunosuppressive effect of CTLA4 Ig fusion rotein, referring to (1992) Proc.Natl.Acad.Sci U.S.A. such as (1992) Science 257:789-92 such as Lenschow and Turka, 89:11102-05.Also can for example in mouse model, estimate the IL-21/IL-21R antagonist at other animal model of vascularization cardiac allograft and through thickness skin allograft.These models can be measured tissue rejection (described tissue has complete MHC mispairing) and the IL-21 blocking-up can be transferred with the donor specific lymphocyte and combine.In addition, the mouse model of GVHD (referring to for example Paul (writing), Fundamental Immunology, Raven Press, New York (1989) 846-47 pages or leaves) can be used for determining that the IL-21/IL-21R antagonist is in vivo to GVHD or SLE Influence and Development.Also can estimate IL-21/IL-21R antagonist and the effect of other curative coupling in prevention of organ transplant rejection or GVHD, described curative is immunosuppressant for example, for example rapamycin, cyclosporin or CTLA4Ig.

[0169] the IL-21/IL-21R antagonist also treatability be used for the treatment of autoimmune disease.Thereby the cause of many autoimmune diseasees is cytokine and autoantibody that the activation of T cellular abnormality and autologous tissue's reaction and promotion produce involved in diseases pathology.Prevent the autoreactive T cell activation, can reduce or eliminate disease symptoms.Give IL-21/IL-21R antagonist, separately or with other medicines (medicine for example as herein described) coupling, can be used for suppressor T cell activation and prevention and produce autoantibody or the cell-derived cytokine of T, but these antibody or cytokine involved in diseases process.In addition, IL-21/IL-21R antagonist list with or with other medicines (medicine for example as herein described) coupling, increased autoreactive T cell the antigenic specificity toleration and can long-term remission disease.Can use the animal model of the various human autoimmune diseases that clearly characterize, measure the effect of these medicines in prevention or alleviation autoimmune disease.Example comprises Mus experimental autoimmune encephalomyelitis, the systemic lupus erythematosus (sle) of MRL/lpr/lpr mice or NZB hybridize mice, the Mus autoimmunity collagen induced arthritis of NOD mice and BioBreeding rat, diabetes, and the experimental myasthenia gravis of Mus is (referring to for example Paul (writing), Fundamental Immunology, Raven Press, New York (1989) 840-56 pages or leaves).

[0170] in one embodiment, IL-21/IL-21R antagonist, for example its pharmaceutical composition, in conjoint therapy, give, promptly with for example curative coupling of other medicines, described medicine is used for the treatment of pathological disorders or disease, for example immune disease and inflammatory diseases.Term " coupling " in this article refers to basically simultaneously (together or sequential) administration.If sequential administration, when giving second kind of chemical compound, first kind in preferred two kinds of chemical compounds still has detectable valid density in therapentic part or patient's body.

[0171] for example, conjoint therapy can comprise that one or more IL-21/IL-21R antagonisies are (for example at the antibody of IL-21 or IL-21 receptor or its Fab (chimeric antibody for example, humanized antibody, the antibody of people's antibody or external generation or its Fab), the IL-21 fusion rotein, solubility IL-21 receptor, inhibitor peptides or micromolecular inhibitor), prepare and/or administration together with one or more other curatives, described other curative is one or more cytokine inhibitors and growth factor receptor inhibitors in greater detail in this article for example, immunosuppressant, anti-inflammatory agent, metabolic poison, enzyme inhibitor and/or cytotoxic agent or cytostatics.In addition, one or more IL-21/IL-21R antagonisies as herein described can be used for and two or more curative couplings as herein described.Described conjoint therapy can be advantageously used in and give the low dose therapy medicine, thereby avoids possible toxicity or the relevant complication of each monotherapy.In addition, curative action pathway disclosed herein is different with the IL-21/IL-21R receptor pathway, thereby is expected to strengthen the effect of IL-21/IL-21R antagonist and/or produces synergism with the IL-21/IL-21R antagonist.

[0172] be that those disturb not mutually medicine simultaneously of autoimmune and inflammatory reaction subsequently with the preferred curative of IL-21/IL-21R antagonist coupling.In one embodiment, one or more IL-21/IL-21R antagonisies as herein described can be prepared and/or administration together with one or more other curatives; Described curative is other cytokine antagonist or growth factor antagonist (for example soluble recepter, inhibitor peptides, micromolecule, part fusions) for example; Or with the bonded antibody of other target or its Fab (for example with other cytokine or somatomedin, its receptor or the bonded antibody of other cell surface molecule); With anti-inflammatory cytokines or its agonist.Can include but not limited to the antagonist of one or more interleukins (IL) or its receptor with the limiting examples of the medicine of IL-21/IL-21R antagonist described herein coupling, for example the antagonist of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18 and IL-22; The antagonist of cytokine or somatomedin or its receptor, for example antagonist of tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGF and PDGF.The IL-21/IL-21R antagonist also can with the inhibitor coupling at antibody of following cell surface molecule etc.: CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90 or their part comprise CD154 (gp39 or CD40L) or LFA-1/ICAM-1 and VLA-4/VCAM-1 (Yusuf-Makagiansar etc. (2002) Med.Res.Rev.22 (2): 146-67).The antagonist that can comprise IL-1, IL-6, IL-12, TNF α, IL-15, IL-17, IL-18 and IL-22 with the preferred antagonist of IL-21/IL-21R antagonist as herein described coupling.

[0173] example of these medicines comprises the IL-12 antagonist, for example with the antibody (or its Fab) of the bonded chimeric antibody of IL-12 (preferred people IL-12), humanized antibody, people's antibody or external generation, described antibody is for example at WO 00/56772, disclosed antibody among the GeneticsInstitute/BASF; The IL-12 acceptor inhibitor, the antibody of for example anti-people IL-12 receptor; Soluble fragments with IL-12 receptor (for example people IL-12 receptor).The example of IL-6 antagonist comprises at the antibody of IL-6 or its receptor (or its Fab), for example at the antibody of chimeric antibody, humanized antibody, people's antibody or the external generation of people IL-6 or its receptor, the soluble fragments and the IL-6 of IL-6 receptor are conjugated protein.The example of IL-15 antagonist comprises such antibody: at the antibody (or its Fab) of IL-15 or its receptor, for example at the antibody of chimeric antibody, humanized antibody, people's antibody or the external generation of human IL-15 or its receptor, the soluble fragments and the IL-15 of IL-15 receptor are conjugated protein.The example of IL-18 antagonist comprises such antibody: for example at the antibody (or its Fab) of chimeric antibody, humanized antibody, people's antibody or the external generation of human il-18, the soluble fragments and the IL-18 of IL-18 receptor conjugated protein (IL-18BP, Mallat etc. (2001) Circ.Res.89:e41-45).The example of IL-1 antagonist comprises for example Vx740, IL-1 antagonist IL-1RA (ANIKINRA for example of il-1 invertase (ICE) inhibitor ^TM, Amgen), sIL1RII (Immunex) and anti-IL-1 receptor antibody (or its Fab).

[0174] example of TNF antagonist comprises the antibody (or its Fab) at chimeric antibody, humanized antibody, people's antibody or the external generation of TNF (for example human TNF alpha), for example D2E7 (human TNF alpha antibody, U.S.6,258,562; BASF), CDP-571/CDP-870/BAY-10-3356 (humanization anti-TNF alpha antibodies; Celltech/Pharmacia), cA2 (chimeric anti-TNF alpha antibodies; REMICADE ^TM, Centocor); Anti-TNF antibodies fragment (for example CPD870); The soluble fragments of TNF receptor, for example p55 or p75 people TNF receptor or derivatives thereof, for example 75kdTNFR-IgG (75kD TNF receptor-IgG fusion rotein, ENBREL ^TMImmunex; Referring to for example Arthritis ﹠amp; Rheumatism (1994) the 37th volume, S295; J.Invest.Med. (1996) the 44th roll up, 235A), p55kdTNFR-IgG (55

KD TNF receptor-IgG fusion rotein (Lenercept)); Enzyme antagonist, for example TNF α invertase (TACE) inhibitor (for example alpha sulfonyl hydroxamic acid derivatives, WO 01/55112 and N-hydroxyformamide tace inhibitor GW 3333 ,-005 or-022); And TNF-bp/s-TNFR (soluble TNF is conjugated protein; Referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S284; Amer.J.Physiol.-Heart and CirculatoryPhysiology (1995) the 268th volume, the 37-42 page or leaf).Preferred TNF antagonist is the soluble fragments of TNF receptor, for example p55 or p75 people TNF receptor or derivatives thereof, for example 75kdTNFR-IgG and TNF α invertase (TACE) inhibitor.

[0175] in other embodiments, IL-21-/IL-21R antagonist as herein described can with one or more following drug combinations: IL-13 antagonist, for example solubility IL-13 receptor (sIL-13) and/or anti-il-13 antibody; IL-2 antagonist, for example DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion rotein; Seragen; Referring to for example Arthritis ﹠amp; Rheumatism (1993) the 36th volume, 1223), and/or anti-IL-2R antibody, for example anti-Tac (the anti-IL-2R of humanization; Protein Design Labs, Cancer Res.1990 March 1; 50 (5): 1495-502).Another kind of conjoint therapy comprises IL-21 antagonist and non-expendable anti-CD4 inhibitor (IDEC-CE9.1/SB 210396 (non-expendable sensitization (primatized) anti-CD 4 antibodies; IDEC/SmithKline) coupling.Some preferred combination medicines comprise common stimulation pathway antagonists CD80 (B7.1) or CD86 (B7.2) again, and described antagonist comprises antibody, soluble recepter or antagonism part; And p-selects protein sugar protein ligands (PSGL), anti-inflammatory cytokines, for example IL-4 (DNAX/Schering); (SCH 52000 for IL-10; Recombinant il-10 DNAX/Schering); IL-13 and TGF, and agonist (for example agonist antibody).

[0176] in other embodiments, one or more IL-21/IL-21R antagonisies can be prepared and/or give together with one or more anti-inflammatory agents, immunosuppressant or metabolic poison or enzyme inhibitor.Can include but not limited to one or more following medicines with the medicine of IL-21 antagonist combination use as herein described or the limiting examples of inhibitor: NSAID (non-steroidal anti-inflammatory drug) (NSAID), for example ibuprofen, tenidap are (referring to for example Arthritis ﹠amp; Rheumatism (1996), the 39th volume, the 9th phase (supplementary issue), S280)), naproxen (referring to for example Neuro Report (1996) the 7th volume, 1209-1213 page or leaf), meloxicam, piroxicam, diclofenac and indomethacin; Sulfasalazine is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S281); Corticosteroid, for example prednisolone; Cell factor inhibiting anti-inflammatory agent (CSAID); The nucleotide biosynthesis inhibitor, for example purine biosynthesis inhibitor, antifol (for example methotrexate (N-[4-[[(2,4-diaminourea-6-pteridyl) methyl] methylamino] benzoyl]-L-glutamic acid); With the pyrimidine biosynthesis inhibitor, for example (for example leflunomide is (referring to for example Arthritis ﹠amp for dihydroorate dehydrogenase (DHODH) inhibitor; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S131; Inflammation Research (1996) the 45th volume, the 103-107 page or leaf).Be used for drawing together NSAID, CSAID, (DHODH) inhibitor (for example leflunomide) and antifol (for example methotrexate) with the preferred therapeutic medicated bag of IL-21/IL-21R antagonist coupling.

[0177] example of other inhibitor comprises one or more following medicines: corticosteroid (oral, suction and local injection); Immunosuppressant, for example cyclosporin, tacrolimus (FK-506); With the mTOR inhibitor, for example sirolimus (rapamycin) or rapamycin derivative, solubility rapamycin derivative (for example rapamycin ester derivant, for example CCI-779 (Elit. (2002) Current Opinion Investig.Drugs 3 (8): 1249-53 for example; Huang etc. (2002) Current Opinion Investig.Drugs 3 (2): 295-304); Disturb the medicine of proinflammatory cytokine signal transduction, described proinflammatory cytokine is TNF α or IL-1 (for example IRAK, NIK, IKK, p38 or map kinase inhibitor) for example; The COX2 inhibitor, for example celecoxib and variant thereof, MK-966 is referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S81); Phosphodiesterase inhibitor, for example R973401 (phosphodiesterase IN type inhibitor; Referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S282)); Inhibitor of phospholipase enzymes, for example cPLA2 2 (cPLA2) inhibitor (for example fluoroform keto analog (U.S.6,350,892)); The inhibitor of vascular endothelial cell growth factor or growth factor receptors, for example VEGF inhibitor and/or VEGF-R inhibitor; And angiogenesis inhibitor.Comprise immunosuppressant with the preferred curative of IL-21/IL-21R antagonist coupling, for example cyclosporin, tacrolimus (FK-506); With the mTOR inhibitor, for example sirolimus (rapamycin) or rapamycin derivative, for example solubility rapamycin derivative (for example rapamycin ester derivant, for example CCI-779; COX2 inhibitor, for example celecoxib and variant thereof; And inhibitor of phospholipase enzymes, for example cPLA2 2 (cPLA2) inhibitor (for example fluoroform keto analog).

[0178] example of other curative that can use with the IL-21/IL-21R antagonist combination comprises one or more following medicines: Ismipur (6-MP); Azathioprine sulfasalazine (sulphasalazine); Mesalazine; Olsalazine chloroquine/oxychloroquine; Penicillamine; Gold sulfur fourth salt (aurothiornalate) (intramuscular and oral); Azathioprine; Colchicine (colchicine); Beta-2-adrenoceptor agonist (albuterol, terbutaline, salmaterol (salmeteral)); Xanthine (theophylline, aminophylline); Cromoglycate; Nedocromil; Ketotifen; Different third atropic ammonium and oxygen holder ammonium; Mycophenolate mofetil; Adenosine agonists; Antithrombotic; Complement inhibitor; And beta adrenergic agent.

[0179] further going through IL-21/IL-21R antagonist disclosed herein and other curative below unites and is used for the treatment of or prevents concrete immune disease.

[0180] can with the limiting examples of medicine of IL-21/IL-21R antagonist coupling with treatment or prevention arthritis (for example RA, inflammatory arthritis, teenager RA, OA and psoriasis arthropathica), comprise one or more following medicines: IL-12 antagonist as herein described, NSAID; CSAID; TNF antagonist TNF alpha-2 antagonists for example as herein described; Non-expendable anti-CD 4 antibodies as herein described; IL-2 antagonist as herein described; Anti-inflammatory cytokines, for example IL-4, IL-10, IL-13 and TGF α, or its agonist; IL-1 antagonist as herein described or IL-1 receptor antagonist); Phosphodiesterase inhibitor as herein described; Cox 2 inhibitor as herein described; Iloprost is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S82); Methotrexate; Thalidomide is (referring to for example Arthritis﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue) is S282) with Thalidomide related drugs (for example Celgen); Leflunomide; The plasminogen activation inhibitor, for example tranexamic acid is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S284); Cytokine inhibitor, for example T-614; Referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S282); PGE1 is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S282); Azathioprine is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S281); Il-1 invertase (ICE) inhibitor; Zap-70 and/or lck inhibitor (tyrosine kinase zap-70 or lck inhibitor); Vascular endothelial cell growth factor inhibitor as herein described or vascular endothelial growth factor receptor inhibitor; Angiogenesis inhibitor as herein described; Corticosteroid anti-inflammatory agent (for example SB203580); The TNF converting enzyme inhibitor; Interleukin-11 is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S296); Interleukin-13 is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S308); The interleukin-17 inhibitor is (referring to for example Arthritis ﹠amp; Rheumatism (1996) the 39th volume, the 9th phase (supplementary issue), S120); Gold; Penicillamine; Chloroquine; Oxychloroquine; Chlorambucil; Cyclophosphamide; Cyclosporin; Total lymph radiation; Antithymocyte globulin; The CD5-toxin; Oral peptide and collagen; Lobenzarit Disodium; Cytokine modulators (CRA) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); (ISIS 2302 for ICAM-1 antisense phosphorothioate oligodeoxynucleotide; IsisPharmaceuticals, Inc.); Soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); Prednisone; Orgotein; Poly-sulphuric acid glycosaminoglycans; Minocycline; Anti-IL2R antibody; Deep sea fish oil and vegetable lipid (fish and plant seed fatty acid; Referring to (1995) Rheum.Dis.Clin.North Am.21:759-777 such as for example DeLuca); Auranofin; Phenylbutazone; Meclofenamic acid; Flufenamic acid; The intravenous immunoglobulin; Zileuton; Mycophenolic Acid (RS-61443); Tacrolimus (FK-506); Sirolimus (rapamycin); Amiprilose (therafectin); Carat Qu Bin (2-chlorodeoxyadenosine); And azaribine.Preferred combination medicine comprises the combination of one or more IL-21 antagonisies and methotrexate or leflunomide, and under the situation of moderate or severe rheumatoid arthritis with the cyclosporin coupling.

[0181] is used for the treatment of the example of arthritic preferred inhibitor with the IL-21/IL-21R antagonist combination, comprises that the TNF antagonist is (for example with antibody or its Fab of the bonded chimeric antibody of TNF, humanized antibody, people's antibody or external generation; The soluble fragments of TNF receptor, for example p55 or p75 people TNF receptor or derivatives thereof, for example 75kdTNFR-IgG (75kD TNF receptor-IgG fusion rotein, ENBREL ^TM), p55kDTNF receptor-IgG fusion rotein; TNF enzyme antagonist, for example TNF α invertase (TACE) inhibitor); The antagonist of IL-6, IL-12, IL-15, IL-17, IL-18, IL-22; T cell and B cell consumption agent (depleting agent) (for example anti-CD 4 antibodies or anti-CD22 antibody); Micromolecular inhibitor, for example methotrexate and leflunomide; Sirolimus (rapamycin) and analog, for example CCI-779; Cox-2 and cPLA2 inhibitor; NSAID; P38 inhibitor, TPL-2 inhibitor, Mk-2 inhibitor and NF κ b inhibitor; RAGE or solubility RAGE; P-selects albumen or PSGL-1 inhibitor (for example anti-P-selects proteic antibody for micromolecular inhibitor for example, its antibody); Erss (ERB) agonist or ERB-NFkb antagonist.Most preferred other curative that can prepare and/or give together with one or more IL-21/IL-21R antagonisies, comprise one or more following medicines: the soluble fragments of TNF receptor, for example p55 or p75 people TNF receptor or derivatives thereof, 75kdTNFR-IgG (75kD TNF receptor-IgG fusion rotein, ENBREL for example ^TM); Methotrexate, leflunomide or sirolimus (rapamycin) or its analog, for example CCI-779.

[0182] can comprise following medicine with the limiting examples of medicine of treatment or prevention multiple sclerosis with the coupling of IL-21-/IL-21R antagonist: interferon, for example interferon-' alpha ' 1a (AVONEX for example ^TMBiogen) and IF1 b (BETASERON ^TMChiron/Berlex); Copolymer 1 (Cop-1; COPAXONE ^TMTeva PharmaceuticalIndustries, Inc.); Hyperbaric oxygen; The intravenous immunoglobulin; Carat Qu Bin (clabribine); TNF antagonist as herein described; Corticosteroid; Prednisolone; Methylprednisolone; Azathioprine; Cyclophosphamide; Cyclosporin; Methotrexate; 4-aminopyridine; And tizanidine.Can comprise the antibody of other human cell factor or somatomedin or the antagonist of other human cell factor or somatomedin with other antagonist that IL-21 unites use, described cytokine or somatomedin be TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF and PDGF for example.IL-21 antagonist as herein described can with the antibody coupling, described antibody is at following cell surface molecule: for example CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or its part.The IL-21 antagonist also can with following drug combination: for example aforesaid methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAID, for example ibuprofen, corticosteroid is prednisolone for example, phosphodiesterase inhibitor, adenosine agonists, antithrombotic, complement inhibitor, beta adrenergic agent, disturb the medicine of proinflammatory cytokine signal transduction as herein described, IL-1b converting enzyme inhibitor (for example Vx740), anti-P7s, PSGL, tace inhibitor, T cell signalling inhibitor is inhibitors of kinases for example, inhibitors of metalloproteinase, sulfasalazine, azathioprine (azathloprine), Ismipur, angiotensin-convertion enzyme inhibitor, soluble cytokine receptor and derivant thereof and anti-inflammatory cytokines (IL-4 for example, IL-10, IL-13 and TGF).

[0183] can comprise interferon-beta, for example IFN β-1a and IFN β-1b with the preferred embodiment that the IL-21 antagonist combination is used for the curative of multiple sclerosis; Ke Pasong (copaxone), corticosteroid, IL-1 inhibitor, tnf inhibitor, antibody, IL-12 antagonist at CD40 part and CD80.

[0184] can be used for the treatment of with the IL-21/IL-21R antagonist combination or prophylaxis of inflammatory bowel disease (Crohn disease; The limiting examples of medicine ulcerative colitis) comprises following medicine: budesonide (budenoside); Epidermal growth factor; Corticosteroid; Cyclosporin, sulfasalazine; Aminosalicylate; Ismipur; Azathioprine; Metronidazole; Lipoxidase inhibitor; Mesalazine; Olsalazine; Balsalazide; Antioxidant; The thromboxane inhibitor; The IL-1 receptor antagonist; Anti-IL-1 monoclonal antibody; Anti-IL-6 monoclonal antibody; Somatomedin; Elastase inhibitor; Pyridine radicals-imidazolium compounds; TNF antagonist as herein described; IL-4, IL-10, IL-13 and/or TGFb cytokine or its agonist (for example agonist antibody); IL-11; The prodrug of prednisolone, dexamethasone or the budesonide of glucosiduronic acid or glucosan-put together; ICAM-1 antisense phosphorothioate oligodeoxynucleotide (ISIS2302; Isis Pharmaceuticals, Inc.); Soluble complement receptor 1 (TP10; T CellSciences, Inc.); The spacetabs type mesalazine; Methotrexate; Platelet activating factor (PAF) antagonist; Ciprofloxacin; And lignocaine.

[0185] in one embodiment, the IL-21/IL-21R antagonist can with one or more antibody couplings, described antibody is at other target that participates in regulating immunne response, and described immunne response is transplant rejection, graft versus host disease or other immunne response relevant disease for example.Can be used for the treatment of with IL-21/IL-21R antagonist combination of the present invention or the limiting examples of the medicine that epidemic prevention is replied comprises following medicine:, include but not limited to CD25 (IL-2 receptor-a), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD40, CD40L, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2) at the antibody of cell surface molecule or its part.In another embodiment, the IL-21/IL-21R antagonist can with following drug combination: corticosteroid; Sirolimus (rapamycin) and its analog, for example CCI-779; Cyclosporin A; FK506; FTY720; Azathioprine; Cyclophosphamide; Methotrexate; Anti-IL-2R antibody, for example basiliximab, dacliximab; CA2 (chimeric anti-TNF alpha antibodies; REMICADE ^TM, Centocor); Anti-cd 3 antibodies (for example muromonab-CD3); Copolymer 1 (Cop-1; COPAXONE ^TMTeva Pharmaceutical Industries, Inc.); Deoxyspergualin; And mycophenolate mofetil.

[0186] can be used for the treatment of or prevent the limiting examples of psoriasis and other dermopathic medicine to comprise one or more following medicine: CD2 or LFA-3 interaction inhibitor (for example solubility CD2-or LFA-polypeptide with the IL-21/IL-21R antagonist combination, Fc fusions for example, or at the antibody of CD2 or LFA-3), cyclosporin A, prednisone, FK506, methotrexate, PUVA, UV line, steroidal, retinoid, interferon or chlormethine.Can comprise cyclosporin A and methotrexate with the example of the preferred agents of IL-21/IL-21R antagonist coupling.

[0187] can be used for the treatment of with the IL-21/IL-21R antagonist combination or the limiting examples of the medicine of prevention of asthma comprises one or more following medicines: suck and use bronchodilator, for example pirbuterol, bitolterol, orciprenaline; Beta-2-adrenoreceptor agonists, for example albuterol, terbutaline, salmaterol, formoterol; Antimuscarinic drug, for example ipratropium, oxygen holder ammonium; Systematicness corticosteroid, for example prednisone, prednisolone, dexamethasone; Suck and use corticosteroid, for example fluticasone, budesonide, beclometasone, mometasone; Leukotriene antagonist, for example Menglusitena, zafirlukast; Mast cell stabilizers, for example disodium cromoglycate, nedocromil; Omalizumab (XOLAIR ^TMGenentech/Novartis); Or cox 2 inhibitor as herein described.

[0188] can be used for the treatment of or prevent the limiting examples of the medicine of lupus (for example SLE) to comprise one or more following medicines with the IL-21/IL-21R antagonist combination: IL-6/IL-6R antagonist, for example anti-IL-6 antibodies or anti-IL-6R antibody; NSAID; Corticosteroid, for example dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone; Azathioprine, cyclophosphamide, hydroxychloroquine or chloroquine.

[0189] therefore, another aspect of the present invention relates to medicine box, and described medicine box is used to unite and gives IL-21/IL-21R antagonist and other therapeutic compound.In one embodiment, medicine box comprises for example curative of one or more bonding agent of being formulated in the pharmaceutical carrier and at least a medicine, and it is formulated as one or more suitable independently pharmaceutical preparatioies.

Exemplary disease

[0190] rheumatoid arthritis is to cause arthralgia, swelling, stiff and hypokinetic autoimmunity inflammatory diseases.Rheumatoid arthritis symmetry usually exists.This disease is involved carpal joint and near the articulations digitorum manus of hands.It also can involve other position outside the joint.In addition, the rheumatoid arthritis people can have fatigue, once in a while the heating and general malaise.The positive factor of diagnosis of rheumatoid arthritis comprises " rheumatoid factor " blood antibody and citrulline antibody.The IL-21/IL-21R antagonist can be used for treatment, prevention or rheumatoid arthritis or one or more symptoms of rheumatoid arthritis.

[0191] systemic lupus erythematosus (sle) (SLE) is the autoimmune disease that causes different bodily tissue inflammation and damage.SLE is mediated by the autoantibody at self DNA.Lupus can be involved many body parts, comprises joint, skin, kidney, heart, lung, blood vessel and brain.Although different symptoms can occur, that modal symptom comprises is extremely tired, arthralgia or swelling (arthritis), not clear heating, erythra and kidney problems (for example glomerulonephritis).Exemplary lupus symptom comprises arthralgia or swelling, fails to understand heating and extremely tired.Distinctive red rash can occur on nose and cheek.Erythra also can and occur on hand at face and ear, upper limb, shoulder, breast.Other symptom of lupus comprises that chest pain, alopecia, anemia, oral ulcer, finger and toe are because of cold and stress be pale or purplish red.Some people also has headache, dizzy, depressed, mental disorder or epilepsy.The positive factor of SLE diagnosis comprises circulation antinuclear antibody, anti-DNA antibody and anti-Sm antibody.The IL-21/IL-21R antagonist can be used for treatment, improves (alleviation) or prevention SLE or one or more SLE symptoms.

[0192] ankylosing spondylitis is an autoimmune disease, and it not only involves vertebra but also involves buttocks, shoulder, and also inflammation of knee joint (as bone and periarticular tendon and ligament), causes pain and stiff.Ankylosing spondylitis tends to involve above the average age for marriage teenager and the people who is only just of age.The IL-21/IL-21R antagonist can be used for treatment, prevention or alleviates ankylosing spondylitis or its one or more symptoms.

[0193] inflammatory bowel (IBD) is the general designation that causes the disease of enteritis.Two examples of inflammatory bowel are Crohn disease and ulcerative colitis.The IL-21/IL-21R antagonist can be used for treatment, prevention or alleviates inflammatory bowel or one or more inflammatory bowel symptoms.

[0194] Crohn disease causes the small intestinal inflammation.Crohn disease takes place at small intestinal hypomere (ileum) usually, but it also involves the gastral any position from the mouth to the anus.Its inflammation can be developed to and reach the organ liner of getting involved deeply, causes pain and makes the frequent emptying of intestinal, causes diarrhoea.The common sympton of Crohn disease is stomachache (usually at the bottom right abdomen) and diarrhoea.Hemorrhage of rectum also can occur, lose weight and generate heat.Hemorrhage can be serious and lasting, causes anemia.Intestinal is directly estimated, can determine the inflammation scope.

[0195] ulcerative colitis is for causing inflammation and the disease of ulcer at the large intestine liner.This inflammation takes place at rectum and colon hypomere usually, but also can involve whole colon.Except small intestinal latter end (being called terminal ileum), the less small intestinal that involves of ulcerative colitis.Inflammation makes the frequent emptying of colon, causes diarrhoea.The position of killing and wounding the colon cell liner in inflammation forms ulcer; Ulcer can hemorrhage and generation pus.The common sympton of ulcerative colitis is stomachache and bloody diarrhea.The patient also has fatigue, loses weight, loss of appetite, hemorrhage of rectum and body fluid and nutritive loss.About half patient has mild.Other then frequently heating, bloody diarrhea, nauseating and serious abdominal cramps.Ulcerative colitis also can cause problems such as arthritis, eye inflammation, hepatopathy (hepatitis, liver cirrhosis and primary sclerosing cholangitis), osteoporosis, erythra and anemia.The diagnosis of ulcerative colitis depends on that usually the discriminating of courageous and upright feces and colon directly estimate.

[0196] psoriasis is the chronic dermatosis that squama is peeled off (scaling) and inflammation.When Skin Cell produces fast from the source below the skin surface and just piled up from the teeth outwards before maturation, psoriasis can take place.Usually such process (being also referred to as renewal) needs one month approximately, but in psoriasis, only needs just take place in several days.The psoriasis of canonic form causes that the silver color squama covers thickens, the skin speckle of inflammation.These specklees (being also referred to as plaque sometimes) are understood pruritus or feels pain usually.They usually occur in other position, lower back, face, palm and the sole of elbow, knee joint, lower limb, but they also can occur on the skin at any position of health.The main basis of psoriatic diagnosis is these characteristic symptoms.Skin biopsy can be used for diagnosis.The IL-21/IL-21R antagonist can be used for treatment, prevention or alleviates psoriasis or one or more psoriasis symptoms.Psoriasis arthropathica betides some psoriasis (a kind of squama exuviae skin disease) patient.Psoriasis arthropathica involves the finger and the joint of toe far-end usually, and with the variation of fingernail and toenail.As then backache can take place when involving spinal column.The IL-21/IL-21R antagonist can be used for treatment, prevention or alleviates psoriasis or one or more psoriasises or psoriasis arthropathica symptom.

[0197] glomerulopathy comprises propagation and non-proliferative disease.Glomerulonephritis is to have the disease of interior inflammation of glomerule and cell proliferation (referring to (1998) New Eng.J.Med.339:888-99 such as for example Hricik.Non-proliferative and hardening glomerulopathy comprise membranous glomerulopathy, diabetic nephropathy, FSG, thin basement membrane disease, amyloidosis, light chain nephropathy, HIV nephropathy, Alport syndrome (Alport ' s syndrome), drug-induced glomerulopathy and minute lesion (minimal-change disease).The generation of following the inflammation of glomerulopathy mainly is because the antibody-mediated glomerular injury due to the autoimmune.Humoral immunization activation can cause producing at messangial cell surface (for example basement membrane) antibody, and circulating antigen-antibody complex is deposited on the glomerule, it is reported to cause glomerulonephritis.Therefore, glomerular injury and glomerulonephritis are normally by due to the bigger systemic autoimmune disease (for example SLE, hepatitis and fibre modification disease).Glomerulonephritis is also relevant with following disease: IgA nephropathy, purpura,Henoch-Schonlein (Henoch-Schonlein purpura), infect (by antibacterial, virus, protozoacidies etc. cause), vasculitis, cryoglobulinemia, hereditary nephritis, granulomatosis (Wegner granulomatosis for example, many vasculitises under the mirror (microscopicpolyan gitis) and Qiu-Shi syndrome (Churg-Strauss syndrome)), the glomerular basement membrane disease, goodpasture's syndrome (Goodpasture ' s syndrome), the nephritic syndrome (betides for example diabetes, lupus (for example SLE), amyloidosis, drug use, cancer and infection), lipodystrophy, sickle-cell disease, complement deficiency, membranoprolifer ative glomerulonephritis, lupus nephritis and lupus membranous nephropathy.The IL-21/IL-21R antagonist can be used for treating, improving or prevent the symptom of glomerulonephritis or one or more glomerulonephritiies and other glomerulopathy.

[0198] the IL-21/IL-21R antagonist can be used for prevention or treated tissue/transplant rejection or repels related symptoms, for example before organ, tissue or cell (for example heart, lung, liver, kidney, pancreas or bone marrow) are transplanted, during or afterwards.When the immune system of host living beings produced immunne response at the non-autoantigen in transplanted tissue's (consubstantiality for example of the same race, allogeneic or heterogenous allosome tissue), transplant rejection can take place.Repulsion can be by antibody for example, cell mediated or simultaneously by these two kinds of mediations, repulsion can show as different modes, comprise hyperacute rejection for example (for example after transplanting early stage), acute cellular rejection and chronic rejection (normally slowly the process of development, graft function is faded).Repel and follow inflammation usually, and can cause transplanted tissue or organ injury and/or failure, for example angiopathy, fibrosis or organ dysfunction go down.During repelling, the host has general malaise, transplantation site pain or swelling and/or heating.Can repel sign or, monitor the rejection of organ and tissue grafts by biopsy by estimating organ dysfunction.The histopathology sign of repelling comprises that for example II class HLA antigen presentation increases, for example after the renal transplantation in renal tubular cell.Can estimate liver function by for example measuring bilirubin regulating liver-QI enzyme (for example alkali phosphatase) serum levels; Can come evaluate renal function by for example measuring the serum creatine level.

[0199] feature of osteoarthritis (OA) is that articular cartilage is destroyed.This makes bone friction each other under cartilage, causes arthralgia, swelling and hypokinesis.Prolong in time, the joint can lose normal shape, grows bony spur or hyperosteogeny at the edge, joint.In addition, minority bone or cartilage meeting fragmentation also swim in the joint space, cause more pain and damage.Patient OA has arthralgia and limitation of movement usually.Different with the arthritis of other type, OA only involves the joint, does not involve internal organs.The positive factor of OA diagnosis comprises through the visible cartilage of X-ray loses.The IL-21/IL-21R antagonist can be used for treating, preventing or alleviate the symptom of OA or one or more OA.

Respiratory disease

[0200] the IL-21/IL-21R antagonist can be used for treating respiratory disease, includes but not limited to asthma (for example allergia and anallergic asthma); Bronchitis (for example chronic bronchitis); Chronic obstructive pulmonary disease (COPD) (emphysema for example, for example the medicated cigarette emphysema of bringing out); The too much mucus of disease, hypereosinophilic syndrome, fibrosis and generation that relates to airway inflammation, for example cystic fibrosis, pulmonary fibrosis and allergic rhinitis.

[0201] method of treatment or prevention of asthma comprises the method for treatment or prevention extrinsic asthma (being also referred to as allergic asthma or atopic asthma), intrinsic asthma (being also referred to as anallergic asthma or ergotropy asthma) or the two combination (being called mixed asthma).Exogenous or allergic asthma comprises by allergen and causes or the disease relevant with allergen that described allergen is pollen, spore, Caulis et Folium Oryzae or weeds, house pet squama, dust, acarid etc. for example.Because the different time points in a year exists distinctive allergen and other stimulus object, so this class disease is also referred to as seasonal asthma.Extrinsic asthma also comprises bronchial asthma and allergic bronchopulmonary aspergillosis.

[0202] the inventive method asthma that can treat or alleviate comprises the asthma that is caused by infectant, and described infectant is virus (for example flu and influenza virus, respiratory syncytial virus (RSV), paramyxovirus, rhinovirus and influenza virus) for example.RSV, rhinovirus and influenza infection are common in the child, and viral infection is the main cause of infant respiratory tract disease.Viral bronchiolitis child can develop into chronic stridulating and asthma, and these sick available methods of the present invention are treated.Also comprise the asthma that some asthmatic patient can cause when motion and/or chance cold air.These methods can be used for smoking expose relevant asthma (for example medicated cigarette bring out and industrial smog), and industry and occupational exposure, for example smog; Ozone; Harmful gas; Sulfur dioxide; Nitrous oxide; Flue dust comprises isocyanates, from paint, plastics, polyurethane, varnish etc.; Wood, plant or other organic dust etc.Described method also can be used for the relevant asthma of food additive, antiseptic or pharmacological agents.Also comprise treatment, suppress or alleviate the method for asymptomatic asthma (silent asthma) or cough variant asthma asthma types such as (cough variant asthma).

[0203] method disclosed herein also can be used for treatment and alleviates the relevant asthma of gastroesophageal reflux (GERD), and so anti-stream can stimulate bronchoconstriction.GERD (follow lasting humoral selection, antitussive and be exposed to allergen and stimulus object in the bedroom) can cause asthma sexually transmitted disease (STD) disease, is referred to as asthma at night.In treatment, suppress or alleviate in the method for the relevant asthma of GERD, can be by the GERD curative coupling of IL-21/TL-21R antagonist and the medicinal effective dose with medicinal effective dose described herein.These medicines include but not limited to proton pump depressant, for example PROTONIX ^Board postpones pantoprazole sodium tablet, the PRILOSEC of release ^Board omeprazole (omeprazole) postpones release capsule agent, ACIPHEX ^Board RABEPRAZOLE SODIUM (rebeprazolesodium) delayed-release tablet or PREVACID ^Board postpones lansoprazole (lansoprazole) capsule of release.

Atopic diseases and symptom thereof

[0204] " atopy " is meant and has the one group of disease that develops into allergic inherent trend.The example of atopic diseases comprises allergy, allergic rhinitis, atopic dermatitis and pollinosis.Can give the IL-21/IL-21R pathway antagonists, to improve atopic diseases or its one or more symptoms.

[0205] symptom of allergic rhinitis (pollinosis) comprises that pruritus, watery nasal discharge, sneeze or nasal obstruction and eyes itch.Can give the IL-21/IL-21R pathway antagonists, to improve one or more these symptoms.

[0206] atopic dermatitis is the chronic disease of involving skin.The information of relevant atopic dermatitis is from for example NIH Publication No.03-4272.In atopic dermatitis, skin becomes and itches very much, causes hyperemia, swelling, chaps, oozes out transparency liquid, finally causes incrustation and squama to come off.In many cases, when disease degenerates (be called worsen or burst) a period of time, a period of time is that skin improves or improves fully (being called alleviation) again.Atopic dermatitis often is called " eczema ", and this term is the general designation of a few class scytitiss.Atopic dermatitis is modal in the multiple eczema.The example of atopic dermatitis comprises: allergic contact eczema or dermatitis (for example show as red, the reaction of itching, shed tears sometimes, when contact skin allogenic material for example during some antiseptic in Rhus toxicodendron or emulsifiable paste and the lotion); Contact eczema (for example comprises local responses such as rubescent, pruritus and burning sensation, when contact skin allergen or stimulus object (for example acid, cleaning agent or other chemicals); Dyshidrotic eczema (for example to the stimulation of palm and sole skin, feature is transparent, dark blister, has gargalesthesia and burn feeling); Neurodermatitis (for example on head, shank, wrist or the forearm because of the part scaling of skin speckle that (for example insect bite) cause (more can stimulate them when the scratching) of itching); Nummular eczema (for example the cutaneous manifestations of irriate is that coin shape speckle is determined, and usually at arm, the back of the body, buttocks and shank, has sclerderm, squama, and itches especially); Seborrheic eczema (for example at scalp, face, the skin speckle that shows as yellow, oily, have squama at other position of health once in a while).Other concrete symptom also comprises stasis dermatitis, atopy fold (for example Dennie-Morgan pleat), cheilitis, hyperlinear palms, superpigmentation eyelid (eyelid darkens), ichthyosis, keratosis pilaris, lichenification, pimple and urticaria due to inflammation or pollinosis.Can give the IL-21/IL-21R pathway antagonists, to improve one or more these symptoms.

The fibre modification disease

[0207] although the generation of collagen is the high degree of controlled process, its disorder can cause the tissue fibering development.The abnormal accumulation of fibrous material causes organ failure (Border etc. (1994) New Engl.J.Med.331:1286-92) the most at last.Any organ injury all can cause stereotypy (stereotypical) physiological reaction: the hemostasis of platelet induced then is that inflammatory cell flows into and the activation fibroblast.Cytokine from these cell types drives formation new extracellular matrix and blood vessel (granulation tissue).The generation of granulation tissue is a careful and complicated process, and wherein the expression of protease inhibitor and extracellular matrix protein is raised, and the expression decreased of protease can cause the accumulation of extracellular matrix.

[0208] development of fibre modification symptom, that no matter bring out or spontaneous, at least all cause by active stimulation of fibroblast.Inflammatory cell and activation fibroblast flow into the damaged organ, depend on the interactional ability of these cell types and a matter substrate, and described matter substrate mainly contains collagen.The relevant numerous disease of fibrous tissue propagation is chronic, makes for example scleroderma deterioration of dermatosis usually.Some fibrosis that comprises pulmonary fibrosis may be fatal, this part is because this fact: the existing treatment of this disease has apparent side effect, usually to slowing down or stopping fibrosis process invalid (Nagler etc. (1996) Am.J.Respir.Crit.Care Med.154:1082-86).

[0209] the fibre modification disease comprises the disease that turns to feature with fiber, for example visceral nerve fiberization, dermatofibrosis disease and eye fibre modification disease.Internal organs (for example liver, lung, kidney, cardiovascular, gastrointestinal tract) fibre modification takes place in following disease: for example pulmonary fibrosis, myelofibrosis, liver cirrhosis, mesentery proliferative glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, kidney region fibrosis, accept the patient's of cyclosporin renal fibrosis and HIV associated kidney disease.

[0210] fibrosis of skin disease comprises for example scleroderma, morphea, keloid, hypertrophic scar, familial cutaneous collagenoma and collagen type connective-tissue nevus.Eye fibre modification disease comprises for example diabetic retinopathy, postoperative scar scar formation (for example after the glaucoma filtration surgery and squinting eye operation back) and proliferative vitreoretinopathy.

[0211] medicable other fibrotic conditions of the inventive method comprises: the arthralgia of rheumatoid arthritis, prolongation and deterioration joint relevant disease, systemic sclerosis (comprising progressive systemic sclerosis), polymyositis, dermatomyositis, diffuse fasciitis with eosinophilia, morphea (local scleroderma), Raynaud syndrome (Raynaud ' s syndrome) and nasal polyp.

[0212] can give the IL-21/IL-21R pathway antagonists,, or improve the symptom of one or more these diseases with treatment or prevention fibre modification disease.

Measure of the active test of IL-21/IL-21R antagonist as cytokine generation and cell proliferation/differentiation regulator

[0213] can use any conventional factor dependent cell proliferation test, to including but not limited to following cell line, measure the activity of IL-21/IL-21R antagonist: 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK as cytokine generation and cell proliferation/differentiation regulator.

[0214] algoscopy of T cell or thymocyte proliferation includes but not limited to the method described in the following document: Current Protocols in Immunology, J.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach, W Strober (writing), Pub.Greene Publishing Associates and Wiley-Interscience (the 3rd chapter, In vitroassays for Mouse Lymphocyte Function 3.1-3.19; The 7th chapter, Immunologicstudies in Humans); Takai etc. (1986) J.Immunol.137:3494-500; Bertagnolli etc. (1990) J.Immunol.145:1706-12; Bertagnolli etc. (1991) Cellular Immunology 133:327-341; Bertagnolli etc. (1992) J.Immunol.149:3778-3783; Bowman etc. (1994) J.Immunol.152:1756-61.The assay method of cytokine generation and/or splenocyte, lymph-node cell or thymocyte proliferation includes but not limited to the method introduced in the following document: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. be stated from Current Protocols in Immunology.J.E.e.a.Coligan (writing), the 1st volume, the 3.12.1-3.12.14 page or leaf, John Wiley and Sons, Toronto.1994; With Measurement of mouse and human Interferon gammma, Schreiber, R.D. be stated from Current Protocols in Immunology.J.E.e.a.Coligan (writing), the 1st volume, the 6.8.1-6.8.8 page or leaf, John Wiley and Sons, Toronto.1994.

[0215] assay method of hematopoietic cell and lymphocyte cellulation propagation and differentiation includes but not limited to the method described in the following document: Measurement of Human andMurine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. be stated from Current Protocols in Immunology.J.E.e.a.Coligan (writing), the 1st volume, the 6.3.1-6.3.12 page or leaf, John Wiley and Sons, Toronto.1991; DeVries etc. (1991) J.Exp.Med.173:1205-11; Moreau etc. (1988) Nature336:690-92; Greenberger etc. (1983) Proc.Natl.Acad. Sci.U.S.A.80:2931-38; Measurement of mouse and human interleukin 6, Nordan, R. are stated from Current Protocols in Immunology.J.E.e.a.Coligan (writing), the 1st volume, the 6.6.1-6.6.5 page or leaf, John Wiley and Sons, Toronto.1991; Smith etc. (1986) Proc.Natl.Acad.Sci.U.S.A.83:1857-61; Measurement of humanInterleukin 11, Bennett, F., Giannotti, J., Clark, S.C. and Turner, K.J. are stated from Current Protocols in Immunology.J.E.e.a.Coligan (writing), the 1st volume, the 6.15.1 page or leaf, John Wiley and Sons, Toronto.1991; Measurement of mouseand human interleukin 9, Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. are stated from Current Protocols in Immunology.J.E.e.a.Coligan (writing), the 1st volume, the 6.13.1 page or leaf, John Wiley and Sons, Toronto.1991.

[0216] is used for responding antigenic T cell clone algoscopy (evaluation wherein influences the albumen of APC-T cell interaction and instructs the T cytological effect by measuring the generation of propagation and cytokine) and includes but not limited to the method that following document is described: Current Protocols inImmunology, J.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach, W Strober (writing), Pub.Greene Publishing Associates and Wiley-Interscience (the 3rd chapter, In vitro assays for Mouse Lymphocyte Function; The 6th chapter, Cytokines and their cellular receptors; The 7th chapter, Immunologicstudies in Humans); Weinberger etc. (1980) Proc.Natl.Acad.Sci.U.S.A.77:6091-95; Weinberger etc. (1981) Eur.J.Immun.11:405-11; Takai etc. (1986) J.Immunol.137:3494-500; Takai etc. (1988) J.Immunol.140:508-12.

Embodiment

[0217] the present invention is further specified by following non-limiting example.

Embodiment 1: separation and the sign of Mus MU-1 cDNA

[0218] by PCR, use oligonucleotide from the human sequence, isolate the part fragment of MU-1 receptor Mus congener.Isolation of RNA from 17 age in days Mus thymus and Mus 2D6 T cell line, and by its preparation cDNA.By PCR,, use following oligonucleotide: correspond respectively to human cDNA sequence's (corresponding to SEQ ID NO:1) the 584-603 of Fig. 1 and the zone of 876-896 from the increase dna fragmentation of about 300 nucleotide of cDNA:

AGCATCAAGCCGGCTCCCCC(5p)(SEQ ID NO：11)

CTCCATTCACTCCAGGTCCC(3p)(SEQ ID NO：12)

Increase with Taq polymerase/1X Taq buffer (containing the 1.5mM magnesium chloride), 30 times the circulation, 94 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 1 minute.Measure this segmental DNA, two oligonucleotide are all from this segmental inside, and its sequence is as follows:

TTGAACGTGACTGRGGCCTT(5P)(SEQ ID NO：13)

TGAATGAAGTGCCTGGCTGA(3P)(SEQ ID NO：14)

[0219] oligonucleotide is used to increase the fragment of 262 nucleotide in inside of former PCR product (corresponding to the nucleotide 781-1043 of the Mus cDNA sequence of Fig. 1, and SEQ ID NO:9), so that, screen from the isolating cDNA of 2D6T cell line library as hybridization probe.With standard 5X SSC hybridization conditions, make filter membrane (Filters) hybridization at 65 ℃, and in SSC 65 ℃ of washings.20 clones of screening and separation and probe hybridization in 426,000 clones.Measure DNA sequence with two independent clones.Clone #6 full length sequence confirms that it is the total length Mus homologous sequence (SEQ ID NO:9) of people MU-1 really.

[0220] the full length nucleotide sequence of Mus MU-1 is seen Fig. 1 (corresponding to SEQ ID NO:9).This nucleotides sequence is listed in the targeting sequencing that nucleotide 407-464 has prediction, and 407-1993 has coded sequence at nucleotide, and 1994-1996 has termination codon at nucleotide.Nucleotide 1-406 is corresponding to 5 ' untranslated region, and nucleotide 1997-2628 is corresponding to 3 ' untranslated region (SEQ ID NO:9).

[0221] the predicted protein matter sequence of Mus MU-1 is seen Fig. 2 (corresponding to SEQ ID NO:10).This Mus MU-1 albumen is measured targeting sequencing (score=10.1) (corresponding to the amino acid/11-19 of SEQ ID NO:10) and the predicted transmembrane domain (corresponding to the aminoacid 237-253 of SEQ IDNO:10) that contains prediction through SPScan.The prediction signal motif comprises following each district among Fig. 2 B: the aminoacid 265-274 of box 1:SEQ ID NO:10; The aminoacid 310-324 of box 2:SEQ ID NO:10, six tyrosine are positioned at the amino acid position 281,319,361,368,397 and 510 of SEQ ID NO:10.Potential STAT berth comprises: STAT5:EDDGYPA (SEQ ID NO:20); STAT3:YLQR.

Embodiment 2: the comparison of people and Mus MU-1

[0222] the GAP algorithm is used for comparison people and Mus MU-1 aminoacid.70 aminoacid districts (SEQ ID NO:3) search GenBank data base of personnel selection IL-5 receptor is with PCR primer (SEQ ID NO:4 and 5) and hybridization oligonucleotide (SEQ ID NO:6 and 7) human cloning MU-1.Mus and people's predicted protein matter sequence relatively see Fig. 4.Use the GAP algorithm, its aminoacid has 65.267% homogeneity.Produce comparison (Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.U.S.A.89:10915-19) by BLOSUM62 aminoacid replacement matrix.Room parameter=room weight: 8, average coupling=2.912, length weight=2, average mispairing=-2.003; % similarity=69.466.

[0223] people and Mus cDNA nucleotide sequence relatively sees Fig. 3.DNA sequence has 66.116% homogeneity, when comparing with GAP.The room parameter: room weight=50, on average mate 10.000, length weight=3, average mispairing=0.000, % similarity=66.198.

[0224] people and mice MU-1 protein all are I cytokines receptor superfamily members.Mus and people MU-1 sequence are estimated, disclosed the existence of potential box-1 and box-2 signal motifs.Six tyrosine residues are present in cytoplasmic region, and it has important function on MU-1 signal conduction function.Sequence and other member of this family of MU-1 are compared, show the existence in the potential berth of STAT 5 and STAT 3.

Embodiment 3: the mensuration of the STAT signal transduction path that people MU-1 is used

[0225] to express the chimeric cell factor acceptor, be made up of human EPO receptor extracellular domain and MU-1 receptor born of the same parents internal area by this receptor through genetic engineering modified for the BAF-3 cell.Express the BAF-3 cellular response human soluble EPO of huEPORJMU-1 (cyto) Chimerical receptor and breed.Analyze these cells and in response EPO signal conduction back phosphorylation takes place to determine which kind of STAT molecule.In brief, in containing the growth medium of IL-3, contrast unmodified parental generation BAF-3 cell and the chimeric BAF-3 cell of EPOR/MU are tranquillization, stimulate again 0 minute, 15 minutes, 30 minutes and 60 minutes with IL-3 or EPO.Sedimentation cell is resuspended in the ice-cold lysis buffer that contains former alum salts, to preserve phosphorylated tyrosine.The cell pyrolysis liquid of equivalent carries out the SDS-PAGE electrophoresis, is transferred on the nitrocellulose filter again, carries out protein analysis.By using STAT molecule to have specific antibody, the STAT 1,3,5 and the 6 double traces of phosphorylation and non-phosphorylating form are dyeed to phosphorylation and non-phosphorylating form.Non-activated and with the activatory HELA cell of alpha-interferon as positive control.

[0226] these results show under these concrete conditions, and the signal by MU-1 causes STAT 5 phosphorylations of all testing time points (T=0 ', T=15 ', T=30 ', T=60 ').Handle contrast or chimeric BAF-3 cell with IL-3, cause the phosphorylation of STAT 3 rather than STAT1 or 5.

Embodiment 4: the tissue expression of Mus and people MU-1

Embodiment 4.1:RNA analyzes

[0227] (CA) method of Tui Jianing is to the polyA from different tissues for Clonetech, PaloAlto according to manufacturer ⁺RNA carries out the RNA trace.For the Mus trace, be used for hybridization corresponding to the nucleotide 781-1043 of Fig. 1 and 262 nucleotide fragments of SEQ ID NO:9.

[0228] in spleen, lung and the heart tissue of adult rats, detects a kind of Mus MU-1 transcript.Observed bigger transcript is not seen in mouse tissue in people's tissue.

[0229] in adult lymphoid tissue, PBL, thymus, spleen and lymph node and fetus lung, detects two kinds of transcripies of people MU-1.

Embodiment 4.2: in situ hybridization

[0230] (Columbus OH) carries out in situ hybridization research (according to described methods of (1990) J.Cell.Biol.111:2427-36 such as Lyons) by Phylogency Inc..In brief, successive 5-7 micron paraffin section de-waxing is fixing, use protease K digesting, handle and dehydration with triethanolamine.Prepare cRNA from wire cDNA template, produce antisense and adopted probe is arranged.According to synthetic cRNA transcript of the condition (Ambion) of manufacturer and usefulness ³⁵The S-UTP labelling.Fragment hybridization is spent the night, wash under stringency, reuse RNA enzyme A handles, and is immersed in then to expose 2-3 week in the nuclear spike emulsion (nuclear track emulsion).Contrast fragment and adopted probe hybridization is arranged, to indicate the background level of this process.The Mus probe is made up of the 186-bp fragment (SEQ ID NO:9) corresponding to nucleotide 860-1064.People's probe is the 23-bp PCR product from people MU-1DNA.

[0231] in the lymph node of small intestinal germinal center of growing up, observes the expression of Mus MU-1.The lymph node of specialization and Pai Shi knot (Peyers patches) also demonstrate the expression of Mus MU-1.

[0232] detects the expression of people MU-1 at the cortex germinal center of lymph node.The medullary substance (medulla) that contains macrophage is a people MU-1 feminine gender.Detect the expression of people MU-1 in the white pulp district (rather than red pulp district) of people's spleen.

Embodiment 5: the expression of people MU-1 in cell and cell line

[0233], and on the T cell line Jurkat, carries out the analysis of RNA enzyme protection at tranquillization and activation human T-cell and B cell line Raji and RPMI 8866.The human T-cell activates with anti-CD3 and anti-CD28.Cell line activates with phorbol ester and ionomycin.By 23-bp PCR product is inserted into pGEM3zf (-) (Promega, Madison, WI) BamHI of carrier and HindIII site, make up the plasmid (, carrying out PCR) that produces the MU-1 riboprobe with 5 ' primer CACAAAGCTTCAGTATGAGCTGCAGTACAGGAACCGGGGA (SEQ ID NO:15) and 3 ' primer CACAGGATCCCTTTAACTCCTCTGACTGGGTCTGAAAGAT (SEQID NO:16).In order to prepare riboprobe, the plasmid HindIII linearisation of ribonucleic acid will be produced.Gained DNA is through phenol/chloroform extracting and ethanol precipitation.(CA) Jian Yi scheme prepares riboprobe with the T7 RNA polymerase for PharMingen, San Diego according to distributors.RIBOQUANT with PharMingen ^TMMulti-ProbeRibonuclease Protection Assay system carries out the RNA enzyme protection and measures.Each RPA reaction contains the total RNA of 2.0 μ g, and after the RNA enzymic digestion, shielded riboprobe is used for QUICKPOINT ^TMQuick separate nucleic acid system (Novex, San Diego, CA).According to the explanation of distributors, desiccant gel also exposes.

[0234] compares with stimulated cells colony not, at anti-CD3 ⁺People's purification CD3 that anti-CD28 stimulates ⁺In the cell, people MU-1 RNA is raised.In Th1 and Th2-skewed T cell colony, after stimulating once more, MU-1 is also raised.MU-1 is expressed on B cell line RPMI 8866 and Raji composing type ground, and Jurkat T cell line is not like this.

Embodiment 6: people MU-1 combines with known cytokine

[0235] makes up people and Mus Ig fusion rotein and be fixed on the Biacore chip, attempt the part of identification of M U-1.Estimate various cell culture with conditioned mediums and one group of known cytokine binding ability to MU-1.Also measured the combination of other receptor chain of some cytokines and this family, needed second receptor chain to be used for the bonded probability of part so that consider MU-1.Measured following cytokine, combination is negative: mIL-2, hIL-2, hTL-15, mIL-7, TSLP, TSLP+IL-7, TSLP+IL-7R, TSLP+IL-7g, TSLP+IL-2, TSLP+IL-2+IL-2R β, IL2-R β, IL-2R γ, IL-7R, IL-2+IL-2R β, IL-2+IL-2R γ, BL-15+IL-2R β, IL-15+IL-2R γ, IL-7+IL-2R γ, IL-2+IL-7R, IL-15+IL-7R, IL-7+IL-7R to MU-1 to find them.Known receptor also is fixed and detects the MUFc combination, obtains negative findings.IL-15 can combine with IL-2Rb, but does not combine with IL-2Rg or MUFc.

Embodiment 7: in collagen-induced arthritis (CIA) mice, suppress the order of severity that the IL-21/IL-21R activity can be improved symptom

[0236] this embodiment shows, in the CIA mouse model, and the IL-21R antagonist, for example IL-21R-Ig fusion rotein (Mus IL-21RFc albumen or " muIL-21RFc ") or anti-IL-21R antibody improve its symptom.

[0237] male DBA/1 (Jackson Laboratories, BarHarbor, ME) mice are all used in all experiments.Arthritis is that (Chondrex, Redmond WA) bring out with II type bovine collagen.II type bovine collagen (Chondrex) is dissolved in the 0.1M acetic acid, and emulsifying in isopyknic complete Freund's adjuvant (Sigma) that contains 1mg/ml mycobacterium tuberculosis (Mycobacterium tuberculosis) bacterial strain H37RA.The 0th day, at tail end subcutaneous injection 100 μ g bovine collagens.The 21st day, use and the blended 0.1M acetic acid solution that contains 100 μ g bovine collagens of isopyknic incomplete Freund's adjuvant (Sigma) for the tail end subcutaneous injection of mice.Be used to the animals received of testing on the same group injection mutually first, just do not have collagen.Dosage regimen is seen the sketch map of Figure 16.Preventative or therapeutic gives DBA mice with MuIL-21RFc.In therapeutic scheme,, then begin to treat if all observed mice and fall ill in continuous two days.

[0238] monitors mice at least three times weekly, observe disease process.According to following index, carry out clinical score to extremity: 0=is normal, no swelling; The visible erythema of 1=with 1-2 enlargement toe head, or has mild swelling at ankle; The obvious erythema of 2=, feature are that claw swelling and/or two toe heads are slightly to moderate swelling; The swelling on a large scale of the whole claw of 3=extends to ankle joint or carpal joint; 4=swelling is disappeared, the claw ankylosis; Extremity use difficulty or ankylosis.Therefore, arbitrary given mice extremity scoring sum, the maximum total health scoring that draws is 16.

[0239] in the different phase of disease, implements euthanasia, cut tissue to animal, with claw fixing in 10% formalin (using) for histologic analysis, perhaps fixing at 4% paraformaldehyde (pH 7.47), use 20%EDTA (pH 8.0) decalcification, then paraffin embedding (using) in situ hybridization.With optical microscope and 5 grades of methods of marking (0-4) claw is marked, to characterize arthritic intensity and scope.Except other inflammatory associated change was used for scoring, inflammatory infiltration also was used for scoring, and for example pannus formation of described inflammatory associated change, synovial membrane fibrosis, articular cartilage corrode and/or subchondral bone destroys.Histological grade is determined with the reading of each claw: NAD=0 or no abnormal; 1: slightly to moderate; 2: slightly to moderate; 3: obviously; 4: a large amount of.

[0240] stimulates the previous day once more from collagen, give CIA mice muIL-21RFc (100 μ g or 200 μ g) every other day through intraperitoneal (IP) and carry out prophylactic treatment, after the treatment, observe the order of severity decline (data not shown) of symptom.

[0241] muIL-21RFc (200 μ g) mice, 3 times/week) effect of double therapeutic CIA mice is the function of treatment back natural law, sees Figure 17.With mice Ig (200 μ g/ mices, 3 times/week) in contrast.From treating back 7 days, visible severity scale descends.

[0242] these experiment showed, with the IL-21R antagonist for example the IL-21R-Fc fusion rotein give CIA mice, no matter be preventative or half therapeutic, all significantly improved arthritic symptom.

The in situ hybridization of embodiment 8:IL-21R transcript

[0243] expression of suffering from IL-21R mRNA in the arthritic claw of mensuration CIA mice.Use antisense Mus IL-21R riboprobe (Figure 18 A); Adopted probe is arranged as negative control (Figure 18 B).According to the explanation of manufacturer, (RocheDiagnostics, Mannheim Germany), prepare the probe of Digitoxin labelling to use DIG RNA labelling mixture.In macrophage, neutrophil cell, fibroblast, lymphocyte subgroup, synovial cell and epidermis, detect the expression (Figure 18 A) of IL-21 receptor mrna.In the contrast claw or use and to have adopted probe to can be observed dyeing to reduce (Figure 18 B).MIL-21R mRNA positive cell is: neutrophil cell (N) and macrophage (M).In situ hybridization shows that the expression of IL-21R in the claw of arthritis mice increases.

Embodiment 9: in the HLA-B27 rat model, to the active inhibition of IL-21/IL-21R, improved the order of severity of IBD sample symptom

[0244] this embodiment shows, in the HLA-B27 rat model, and the IL-21R antagonist, for example IL-21R-Ig fusion rotein (Mus IL-21RFc albumen or " muIL-21RFc ") or anti-IL-21R antibody have improved IBD sample symptom.

[0245] as described herein, produce Mus IL-21 receptor-Fc fused polypeptide (MuIL-21RFc), and in the HLA-B27 rat model, estimate the ability that it alleviates enteritis.The HLA-B27 rat model has been widely used in estimates the IBD therapy, because observe some clinical, histology and identical (relevant summarize referring to (1995) Gastroenterologys such as for example Elson, 109:1344-67 of immune characteristic of the enteritis in road in this model with human IBD; Blanchard etc. (2001) European Cytokine Network 12:111-18; Kim etc. (1999) Arch.Pharm.Res.22:354-60).For example, human major histocompatibility complex I equipotential B27 of HLA-B27 rat overexpression and B2 microglobulin gene product.Such gene outcome and the chronic inflammatory disease for example development of IBD are relevant.

[0246] rat that is used to study has suffered from chronic gastrointestinal tract (GI) inflammation, and evidence is to continue the diarrheal clinical sign.According to following index feces is carried out clinical score (0-3): 0=is normal, has shaping feces; 1=is soft, has shaping feces; 2=is loose, and feces is not shaped; With 3=watery diarrhea (referring to Figure 19).In disease process, estimate during the feces, rat is monitored reach 18 days.Clinical score is 3 to show and continue diarrhoea (being shown as the IgG contrast).MuIL-21RFc is given (6mg/kg IP, 3 times/week) 5 HLA-B27 transgenic rat/groups, reach 18 days.Another group gives 6mg/ml mEnbrel (soluble TNF-receptor Fc fusions), i.e. positive control.Give the same number of the 3rd group of mice by identical mode and dosage with IgG, in contrast.

[0247] compares with IgG contrast, in each group, detect clinical score obviously descend (referring to Figure 19 and Figure 20) with MuIL-21RFc and mEnbrel treatment.Aspect alleviation EBD sample symptom, give MuIL-21RFc and demonstrate and give mEnbrel similar effect.This result of study proves, in the HLA-B27 rat model, compares with the rat that contrasts IgG, gives MuIL-21RFc and has reduced enteritis, and its effect is similar to mEnbrel (referring to Figure 19 and Figure 20).

[0248] histologic analysis has confirmed that the stools scored to improve is the doing well,improving of representative.With regard to ulcer, inflammation, the focus degree of depth and fibrosis, through the scoring of MuIL-21RFc treatment rat, than scoring obviously lower (referring to Figure 21) with contrast IgG treatment rat.As mentioned above, carry out histologic analysis, clinical score 0-2 or 0-3, the bright order of severity rising in rat IBD model of wherein higher grade form.For contrast, in all groups, all detect the obvious minimizing of enteritis with MuIL-21RFc and mEnbrel treatment.Improving aspect histology's sign of disease severity, MuIL-21RFc shows the similar effect with mEnbrel.In order to support to expand above result to the mankind, Figure 19 (right figure) is presented at the in situ hybridization of MU-1 mRNA in normal HE lymphocyte and the lymph node, shows the expression of MU-1 mRNA in the organ relevant with disease.

Embodiment 10: suppress the IL-21/IL-21R activity, the allogeneic skin graft that can postpone mice repels

[0249] this embodiment shows, the IL-21R antagonist, and for example IL-21R-Ig fusion rotein (Mus IL-21RFc albumen or " muIL-21RFc ") or anti-IL-21R antibody postpone the mice allogeneic skin graft and repel, therefore the survival period that has prolonged graft.

[0250] in the mice of the T cell of having injected the retrovirus transduction, gives MuIL-21RFc and can postpone the allogeneic skin graft repulsion.Figure 22 is a line graph, shows the variation of the natural law after graft survival percentage ratio is with adoptive transfer.In this model, nude mouse shows the rehabilitation allogeneic skin graft, because mice does not have detectable T cell.When to nude mouse injection activated b 6 T cells (through the retrovirus through engineering approaches with secretion contrast GFP or IL-21), graft will be ostracised (referring to Figure 22).If the T cell through genetic engineering modified with secretion MuIL-21RFc (its IL-21 that these cells produce that is expected to neutralize), the graft survival longer time, see Figure 22 (shown in IL-21R-Fc, with GFP with IL-21 contrast compare).10 mices are respectively applied for GFP and MuIL-21RFc; 15 mices are used for the IL-21 contrast.These results have proved that the IL-21R antagonist is prolonging the effect of graft survival on the phase.

Embodiment 11: at CD45RB ^HiIn the adoptive transfer model, suppress the active meeting of IL-21/TL-21R and reduce disease symptoms

[0251] this embodiment shows, in psoriasis and inflammatory bowel (IBD) mouse model, IL-21R antagonist for example IL-21R-Ig fusion rotein (Mus IL-21RFc albumen or " muIL-21RFc ") or anti-IL-21R antibody capable improves symptom.

[0252] with CD45RB ^HiCD4 ⁺Inmature T cell transfer is given severe combined immunodeficiency (SCID) mice, can cause colitis and/or skin lesion parapsoriasis, and this depends on the condition of raising in cages.At first, by selecting CD4 ⁺Carry out feminine gender on the pillar of T cell and select, from splenocyte, sub-elect BALBc CD45RB ^HiCD4 ⁺T cell (inmature colony) is then in the further sorting of flow cytometry by selecting high CD45 to express.With 4 * 10 ⁵This colony of cell is transferred to female C.B-17 SCID mice, and the clinical sign of the psoriasis of mice and IBD is marked reaches several weeks.Inflammatory bowel takes place in the mice under static state is raised in cages condition; And psoriasis also takes place in the mice of supporting in the normal condition ShiShimonoseki that has the circulation of air variation.According to the scale of 1-6, the psoriasis of mice is marked: the health of the light moderate erythema of 1=(normally eyelid and ear)＜2%; The health of slight squama of 2=and middle severe erythema (normally ear and face) 2-10%; The health of serious erythema of 3=and squama (ear, face and trunk) 10-20%; 4=is erythema very seriously, accounts for the 20-40% of whole health; 5=is erythema very seriously, accounts for the 40-60% of whole health; 6=is erythema very seriously, accounts for the 60-100% of whole health.By losing weight and stools scored is marked to the IBD of mice: 0=is normal; 1=is soft; 2=diarrhoea; 3=has blood and mucous diarrhoea.

[0253] treat with muIL-21RFc, effective to improving the psoriasiform symptom.In suffering from the mice of scytitis, compare with the control mice for the treatment of, at CD45RB with resisting Eimeria tenella Ig ^HiThe beginning of 8 weeks is treated by peritoneal injection 200 μ gmuIL-21RFc (3 times/week) after the cell transfer, and erythema, squama and alopecia as a result all reduces (Figure 23).Compared with the control, in the transcellular while, with the CD45RB of 200 μ g muIL-21RFc (3 times/week) treatment ^HiThe receptor mice, the result is in whole experiment, and the order of severity that psoriasis postpones outbreak and clinical disease reduces (Figure 35).Experimental results reduction is at Figure 36.

[0254] treat with muIL-21RFc, also effective to improving inflammatory bowel to symptom.Compare with Ig contrast treatment mice, in the transcellular while, with 200 μ g or 400 μ gmuIL-21RFc treatment CD45RB ^HiThe receptor mice, 3 times weekly, the result determines that by lose weight (Figure 37) and stools scored (Figure 38) clinical sign of colitis obviously reduces.The result is summarized in Figure 39.CD45RB to the contrast treatment ^HiThe colon of receptor carries out the naked eyes evaluation, show seriously to thicken and swelling, and these phenomenons is almost suppressed fully in the mice with the muIL-21RFc treatment.On micro-, to compare with the mice of muIL-21RFc treatment, lamina propria/tela submucosa of the mice of contrast treatment also demonstrates epithelial hyperplasia and leukocyte infiltration greatly.In addition, measure the serum cytokines of contrast treatment mice and muIL-21RFc treatment mice.Survey in the cytokine several, only can detect serum r interferon (IFN-γ).Compare with Ig contrast treatment mice, treat with the muIL-21RFc of 200 μ g or 400 μ g dosage, the result has significantly reduced the serum levels (Figure 40) of IFN-γ.IFN-γ can be used as the biomarker of the effect of IL-21R antagonist in IBD.

[0255], detects CD45RB by proliferation assay ^Hi(naivety) subgroup and CD45RB ^Lo(memory) subgroup is to the response of IL-21.In the IBD metastasis model, only juvenile cell causes disease, and disease can be suppressed by adding memory colony.In this test, use with the bonded anti-CD3 stimulation purification colony of flat board and be determined as response IL-21 and mix ³The H-thymidine.With memory cluster bulk phase ratio, the significant reaction that inmature colony shows response IL-21 increases (Figure 41).This shows that IL-21 is an important cytokine for amplification in the body of this colony.

[0256] IL-21 is joined activation CD4 in the culture ⁺CD45RB ^HiIn the cell, induce the secretion of the various kinds of cell factor.The CD45RB that anti-CD3 stimulates ^HiCD4 ⁺The T cell is handled with 100 units/ml IL-2 or 1ng/ml, 10ng/ml or 100ng/ml IL-21.In order to respond IL-21, CD45RB ^HiThe level rising (Figure 42) of emiocytosis IL-2, IL-4, IL-10, IL-17, IL-18, IL-22, IFN-γ and TNF α.With compare with the culture of Ig control treatment, by adding 50 μ g/ml or 100 μ g/ml muIL-21RFc, blocking-up endogenous IL-21, these cells in culture factor levels descend (Figure 43) as a result.

[0257] in a word, these results show that IL-21 has potent latent effect in the inflammatory response of this model, and the IL-21R antagonist can play the therapeutic benefit in the disease (for example Crohn disease and psoriasis) of Th1 mediation.

Embodiment 12: the airway inflammation that the mice of shortage IL-21R shows antigen induced alleviates

[0258] this embodiment shows, the transgenic that lacks the IL-21 receptor is pounded out the reaction that mice (IL-21R-/-) has obvious minimizing to the airway inflammation and the air flue overreaction of antigen induced.

[0259] the 0th day and the 14th day, IL-21R-/-and wild type (WT+ /+) C57BL/6 mice (8-12 age in week) through peritoneal injection with 2.25mg aluminum potassium sulfate (Alum Inject; Pierce) emulsive 20 μ g OVA.26th, 27 and 28 days, the aerosol challenge of air flue usefulness 5%OVA/PBS 30 minutes.Last OVA attacked back 48 hours, estimated aerosolized methacholine chloride to the variation of animal on lung resistance and Cdgn dyanamic compliance.The WT+ that attacks with the PBS of OVA sensitization /+mice compares, OVA sensitization and attack WT+ /+mice in, after giving the methacholine chloride aerosol, cause the air flue overreaction obviously to increase (Figure 24).Yet, the IL-21R-that OVA-sensitization/OVA is attacked/-WT+ of mice and OVA sensitization/OVA attack /+mice compares, and the air flue overreaction of the aerosolized methacholine chloride of all dosage ranges is not all had difference (Figure 24).

[0260] put to death animal then, collection blood and bronchoalveolar lavage fluid (BALF) are used to analyze pneumonia, cytokine levels and total titer and anti-OVA IgE tires.Carry out bronchoalveolar lavage with 3 * 0.7ml PBS, collect BALF.With IL-21R-/-contrast that the PBS-of animal attacks compares, after OVA attacks, WT+ /+total BALF cell number of mice increases about 36 times, by contrast, the contrast that PBS-attacks increases by 3 times (Figure 25 A).In addition, the IL-21R-that OVA sensitization/OVA is attacked/-the BALF inner cell sum of mice, be starkly lower than WT+ that OVA sensitization/OVA attacks /+animal in viewed total cellular score.The IL-21R-that OVA sensitization/PBS is attacked/-and WT+ /+the BALF total cellular score of mice do not have difference (Figure 25 A).With same sensitization but compare through the contrast that PBS attacks, WT+ /+and IL-21R-/-mice in, the OVA attack causes the BALF eosinophilic granulocyte obviously to rise.The WT+ that attacks with OVA sensitization/OVA /+animal compares, IL-21-/-animal in, BALF eosinophilic granulocyte's absolute quantity significantly descend (Figure 25 B).Disappearance IL-21R also can obviously reduce the increase on BALF lymphocyte number (Figure 25 C) and the neutrophil cell number (Figure 25 D) after OVA attacks.

[0261] compare with the contrast that PBS-attacks, the WT+ of OVA sensitization/attack /+BALF of mice in IL-5, IL-13 and TNF alpha levels obviously raise (Figure 26 and 27).By contrast, compare with the contrast that PBS-attacks, OVA sensitization and attack IL-21R-/-bring out the very small amount of rising of these cytokine levels in the BALF in the mice, and its level is than viewed level (Figure 26 and 27) obviously on the low side in the WT animal of attacking at OVA sensitization/OVA.TNF α and IL-5 level cytometer beads array test kit (cytometricbead array kit) (mice Th1/Th2 cytokine CBA, BD Biosciences, San Diego, CA) quantitative assay among the BALF.IL-13 level among the BALF is by the ELISA quantitative assay.

[0262] shown in Figure 28 A-B, after OVA sensitization/OVA is attacked, IL-21R-/-serum total Ig E and anti-OVA IgE level and identical treatment WT+ /+mice is compared much lower.Yet, after PBS attacks when relatively more total IgE or OVA specific IgE level, IL-21R-/-and WT+ /+but do not have significant difference in the mice.

[0263] these results show, suppress the reaction of IL-21 mediation, have therapeutic value in treatment allergy and asthma.

Embodiment 13: at MRL-Fas ^LprIn the lupus model, suppress the order of severity that the IL-21/IL-21R activity can be improved symptom

[0264] this embodiment shows, at MRL-Fas ^LprIn the mouse model, IL-21R antagonist for example IL-21R-Ig fusion rotein (Mus IL-21RFc albumen or " muIL-21RFc ") or anti-IL-21R antibody capable improves systemic lupus erythematosus (sle) (SLE) sample symptom.

[0265] male MRL-Fas is all used in all experiments ^LprMice.These mices have the multiple symptom that is similar to people SLE, comprise the DNA autoantibody, many disorganizations, and ICG.In 10 ages in week, begin the contrast of 400 μ g MuIL-21RFc or isotype through peritoneal injection, 3 times weekly, analyze the disease progression of mice weekly.Put to death mice in the 15th week and be used for further analysis.Each treatment group has 10 mices.

[0266] at MRL-Fas ^LprIn the mice, detect through ELISA, the MuIL-21RFc treatment can obviously reduce circulation anti-dsDNA autoantibody level (Figure 29) and serum total Ig G level (Figure 30).In brief, for the detection of anti-dsDNA autoantibody, dsDNA is coated on the titer plate, adds serum antibody, the second antibody of the anti-mice of reuse detects described antibody.For the detection of total IgG, serum is adsorbed onto on the titer plate, the second antibody of the anti-mice of reuse detects.

[0267] treats with MuIL-21RFc, also can reduce the IgG deposit at MRL-Fas ^LprAccumulation in the mouse kidney.The 15th week, put to death mice, freezing kidney segment (5 μ m) is dyeed with goat anti-mouse IgG-FITC.According to scale record fluorescence intensity from 0-3.Figure 31 shows the total fluorescence intensity of measuring in treatment and control mice kidney.

[0268] these results show, carry out therapeutic treatment with the IL-21R antagonist and can alleviate the lupoid acne symptom.

Embodiment 14: lupus and GVHD animal model: in the IL-21R deficient mice kidney of transplanting B6 bm12 splenocyte, lack autoantibody formation and IgG deposition

[0269] experimentize, research IL-21R pounds out the reaction (Chen etc. (1998) J.Immunol.161:5880-85) of (KO) mice in chronic graft versus host disease (GVHD) model of systemic lupus erythematosus (sle) (SLE).This model comprises the representative aspect of SLE and GVHD.

[0270] used animal is: B6.C-H2＜bm12 〉/KhEG (bm12), JacksonLabs (splenocyte); IL-21R-2 KO mice, Charles River Labs (CRL); C57/B6 wild type (WT) mice, Charles River Labs; With the C57/B6 wild-type mice, and Taconic (TAC) (Germantown, NY).

[0271] on the same day that induces an illness, passes through CO ₂Expose and put to death suitable donor mice.The results spleen is also built.Each spleen 0.16M NH ₄The 1ml lysate splitting erythrocyte of Cl: 0.17M TrisCl (9: 1), copyrolysis 5 minutes mixes once in a while.With trypan blue pair cell suspension counting, and to be adjusted to final concentration with aseptic phosphate buffered saline(PBS) be 2 * 10 ⁸Cell/ml.Give suitable receptor mice through the suitable cell suspension of peritoneal injection 0.5ml (seeing the following form 2) again.Then, monitor urine protein and the body weight increase and decrease of receptor mice weekly.Every two weeks, give every mice by (retro-orbital sinus) blood-letting behind the eye socket hole, store serum and be used for further analysis.All serum that each time point is collected carry out ELISA mensuration (as described in Zouali and Stollar (1986) J.Immunol.Methods 90:105-10), measure the autoantibody at double-stranded DNA.

[0272] the 12nd week after disease is brought out makes every group half animal euthanasia, collects spleen and kidney.Left side kidney is preserved (complete) in 10% non-buffered formalin, uses H﹠amp; E dyeing.According to (ibid) described methods such as Chen, dyeing is marked.Grading parameters comprises: blood vessel peripheral lymphoid cellularity is soaked into, a matter lymphocytic infiltrate, hypercellularity (hypercellularity) and basement membrane thickened.Right kidney longitudinal section, each is partly all organized section in the enclosure to being placed down in.Reuse immunohistochemistry technology analyzes the existence of immune deposit, especially IgG, IgM and C3 in the right kidney.

Table 2

	Group	Donor	Receptor	n
						1	IL-21R KO	bm12	CRL IL-21R KO	8
2	CRL-GVHD(C-GVHD)	bm12	CRL B6		10
						3	TAC-GVHD(T-GVHD)	bm12	TAC B6	10
4	CRL-Control(C-Control)	CRL B6	CRL B6		5
						5	TAC-Control(T-Control)	TAC B6	TAC B6		5

[0273] these experimental results are seen Figure 44.Pound out any time point of mice at any IL-21R, all do not detect anti-dsDNA autoantibody (Figure 44 A).In addition, Figure 44 B shows, 20 weeks after disease is brought out, compare with the GVHD mice, and in IL-21R deficient mice kidney, do not see the IgG deposition.Therefore, in the GVHD-SLE model, the mice that lacks IL-21R does not produce autoantibody, does not form the IgG deposit in kidney yet.Therefore, with IL-21/IL-21R antagonist for treating individuality, all can provide effective therapy to SLE and GVHD.

[0274] all lists of references of quoting of this specification, unexamined patent application (comprise 60/599 of application on August 5th, 2004, the application in 23, of 086 and 2004 on December 60/639,176), the content of the patent application of having announced (comprise application on October 4th, 2002 2003/0108549) and the patent announced all is attached to herein by reference.Be equal to embodiment

[0275] it will be understood to those of skill in the art that or need only many embodiments that are equal to that the use normal experiment just can be determined invention specific embodiments described herein.Comprise the described embodiment that is equal in the appended claims.

Sequence table

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cagcctacac aggtctggcc acaacaccac acatatatgg tacacgtgcc atatgcgctt 660

gtctcaattc ctgtccgatg aagttttcat tgtcaatgtg acggaccagt ctggcaacaa 720

ctcccaagag tgtggcagct ttgtcctggc tgagagcatc aaaccagctc cccccttgaa 780

cgtgactgtg gccttctcag gacgctatga tatctcctgg gactcagctt atgacgaacc 840

ctccaactac gtgctgaggg gcaagctaca atatgagctg cagtatcgga acctcagaga 900

cccctatgct gtgaggccgg tgaccaagct gatctcagtg gactcaagaa acgtctctct 960

tctccctgaa gagttccaca aagattctag ctaccagctg caggtgcggg cagcgcctca 1020

gccaggcact tcattcaggg ggacctggag tgagtggagt gaccccgtca tctttcagac 1080

ccaggctggg gagcccgagg caggctggga ccctcacatg ctgctgctcc tggctgtctt 1140

gatcattgtc ctggttttca tgggtctgaa gatccacctg ccttggaggc tatggaaaaa 1200

gatatgggca ccagtgccca cccctgagag tttcttccag cccctgtaca gggagcacag 1260

cgggaacttc aagaaatggg ttaatacccc tttcacggcc tccagcatag agttggtgcc 1320

acagagttcc acaacaacat cagccttaca tctgtcattg tatccagcca aggagaagaa 1380

gttcccgggg ctgccgggtc tggaagagca actggagtgt gatggaatgt ctgagcctgg 1440

tcactggtgc ataatcccct tggcagctgg ccaagcggtc tcagcctaca gtgaggagag 1500

agaccggcca tatggtctgg tgtccattga cacagtgact gtgggagatg cagagggcct 1560

gtgtgtctgg ccctgtagct gtgaggatga tggctatcca gccatgaacc tggatgctgg 1620

ccgagagtct ggccctaatt cagaggatct gctcttggtc acagaccctg cttttctgtc 1680

ttgcggctgt gtctcaggta gtggtctcag gcttggaggc tccccaggca gcctactgga 1740

caggttgagg ctgtcatttg caaaggaagg ggactggaca gcagacccaa cctggagaac 1800

tgggtcccca ggagggggct ctgagagtga agcaggttcc ccccctggtc tggacatgga 1860

cacatttgac agtggctttg caggttcaga ctgtggcagc cccgtggaga ctgatgaagg 1920

accccctcga agctatctcc gccagtgggt ggtcaggacc cctccacctg tggacagtgg 1980

agcccagagc agctagcata taataaccag ctatagtgag aagaggcctc tgagcctggc 2040

atttacagtg tgaacatgta ggggtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 2100

tgtgtgtgtg tgtgtgtgtg tgtcttgggt tgtgtgttag cacatccatg ttgggatttg 2160

gtctgttgct atgtattgta atgctaaatt ctctacccaa agttctaggc ctacgagtga 2220

attctcatgt ttacaaactt gctgtgtaaa ccttgttcct taatttaata ccattggtta 2280

aataaaattg gctgcaacca attactggag ggattagagg tagggggctt ttgagttacc 2340

tgtttggaga tggagaagga gagaggagag accaagagga gaaggaggaa ggagaggaga 2400

ggagaggaga ggagaggaga ggagaggaga ggagaggaga ggagaggaga ggctgccgtg 2460

aggggagagg gaccatgagc ctgtggccag gagaaacagc aagtatctgg ggtacactgg 2520

tgaggaggtg gccaggccag cagttagaag agtagattag gggtgacctc cagtatttgt 2580

caaagccaat taaaataaca aaaaaaaaaa aaaagcggcc gctctaga 2628

<210>10

<211>529

<212>PRT

＜213〉mice

<400>10

Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly

1 5 10 15

Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr

20 25 30

Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser

35 40 45

Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe

50 55 60

Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His Ile Trp Tyr Thr

65 70 75 80

Cys His Met Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val

85 90 95

Asn Val Thr Asp Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser Phe

100 105 110

Val Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Leu Asn Val Thr Val

115 120 125

Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu

130 135 140

Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys

180 185 190

Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro Gln Pro Gly Thr

195 200 205

Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp Asp Pro His Met Leu Leu

225 230 235 240

Leu Leu Ala Val Leu Ile Ile Val Leu Val Phe Met Gly Leu Lys Ile

245 250 255

His Leu Pro Trp Arg Leu Trp Lys Lys Ile Trp Ala Pro Val Pro Thr

260 265 270

Pro Glu Ser Phe Phe Gln Pro Leu Tyr Arg Glu His Ser Gly Asn Phe

275 280 285

Lys Lys Trp Val Asn Thr Pro Phe Thr Ala Ser Ser Ile Glu Leu Val

290 295 300

Pro Gln Ser Ser Thr Thr Thr Ser Ala Leu His Leu Ser Leu Tyr Pro

305 310 315 320

Ala Lys Glu Lys Lys Phe Pro Gly Leu Pro Gly Leu Glu Glu Gln Leu

325 330 335

Glu Cys Asp Gly Met Ser Glu Pro Gly His Trp Cys Ile Ile Pro Leu

340 345 350

Ala Ala Gly Gln Ala Val Ser Ala Tyr Ser Glu Glu Arg Asp Arg Pro

355 360 365

Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Gly Asp Ala Glu Gly

370 375 380

Leu Cys Val Trp Pro Cys Ser Cys Glu Asp Asp Gly Tyr Pro Ala Met

385 390 395 400

Asn Leu Asp Ala Gly Arg Glu Ser Gly Pro Asn Ser Glu Asp Leu Leu

405 410 415

Leu Val Thr Asp Pro Ala Phe Leu Ser Cys Gly Cys Val Ser Gly Ser

420 425 430

Gly Leu Arg Leu Gly Gly Ser Pro Gly Ser Leu Leu Asp Arg Leu Arg

435 440 445

Leu Ser Phe Ala Lys Glu Gly Asp Trp Thr Ala Asp Pro Thr Trp Arg

450 455 460

Thr Gly Ser Pro Gly Gly Gly Ser Glu Ser Glu Ala Gly Ser Pro Pro

465 470 475 480

Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Ala Gly Ser Asp Cys

485 490 495

Gly Ser Pro Val Glu Thr Asp Glu Gly Pro Pro Arg Ser Tyr Leu Arg

500 505 510

Gln Trp Val Val Arg Thr Pro Pro Pro Val Asp Ser Gly Ala Gln Ser

515 520 525

Ser

<210>11

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: PCR primer

<400>11

agcatcaagc cggctccccc 20

<210>12

<211>20

<212>DNA

＜213〉artificial primer

<220>

＜223〉description of artificial sequence: PCR primer

<400>12

ctccattcac tccaggtccc 20

<210>13

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: PCR primer

<400>13

ttgaacgtga ctgrggcctt 20

<210>14

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the inner oligonucleotide of Mus MU-1 cDNA

<400>14

tgaatgaagt gcctggctga 20

<210>15

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: 5 ' PCR primer

<400>15

cacaaagctt cagtatgagc tgcagtacag gaaccgggga 40

<210>16

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: 3 ' PCR primer

<400>16

cacaggatcc ctttaactcc tctgactggg tctgaaagat 40

<210>17

<211>224

<212>PRT

＜213〉unknown

<220>

＜221〉unknown

<222>(1)..(224)

＜223〉description of unknown: second polypeptide that comprises the Fc district

<400>17

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu Gly Ala Pro Ser

1 5 10 15

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

20 25 30

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

35 40 45

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

50 55 60

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

65 70 75 80

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

85 90 95

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile Glu Lys Thr

100 105 110

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

115 120 125

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

130 135 140

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

145 150 155 160

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

165 170 175

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

180 185 190

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

195 200 205

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>18

<211>617

<212>DNA

＜213〉people

<400>18

gctgaagtga aaacgagacc aaggtctagc tctactgttg gtacttatga gatccagtcc 60

tggcaacatg gagaggattg tcatctgtct gatggtcatc ttcttgggga cactggtcca 120

caaatcaagc tcccaaggtc aagatcgcca catgattaga atgcgtcaac ttatagatat 180

tgttgatcag ctgaaaaatt atgtgaatga cttggtccct gaatttctgc cagctccaga 240

agatgtagag acaaactgtg agtggtcagc tttttcctgc tttcagaagg cccaactaaa 300

gtcagcaaat acaggaaaca atgaaaggat aatcaatgta tcaattaaaa agctgaagag 360

gaaaccacct tccacaaatg cagggagaag acagaaacac agactaacat gcccttcatg 420

tgattcttat gagaaaaaac cacccaaaga attcctagaa agattcaaat cacttctcca 480

aaagatgatt catcagcatc tgtcctctag aacacacgga agtgaagatt cctgaggatc 540

taacttgcag ttggacacta tgttacatac tctaatatag tagtgaaagt catttctttg 600

tattccaagt ggaggag 617

<210>19

<211>162

<212>PRT

＜213〉people

<400>19

Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met

1 5 10 15

Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln

20 25 30

Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln

35 40 45

Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro

50 55 60

Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln

65 70 75 80

Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile

85 90 95

Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala

100 105 110

Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr

115 120 125

Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu

130 135 140

Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu

145 150 155 160

Asp Ser

<210>20

<211>7

<212>PRT

＜213〉people

<400>20

Glu Asp Asp Gly Tyr Pro Ala

1 5

<210>21

<211>16

<212>PRT

＜213〉people

<400>21

Met Pro Leu Leu Leu Leu Leu Leu Leu Leu Pro Ser Pro Leu His Pro

1 5 10 15

<210>22

<211>786

<212>DNA

＜213〉people

<400>22

atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tgtacatttc ttacatctat 60

gccggcagcg gacaccacca tcatcaccac ggtagcggcg actataaaga cgatgacgat 120

aagggttccg gatgccccga cctcgtctgc tacaccgatt acctccagac ggtcatctgc 180

atcctggaaa tgtggaacct ccaccccagc acgctcaccc ttacctggca agaccagtat 240

gaagagctga aggacgaggc cacctcctgc agcctccaca ggtcggccca caatgccacg 300

catgccacct acacctgcca catggatgta ttccacttca tggccgacga cattttcagt 360

gtcaacatca cagaccagtc tggcaactac tcccaggagt gtggcagctt tctcctggct 420

gagagcatca agccggctcc ccctttcaac gtgactgtga ccttctcagg acagtataat 480

atctcctggc gctcagatta cgaagaccct gccttctaca tgctgaaggg caagcttcag 540

tatgagctgc agtacaggaa ccggggagac ccctgggctg tgagtccgag gagaaagctg 600

atctcagtgg actcaagaag tgtctccctc ctccccctgg agttccgcaa agactcgagc 660

tatgagctgc aggtgcgggc agggcccatg cctggctcct cctaccaggg gacctggagt 720

gaatggagtg acccggtcat ctttcagacc cagtcagagg agttaaagga aggctggaac 780

taatga 786

<210>23

<211>260

<212>PRT

＜213〉people

<400>23

Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile

1 5 10 15

Ser Tyr Ile Tyr Ala Gly Ser Gly His His His His His His Gly Ser

20 25 30

Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gly Cys Pro Asp Leu

35 40 45

Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys Ile Leu Glu Met

50 55 60

Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp Gln Asp Gln Tyr

65 70 75 80

Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu His Arg Ser Ala

85 90 95

His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met Asp Val Phe His

100 105 110

Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr Asp Gln Ser Gly

115 120 125

Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala Glu Ser Ile Lys

130 135 140

Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser Gly Gln Tyr Asn

145 150 155 160

Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe Tyr Met Leu Lys

165 170 175

Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg Gly Asp Pro Trp

180 185 190

Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp Ser Arg Ser Val

195 200 205

Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser Tyr Glu Leu Gln

210 215 220

Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln Gly Thr Trp Ser

225 230 235 240

Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser Glu Glu Leu Lys

245 250 255

Glu Gly Trp Asn

260

<210>24

<211>1426

<212>DNA

＜213〉people

<400>24

gcggccgcac caccatgccg cgtggctggg ccgccccctt gctcctgctg ctgctccagg 60

gaggctgggg ctgccccgac ctcgtctgct acaccgatta cctccagacg gtcatctgca 120

tcctggaaat gtggaacctc caccccagca cgctcaccct tacctggcaa gaccagtatg 180

aagagctgaa ggacgaggcc acctcctgca gcctccacag gtcggcccac aatgccacgc 240

atgccaccta cacctgccac atggatgtat tccacttcat ggccgacgac attttcagtg 300

tcaacatcac agaccagtct ggcaactact cccaggagtg tggcagcttt ctcctggctg 360

agagcatcaa gccggctccc cctttcaacg tgactgtgac cttctcagga cagtataata 420

tctcctggcg ctcagattac gaagaccctg ccttctacat gctgaagggc aagcttcagt 480

atgagctgca gtacaggaac cggggagacc cctgggctgt gagtccgagg agaaagctga 540

tctcagtgga ctcaagaagt gtctccctcc tccccctgga gttccgcaaa gactcgagct 600

atgagctgca ggtgcgggca gggcccatgc ctggctcctc ctaccagggg acctggagtg 660

aatggagtga cccggtcatc tttcagaccc agtcagagga gttaaaggaa ggctggaacg 720

gctccggctc tagagacaaa actcacacat gcccaccgtg cccagcacct gaactcctgg 780

ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga 840

cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca 900

actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt 960

acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg 1020

gcaaggagta caagtgcaag gtctccaaca aagccctccc agtccccatc gagaaaacca 1080

tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg 1140

aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg 1200

acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc 1260

ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg gacaagagca 1320

ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact 1380

acacgcagaa gagcctctcc ctgtccccgg gtaaatgagt gaattc 1426

<210>25

<211>467

<212>PRT

＜213〉people

<400>25

Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly

1 5 10 15

Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr

20 25 30

Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr

35 40 45

Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser

50 55 60

Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr

65 70 75 80

Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val

85 90 95

Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe

100 105 110

Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val

115 120 125

Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp

130 135 140

Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys

180 185 190

Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser

195 200 205

Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Gly Ser Gly Ser Arg

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210>26

<211>1499

<212>DNA

＜213〉people

<400>26

gcggccgcac caccatgccg cgtggctggg ccgccccctt gctcctgctg ctgctccagg 60

gaggctgggg ctgccccgac ctcgtctgct acaccgatta cctccagacg gtcatctgca 120

tcctggaaat gtggaacctc caccccagca cgctcaccct tacctggcaa gaccagtatg 180

aagagctgaa ggacgaggcc acctcctgca gcctccacag gtcggcccac aatgccacgc 240

atgccaccta cacctgccac atggatgtat tccacttcat ggccgacgac attttcagtg 300

tcgacatcac agaccagtct ggcaactact cccaggagtg tggcagcttt ctcctggctg 360

agagcatcaa gccggctccc cctttcaacg tgactgtgac cttctcagga cagtataata 420

tctcctggcg ctcagattac gaagaccctg ccttctacat gctgaagggc aagcttcagt 480

atgagctgca gtacaggaac cggggagacc cctgggctgt gagtccgagg agaaagctga 540

tctcagtgga ctcaagaagt gtctccctcc tccccctgga gttccgcaaa gactcgagct 600

atgagctgca ggtgcgggca gggcccatgc ctggctcctc ctaccagggg acctggagtg 660

aatggagtga cccggtcatc tttcagaccc agtcagagga gttaaaggaa ggctggaacg 720

gctccggctc tagagacaaa actcacacat gcccaccgtg cccagcacct gaaotcctgg 780

ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga 840

cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca 900

actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt 960

acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg 1020

gcaaggagta caagtgcaag gtctccaaca aagccctccc agtccccatc gagaaaacca 1080

tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg 1140

aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg 1200

acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc 1260

ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg gacaagagca 1320

ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact 1380

acacgcagaa gagcctctcc ctgtccccgg gtaaatcagg aatggcatca atgacaggag 1440

gtcaacaaat gggttctgga tctcatcatc atcatcatca ttctggaggt tgagaattc 1499

<210>27

<211>492

<212>PRT

＜213〉people

<400>27

Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly

1 5 10 15

Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr

20 25 30

Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr

35 40 45

Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser

50 55 60

Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr

65 70 75 80

Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val

85 90 95

Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe

100 105 110

Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val

115 120 125

Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp

130 135 140

Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys

180 185 190

Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser

195 200 205

Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Gly Ser Gly Ser Arg

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Val Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys Ser Gly Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly

465 470 475 480

Ser Gly Ser His His His His His His Ser Gly Gly

485 490

<210>28

<211>1426

<212>DNA

＜213〉people

<400>28

gcggccgcac caccatgccg cgtggctggg ccgccccctt gctcctgctg ctgctccagg 60

gaggctgggg ctgccccgac ctcgtctgct acaccgatta cctccagacg gtcatctgca 120

tcctggaaat gtggaacctc caccccagca cgctcaccct tacctggcaa gaccagtatg 180

aagagctgaa ggacgaggcc acctcctgca gcctccacag gtcggcccac aatgccacgc 240

atgccaccta cacctgccac atggatgtat tccacttcat ggccgacgac attttcagtg 300

tcaacatcac agaccagtct ggcaactact cccaggagtg tggcagcttt ctcctggctg 360

agagcatcaa gccggctccc cctttcaacg tgactgtgac cttctcagga cagtataata 420

tctcctggcg ctcagattac gaagaccctg ccttctacat gctgaagggc aagcttcagt 480

atgagctgca gtacaggaac cggggagacc cctgggctgt gagtccgagg agaaagctga 540

tctcagtgga ctcaagaagt gtctccctcc tccccctgga gttccgcaaa gactcgagct 600

atgagctgca ggtgcgggca gggcccatgc ctggctcctc ctaccagggg acctggagtg 660

aatggagtga cccggtcatc tttcagaccc agtcagagga gttaaaggaa ggctggaacg 720

gctccggctc tagagacaaa actcacacat gcccaccgtg cccagcacct gaagccctgg 780

gggcaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg atctcccgga 840

cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag gtcaagttca 900

actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg gaggagcagt 960

acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg 1020

gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc gagaaaacca 1080

tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccatcccggg 1140

aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc tatcccagcg 1200

acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag accacgcctc 1260

ccgtgctgga ctccgacggc tccttcttcc tctatagcaa gctcaccgtg gacaagagca 1320

ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg cacaaccact 1380

acacgcagaa gagcctctcc ctgtccccgg gtaaatgagt gaattc 1426

<210>29

<211>467

<212>PRT

＜213〉people

<400>29

Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly

1 5 10 15

Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr

20 25 30

Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr

35 40 45

Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser

50 55 60

Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr

65 70 75 80

Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val

85 90 95

Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe

100 105 110

Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val

115 120 125

Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp

130 135 140

Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys

180 185 190

Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser

195 200 205

Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Gly Ser Gly Ser Arg

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Leu Gly

245 250 255

Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210>30

<211>741

<212>DNA

＜213〉people

<400>30

atgccgcgtg gctgggccgc ccccttgctc ctgctgctgc tccagggagg ctggggctgc 60

cccgacctcg tctgctacac cgattacctc cagacggtca tctgcatcct ggaaatgtgg 120

aacctccacc ccagcacgct cacccttacc tggcaagacc agtatgaaga gctgaaggac 180

gaggccacct cctgcagcct ccacaggtcg gcccacaatg ccacgcatgc cacctacacc 240

tgccacatgg atgtattcca cttcatggcc gacgacattt tcagtgtcaa catcacagac 300

cagtctggca actactccca ggagtgtggc agctttctcc tggctgagag catcaagccg 360

gctccccctt tcaacgtgac tgtgaccttc tcaggacagt ataatatctc ctggcgctca 420

gattacgaag accctgcctt ctacatgctg aagggcaagc ttcagtatga gctgcagtac 480

aggaaccggg gagacccctg ggctgtgagt ccgaggagaa agctgatctc agtggactca 540

agaagtgtct ccctcctccc cctggagttc cgcaaagact cgagctatga gctgcaggtg 600

cgggcagggc ccatgcctgg ctcctcctac caggggacct ggagtgaatg gagtgacccg 660

gtcatctttc agacccagtc agaggagtta aaggaaggct ggaacaaaac cgaaacctcc 720

caggttgctc cggcataatg a 741

<210>31

<211>245

<212>PRT

＜213〉people

<400>31

Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly

1 5 10 15

Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr

20 25 30

Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr

35 40 45

Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser

50 55 60

Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr

65 70 75 80

Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val

85 90 95

Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe

100 105 110

Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val

115 120 125

Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp

130 135 140

Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys

180 185 190

Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser

195 200 205

Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Lys Thr Glu Thr Ser

225 230 235 240

Gln Val Ala Pro Ala

245

<210>32

<211>1413

<212>DNA

＜213〉people

<400>32

atgccgcgtg gctgggccgc ccccttgctc ctgctgctgc tccagggagg ctggggctgc 60

cccgacctcg tctgctacac cgattacctc cagacggtca tctgcatcct ggaaatgtgg 120

aacctccacc ccagcacgct cacccttacc tggcaagacc agtatgaaga gctgaaggac 180

gaggccacct cctgcagcct ccacaggtcg gcccacaatg ccacgcatgc cacctacacc 240

tgccacatgg atgtattcca cttcatggcc gacgacattt tcagtgtcaa catcacagac 300

cagtctggca actactccca ggagtgtggc agctttctcc tggctgagag catcaagccg 360

gctccccctt tcaacgtgac tgtgaccttc tcaggacagt ataatatctc ctggcgctca 420

gattacgaag accctgcctt ctacatgctg aagggcaagc ttcagtatga gctgcagtac 480

aggaaccggg gagacccctg ggctgtgagt ccgaggagaa agctgatctc agtggactca 540

agaagtgtct ccctcctccc cctggagttc cgcaaagact cgagctatga gctgcaggtg 600

cgggcagggc ccatgcctgg ctcctcctac caggggacct ggagtgaatg gagtgacccg 660

gtcatctttc agacccagtc agaggagtta aaggaaggct ggaacgatga cgatgacaag 720

ggctccggcg acaaaactca cacatgccca ccgtgcccag cacctgaagc cctgggggca 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1140

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc cccgggtaaa tga 1413

<210>33

<211>470

<212>PRT

＜213〉people

<400>33

Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu Gln Gly

1 5 10 15

Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr

20 25 30

Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr

35 40 45

Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser

50 55 60

Cys Ser Leu His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr

65 70 75 80

Cys His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val

85 90 95

Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe

100 105 110

Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val

115 120 125

Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp

130 135 140

Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys

180 185 190

Asp Ser Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser

195 200 205

Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Asp Asp Asp Asp Lys

225 230 235 240

Gly Ser Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Ala Leu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210>34

<211>1754

<212>DNA

＜213〉mice

<400>34

atgccccggg gcccagtggc tgccttactc ctgctgattc tccatggagc ttggagctgc 60

ctggacctca cttgctacac tgactacctc tggaccatca cctgtgtcct ggagacacgg 120

agccccaacc ccagcatact cagtctcacc tggcaagatg aatatgagga acttcaggac 180

caagagacct tctgcagcct acacaggtct ggccacaaca ccacacatat atggtacacg 240

tgccatatgc gcttgtctca attcctgtcc gatgaagttt tcattgtcaa tgtgacggac 300

cagtctggca acaactccca agagtgtggc agctttgtcc tggctgagag catcaaacca 360

gctcccccct tgaacgtgac tgtggccttc tcaggacgct atgatatctc ctgggactca 420

gcttatgacg aaccctccaa ctacgtgctg aggggcaagc tacaatatga gctgcagtat 480

cggaacctca gagaccccta tgctgtgagg ccggtgacca agctgatctc agtggactca 540

agaaacgtct ctcttctccc tgaagagttc cacaaagatt ctagctacca gctgcaggtg 600

cgggcagcgc ctcagccagg cacttcattc agggggacct ggagtgagtg gagtgacccc 660

gtcatctttc agacccaggc tggggagccc gaggcaggct gggacggctc cggctctaga 720

gagccccgcg gaccgacaat caagccctgt cctccatgca aatgcccagg taagtcacta 780

gaccagagct ccactcccgg gagaatggta agtgctataa acatccctgc actagaggat 840

aagccatgta cagatccatt tccatctctc ctcatcagca cctaacctcg agggtggacc 900

atccgtcttc atcttccctc caaagatcaa ggatgtactc atgatctccc tgagccccat 960

agtcacatgt gtggtggtgg atgtgagcga ggatgaccca gatgtccaga tcagctggtt 1020

tgtgaacaac gtggaagtac acacagctca gacacaaacc catagagagg attacaacag 1080

tactctccgg gtggtcagtg ccctccccat ccagcaccag gactggatga gtggcaaggc 1140

tttcgcatgc gccgtcaaca acaaagacct cccagcgccc atcgagagaa ccatctcaaa 1200

acccaaaggt gagagctgca gcctgactgc atgggggctg ggatgggcat aaggataaag 1260

gtctgtgtgg acagccttct gcttcagcca tgacctttgt gtatgtttct accctcacag 1320

ggtcagtaag agctccacag gtatatgtct tgcctccacc agaagaagag atgactaaga 1380

aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt tacgtggagt 1440

ggaccaacaa cgggaaaaca gagctaaact acaagaacac tgaaccagtc ctggactctg 1500

atggttctta cttcatgtac agcaagctga gagtggaaaa gaagaactgg gtggaaagaa 1560

atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg actaagagct 1620

tctcccggac tccgggtaaa tgagctcagc acccacaaaa ctctcaggtc caaagagaca 1680

cccacactca tctccatgct tcccttgtat aaataaagca cccagcaatg cctgggacca 1740

tgtaatagga attc 1754

<210>35

<211>240

<212>PRT

＜213〉mice

<400>35

Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly

1 5 10 15

Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr

20 25 30

Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser

35 40 45

Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe

50 55 60

Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His Ile Trp Tyr Thr

65 70 75 80

Cys His Met Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val

85 90 95

Asn Val Thr Asp Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser Phe

100 105 110

Val Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Leu Asn Val Thr Val

115 120 125

Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu

130 135 140

Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys

180 185 190

Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro Gln Pro Gly Thr

195 200 205

Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp Asp Gly Ser Gly Ser Arg

225 230 235 240

<210>36

<211>795

<212>DNA

＜213〉mice

<400>36

ctgcaggtcg acaccaccat gccccggggc ccagtggctg ccttactcct gctgattctc 60

catggagctt ggagctgcct ggacctcact tgctacactg actacctctg gaccatcacc 120

tgtgtcctgg agacacggag ccccaacccc agcatactca gtctcacctg gcaagatgaa 180

tatgagggac ttcaggacca agagaccttc tgcagcctac acaggtctgg ccacaacacc 240

acacatatat ggtacacgtg ccatatgcgc ttgtctcaat tcctgtccga tgaagttttc 300

attgtcaatg tgacggacca gtctggcaac aactcccaag agtgtggcag ctttgtcctg 360

gctgagagca tcaaaccagc tccccccttg aacgtgactg tggccttctc aggacgctat 420

gatatctcct gggactcagc ttatgacgaa ccctccaact acgtgctgag gggcaagcta 480

caatatgagc tgcagtatcg gaacctcaga gacccctatg ctgtgaggcc ggtgaccaag 540

ctgatctcag tggactcaag aaacgtctct cttctccctg aagagttcca caaagattct 600

agctaccagc tgcaggtgcg ggcagcgcct cagccaggca cttcattcag ggggacctgg 660

agtgagtgga gtgaccccgt catctttcag acccaggctg gggagcccga ggcaggctgg 720

gacggcagcg gacaccacca tcatcaccac ggtagcggcg actataaaga cgatgacgat 780

aagtagtgag aattc 795

<210>37

<211>255

<212>PRT

＜213〉mice

<400>37

Met Pro Arg Gly Pro Val Ala Ala Leu Leu Leu Leu Ile Leu His Gly

1 5 10 15

Ala Trp Ser Cys Leu Asp Leu Thr Cys Tyr Thr Asp Tyr Leu Trp Thr

20 25 30

Ile Thr Cys Val Leu Glu Thr Arg Ser Pro Asn Pro Ser Ile Leu Ser

35 40 45

Leu Thr Trp Gln Asp Glu Tyr Glu Glu Leu Gln Asp Gln Glu Thr Phe

50 55 60

Cys Ser Leu His Arg Ser Gly His Asn Thr Thr His Ile Trp Tyr Thr

65 70 75 80

Cys His Met Arg Leu Ser Gln Phe Leu Ser Asp Glu Val Phe Ile Val

85 90 95

Asn Val Thr Asp Gln Ser Gly Asn Asn Ser Gln Glu Cys Gly Ser Phe

100 105 110

Val Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Leu Asn Val Thr Val

115 120 125

Ala Phe Ser Gly Arg Tyr Asp Ile Ser Trp Asp Ser Ala Tyr Asp Glu

130 135 140

Pro Ser Asn Tyr Val Leu Arg Gly Lys Leu Gln Tyr Glu Leu Gln Tyr

145 150 155 160

Arg Asn Leu Arg Asp Pro Tyr Ala Val Arg Pro Val Thr Lys Leu Ile

165 170 175

Ser Val Asp Ser Arg Asn Val Ser Leu Leu Pro Glu Glu Phe His Lys

180 185 190

Asp Ser Ser Tyr Gln Leu Gln Val Arg Ala Ala Pro Gln Pro Gly Thr

195 200 205

Ser Phe Arg Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln

210 215 220

Thr Gln Ala Gly Glu Pro Glu Ala Gly Trp Asp Gly Ser Gly His His

225 230 235 240

His His His His Gly Ser Gly Asp Tyr Lys Asp Asp Asp Asp Lys

245 250 255

<210>38

<211>792

<212>DNA

＜213〉mice

<400>38

atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tgtacatttc ttacatctat 60

gccggcagcg gacaccacca tcatcaccac ggtagcggcg actataaaga cgatgacgat 120

aagggttccg gatgcctgga cctcacttgc tacactgact acctctggac catcacctgt 180

gtcctggaga cacggagccc caaccccagc atactcagtc tcacctggca agatgaatat 240

gaggaacttc aggaccaaga gaccttctgc agcctacaca ggtctggcca caacaccaca 300

catatatggt acacgtgcca tatgcgcttg tctcaattcc tgtccgatga agttttcatt 360

gtcaatgtga cggaccagtc tggcaacaac tcccaagagt gtggcagctt tgtcctggct 420

gagagcatca aaccagctcc ccccttgaac gtgactgtgg ccttctcagg acgctatgat 480

atctcctggg actcagctta tgacgaaccc tccaactacg tgctgagggg caagctacaa 540

tatgagctgc agtatcggaa cctcagagac ccctatgctg tgaggccggt gaccaagctg 600

atctcagtgg actcaagaaa cgtctctctt ctccctgaag agttccacaa agattctagc 660

taccagctgc aggtgcgggc agcgcctcag ccaggcactt cattcagggg gacctggagt 720

gagtggagtg accccgtcat ctttcagacc caggctgggg agcccgaggc aggctgggac 780

tagtgagaat tc 792

<210>39

<211>260

<212>PRT

＜213〉mice

<400>39

Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr Ile

1 5 10 15

Ser Tyr Ile Tyr Ala Gly Ser Gly His His His His His His Gly Ser

20 25 30

Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Gly Cys Leu Asp Leu

35 40 45

Thr Cys Tyr Thr Asp Tyr Leu Trp Thr Ile Thr Cys Val Leu Glu Thr

50 55 60

Arg Ser Pro Asn Pro Ser Ile Leu Ser Leu Thr Trp Gln Asp Glu Tyr

65 70 75 80

Glu Glu Leu Gln Asp Gln Glu Thr Phe Cys Ser Leu His Arg Ser Gly

85 90 95

His Asn Thr Thr His Ile Trp Tyr Thr Cys His Met Arg Leu Ser Gln

100 105 110

Phe Leu Ser Asp Glu Val PheIle Val Asn Val Thr Asp Gln Ser Gly

115 120 125

Asn Asn Ser Gln Glu Cys Gly Ser Phe Val Leu Ala Glu Ser Ile Lys

130 135 140

Pro Ala Pro Pro Leu Asn Val Thr Val Ala Phe Ser Gly Arg Tyr Asp

145 150 155 160

Ile Ser Trp Asp Ser Ala Tyr Asp Glu Pro Ser Asn Tyr Val Leu Arg

165 170 175

Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Leu Arg Asp Pro Tyr

180 185 190

Ala Val Arg Pro Val Thr Lys Leu Ile Ser Val Asp Ser Arg Asn Val

195 200 205

Ser Leu Leu Pro Glu Glu Phe His Lys Asp Ser Ser Tyr Gln Leu Gln

210 215 220

Val Arg Ala Ala Pro Gln Pro Gly Thr Ser Phe Arg Gly Thr Trp Ser

225 230 235 240

Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ala Gly Glu Pro Glu

245 250 255

Ala Gly Trp Asp

260

NY_Main 516391_1

Claims (32)

1. a treatment, improve or the autoimmune disease of prevention mammalian subject or the method for inflammatory diseases, described method comprises that giving described patient is selected from following IL-21/IL-21R antagonist: anti-IL-21R antibody, anti-IL-21 antibody, anti-IL-21R antigen-binding fragments of antibodies, anti-IL-21 antigen-binding fragments of antibodies and IL-21R soluble fragments, its consumption are enough to treatment, improve or prevent described disease.

2. a treatment, improve or the method for the disease of prevention mammalian subject, described disease is selected from: arthritis, atopic diseases, respiratory disease, inflammatory disease of the skin, inflammatory bowel disease, fibre modification disease, systemic lupus erythematosus (sle), transplant rejection and transplant rejection relevant disease; Described method comprises that giving described patient is selected from following IL-21/IL-21R antagonist: anti-IL-21R antibody, anti-IL-21 antibody, anti-IL-21R antigen-binding fragments of antibodies, anti-IL-21 antigen-binding fragments of antibodies and IL-21R soluble fragments, its consumption are enough to treatment, improve or prevent described disease.

3. the method for claim 2, wherein said anti-IL-21R antibody can be in conjunction with IL-21R, and are made up of the aminoacid sequence that has 90% homogeneity at least with sequence shown in the SEQ ID NO:2, and wherein said IL-21R can be in conjunction with IL-21.

4. the method for claim 3, wherein said arthritis is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriasis arthropathica and ankylosing spondylitis.

5. the method for claim 4, wherein said arthritis is rheumatoid arthritis.

6. the method for claim 3, wherein said respiratory disease is asthma or chronic obstructive pulmonary disease.

7. the method for claim 3, wherein said fibre modification disease are selected from visceral nerve fiberization, dermatofibrosis disease, eye fibre modification disease, systemic sclerosis, polymyositis, dermatomyositis, diffuse fasciitis with eosinophilia, Raynaud syndrome, glomerulonephritis and nasal polyp.

8. the method for claim 3, wherein said inflammatory bowel disease is selected from inflammatory bowel, ulcerative colitis and Crohn disease.

9. the method for claim 3, wherein said inflammatory disease of the skin is a psoriasis.

10. the method for claim 3, wherein said atopic diseases is selected from allergic asthma, atopic dermatitis, urticaria, eczema, allergic rhinitis and allergia gastroenteritis.

11. the method for claim 10, wherein said atopic diseases is an allergic asthma.

12. the method for claim 3, wherein said transplant rejection relevant disease is a graft versus host disease.

13. the method for claim 3, wherein said disease is a transplant rejection.

14. the method for claim 3, wherein said disease is a systemic lupus erythematosus (sle).

15. the method for claim 2, wherein said mammalian subject is behaved.

16. the method for claim 2, wherein said IL-21R soluble fragments is made up of IL-21R extracellular domain and Fc immunoglobulin fragment.

17. the method for claim 16, wherein said IL-21R extracellular domain comprises about amino acid/11-235 of SEQ ID NO:2.

18. the method for claim 2, wherein said IL-21R soluble fragments is made up of the aminoacid sequence that has 90% homogeneity at least with sequence shown in the SEQ IDNO:29.

19. the method for claim 2, wherein said IL-21/IL-21R antagonist are anti-IL-21R antibody or its Fab.

20. the method for claim 2, wherein said IL-21/IL-21R antagonist are anti-IL-21 antibody or its Fab.

21. a fusion rotein, it is made up of IL-21R extracellular domain and Fc immunoglobulin fragment, and sequence has at least 90% homogeneity shown in the aminoacid sequence of wherein said IL-21R and the SEQ ID NO:2, and wherein said fusion rotein can be in conjunction with IL-21.

22. the fusion rotein of claim 21, it is made up of the aminoacid sequence that has 90% homogeneity with sequence shown in the SEQ ID NO:29 at least.

23. a carrier, it has the nucleotide sequence of the fusion rotein of coding claim 21.

24. a recombinant host cell, it comprises the carrier of claim 23.

25. a method for preparing fusion rotein, described method comprises:

(a) can express under the condition of described fusion rotein, cultivate the recombinant host cell of claim 24; With

(b) reclaim described fusion rotein.

26. a pharmaceutical composition, it comprises IL-21/IL-21R antagonist and pharmaceutically acceptable carrier.

27. the pharmaceutical composition of claim 26, wherein said IL-21/IL-21R antagonist are selected from anti-IL-21R antibody, anti-IL-21 antibody, anti-IL-21R antigen-binding fragments of antibodies, anti-IL-21 antigen-binding fragments of antibodies and IL-21R soluble fragments.

28. the pharmaceutical composition of claim 27, wherein said IL-21R soluble fragments is made up of IL-21R extracellular domain and Fc immunoglobulin fragment.

29. one kind organ, tissue, cell or cell mass transplanted method to mammalian subject, described method comprises the steps:

(a) give described patient and be selected from following IL-21/IL-21R antagonist: anti-IL-21R antibody, anti-IL-21 antibody, anti-IL-21R antigen-binding fragments of antibodies, anti-IL-21 antigen-binding fragments of antibodies and IL-21R soluble fragments, its consumption is enough to reduce the danger of transplant rejection; With

(b) organ, tissue, cell or cell mass are transplanted to described patient,

Wherein said transplanting step (b) dosing step (a) before, during or carry out afterwards.

30. the method for claim 29, wherein said transplanted organ, tissue, cell or cell mass are selected from heart, kidney, liver, lung, pancreas, bone marrow, cartilage, cornea, neuronal tissue and cell thereof.

31. a treatment, prevent or improve the method for the transplant rejection of mammal transplant recipient, described method comprises:

(a) the transplant rejection symptom of detection transplant recipient; With

(b) give described transplant recipient and be selected from following IL-21/IL-21R antagonist: anti-IL-21R antibody, anti-IL-21 antibody, anti-IL-21R antigen-binding fragments of antibodies, anti-IL-21 antigen-binding fragments of antibodies and IL-21R soluble fragments.

32. the method for claim 31, wherein said transplant rejection symptom are selected from, and inflammation, organ dysfunction go down, repulsion sign and fibrosis in the biopsy.

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