CN102784392A

CN102784392A - Methods and compositions for use in treatment of patients with autoantibody positive disease

Info

Publication number: CN102784392A
Application number: CN2012102886855A
Authority: CN
Inventors: M·谢弗瑞尔; W·弗里穆特; 钟振劭; D·奥登海姆尔; M·D·珀金斯
Original assignee: Human Genome Sciences Inc
Current assignee: Human Genome Sciences Inc
Priority date: 2005-10-13
Filing date: 2006-10-05
Publication date: 2012-11-21

Abstract

The present invention relates to methods and compositions for use in treatment of patients with autoantibody positive disease. In a specific embodiment, the present invention relates to a method of treating a patient that has an ANA titer of 1:80 or greater and/or greater than or equal to 30 IU/ml of anti-dsDNA antibodies in his/her blood plasma or serum comprising administering a therapeutically effective amount of an immunomodulatory agent, such as an antagonist of Neutrokine-alpha. Additionally provided is a method of reducing the frequency and/or quantity of corticosteroid administration to patients. In preferred embodiments, the patient has systemic lupus erythematosus. Methods for determining if a lupus patient is responding to medical treatment are also provided.

Description

Method and composition for treating autoantibodies Disease

The application is the divisional application of the application of the Application No. 200680047110.7 that the applying date is on October 05th, 2006 entitled " being used for the method and composition for treating autoantibodies Disease "

Background technology

Neutrokine- α albumen (SEQ ID NO:2) be tnf ligand family a member, its with APRIL (28.7%, SEQ ID NO:4), TNF α (16.2%) and Lymphotoxin-α (LT α) (14.1%) enjoy Amino acid sequence identity (Moore, et al., (1999) Science 285:260-263).Neutrokine- α have multiple titles in science and technology and patent document, including bone-marrow-derived lymphocyte stimulant (BLyS), B cell activation factor (BAFF), TNF and ApoL correlations leucocyte expression part -1 (TALL-1) (Moore, et al., (1999) Science 285:260-263；Schneider et al.,(1999)J.Exp.Med.189:1747-1756；With Khare et al., (2000) Proc.Natl.Acad.Sci.97:3370-3375).Neutrokine- α formal name is TNF (part) superfamily member 13B (TNFSF13b).The polypeptide of total length Neutrokine- α 285 amino acid of gene code, its 47th to being transmembrane region between 73 amino acids, be the non-hydrophobic sequence of II type embrane-associated protein characteristics before transmembrane region.As other members of TNF families, Neutrokine- α are played a role in the form of trimer protein.After Neutrokine- α are in cell surface expression, the cell lysis outskirt at the 134th amino acids, to discharge the tripolymer of biological activity.

Isoacceptor is not combined known Neutrokine- α with three kinds from tumor necrosis factor receptor super family.They are cross-film activator and CAML interactions body (TACI, GenBank numbering AAC51790, SEQ ID NO:6), ripe antigen (BCMA, GenBank numbering NP_001183, SEQ the ID NO of B cell activating factor receptor, B cell:8) with (BAFF-R, GenBank numbering NP_443177SEQ ID NO:10).(Gross,etal.,(2000)Nature 404:995-999；Thompson et al.,(2001)Science 293:2108-2111；With Yan et al., (2000) Nature Immunol.1:252-256).The expression of acceptor is principally limited to bone-marrow-derived lymphocyte (Moore, et al., (1999) Science 285:260-263).Believe that Neutrokine- α most of effect is all mediated by BAFF-R, because unobvious (the Schieman in TACI or BCMA deficient mices of the major defect in Neutrokine- alpha expressions defect or the B cell component of BAFF-R expression deficient mices, et al., (2001) Science 292:2111-2114；Gross et al.,(2001)Immunity15:289-302；With Yan et al., (2000) Nature Immunol.1:252-256).

When detecting Neutrokine- α albumen in vitro and in vivo, Neutrokine- α are shown to promote the propagation of B cell, differentiation and survived.In addition, Neutrokine- α also show that some effect (MacKay et al., (1999) J.Exp.Med.190 to T cell:1697-1710；Huard et al.,(2001)J.Immunol.167:6225-6231；Huard et al.,(2004)Int.Immunol.16:467-475；Ng et al.,(2004)J.Immunol.173:807-817).The immunoglobulin concentrations that the mouse for being melted into overexpression Neutrokine- α by genetic engineering has the periphery B cell of increase number and increased.In addition, Neutrokine- α transgenic mices show autoimmune phenotype, and it is similar with what is seen in people's systemic loupus erythematosus, including occur autoantibody and glomerulonephritis related symptoms (Moore, et al., (1999) Science 285:260-263；MacKay,et al.,(1999)J.Exp.Med.192:129-135).The Neutrokine- alpha levels that later research is shown in the serum and/or synovial fluid of autoimmunity disease such as systemic loupus erythematosus, rheumatoid arthritis and Patients with Sjogren Syndrome are also (the Cheema et al., (2001) Arthritis Rheum.44 raised:1313-1319；Groom et al.,(2002)J.Clin.Invest.109:59-68；Mariette et al.,(2003)Ann.Rheum.Dis 62:168-171).Therefore, it is that Neutrokine- alpha-2 antagonists have therapeutic action in terms of autoimmunity disease is treated in the universal view of scientific circles.

Systemic loupus erythematosus (SLE or " lupus ") is a kind of autoimmunity disease, and its symptom is extremely heterogeneous.Current SLE diagnostic criteria includes 11 standards：(1) cheek " butterfly " erythema；(2) plate-like erythema；(3) photosensitization；(4) canker sore；(5) arthritis；(6) scrositis；(7) kidney trouble；(8) nervous system abnormality；(9) hematological abnormalities；(10) crucial immunological abnormality；(11) there is antinuclear antibodies.In Tan et al., (1982) Arthritis Rheum.25:1271-1277；With Hochberg et al., Arthritis Rheum. (1997) 40:These standards are explained in more detail in 1725.SLE can be just diagnosed as by meeting the patient of 4 standards in this 11 standards.Therefore, clinical diagnosis can have non-overlapping symptom for SLE individual.In addition, the symptom of multiple lupus symptoms and other diseases overlaps.For example, rheumatoid arthritis, Dermatomyositis, systemic sclerosis (or chorionitis), Sjogren syndrome and various forms of vasculitises all have some symptoms, including one or more following characteristics with lupus：Autoantibody occur includes antinuclear antibodies and Anti-hCG action, arthralgia and arthroncus, fash and organ involvement.Therefore, it is often difficult to be diagnosed to be the patient of lupus patient disease similar with other in clinical practice exactly.Causing the other factors of lupus difficult diagnosis to include disease does not have rapid progress, and patient accumulates symptom step by step within a certain period of time.In addition, SLE is a kind of disease for possessing variable activity in patient's body.Disease is sometimes static, and patient shows the increase of symptom number and severity at other, i.e., broken out in " activity ".Finally, neither one laboratory examination can make a definite diagnosis lupus.Therefore, this area be required to determine with characteristic symptom lupus patient subgroup and determine these patient subgroups and most probable so that these subgroups patient benefit treatment between correlation.

The application identifies special Serum of Patients With Autoimmune Diseases subgroup, and its most probable benefits from immunomodulator treatment.

The content of the invention

In 2 clinical trial phases, it is found that with the Antybody therapy lupus patient (the 0th, 14,28 days intravenous infusions, every 4 weeks intravenous infusions 1 time afterwards, until the 52nd week) for neutralizing Neutrokine- α albumen, it is 1 significantly to alleviate ANA titres:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to the symptom related to lupus in 30IU/mL patient subgroups (see embodiment 1).Surprisingly, from unlike the patients of whole selected clinical test, the statistically significant improvement for the clinical endpoint for determining disease activity is only obtained in patient's subgroup (such as SELENA SLEDA scorings decline, below by explaining in more detail).Therefore, the present invention relates to identify most probable response in patient's subgroup of the immunomodulator such as treatment of Neutrokine- α antagonist.

Therefore, in one embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient method, include apply therapeutically effective amount immunomodulator.Immunomodulator is described in greater detail below.In a specific embodiment, immunomodulator is Neutrokine- α antagonist, including but not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins or its fragment or variant, antibody or its antigen-binding fragment, Neutrokine- alpha bindings or polypeptide, Neutrokine- α and/or APRIL polypeptide variants (such as Neutrokine- α and/or APRIL dominant negative regulation thing (dominant negative) form) with reference to Neutrokine- α acceptors.Small molecular antagonists, Neutrokine- α peptide mimics, the antisense RNA that targets Neutrokine- α and short interfering rna (siRNA) of Neutrokine- α other antagonists including Neutrokine- α, the antisense RNA and short interfering rna (siRNA), the antisense RNA and short interfering rna (siRNA) that target Neutrokine- α acceptor and/or APRIL acceptor that target APRIL.Neutrokine- α acceptors include such as cross-film activator and CAML interactions body (TACI, GenBank numbering AAC51790, SEQ ID NO:6), ripe antigen (BCMA, GenBank numbering NP_001183, SEQ the ID NO of B cell activating factor receptor, B cell:8) with (BAFF-R, GenBank numbering NP 443177SEQ ID NO:10).

In another embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL Serum of Patients With Autoimmune Diseases method, include apply therapeutically effective amount immunomodulator.It is 1 that ANA titres, which can wherein be identified,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to the 30IU/mL autoimmunity disease example of patient and include, but are not limited to systemic loupus erythematosus (SLE), rheumatoid arthritis, Sjogren syndrome, chorionitis, Dermatomyositis, Felty syndromes, mixed connective tissue disease, raynaud's syndrome, juvenile chronic arthritis, glomerulonephritis, ITP and IgA nephrosis.

In a specific embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL Patients with Sjogren Syndrome method, include apply therapeutically effective amount immunomodulator.In another embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL Patients with Sjogren Syndrome method, include apply therapeutically effective amount Neutrokine- α antagonist.

In a specific embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL rheumatoid arthritis patients method, include apply therapeutically effective amount immunomodulator.In another embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL rheumatoid arthritis patients method, include apply therapeutically effective amount Neutrokine- α antagonist.

In a specific embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL systemic loupus erythematosus (SLE) patient method, include apply therapeutically effective amount immunomodulator.In another embodiment, it is 1 that the present invention, which provides treatment ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL systemic loupus erythematosus (SLE) patient method, include apply therapeutically effective amount Neutrokine- α antagonist.It is SLE (see such as Tan et al., Arthritis Rheum.25 by clinical diagnosis according to American society of rheumatism (ACR) standard lupus patient in a specific embodiment:1271-7,(1982)；With Hochberg et al., Arthritis Rheum.40:1725, (1997) are incorporated into the application by quoting entire contents herein).

Present invention provides the method for the treatment of patient, it is 1 to be included in using the ANA titres determined before immunomodulator in patients blood plasma or serum:80 or higher and/or Anti-hCG action be more than or equal to 30IU/mL.Present invention provides the method for the treatment of patient, it is 1 that the ANA titres in patients blood plasma or serum are determined before being included in the antagonist using Neutrokine- α:80 or higher and/or Anti-hCG action be more than or equal to 30IU/mL.

In other embodiments, the method that the present invention provides treatment lupus patient, be included in has one or more following characteristics using determination patient before immunomodulator：Foundation American society of rheumatism (ACR) standard clinical diagnosis is SLE (see such as Tan et al., Arthritis Rheum.25:1271-7,(1982)；With Hochberg et al., Arthritis Rheum.40:1725,(1997))；SELENA SLEDAI scorings >=6；Blood plasma or the level of complement of change of serum C 4 decline；Blood plasma or the level of complement of change of serum C 3 decline；ANA titres are 1:80 or higher；Blood plasma or anti-ds-DNA antibody are more than or equal to 30IU/mL；Receive >=metacortandracin of 7.5mg/ days or other be used to treat the related indication glucocorticoid of lupus；Receiving or previously-accepting mistake is used to treat the related indication immunodepressant of lupus.In a specific embodiment, clinician is determined according to the evaluation that patient cases record.In another particular embodiment of the invention, clinician is determined according to laboratory examination results.In a specific embodiment, the laboratory examination results that clinician includes obtaining before immunomodulator described herein (antagonist for including Neutrokine- α) drug therapy of administration therapeutically effective amount according to the drug therapy (drug therapy for including immunomodulator) of the last time lupus since patient and are determined.

In another embodiment, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient glucocorticoid frequency and/or amount method, include apply therapeutically effective amount immunomodulator.

In a specific embodiment, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL Patients with Sjogren Syndrome glucocorticoid frequency and/or amount method, include apply therapeutically effective amount immunomodulator.In another particular embodiment of the invention, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL Patients with Sjogren Syndrome glucocorticoid frequency and/or amount method, include apply therapeutically effective amount Neutrokine- α antagonist.

In a specific embodiment, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL rheumatoid arthritis patients glucocorticoid frequency and/or amount method, include apply therapeutically effective amount immunomodulator.In another particular embodiment of the invention, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL rheumatoid arthritis patients glucocorticoid frequency and/or amount method, include apply therapeutically effective amount Neutrokine- α antagonist.

In a specific embodiment, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL systemic loupus erythematosus (SLE) patient glucocorticoid frequency and/or amount method, include apply therapeutically effective amount immunomodulator.In another particular embodiment of the invention, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL systemic loupus erythematosus (SLE) patient glucocorticoid frequency and/or amount method, include apply therapeutically effective amount Neutrokine- α antagonist.It is SLE (see such as Tan et al., Arthritis Rheum.25 by clinical diagnosis according to American society of rheumatism (ACR) standard lupus patient in a specific embodiment:1271-7,(1982)；With Hochberg et al., Arthritis Rheum.40:1725, (1997) are incorporated into the application by quoting entire contents herein).

In further embodiment, it is 1 that the present invention, which provides reduction and is administered to ANA titres,:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient glucocorticoid amount method, the amount of the glucocorticoid is reduced by least 25% to≤7.5mg/ days.In a specific embodiment, glucocorticoid is selected from the group being made up of metacortandracin, prednisolone, hydrocortisone, methylprednisolone and dexamethasone.In further specific embodiment, glucocorticoid is metacortandracin.In another embodiment there is provided the method for the frequency and/or amount for reducing the glucocorticoid for being administered to Serum of Patients With Autoimmune Diseases, including apply the anti-Neutrokine- Alpha antibodies of therapeutically effective amount.

In another 2 clinical trial phase (embodiment 3), rheumatoid arthritis patients receive the treatment for the antibody for neutralizing Neutrokine- α albumen, method of application is the 0th, 14, venoclysises in 28 days 1 time, then once until the 24th week, the treatment most probable is alleviated is more than the rheumatoid arthritis related symptoms in 5.1 patient, the patient for not received anti-TNF treatments, and/or blood plasma and/or the serum rheumatoid factor (SRF) positive previously in its DAS28 scorings before starting to neutralize Neutrokine- α Antybody therapy within every 4 weeks.Show most probable response in neutralize Neutrokine- α albumen Antybody therapy other subgroups or rheumatoid arthritis patients include male patient, blood plasma and/or serum have anti-CCP (ring-type citrulling peptide (cycliccitrullinated pEptide the)) patient of antibody.Receiving to neutralize the antibody of Neutrokine- α albumen while receiving patient, the patient of previous methopterin Endodontic failure, and/or previous methopterin treatment and the patient of other at least one DMARD Endodontic failures of methopterin.

In another embodiment, the present invention provides a kind of SELENA SLEDAI, BILAG and PGA scoring for determining whether lupus patient replys the method in drug therapy, being included in using determination patient before drug therapy；Using drug therapy；And SELENA SLEDAI, BILAG and PGA scoring of patient is determined after drug therapy is applied.In this approach, as long as situations below patient occur is regarded as having response to drug therapy：Scored low 4 points or more points than the SELENA SLEDAI before drug therapy is applied in the SELENA SLEDAI scorings of the patient determined after applying drug therapy；Compared with the BILAG scorings determined before drug therapy is applied, the BILAG index scores of the patient determined after drug therapy is applied do not include the new scoring of BILAG A organ areas or 2 new BILAG B organ areas scorings；It is higher by with the PGA scorings determined after applying drug therapy than the PGA scorings determined before drug therapy is applied less than 0.3 point.

Therefore, in one embodiment, the method that the present invention provides treatment rheumatoid arthritis patients, be included in has one or more following characteristics using determination rheumatoid arthritis patients before immunomodulator：Patient had not received anti-TNF treatments such as infliximab (Infliximab) (also referred to as Remicade previously^TMCentocor, Inc.), adalimumab (adalimumab) (Abbott laboratories

) or Etanercept (etanercept) (There is rheumatoid factor in the blood plasma and/or serum of patient；Patient has measurable blood plasma and/or the anti-CCP of serum (ring-type citrulling peptide) antibody；Patient has the high-caliber blood plasma of increasing and/or serum CA125 (c reactive protein) level；Patient is previously through one or more modifying antirheumatic drug Endodontic failures for alleviating disease；Patient has high modification disease activity score (DAS28)；Patient has the joint of swelling and tenderness；Patient has morning stiffness；The erythrocyte sedimentation rate (ESR) of patient is accelerated and/or patient is male.

In another embodiment, the present invention provides a kind of aqueous pharmaceutical preparations, antibody including therapeutically effective amount, amount from about 5mM to about 50mM buffer, amount from about 150mM to about 500mM NaCl, measure from about 0.003% to about 0.05% surfactant, and pH value is from about 5.5 to about 6.5.In a specific embodiment, the antibody in above-mentioned preparation is the antibody with IgG1/ λ isotypes.In further embodiment, antibody in above-mentioned preparation is the human antibody for having IgG1/ λ isotypes, buffer in above-mentioned preparation is 10mM histidines or sodium citrate, the polyoxyethylene sorbitan monoleate (polysorbate80) that the surfactant amount of being in above-mentioned preparation is 0.01%w/v, NaCl concentration in above-mentioned preparation is about 150mM, and the pH value of preparation is 6.0.In other specific embodiments, above-mentioned preparation can keep stable at least 1 year in about 2-8 °C of temperature.In another embodiment, the amount of the antibody in above-mentioned preparation is 100mg/ml.

In a specific embodiment, the present invention provides aqueous pharmaceutical preparations, including 100mg/mlIgG1/ λ antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one (L-histidine monohydrochloride), 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleates, the wherein pH value of preparation is 6.0.

Brief description of the drawings

Following accompanying drawing is only intended to illustrate embodiments of the present invention, is not offered as limiting the scope of the present invention that claims are covered.

Fig. 1 shows that baseline ANA antibody is 1:80 or higher, and/or Anti-hCG action be more than or equal to 30IU/mL patient at the 52nd week SELENA SLEDAI decline percentage average value.Examined with t and determine p value.

Definition

In one aspect, present invention relates generally to the method for treating autoantibodies Disease by applying the immunomodulator of therapeutically effective amount.Autoantibodies Disease is the patient with detectable autoantibody titer in one or more biological body fluid sample such as blood plasma or serum or synovial fluid.

" immunomodulator " referred to herein as major class can stimulate or suppress the medical compounds of immune system.The working Examples of the application describe successful application of the anti-Neutrokine- Alpha antibodies of Antagonism in treatment lupus patient subgroup.Therefore, term " immunomodulator " be specifically intended to covering can as Neutrokine- α antagonist medical compounds or molecule.Neutrokine- α antagonist includes the composition of anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins or its fragment or variant, the antibody with reference to Neutrokine- α acceptors or its antigen-binding fragment and Neutrokine- alpha bindings or polypeptide.Neutrokine- α acceptors include such as cross-film activator and CAML interaction bodies (TACI, GenBank numbering AAC51790), BAFF-R (GenBank encode NP_443177) and the ripe antigen (BCMA, GenBank numbering NP_001183) of B cell.Particularly useful Neutrokine- α receptor type fashions include the cell outskirt of soluble form.Neutrokine- α acceptors or its fragment or variant and Neutrokine- α Binding peptides are used as fusion protein such as Fc or human serum albumins (HAS) fusion protein.Neutrokine- α other antagonists include Neutrokine- α small molecular antagonists, Neutrokine- α peptide mimicses, Neutrokine- α and/or APRIL polypeptide variants (such as Neutrokine- α and/or APRIL dominant negative regulation thing form).This Neutrokine- α and/or APRIL polypeptide variants can with antagonism Neutrokine- α functions, for example by disturb Neutrokine- α and/or APRIL with or heteromultimeric.Or, Neutrokine- α and/or APRIL polypeptide variants can prevent to include polypeptide combination Neutrokine- α acceptors such as TACI, BCMA and BAFF-R of the polypeptide and/or the signal transduction through the Neutrokine- α acceptors.Small molecular antagonists, Neutrokine- α peptide mimics, the antisense RNA that targets Neutrokine- α and short interfering rna (siRNA) of Neutrokine- α other antagonists including Neutrokine- α, the antisense RNA and short interfering rna (siRNA), the antisense RNA and short interfering rna (siRNA) that target Neutrokine- α acceptors and/or april receptor that target APRIL.Neutrokine- α antagonist is described in more detail below.

Believe Neutrokine- Alpha antibodies by reducing B cell number and/or the active such as immunoglobulin secretion of B cell to play a role.Therefore, term " immunomodulator " is also specifically intended to cover B cell conditioning agent, specifically directly or indirectly suppresses or reduces the drug molecule and compound of B cell activity (such as B cell proliferation, differentiation, existence or immunoglobulin secretion) and/or B cell number.In a specific embodiment, the B cell conditioning agent that uses of method that the present invention can be combined be reduce all B cells, activating B cell, pure B cell (

B cell), memory B cell, slurry property B cell (plasma B cell) and Plasmacytoid B cell (plasmacytoid B cell), the activity of CD19+B cells and/or CD20+B cells or the medicine of number.

Immune system is the complicated network system of cell and cell factor interaction.For example, antigen presenting cell (APC, such as macrophage and BMDC) and T cell (particularly CD4+T auxiliary (Th) cell) serve effect in terms of activation B cell proliferation and secretory antibody (being included in the autoantibody under some morbid states).Therefore, it is also possible to suppress B cell activity by reducing or suppressing APC or Th cell numbers or activity.Likewise, it is known that by such as Th1 and Th2 responses of different types of immune response.Can be used for the present invention immunomodulator can promote a type of immune response exceed it is another, therefore to treatment autoantibodies patient have positive effect.Therefore, term " immunomodulator " is most broadly specifically intended to covering stimulation or suppresses the activity or the drug molecule or composition of quantity of the one or more cells, cell surface molecule (such as cell-surface signal transduction molecule) and/or cell factor of immune system, including is used as congenital and/or a part of adaptive immune system cell, cell surface molecule (such as cell-surface signal transduction molecule) and cell factor.The cell of immune system includes, but are not limited to B cell, T cell, BMDC, monocyte, macrophage, neutrophil leucocyte, eosinophil, basophilic granulocyte, mast cell and natural killer (NK) cell.T cell differentiation antigen such as CD20 can be included but is not limited to by the cell surface molecule on the cell surface for the immune system that immunomodulator is stimulated or is suppressed.Important cell factor includes but is not limited to tnf ligand superfamily member in immune system, and the latter includes but is not limited to Neutrokine- α, APRIL and CD40L.

Embodiment

In the II clinical trial phases of Patients with SLE, it is found by the applicant that with the antibody of neutralization Neutrokine- α albumen, (method of application is the 0th, 14,28 days intravenous infusions, then every 4 weeks intravenous infusions 1 time, until the 52nd week) treatment lupus patient, it is 1 significantly to eliminate ANA titres:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to the symptom related to lupus in 30IU/mL patient (see embodiment 1).

Therefore, the specific embodiment of the present invention provides and its blood plasma or serum ANA titre >=1 is treated with Neutrokine- α antibody antagonists:80 and/or Anti-hCG action be more than or equal to 30IU/mL Patients with SLE method.But, those skilled in the art be readily apparent that antibody molecule be it is various can be as the one of which in the molecule of Neutrokine- α antagonist.Therefore, another embodiment of the invention provides its blood plasma of antagonist for treating with Neutrokine- α or serum ANA titre >=1:80 and/or Anti-hCG action be more than or equal to 30IU/mL Patients with SLE method.

Neutrokine- α antagonist includes the composition of anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins or its fragment or variant, the antibody with reference to Neutrokine- α acceptors or its antigen-binding fragment and Neutrokine- alpha bindings or polypeptide.Neutrokine- α acceptors include such as cross-film activator and CAML interaction bodies (TACI, GenBank numbering AAC51790), the ripe antigen (BCMA, GenBank numbering NP_001183) of BAFF-R (GenBank numbering NP_443177) and B cell.Particularly useful Neutrokine- α receptor type fashions include that the cell outskirt of Neutrokine- α soluble form can be combined.Neutrokine- α acceptors or its fragment or variant and Neutrokine- α Binding peptides are used as fusion protein such as Fc or human serum albumins (HAS) fusion protein.In a specific embodiment, Neutrokine- α antagonist is TACI-Fc albumen.One example of TACI-Fc albumen is SEQ ID NO:6 the 1 to 154th amino acids are blended with the Fc areas of IgG1 immunoglobulin molecules.In a specific embodiment, Neutrokine- α antagonist is BAFF-R-Fc albumen.One example of BAFF-R-Fc albumen is SEQ ID NO:10 the 1 to 70th amino acids are blended with the Fc areas of IgG1 immunoglobulin molecules.Optionally, the 27th amino acids (leucine) in BAFF-R are replaced with the 20th amino acids (valine) in asparagine substitution BAFF-R and with proline.SEQ IDNO:26 show the BAFF-R changed with the two amino acid the 1 to 70th amino acids.

Neutrokine- α other antagonists include Neutrokine- α small molecular antagonists, Neutrokine- α peptide mimicses, Neutrokine- α and/or APRIL polypeptide variants (such as Neutrokine- α and/or APRIL dominant negative regulation thing form).Such Neutrokine- α and/or APRIL polypeptide variants can with antagonism Neutrokine- α functions, for example by disturb Neutrokine- α and/or APRIL with or heteromultimeric.In a specific embodiment, Neutrokine- α antagonist is the Neutrokine- α protein fragments or its variant that play dominant negative regulation thing function.Or, Neutrokine- α and/or APRIL polypeptide variants can prevent to include polypeptide combination Neutrokine- α acceptors such as TACI, BCMA and BAFF-R of the polypeptide and/or the signal transduction through the Neutrokine- α acceptors.Small molecular antagonists, Neutrokine- α peptide mimics, the antisense RNA that targets Neutrokine- α and short interfering rna (siRNA) of Neutrokine- α other antagonists including Neutrokine- α, the antisense RNA and short interfering rna (siRNA), the antisense RNA and short interfering rna (siRNA) that target Neutrokine- α acceptors and/or april receptor that target APRIL.Neutrokine- α antagonist is described in more detail below.

Equally, those skilled in the art understand that other immunomodulators (specifically including that can adjust B cell activity or number molecules of interest) can be used for the present invention.In a particular embodiment, the B cell conditioning agent that is used together of method that can combine the present invention is can directly or indirectly to suppress or reduce the medicine of B cell activity (such as B cell proliferation, differentiation, survive or immunoglobulin secretion) and/or B cell number.In another embodiment, the B cell conditioning agent that is used together of method that can combine the present invention is can to reduce the activity or the medicine of number of all B cells, activating B cell, pure B cell, memory B cell, slurry property B cell and Plasmacytoid B cell, CD19+B cells and/or CD20+B cells.The immune modulatory molecules and B cell regulation molecule that can be used for the present invention are known for those skilled in the art, and are described in more detail below.

In another embodiment, the present invention provides the method for the patient that treatment is classified as in the Patients with SLE subgroup with " activity " systemic loupus erythematosus (SLE or " lupus ") lesion, including applies the Neutrokine- α of therapeutically effective amount antibody antagonists.In a particular embodiment, the present invention provides treatment and is previously diagnosed as lupus and has the method for the patient of activity lupus, including applies the Neutrokine- α of therapeutically effective amount antibody antagonists.The method that the present invention provides the patient that treatment is classified as in the Patients with SLE subgroup with " activity " systemic loupus erythematosus (SLE or " lupus ") lesion, determines that patient has " activity lupus " before being included in the antibody antagonists using the Neutrokine- α of therapeutically effective amount.In a particular embodiment, the present invention provides treatment and is previously diagnosed as lupus and has the method for the patient of activity lupus, determines that patient had previously been diagnosed as lupus and with " activity lupus " before being included in the antibody antagonists using the Neutrokine- α of therapeutically effective amount.

In another embodiment, the present invention provides the method for the patient that treatment is classified as in the Patients with SLE subgroup with " activity " systemic loupus erythematosus (SLE or " lupus ") lesion, including applies the Neutrokine- α of therapeutically effective amount antibody antagonists or other immunomodulators known in the art and/or described herein.In a particular embodiment, the present invention provides treatment and is previously diagnosed as lupus and has the method for the patient of activity lupus, including applies the Neutrokine- α of therapeutically effective amount antibody antagonists or other immunomodulators known in the art and/or described herein.The method that the present invention provides the patient that treatment is classified as in the Patients with SLE subgroup with " activity " systemic loupus erythematosus (SLE or " lupus ") lesion, determines that patient has " activity lupus " before being included in antibody antagonists or other immunomodulators known in the art and/or described herein using the Neutrokine- α of therapeutically effective amount.In a particular embodiment, the present invention provides treatment and is previously diagnosed as lupus and has the method for the patient of activity lupus, determines that patient had previously been diagnosed as lupus and with " activity lupus " before being included in antibody antagonists or other immunomodulators known in the art and/or described herein using the Neutrokine- α of therapeutically effective amount.

In a particular embodiment, activity lupus patient be defined as according to American society of rheumatism (ACR) standard by clinical diagnosis for SLE patient (see such as Tan et al., Arthritis Rheum.25:1271-7,(1982)；With Hochberg et al., Arthritis Rheum.40:1725, entire contents are incorporated herein by (1997) by quoting herein).

In a particular embodiment, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=4.SELENA SLEDAI represent the systemic loupus erythematosus disease activity index revised such as estrogen in the national evaluation test of the security in lupus erythematosus.Clinician can routinely determine that SELENA SLEDAI score using techniques known in the art and methodology, see such as Bombardier, et al., Arthritis Rheum.Jun;35(6):630-40,1992；And Strand, et al., J Rheumatol.Feb;26(2):490-7,1999, entire contents are incorporated herein by quoting herein.Simply, by considering to determine that SELENA SLEDAI score across 24 SLE disease activity projects in 9 tracts.The lesion score of some tracts weights more than the lesion of other tracts.Specifically, the SLE disease activities performance if there is central nervous system and blood vessel is then designated as 8 points；SLE disease activities performance if there is kidney and muscle skeleton is then designated as 4 points；2 points are then designated as if there is the performance of serous cavity, skin and immunologic SLE disease activities；And then it is designated as 1 point if there is whole body and the performance of hematological SLE disease activities.The peak of SELENASLEDAI scorings is 105 in theory, but actually only has small number of patients to be scored above 45 points.

In the SELENA SLEDAI points-scoring systems of standard, if patient has the emerging albuminuria for being more than 0.5g/24 hours or albuminuria to increase recently more than 0.5g/24 hours, 4 points are designated as.In other words, if the high 0.5g of numerical value being measured in the last twenty-four-hour urine liquid sample of the albuminuria numeric ratio patient obtained in a twenty-four-hour urine liquid sample, then the albuminuria one in SELENA SLEDAI grade forms is designated as 4 points.This is usually described albuminuria increase or new appearance " 0.5g/24 hours ".Therefore, in the SELENA SLEDAI points-scoring systems of standard, in baseline because albuminuria is designated as 4 points of patient and will have improved SELENA SLEDAI in follow-up, condition is that the albuminuria numerical value being measured in the last twenty-four-hour urine liquid sample of Urine proteins numeric ratio in current twenty-four-hour urine liquid sample is no more than 0.5g.In other words, patient will deduct 4 points from its total score, even there is stable albuminuria or albuminuria increase≤0.5g/24 hours.Examples 2 describe the revision to SELENA SLEDAI albuminuria code of points.In example 2, albuminuria scoring has been revised so that be persistently designated as 4 points, unless the albuminuria numerical value being measured in the albuminuria being measured in twenty-four-hour urine liquid sample twenty-four-hour urine liquid sample more the last than patient has reduced by more than 0.5g.In addition, if emerging albuminuria or albuminuria increase>0.5g/24 hours, then it is designated as 4 points.If mentioning SELENA SLEDAI grade forms herein, represent that the scoring of albuminuria can be completed with the SELENASLEDAI grade forms of establishing criteria.Preferably, the albuminuria points-scoring system according to embodiment 2 determines the scoring of the albuminuria in the SELENA SLEDAI scorings of patient.

In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=5.In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=6.In further specific embodiment, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=7.In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=8.In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=9.In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=10.In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=11.In other specific embodiments, activity lupus patient is defined as the patient of SELENA SLEDAI scorings >=12.

In other implementations, activity lupus patient is defined as the patient for having Anti-hCG action in its blood plasma or serum.Clinician can routinely determine Anti-hCG action titre, concentration or level using techniques known in the art and method.A kind of example detection method for determining Anti-hCG action titre, concentration or level is the enzyme-linked immuno-sorbent assay (ELISA) of the specific bond of the dsDNA based on Anti-hCG action and immobilization, see such as Halbert, et al., J Lab Clin Med.97:97-111,(1981).Another example detection method for determining dsDNA antibody titers, concentration or level is the indirect immunofluorescene assay method of the specific bond based on Anti-hCG action and crithidia luciliae dsDNA, sees such as Whiteside, et al., Am J Clin Pathol.72:829-35,(1979).Another example detection method for determining dsDNA antibody titers, concentration or level be the dsDNA based on Anti-hCG action and isotope marks specific bond and subsequent Anti-hCG action-isotope marks dsDNA compounds precipitation Farr detection methods, see such as Davis, et al., Am J Clin Pathol., 67:374-8,(1977).In a particular embodiment, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 30 international unit/mL, wherein international unit (IU) is the reference preparation for being dependent on World Health Organization's Anti-hCG action, see such as Feltkamp, et al., Ann.Rheum.Dis., 47:740-746,(1988).The full content of all bibliography cited in this section is incorporated into the application by quoting at this.

In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 40IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 50IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 60IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 75IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 100IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 125IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 150IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 200IU/mL.In other specific embodiments, activity lupus patient is defined as the patient that Anti-hCG action in its blood plasma or serum is more than or equal to 300IU/mL.

In other implementations, activity lupus patient is defined as the patient in its blood plasma or serum with antinuclear antibodies (ANA+).Clinician can routinely determine antinuclear antibodies titre using techniques known in the art and method.An example detection method for determining antinuclear antibodies titre is the indirect immunofluorescene assay method of the specific bond based on antinuclear antibodies Yu HEp-2 human epithelial cells, sees such as Osborn, et al., Arthritis Rheum., 27:1286-9,(1984).In another example detection method, antinuclear antibodies concentration or level can be determined using the ELISA of the specific bond based on ANA and ANA antigens such as dsDNA, Ro/SS-A, La/SS-B, Sm, RNP of immobilization, such as Fenger, et al. is seen, Clin Chem., 50:2141-7,(2004).In Kavanaugh et al., Archives of Pathology ＆ Laboratory Medicine (2000) 124:71-81 and Greidinger, EL and Hoffman, RW, Laboratory Medicine (2003) 34:ANA detections are further described in 113-117.The full content of all bibliography cited in this section is incorporated into the application by quoting at this.

In a preferred embodiment, it is 1 that activity lupus patient, which is defined as ANA titres,:80 or higher patient's (i.e. when the blood plasma of patient or the coefficient of dilution of serum are 80 or higher, the ANA inspection results of the positive can be obtained).Such as titre 1:160、1:320 and 1:640 are both greater than titre 1:80.Other preferred embodiment in, it is 1 that the patient of activity lupus, which is defined as ANA titres,:160 or higher patient.Other preferred embodiment in, activity lupus patient is defined as ANA titres for 1:320 or higher patient.Other preferred embodiment in, activity lupus patient is defined as ANA titres for 1:640 or higher patient.In a specific embodiment, ANA titres are determined using the IIF based on HEp-2 cells.In another particular embodiment of the invention, ANA titres are determined using anti-dsDNA ELISA detection methods.

In other implementations, activity lupus patient is defined as the patient with detectable autoantibody, and the autoantibody includes but is not limited to anti-Ro/SS-A antibody, anti-La/SS-B antibody, anti-RNP antibody, anticardiolipin (anti-phosphatide) antibody, Anti-hCG action, anti-Sm antibody.Clinician can routinely determine the titre, concentration or level of autoantibody using techniques known in the art and methodology.

In other embodiments, activity lupus patient is defined as the patient of blood plasma or change of serum C 3 and/or C4 level of complement with reduction.Those skilled in the art understand that C3 and/or C4 normal level can be with different, depending on the detection method for determining C3 and/or C4.Therefore, the normal level of blood plasma or the complement of change of serum C 3 can be from about 90mg/dL to about 180mg/dL.In other specific embodiments, the scope of the normal level of blood plasma or the complement of change of serum C 3 can also be from about 88mg/dL to about 206mg/dL or from about 88mg/dL to about 252mg/dL.The normal level of blood plasma or the complement of change of serum C 4 can be from about 16mg/dL to about 47mg/dL.In other specific embodiments, the scope of the normal level of blood plasma or the complement of change of serum C 4 can also be from about 12mg/dL to about 72mg/dL or from about 13mg/dL to about 75mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 3 are defined as being less than 90mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 3 are defined as being less than 88mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 3 are defined as being less than 85mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 3 are defined as being less than 80mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 3 are defined as being less than 75mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 16mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 15mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 14mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 13mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 12mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 11mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 10mg/dL.In a particular embodiment, the blood plasma of reduction or the level of complement of change of serum C 4 are defined as being less than 9mg/dL.Clinician for example can routinely determine level of complement using techniques known in the art and methodology using radio-immunity disperse detection method.

In other embodiments, activity lupus patient is defined as the patient of any one or more following features：The foundation U.S. rheumatology association (ACR) standard clinical diagnosis is SLE (see such as Tan et al., Arthritis Rheum.25:1271-7,(1982)；With Hochberg et al., Arthritis Rheum.40:1725,(1997))；SELENA SLEDAI scorings >=6；The blood plasma or the level of complement of change of serum C 4 of reduction；The blood plasma or the level of complement of change of serum C 3 of reduction；ANA titres are 1:80 or higher；Blood plasma or serum Anti-hCG action are more than or equal to 30IU/mL；Receive >=metacortandracin of 7.5mg/ days or other be used to treat the related indication glucocorticoid of lupus；And/or receive or previously-accepting mistake be used for treat the related indication immunosuppressive therapy of lupus.

In other embodiments, activity lupus patient is defined as the patient of any one or more following features：It is SLE according to the U.S. rheumatology association (ACR) standard clinical diagnosis；SELENA SLEDAI scorings >=8；The blood plasma or the level of complement of change of serum C 4 of reduction；The blood plasma or the level of complement of change of serum C 3 of reduction；ANA titres are 1:80 or higher；Blood plasma or serum Anti-hCG action are more than or equal to 30IU/mL；Receiving the up to metacortandracin of 40mg/ days or other is used to treat the related indication glucocorticoid of lupus；And/or receive or previously-accepting mistake be used for treat the related indication immunosuppressive therapy of lupus.

Multiple Disease Activity Index are all well known for this area clinician, and can be used for determining rheumatism mobility such as SLE disease activities degree (see such as Strand, et al., J Rheumatol., 26:490-7,(1999)).In one embodiment, SELENA SLEDAI are used as Disease Activity Index (DAI) (see such as Bombardier, et al., Arthritis Rheum.35:630-40,(1992)).In another embodiment, SLE Flare indexes are used as DAI (see such as Petri, et al., Lupus, 8:685-91,(1999)).In further embodiment, systemic loupus erythematosus international cooperation unit/rheumatology association of U.S. damage index (SLICC/ACR) be used as DAI (see such as Gladman, et al., Arthritis Rheum., 39:363-9,(1996)).In another embodiment, doctor's overall assessment (PGA) is used as DAI, and PGA is a kind of visual analogue scale, and scope is from 0 to 3, wherein 0 is no disease activity, 1 is minor ailment activity, and 2 be medium disease activity, and 3 be serious disease activity.In still another embodiment, medical science lapses to the simple form (Medical Outcomes Survey Short Form 36, SF-36) of investigation and is used as DAI.SF-36 is related quality of life (HRQOL) instrument of general health, all have shown that it has reflected influence (Cooks of the SLE to all aspects of HRQOL in observation property cohort studies and randomized controlled trial, et al., J.Rheumatol., 27:1892-1895,(2000)；Thumboo,et al.,J Rheumatol.,26:97-102,(1999)；Thumboo,et al.,J Rheumatol.,27:1414-1420,(2000)；Ware JE, et al., Med Care, 30:473-483,(1992)；Smolen JS,et al.,J Rheumatol.,26:504-507,(1999)；Gladman,et al.,Lupus,5:190-195,(1996)；Alonso J,et al.,Qual Life Res.,13:283-298,2004；With Gladman et al, J Rheumatol., 27:377-9, their full content is incorporated herein by (1995) by quoting herein).

In further embodiment, EQ-5D (also referred to as EuroQol instruments) is used as DAI.EQ-5D is a kind of related quality of life survey tool of general health.This is a kind of simple, personal questionnaire, and it not only includes descriptive health status categorizing system, additionally it is possible to which generation reflects the composite score or index of the deviation value related to given health status.EQ-5D describes sexual system and is made up of 5 parts：Mobility, Self-Care, daily routines, pain/discomfort and anxiety/depression.There are 3 levels each part, reflects " no health problem ", " medium health problem " and " severe health problem " (see such as Health Policy.1990Dec respectively;16(3):Its content, is incorporated herein by 199-208 by quoting herein).

In further embodiment, the secondary grade forms of functional evaluation-weak (FACIT-F) of the chronic disease treatment in functional evaluation (FACIT) measuring system of chronic disease treatment are used as DAI.FACIT-F level grade form is the combination of the FAQs of 27 projects, is divided into 4 main QOL parts：Condition, society/home state, emotional state and functional status.The survey tool have collected patient on energy level, burnout and start or the ability of completion activity in terms of feedback (see, such as Yellen, S.B., et al., Journal of Pain and Symptom Management, 13:63-74,(1997)；Cella,D.,et al.,94(2):528-538,(2002)；And Cella, D., et al., Journal of Pain ＆ Symptom Management, 24 (6):Entire contents, are incorporated herein by 547-561, (2002) by quoting herein).

In further embodiment, disease activity scoring (DAS28) is used as DAI.DAS28 is the conventional tool for being used to evaluate rheumatic disease mobility that rheumatologist uses.The survey tool calculates index score according to following evaluation：Evaluation (VAS of number, the number in swelling (SW) joint, erythrocyte sedimentation rate (ESR) (ESR) and the patient in tenderness (TEN) joint to disease activity；Mm) (see such as Van der Heijde D.M.F.M., et al., J.Rheumatol, 20:579-8,(1993)；Prevoo M.L.L.,et al.,Arthritis Rheum,38:Entire contents, are incorporated herein by 44-8, (1995) by quoting herein).

In further embodiment, The British Isles's lupus evaluation group (BILAG) is used as DAI (see for example, Isenberg, et al., Rheumatology, 44:902-6,(2005)；Gordon,et al.,Rheumatology,42(11):1372-9,(2003)；Isenberg,et al.,Lupus,9(9):651-4,(2000)；Hay et al.,Q J Med.,86:447-58,(1993)；The BLIPS issued with ADS-Limathon Ltd on April 4th, 2004^TMEntire contents, are incorporated herein by the 3.0th edition software program user's guide by quoting herein).BILAG indexes include the known 8 organ/systems influenceed by lupus, and each scoring is both depended on compared with previous evaluation, and whether clinical manifestation is new, deterioration, identical or improvement.For all 8 organ/systems, the severity of SLE diseases performance is respectively A, B, C, D or E scoring, and wherein A is the most serious (see for example, Hay, ibid).BILAG indexes provide the composite score for evaluating Disease severity and treatment validity.The composite score is combined with all 8 organ/systems impacted in lupus.Using BILAG known in the art or other measuring methods, treatment can target the lupus patient of the disease performance with special organ/system subgroup.

Therefore, in a specific embodiment, if patient has been obtained for the reduction of its SELENASLEDAI scorings, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In a specific embodiment, if scored with the baseline SELENA SLEDAI of same patient, the SELENA SLEDAI scorings being measured to before patient starts immunomodulator treatment compare, patient has been obtained for the reduction of its SELENA SLEDAI scorings, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In a specific embodiment, if patient has been obtained at least 4 points of decline in its SELENA SLEDAI scorings, the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In one specific embodiment, if scored with the baseline SELENA SLEDAI of same patient, the SELENA SLEDAI scorings being measured to before patient starts immunomodulator treatment compare, patient has been obtained at least 4 points of decline in its SELENA SLEDAI scorings, then the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.

In another particular embodiment of the invention, if patient does not show the deterioration of the disease activity determined such as doctor's overall evaluation (PGA), the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In a specific embodiment, if PGA scorings are decreased, still stablize or increase no more than 0.3 point, patient does not show the deterioration of disease activity.In a specific embodiment, if scored with the baseline PGA of same patient, the PGA scorings being measured to before patient starts immunomodulator treatment compare, the PGA scorings of the patient are decreased, still stable or increase no more than 0.3 point, then patient does not show the deterioration of disease activity.

In another particular embodiment of the invention, if patient does not show the deterioration of the disease activity determined such as The British Isles lupus evaluation group (BILAG) disease activity index, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In a specific embodiment, if patient there is no new BILAG A organ areas scoring or there is no 2 new BILAG B organ areas scorings, patient does not show the deterioration of disease activity.In a specific embodiment, if evaluated with the baseline BILAG of same patient, the BILAG being measured to before patient starts immunomodulator treatment, which is evaluated, to compare, patient there is no new BILAG A organ areas scoring or there is no 2 new BILAG B organ areas scorings, then patient does not show the deterioration of disease activity.

In another particular embodiment of the invention, if scored respectively with their baseline SELENASLEDAI, PGA scores to evaluate with The British Isles's lupus evaluation group (BILAG) and compared, patient has been obtained for the decline of its SELENA SLEDAI scorings, the notable deterioration of its PGA scorings is not shown, and do not show the deterioration of disease activity determined such as BILAG disease activity index, the lupus patient for then receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with being evaluated respectively with its baseline SELENA SLEDAI scorings, PGA scorings and BILAG, patient has been obtained at least 4 points of decline in its SELENA SLEDAI scorings, the increase of its PGA scorings no more than 0.3 point and there is no new BILAG A organ areas scoring or there is no 2 new BILAG B organ areas scorings, then the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.

In another particular embodiment of the invention, if the metacortandracin dosage of patient is reduced, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another embodiment, if the baseline metacortandracin dosage with patient, the metacortandracin dosage taken before patient starts immunomodulator treatment compares, the metacortandracin dosage of patient is reduced, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if the metacortandracin dosage of patient has already decreased to few 25%, the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient has already decreased to few 25% and arrived, receiving or receiving the lupus patient that immunomodulator known in the art and/or described herein treats be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient is arrived than baseline metacortandracin dosage reduction at least 25%, receiving or receiving the lupus patient that immunomodulator known in the art and/or described herein treats be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if the metacortandracin dosage of patient has already decreased to few 50%, the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient has already decreased to few 50% and arrived, receiving or receiving the lupus patient that immunomodulator known in the art and/or described herein treats be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient is arrived than baseline metacortandracin dosage reduction at least 50%, receiving or receiving the lupus patient that immunomodulator known in the art and/or described herein treats be considered as response in or have answered that in the treatment.

Lupus patient response can be determined with other measuring methods in the quality for the treatment of.In a specific embodiment, if patient has improved SF-36 health surveies scoring (Health Survey Score), the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline SF-36 health surveies of patient scoring, patient has improved a SF-36 health surveies scoring, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.

In a specific embodiment, if patient has improved an EQ-5D scorings, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline EQ-5D of patient scorings, patient has improved an EQ-5D scorings, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.

In a specific embodiment, if patient shows the shown weak mitigation gone out of FACIT-F scorings through patient, receiving or receiving the lupus patient that immunomodulator known in the art and/or described herein treats be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline FACIT-F of patient scorings, patient shows the shown weak mitigation gone out of FACIT-F scorings through patient, then receiving or receiving the lupus patient that immunomodulator known in the art and/or described herein treats be considered as response in or have answered that in the treatment.

In a specific embodiment, if patient has improved a DAS28 scorings, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline DAS28 of patient scorings, patient has improved a DAS28 scorings, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.

In another particular embodiment of the invention, if patient has the reduction of seizure frequency and/or duration, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if seizure frequency before being treated with immunomodulator and/or compared with the duration, patient has the reduction of seizure frequency and/or duration, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if patient has the mitigation of breaking-out severity, the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the breaking-out severity before being treated with immunomodulator, patient has the reduction of breaking-out severity, then the lupus patient for receiving or receiving immunomodulator known in the art and/or described herein treatment be considered as response in or have answered that in the treatment.SLE breaking-out index assessment lupus symptoms deteriorate the frequency and severity of (breaking-out).Breaking-out is divided into " slight or moderate " or " severe ".The breaking-out of slight or moderate includes following one or more：SELENASLEDAI scorings change 3 points or more；New/plate-like, photosensitization, deep, that veins beneath the skin is scorching or epidermolysis lupus for deteriorating；Pharynx nasalis ulcer；Pleurisy；Pericarditis；Arthritis；Generate heat (SLE)；The increase of strong pine dosage, but no more than 0.5mg/kg/ days；Because disease activity adds NSAID or hydroxychloroquine sulfate (Plaquenil) dosage；And PGA scorings increase to above 1.0, but no more than 2.5.Severe attack includes following one or more：SELENA SLEDAI scorings change more than 12 points；New/CNS-SLE for deteriorating；Vasculitis；Ephritis；Myositis；Plt<60,000；Anaemia (<7% or Hb declines>3%)；Metacortandracin dosage is doubled；Metacortandracin dosage is increased to above 0.5mg/kg/ days；Prescription endoxan；Prescription imuran；Prescription methopterin；(SLE) and PGA scorings increase to above 2.5 points in hospital.In another particular embodiment of the invention, broken out the reduction of seizure frequency and/or severity that assessment of indices arrives through SLE if patient has, the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the seizure frequency and/or severity before being treated with immunomodulator, patient has to break out the reduction of seizure frequency and/or severity that assessment of indices arrives through SLE, then the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if the reduction of seizure frequency and/or severity that the SLE breaking-out assessment of indices that patient has modified version is arrived, the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the seizure frequency and/or severity before being treated with immunomodulator, the reduction of seizure frequency and/or severity that the SLE breaking-out assessment of indices that patient has modified version is arrived, then the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.The SLE breaking-outs index of modified version does not include being scored by SELENA SLEDAI changing the serious attack individually triggered.

Therefore, if patient has shown that the reduction of its SELENA SLEDAI scorings, the antagonist for then receiving or having received Neutrokine- α (includes but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with the SELENA SLEDAI scorings scored with the baseline SELENA SLEDAI of same patient, being measured to before patient starts the treatment of Neutrokine- alpha-2 antagonists, patient has been obtained for the reduction of its SELENA SLEDAI scorings, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In a specific embodiment, if patient has been obtained at least 4 points of decline in its SELENA SLEDAI scorings, the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In one specific embodiment, if compared with the SELENA SLEDAI scorings scored with the baseline SELENA SLEDAI of same patient, being measured to before patient starts the treatment of Neutrokine- alpha-2 antagonists, patient has been obtained at least 4 points of decline in its SELENA SLEDAI scorings, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies (peptibody).In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if patient does not show the deterioration through disease activity determined by doctor's overall evaluation (PGA), then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, if PGA scorings are decreased, still stablize or increase no more than 0.3 point, patient does not show the deterioration of disease activity.In a specific embodiment, if compared with the PGA scorings scored with the baseline PGA of same patient, being measured to before patient starts the treatment of Neutrokine- alpha-2 antagonists, the PGA scorings of the patient are decreased, still stable or increase no more than 0.3 point, then patient does not show the deterioration of disease activity.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if patient does not show the deterioration of the disease activity determined such as The British Isles lupus evaluation group (BILAG) disease activity index, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, if patient there is no new BILAG A organ areas scoring or there is no 2 new BILAG B organ areas scorings, patient does not show the deterioration of disease activity.In a specific embodiment, if compared with the BILAG evaluated with the baseline BILAG of same patient, being measured to before patient starts the treatment of Neutrokine- alpha-2 antagonists is evaluated, patient there is no new BILAG A organ areas scoring or there is no 2 new BILAG B organ areas scorings, then patient does not show the deterioration of disease activity.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if scored respectively with their baseline SELENA SLEDAI, PGA scores to evaluate with BILAG and compared, and patient has been obtained for the decline of its SELENA SLEDAI scorings, the notable deterioration of its PGA scorings is not shown, and do not show the deterioration of disease activity determined such as The British Isles lupus evaluation group (BILAG) disease activity index, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with being evaluated respectively with its baseline SELENA SLEDAI scorings, PGA scorings and BILAG, patient has been obtained at least 4 points of decline in its SELENA SLEDAI scorings, the increase of its PGA scorings no more than 0.3 point and there is no new BILAG A organ areas scoring or there is no 2 new BILAG B organ areas scorings, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if the metacortandracin dosage of patient is reduced, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In another embodiment, if the baseline metacortandracin dosage with patient, the metacortandracin dosage taken before patient starts the treatment of Neutrokine- alpha-2 antagonists compares, the metacortandracin dosage of patient is reduced, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if the metacortandracin dosage of patient has already decreased to few 25%, the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient has already decreased to few 25% and arrived, receiving or receiving the lupus patient that Neutrokine- alpha-2 antagonists treat be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient is arrived than baseline metacortandracin dosage reduction at least 25%, receiving or receiving lupus patient that Neutrokine- alpha-2 antagonists treat be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if the metacortandracin dosage of patient has already decreased to few 50%, the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient has already decreased to few 50% and arrived, receiving or receiving the lupus patient that Neutrokine- alpha-2 antagonists treat be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, be less than or equal to 7.5mg/ days if the metacortandracin dosage of patient is arrived than baseline metacortandracin dosage reduction at least 50%, receiving or receiving lupus patient that Neutrokine- alpha-2 antagonists treat be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

Lupus patient response can be determined with other measuring methods in the quality for the treatment of.In a specific embodiment, if patient has improved SF-36 health surveies scoring, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline SF-36 health surveies of patient scoring, patient has improved SF-36 health surveies scoring, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, if patient has improved EQ-5D scorings, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline EQ-5D of patient scorings, patient has improved EQ-5D scorings, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, if patient shows the weak mitigation gone out shown by the FACIT-F scorings through patient, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline FACIT-F of patient scorings, patient shows the weak mitigation gone out shown by the FACIT-F scorings through patient, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, if patient has improved DAS28 scorings, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the baseline DAS28 of patient scorings, patient has improved DAS28 scorings, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if patient has the reduction of seizure frequency and/or duration, then receive or receiving Neutrokine- alpha-2 antagonists and (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if with Neutrokine- alpha-2 antagonists treat before seizure frequency and/or compared with the duration, patient has the reduction of seizure frequency and/or duration, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if patient has the mitigation of breaking-out severity, the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the breaking-out severity before being treated with Neutrokine- alpha-2 antagonists, patient has the reduction of breaking-out severity, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.SLE breaking-out index assessment lupus symptoms deteriorate the frequency and severity of (breaking-out).Breaking-out is divided into " slight or moderate " or " severe ".The breaking-out of slight or moderate includes following one or more：SELENA SLEDAI scorings change 3 points or more；New/plate-like, photosensitization, deep, that veins beneath the skin is scorching or epidermolysis lupus for deteriorating；Pharynx nasalis ulcer；Pleurisy；Pericarditis；Arthritis；Generate heat (SLE)；The increase of strong pine dosage, but no more than 0.5mg/kg/ days；Because disease activity adds NSAID or hydroxychloroquine sulfate dosage；And PGA scorings increase to above 1.0, but no more than 2.5.Severe attack includes following one or more：SELENA SLEDAI scorings change more than 12 points；New/CNS-SLE for deteriorating；Vasculitis；Ephritis；Myositis；Plt<60,000；Anaemia (<7% or Hb declines>3%)；Metacortandracin dosage is doubled；Metacortandracin dosage is increased to above 0.5mg/kg/ days；Prescription endoxan；Prescription imuran；Prescription methopterin；(SLE) and PGA scorings increase to above 2.5 points in hospital.In another particular embodiment of the invention, broken out the reduction of seizure frequency and/or severity that assessment of indices arrives through SLE if patient has, the lupus patient for receiving or receiving immunomodulator treatment known in the art and/or described herein be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the seizure frequency and/or severity before being treated with immunomodulator, patient has to break out the reduction of seizure frequency and/or severity that assessment of indices arrives through SLE, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if the reduction of seizure frequency and/or severity that the SLE breaking-out assessment of indices that patient has modified version is arrived, the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.In another particular embodiment of the invention, if compared with the seizure frequency and/or severity before being treated with Neutrokine- alpha-2 antagonists, the reduction of seizure frequency and/or severity that the SLE breaking-out assessment of indices that patient has modified version is arrived, then the lupus patient for receiving or receiving the treatment of Neutrokine- alpha-2 antagonists be considered as response in or have answered that in the treatment.The SLE breaking-outs index of modified version does not include being scored by SELENA SLEDAI changing the serious attack individually triggered.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

The state of lupus patient can individually or be jointly evaluated using above-mentioned Disease Activity Index (such as SELENA SLEDAI, PGA, BILAG, SLE breaking-outs index, SF-36 health surveies scoring, EQ-5D, FACIT-F, DAS28).(anti-Neutrokine- Alpha antibodies or its antigen-binding fragment can also be included but is not limited in beginning Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment after score measured value to evaluate the improvement for the patient health state being measured to such as these Disease Activity Index by the previous Disease Activity Index of relatively one or more patients.In addition, in a specific embodiment, if patient remains compared with previous measured value disease activity, scoring has improvement, the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, one or more Disease Activity Index scorings can be evaluated before Neutrokine- alpha-2 antagonists or other immunomodulators are started or at the 1st after starting Neutrokine- alpha-2 antagonists or other immunomodulators, 2,3,4,5,6,7,8,10,11 or 12 weeks.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, it is known in the art or other immunomodulators described herein (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment with Neutrokine- alpha-2 antagonists, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treat the lupus patient that the disease with one or more organ/systems is showed.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a particular embodiment, it is known in the art or disease of other immunomodulators described herein treatment with one or more inherent tracts shows and be with or without the lupus patient that mucocutaneous and/or musculoskeletal system is involved with Neutrokine- alpha-2 antagonists.In a particular embodiment, it is known in the art or disease of other immunomodulators described herein treatment with one or more inherent tracts is showed and without the lupus patient of mucocutaneous and/or musculoskeletal system involvement with Neutrokine- alpha-2 antagonists.In lupus, involve mucocutaneous and/or musculoskeletal system disease performance and include but is not limited to：Plate-like erythema, cheek erythema or other skin maculas, mucosal ulcer, panniculitis, cutaneous vasculitis, skin thrombosis, refer to toe infarct, refer to all erythema of toe thrombosis, baldness, first, pernio, splinter hemorrhages, myositis, panarthritis, arthritis, tendonitis, arthralgia and myalgia.In lupus, the inherent tract that can be influenceed by lupus includes but is not limited to nervous system, the circulatory system, respiratory system, uropoiesis/excretory system, digestive system and eyes.The lupus disease performance of nervous system includes but is not limited to aseptic meningitis, cerebral vasculitis, demyelinating syndromes, myelopathy, acute confusional state, mental disease, acute inflammation demyelinating polyneuropathy root disease, mononeuropathy, cranial neuropathy, plexopathy, DPN, epileptics, status epilepticus, the cerebrovascular disease that non-vascular inflammation is caused, cognitive disorder, ataxia, autonomic neuropathy, cerebellar ataxia, headache, antimigraine, emotional handicap and anxiety disorder.The lupus disease performance of the circulatory system includes but is not limited to myocarditis, heart failure, arrhythmia cordis, new valve insufficiency, scrositis, pericardial tamponade, pleural effusion with expiratory dyspnea, empsyxis, pulmonary vasculitis, chromic fibrous pulmonary alveolitis, interstitial pneumonia, shrinkage lung syndrome, aortitis and coronary artery inflammation.The lupus disease performance of digestive system includes but is not limited to peritonitis, belly scrositis, ascites, lupus enteritis, lupus colitis, malabsorption, protein-losing enteropathy, hepatitis, intestinal pseudo obstruction, acute cholecystitis and acute pancreatitis.The lupus disease performance related to eyes includes but is not limited to the inaccessible disease of eye socket inflammation, keratitis, anterior uveitis, posterior uveitis, retinal vasculitis, episcleritis, sclerotitis, retina/choroidal artery, cutoid bodies, optic neuritis and anterior ischemic optic neuropathy.

It can monitor that patient is known in the art to Neutrokine- alpha-2 antagonists or other immunomodulators described herein (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment by the one or more phase inner evaluation biological markers after treatment is started and the baseline and/or previous measured value that compare the biological marker evaluation of patient and the same biological marker of patient, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment response.The biological marker that can be evaluated includes but is not limited to immunoglobulin level (such as total serum immunoglobulin, and serum IgM, IgG, IgA, and/or IgE levels), autoantibody (such as Anti-hCG action, antiCCP antibody, anti- Ro/SS-A antibody, anti- La/SS-B antibody, anti-RNP antibody, anticardiolipin (anti-phosphatide) antibody and anti-Sm antibody level and ANA titres), B cell number (such as total B cell number, activating B cell number, pure B cell number, memory B cell number, starch sample B cell number, Plasmacytoid B cell number, total CD19+B cells and/or CD20+B cell numbers), C4 level of complement, C3 level of complement.In a specific embodiment, Biological measurement value is evaluated before Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators are started and/or in 1 after starting Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators, 2,3,4,5,6,7,8,9,10,11 or 12 weeks, Month And Year.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, if compared with the baseline measures of the immunoglobulin of patient, patient has immunoglobulin level (such as total serum immunoglobulin of reduction, and serum IgM, IgG, IgA, and/or IgE levels), then receiving or receiving Neutrokine- alpha-2 antagonists or other immunomodulators (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a specific embodiment, if compared with the immunoglobulin measured value that one or many patients are previous, patient has the immunoglobulin level (such as total serum immunoglobulin and serum IgM, IgG, IgA, and/or IgE level) of reduction, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with the immunoglobulin measured value that one or many patients are previous, patient maintains the immunoglobulin level (such as total serum immunoglobulin and serum IgM, IgG, IgA, and/or IgE level) of reduction, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if patient has normal immunoglobulin level (such as total serum immunoglobulin and serum IgM, IgG, IgA, and/or IgE level), the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.

In a specific embodiment, if compared with the baseline measures of the autoantibody of patient, patient has autoantibody (such as Anti-hCG action of reduction, antiCCP antibody, anti- Ro/SS-A antibody, anti- La/SS-B antibody, anti-RNP antibody, anticardiolipin (anti-phosphatide) antibody and anti-Sm antibody level and ANA titres) level, then receiving or receiving Neutrokine- alpha-2 antagonists or other immunomodulators (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a specific embodiment, if compared with one or many previous autoantibody measured values of patient, patient has autoantibody (such as Anti-hCG action, antiCCP antibody, anti-Ro/SS-A antibody, anti-La/SS-B antibody, anti-RNP antibody, anticardiolipin (anti-phosphatide) antibody and anti-Sm antibody level and ANA titres) level of reduction, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with one or many previous autoantibody measured values of patient, patient maintains autoantibody (such as Anti-hCG action, antiCCP antibody, anti-Ro/SS-A antibody, anti-La/SS-B antibody, anti-RNP antibody, anticardiolipin (anti-phosphatide) antibody and anti-Sm antibody level and ANA titres) level of reduction, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if patient obtains normal autoantibody (such as Anti-hCG action, antiCCP antibody, anti-Ro/SS-A antibody, anti-La/SS-B antibody, anti-RNP antibody, anticardiolipin (anti-phosphatide) antibody and anti-Sm antibody level and ANA titres) level, the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, the autoantibody of IgG isotypes is determined.

In a specific embodiment, if compared with the baseline measures of the B cell number of patient, patient has B cell number (total B cell number of reduction, activating B cell number, pure B cell number, memory B cell number, starch sample B cell number, Plasmacytoid B cell number, total CD19+B cells and/or CD20+B cell numbers), then receiving or receiving Neutrokine- alpha-2 antagonists or other immunomodulators (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a specific embodiment, if compared with the previous B cell number measured value with one or many patients, patient has the B cell number (total B cell number, activating B cell number, pure B cell number, memory B cell number, slurry sample B cell number, Plasmacytoid B cell number, total CD19+B cells and/or CD20+B cell numbers) of reduction, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with the previous B cell number measured value with one or many patients, patient maintains the B cell number (total B cell number, activating B cell number, pure B cell number, memory B cell number, slurry sample B cell number, Plasmacytoid B cell number, total CD19+B cells and/or CD20+B cell numbers) of reduction, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if patient obtains normal B cell number (total B cell number, activating B cell number, pure B cell number, memory B cell number, slurry sample B cell number, Plasmacytoid B cell number, total CD19+B cells and/or CD20+B cell numbers), the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.

In a specific embodiment, if compared with the C4 baseline measureses of patient, patient has the serum complement factor C4 levels increased, then receiving or receiving Neutrokine- alpha-2 antagonists or other immunomodulators (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a specific embodiment, if compared with the previous C4 measured values with one or many patients, patient has the C4 levels that increase, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with the previous C4 measured values with one or many patients, patient maintains the C4 levels increased, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if patient obtains normal C4 levels, the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.

In a specific embodiment, if compared with the C3 baseline measureses of patient, patient has the serum complement factor C3 levels increased, then receiving or receiving Neutrokine- alpha-2 antagonists or other immunomodulators (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment lupus patient be considered as response in or have answered that in the treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a specific embodiment, if compared with the previous C3 measured values with one or many patients, patient has the C3 levels that increase, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if compared with the previous C3 measured values with one or many patients, patient maintains the C3 levels increased, then the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.In a specific embodiment, if patient obtains normal C3 levels, the lupus patient for receiving or receiving Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators be considered as response in or have answered that in the treatment.

In a particular embodiment, the present invention provides the method that treatment had previously received the patient of one or more immunosuppressant treatments, Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein including applying therapeutically effective amount, including but not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor.In a particular embodiment, the present invention provides the method that treatment has previously been diagnosed as the patient of systemic loupus erythematosus (lupus) and previously-accepting excessively one or more immunosuppressant treatments, including applies Neutrokine- alpha-2 antagonists or other immunomodulators.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a particular embodiment, the immunodepressant of the previously-accepting mistake of patient is imuran (such as according to lily magnolia^TM), endoxan (for example

CTX), calcineurin inhibitor such as FK506, tacrolimus or cyclosporin be (for example

) and/or

(mycophenolate, its active metabolite is mycophenolic acid).

The treatment method the most general of current lupus and other autoimmunity diseases all utilizes the medicine of the non-specific various Inflammatory Pathways of blocking.Medicine the most dangerous may be exactly glucocorticoid in this treatment method.Although glucocorticoid such as metacortandracin is the essential drugs for controlling disease performance, they also have a variety of ill-effects such as overall immunosupress to cause infection, osteoporosis to cause fracture and atherosclerosis to cause early stage to the health of patient and occur cardiac event and apoplexy.In clinical test, it is found by the applicant that with the treatment of the antibody for neutralizing Neutrokine- α albumen (the 0th, 14 and 28 days intravenous infusions, then every 4 weeks once, until the 52nd week) can efficiently reduce alleviate lupus patient disease performance needed for glucocorticoid metacortandracin dosage.Particularly, the reduction of the metacortandracin amount in the treatment of anti-Neutrokine- Alpha antibodies and treatment phase last 3 months is relevant.It is 1 in baseline ANA titres:80 or higher, and/or anti-dsDNA be more than or equal to 30IU/mL patient in, there is the higher proportion of patient for receiving anti-Neutrokine- Alpha antibodies to reduce their metacortandracin dosage, on the contrary, the patient for receiving placebo treatment of greater number has increased metacortandracin dosage to more than 7.5mg/ days.

Therefore, in one embodiment, the method that the present invention provides the amount for reducing the frequency for being administered to the glucocorticoid treatment of patient and/or glucocorticoid, Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein including applying therapeutically effective amount, including but not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor.In a particular embodiment, glucocorticoid is metacortandracin, prednisolone, hydrocortisone, methylprednisolone or dexamethasone.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a specific embodiment, wherein the patient for reducing glucocorticoid treatment is the patient with inflammation.In another particular embodiment of the invention, the patient for wherein reducing glucocorticoid treatment is the patient with autoimmunity disease, including but not limited to rheumatoid arthritis, lupus, Sjogren syndrome or other autoimmune diseases a kind of autoimmunity disease as described herein.

Therefore, in a specific embodiment, the method that the present invention provides the frequency for the glucocorticoid treatment that reduction is administered to systemic loupus erythematosus (lupus) patient and/or the amount of glucocorticoid, Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein including applying therapeutically effective amount, including but not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor.In another particular embodiment of the invention, the method that the present invention provides the frequency for the Prednisone Therapy that reduction is administered to systemic loupus erythematosus (lupus) patient and/or the amount of metacortandracin, Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein including applying therapeutically effective amount, including but not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor." therapeutically effective amount " referred to herein as reduces the amount of the recipe quantity of the glucocorticoid needed for common alleviation disease performance.These show and are all known to clinician for the methodology of the amount for the antibody/composition for determining effectively to mitigate these severities showed.In a preferred embodiment, the dosage for being administered to the antibody of the invention of patient is 0.1mg to 100mg/kg weight in patients.It is highly preferred that it is between 0.1mg to 20mg/kg weight in patients to be administered to the dosage of patient.In most preferred embodiments, the dosage for being administered to patient is 1,4,10 or 20mg/kg.

In a specific embodiment, (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein when same patient receives simultaneously, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) therapeutic scheme when, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤80mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤40mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to less than 20mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤10mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤8mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤6mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤4mg/ days from previously higher dosage.In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤2mg/ days from previously higher dosage.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount for being administered to the glucocorticoid (such as metacortandracin) of patient has been reduced to≤7.5mg/ days from previously higher dosage.In a specific embodiment, compared with the metacortandracin dosage that patient is taken before starting to include using Neutrokine- alpha-2 antagonists or other immunomodulators, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount finally reduction at least 25% of the glucocorticoid (such as metacortandracin) of patient is administered to.In a specific embodiment, compared with the metacortandracin dosage that patient is taken before starting to include using Neutrokine- alpha-2 antagonists or other immunomodulators, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, the amount finally reduction at least 50% of the glucocorticoid (such as metacortandracin) of patient is administered to.In a specific embodiment, compared with the metacortandracin dosage that patient is taken before starting to include using Neutrokine- alpha-2 antagonists or other immunomodulators, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, being administered to the amount of the glucocorticoid (such as metacortandracin) of patient finally reduces at least 25% to≤7.5mg/ days.In a specific embodiment, compared with the metacortandracin dosage that patient is taken before starting to include using Neutrokine- alpha-2 antagonists or other immunomodulators, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein simultaneously, being administered to the amount of the glucocorticoid (such as metacortandracin) of patient finally reduces at least 50% to≤7.5mg/ days.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a specific embodiment, when same patient receives the therapeutic scheme including applying Neutrokine- alpha-2 antagonists or other immunomodulators simultaneously, patient either temporarily or permanently stopped glucocorticoid (such as metacortandracin) treatment.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In a particular embodiment, the patient that clinical diagnosis is rheumatoid arthritis (RA) is treated with Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein.In a particular embodiment, the rheumatoid arthritis patients treated do not have B cell tumour.In addition, rheumatoid arthritis patients also optionally receive one or more medicine such as salicylates for being applied to treatment RA, non-steroid anti-inflammatory drug such as Indomethacin, benzoxazolone, phenylacetic acid derivatives (such as brufen and fenoprofen), methyl α-naphthyl acetate (Naproxenic acid), pyrrolealkanoic acid (Tometin), heteroauxin (Sulindac), haloanthranilic (meclofenamate sodium), Piroxicam, zomepirac sodium and Diflunisal, antimalarial drugs such as chloroquine, gold salt, penicillamine, or immunodepressant such as methopterin or glucocorticoid, the dosage is the common dose or decrement of these medicines.However, preferably rheumatoid arthritis patients only receive Neutrokine- alpha-2 antagonists or the treatment of other immunomodulators known in the art and/or affiliated herein.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.Dosage regimen as described below applies these immunomodulators to RA patient, and those skilled in the art can be readily determined the dosage regimen.With Paulus indexes (Paulus et al.Arthritis Rheum.33:477-484 (1990)) determine that initial response, i.e. morning stiffness, pain and inflamed joints number, the raising of erythrocyte sedimentation rate (ESR) (ESR) and the 5 points of Disease severity grade forms evaluated through patient and doctor at least improve 2 points.One or more RA symptoms of the RA patient treated as described above will be alleviated using Neutrokine- alpha-2 antagonists or other other immunomodulators known in the art or described herein.

In 2 clinical trial phases (embodiment 3), rheumatoid arthritis patients receive the treatment for the antibody for neutralizing Neutrokine- α albumen, 0th, 14,28 days intravenous infusions, then, every 4 weeks 1 time until the 24th week, treatment is more likely to alleviate symptom related to rheumatoid arthritis in the patient of rheumatoid factor positive in patient of its DAS28 scorings more than 5.1, the patient for not received anti-TNF treatment previously, and/or blood plasma and/or serum before starting to neutralize the Antybody therapy of Neutrokine- α albumen.Show to be more likely to additional subgroup of the response in the treatment for the antibody for neutralizing Neutrokine- α albumen including male patient, the patient of blood plasma and/or serum anti-CCP (cyclic citrullinated peptide) antibody positive, while receiving to neutralize the patient of patient, the patient of previous methopterin Endodontic failure, and/or the treatment of previous methopterin and other at least one DMARD Endodontic failures of the antibody of Neutrokine- α albumen and methopterin.

Therefore, the present invention provides a kind of method that use Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein treat rheumatoid arthritis patients, wherein the rheumatoid arthritis patients have following any one or more of feature：Patient had not received anti-TNF treatments such as infliximab (also referred to as Remicade previously^TMCentocor, Inc.), adalimumab (Abbott laboratories) or Etanercept

The blood plasma and/or the serum rheumatoid factor (SRF) positive of patient；There is detectable anti-CCP (cyclic citrullinated peptide) antibody in the blood plasma and/or serum of patient；Blood plasma and/or serum CA125 (c reactive protein) level that patient has been improved；Patient had previously had one or more disease modification resisting rheumatoid disease medicine failure histories；Patient has high improved disease activity scoring (DAS28)；Patient has the joint of swelling and tenderness；Patient has morning stiffness；Patient has high erythrocyte sedimentation rate (ESR) (ESR) and/or male patient.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In a particular embodiment, the rheumatoid factor of the blood plasma of rheumatoid arthritis patients and/or serum is equal to or more than 12IU/ml.In a particular embodiment, the CRP levels improved are defined as at least 1.5mg/L.In a particular embodiment, the CRP levels improved are defined as at least 5mg/L.In a particular embodiment, the CRP levels improved are defined as at least 6mg/L.In a particular embodiment, the CRP levels improved are defined as at least 9mg/L.In a particular embodiment, the CRP levels improved are defined as at least 10mg/L.In a particular embodiment, the CRP levels improved are defined as at least 20mg/L.In a particular embodiment, the antiCCP antibody of the blood plasma of rheumatoid arthritis patients and/or serum is equal to or more than 10 units.In a particular embodiment, the antiCCP antibody of the blood plasma of rheumatoid arthritis patients and/or serum is equal to or more than 20 units.In a particular embodiment, patient had previously included but is not limited to the Endodontic failure of methopterin, Aminoquinuride, SASP and leflunomide through one or more DMARD.In a particular embodiment, patient is previously through methopterin Endodontic failure.In a particular embodiment, the DAS scorings of patient are more than 5.1.In a particular embodiment, patient has the joint of at least six swelling and the joint of at least eight tenderness.In a particular embodiment, the ESR of patient is more than 28mm/ hours.In a particular embodiment, the morning stiffness time of patient is at least 45 minutes.In a particular embodiment, the morning stiffness time of patient is at least 1 hour.In a particular embodiment, the morning stiffness time of patient is at least 1 and a half hours.In a particular embodiment, the morning stiffness time of patient is at least 2 hours.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

Therefore, the present invention is provided (includes but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment with Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment rheumatoid arthritis patients method.Wherein described rheumatoid arthritis patients have following any one or more of feature：Patient had not received anti-TNF treatments such as infliximab (also referred to as Remicade previously^TMCentocor, Inc.), adalimumab (Abbott laboratories

) or Etanercept

The blood plasma and/or the serum rheumatoid factor (SRF) positive of patient；There is detectable anti-CCP (cyclic citrullinated peptide) antibody in the blood plasma and/or serum of patient；Blood plasma and/or serum CA125 (c reactive protein) level that patient has been improved；Patient had previously had one or more disease modification resisting rheumatoid disease medicine failure histories；Patient has high improved disease activity scoring (DAS28)；Patient has the joint of swelling and tenderness；Patient has morning stiffness；Patient has high erythrocyte sedimentation rate (ESR) (ESR) and/or male patient.In a particular embodiment, the rheumatoid factor of the blood plasma of rheumatoid arthritis patients and/or serum is equal to or more than 12IU/ml.In a particular embodiment, the CRP levels improved are defined as at least 1.5mg/L.In a particular embodiment, the CRP levels improved are defined as at least 5mg/L.In a particular embodiment, the CRP levels improved are defined as at least 6mg/L.In a particular embodiment, the CRP levels improved are defined as at least 9mg/L.In a particular embodiment, the CRP levels improved are defined as at least 10mg/L.In a particular embodiment, the CRP levels improved are defined as at least 20mg/L.In a particular embodiment, the antiCCP antibody of the blood plasma of rheumatoid arthritis patients and/or serum is equal to or more than 10 units.In a particular embodiment, the antiCCP antibody of the blood plasma of rheumatoid arthritis patients and/or serum is equal to or more than 20 units.In a particular embodiment, patient had previously included but is not limited to the Endodontic failure of methopterin, Aminoquinuride, SASP and leflunomide through one or more DMARD.In a particular embodiment, patient is previously through methopterin Endodontic failure.In a particular embodiment, the DAS scorings of patient are more than 5.1.In a particular embodiment, patient has the joint of at least six swelling and the joint of at least eight tenderness.In a particular embodiment, the ESR of patient is more than 28mm/ hours.In a particular embodiment, the morning stiffness time of patient is at least 45 minutes.In a particular embodiment, the morning stiffness time of patient is at least 1 hour.In a particular embodiment, the morning stiffness time of patient is at least 1 and a half hours.In a particular embodiment, the morning stiffness time of patient is at least 2 hours.

In another particular embodiment of the invention, if rheumatoid arthritis patients have been obtained for ACR20 responses, then receiving or receiving that Neutrokine- alpha-2 antagonists are known in the art or other immunomodulators described herein (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment rheumatoid arthritis patients be considered as response in or have answered that in the treatment.ACR20 be rheumatology association of the U.S. (ACR) develop evaluate patient to the index of the response for the treatment of rheumatoid arthritis.ACR20 responses are defined as in addition to 3 evaluations that 5 other symptoms or disease performance are evaluated in (i.e. Patient Pain Assessment, patient global evaluation, doctor's overall assessment, the disabled of patient's self-assessment, acute phase reactant (ESR or CRP)) all at least improve 20%, also want tenderness joint number and swollen joint number at least to reduce 20%.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if rheumatoid arthritis patients have been obtained for ACR50 responses, then receiving or receiving that Neutrokine- alpha-2 antagonists are known in the art or other immunomodulators described herein (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment rheumatoid arthritis patients be considered as response in or have answered that in the treatment.ACR50 be rheumatology association of the U.S. (ACR) develop evaluate patient to the index of the response for the treatment of rheumatoid arthritis.ACR50 responses are defined as in addition to 3 evaluations that 5 other symptoms or disease performance are evaluated in (i.e. Patient Pain Assessment, patient global evaluation, doctor's overall assessment, the disabled of patient's self-assessment, acute phase reactant (ESR or CRP)) all at least improve 50%, also want tenderness joint number and swollen joint number at least to reduce 50%.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

In another particular embodiment of the invention, if rheumatoid arthritis patients have been obtained for ACR70 responses, then receiving or receiving that Neutrokine- alpha-2 antagonists are known in the art or other immunomodulators described herein (include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins (such as TACI, BCMA or BAFF-R) or its fragment or variant, anti- Neutrokine- α acceptors (such as TACI, BCMA or BAFF-R) antibody or its antigen-binding fragment, Neutrokine- α Binding peptides, Neutrokine- α and/or APRIL polypeptide variants, target Neutrokine- α, APRIL, TACI, BCMA, the antisense or siRNA of BAFF-R or other Neutrokine- α and/or APRIL acceptor) treatment rheumatoid arthritis patients be considered as response in or have answered that in the treatment.ACR70 be rheumatology association of the U.S. (ACR) develop evaluate patient to the index of the response for the treatment of rheumatoid arthritis.ACR70 responses are defined as in addition to 3 evaluations that 5 other symptoms or disease performance are evaluated in (i.e. Patient Pain Assessment, patient global evaluation, doctor's overall assessment, the disabled of patient's self-assessment, acute phase reactant (ESR or CRP)) all at least improve 70%, also want tenderness joint number and swollen joint number at least to reduce 70%.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.

Immunomodulator

Offer immunomodulator treatment ANA titres of the present invention are 1:80 or higher and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient method.It discussed above is the implication of " immunomodulator " as used herein.In a specific embodiment, immunomodulator is Neutrokine- alpha-2 antagonists." antagonist " represents to suppress or antagonism Neutrokine- α external and/or internal function affect and/or biological action (for example stimulate differentiation, propagation, and/or the existence of B cell；B cell is stimulated to generate Ig；With combine Neutrokine- α acceptors) medicine.Can occur this inhibitory action when presence or absence of antagonist contact with the direct physics of Neutrokine- α polypeptides (such as antagonist can adjust the upstream effects thing of Neutrokine- alpha actives, to reduce the activity).It there is described herein the detection method for suppressing B cell activity for determining Neutrokine- alpha-2 antagonists.Neutrokine- alpha-2 antagonists include but is not limited to anti-Neutrokine- Alpha antibodies or its antigen-binding fragment, Neutrokine- α receptor proteins or its fragment or variant, antibody or its antigen-binding fragment, Neutrokine- alpha bindings or polypeptide, Neutrokine- α and/or APRIL polypeptide variants (such as Neutrokine- α and/or APRIL dominant negative regulation thing form) with reference to Neutrokine- α acceptors.Other Neutrokine- alpha-2 antagonists include Neutrokine- α small molecular antagonists, Neutrokine- α peptide mimicses, the antisense RNA for targeting Neutrokine- α and short interfering rna (siRNA) and target APRIL antisense RNA and short interfering rna (siRNA), target the antisense RNA and short interfering rna (siRNA) of Neutrokine- α acceptor and/or APRIL acceptor.Every kind of antagonist is described in more detail below.

Neutrokine- alpha-2 antagonists

A.Neutrokine- α and APRIL polypeptides

In a specific embodiment, the Neutrokine- alpha-2 antagonists for the inventive method are Neutrokine- α or APRIL polypeptide fragment or variant.Neutrokine- α polypeptides, APRIL polypeptides and its fragment or variant is described in more detail below.Neutrokine- α albumen (SEQ ID NO:2) be tnf ligand family member, itself and APRIL (SEQ ID NO:4；GenBank numberings AF046888；PCT international publications WO97/33902；Hahne,M.,et al.,J Exp Med.(1998)188(6):1185-90), TNF α and Lymphotoxin Alpha (LT α) (Moore, etal., 1999) enjoy Amino acid sequence identity.Total length Neutrokine- α 285 amino acid polypeptides of gene code, it is with the transmembrane region between the intracellular region between the 1st and 46 residue, the 47th and 73 residue, it is the cell outskirt between the non-hydrophobic sequence of II type embrane-associated protein characteristics, and the 74th and 285 residue before the transmembrane region.As other members of TNF families, Neutrokine- α are played a role in the form of trimer protein.Expressed on cell surface after Neutrokine- α, cell outskirt is cracked out in 134 amino acids, to discharge the tripolymer of biological activity.Structural characterization is found that while that TNF families part shows sequence polymorphism, but they all show the structural homology of height.As the member of other tnf ligand families, Neutrokine- α albumen is 2 layers of β-sandwich structure, and which form the jelly-roll of TNF samples (jellyroll) form.Neutrokine- α albumen is all similar to other tnf ligand families in general structure and yardstick.But, Neutrokine- α receptor binding domain is the deeper ditch in land (Oren, et al., (2002) Nature Structural Biology 9 than being observed in other cell factors:288-292).Neutrokine- α polypeptides are described in further detail in such as international publication WO98/18921, WO00/50597, WO02/1820 and WO03/033658, entire contents are incorporated into the application by quoting herein.

As described above, act as stimulation B cell proliferation, differentiation, survival and the Ig of Neutrokine- α polypeptides secrete.Therefore, nobody can be expected that the Neutrokine- α of native form can be used in the methods of the invention.But, the Neutrokine- α of native form can be used as targeting agent, its medicine (such as cell toxicant motif or albumen) that other can be suppressed to B cell activity carries band close to the position of B cell (see for example, WO00/033658 embodiment 12 and 13, the Neutrokine- α of wherein isotope marks be used to target and kill the cell of expression Neutrokine- α acceptors, and the cell is mainly B cell source).Or, with reference to one or more Neutrokine- α acceptors but not the Neutrokine- α of inducement signal conduction fragment or variant not may be used as Neutrokine- alpha-2 antagonists.The Neutrokine- α fragments or variant of the ability of influence Neutrokine- α formation or the stable homotrimer of holding or heterotrimer are also used as the Neutrokine- alpha-2 antagonists in the inventive method.It therefore, it can include SEQ ID NO for the Neutrokine- α polypeptides in the inventive method:The polypeptide fragment or variant of 2 Neutrokine- α albumen.Polypeptide fragment or variant can be " freestanding (free standing) " or may be included in bigger polypeptide, wherein the fragment form the bigger polypeptide a part or a region, most preferably as single continuum.In a particular embodiment, the Neutrokine- α polypeptides that can be used in the inventive method include polypeptide fragment, and the polypeptide fragment includes Neutrokine- α cells outskirt (the SEQ ID NO of presumption:2 the 73 to 285th amino acids residue) and Neutrokine- α soluble fragments (SEQ ID NO:2 the 134 to 285th amino acids residue) or be made up of them.In another embodiment, the polypeptide fragment or variant that can be used in the inventive method include the polypeptide fragment or variant at least having 80%, 85%, 90%, 92%, 95%, 96%, 97%, the 98% or 99% phase same sex with natural Neutrokine- α recited above polypeptide fragment or are made from it.

In another embodiment, the Neutrokine- α polypeptide variants that can be used in the inventive method include peptide mimics.Analogies be simulate Protein secondary structure element contain peptide molecule.See such as Johnson et al., " Peptide Turn Mimetics " in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., its content, is incorporated herein by Chapman and Hall, New York (1993) by quoting herein.Existed using the peptide backbone that the potential principle of peptide mimics behind is albumen primarily to coming orient amino acid side chains, such as those side chains of antibody and antigen in the way of interaction of molecules is promoted.It is expected that peptide mimics can allow the interaction of molecules similar to natural molecule.These principles can be used for the genetically engineered natural performance with targeting peptides described herein but with the second generation molecule of feature changing, even improveing.

APRIL (SEQ ID NO:4) be tnf ligand family member, itself and Neutrokine- α (SEQ ID NO:2；GenBank numberings NM_006573；Moore,et al.,(1999)Science285:260-263；Schneider et al.,(1999)J.Exp.Med.189:1747-1756；With Khareet al., (2000) Proc.Natl.Acad Sci.97:3370-3375), TNF α and Lymphotoxin-α (LT α) (Moore, etal., 1999) enjoy the phase same sex of amino acid sequence.The polypeptide of total length APRIL 250 amino acid of gene code, it is with the cell outskirt between the transmembrane region and the 50th and 250 residue between the intracellular region between the 1st and 28 residue, the 29th and 49 residue.As other members of TNF families, APRIL is played a role in the form of trimer protein.Expressed on cell surface after APRIL, cell outskirt is cracked out in 105 amino acids, to discharge the tripolymer of biological activity.

In a specific embodiment, the APRIL polypeptides that can be used in the inventive method include SEQ ID NO:The polypeptide fragment or variant of 4 APRIL albumen.Polypeptide fragment or variant can be " freestanding " or may be included in bigger polypeptide, wherein the fragment form the bigger polypeptide a part or a region, most preferably as single continuum.In a particular embodiment, the APRIL polypeptides that can be used in the inventive method include polypeptide fragment, and the polypeptide fragment includes APRIL cells outskirt (the SEQ ID NO of presumption:4 the 50 to 250th amino acids residue) and APRIL soluble fragments (SEQ ID NO:4 the 105 to 250th amino acids residue) or be made from it.In another embodiment, the polypeptide fragment or variant that can be used in the inventive method include the polypeptide fragment or variant at least having 80%, 85%, 90%, 92%, 95%, 96%, 97%, the 98% or 99% phase same sex with natural APRIL recited above polypeptide fragment or are made from it.

In another embodiment, the APRIL polypeptide variants that can be used in the inventive method include peptide mimics.Analogies be simulate Protein secondary structure element contain peptide molecule.See such as Johnson et al., " Peptide Turn Mimetics " in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., its content, is incorporated herein by Chapman and Hall, New York (1993) by quoting herein.Existed using the peptide backbone that the potential principle of peptide mimics behind is albumen primarily to coming orient amino acid side chains, such as those side chains of antibody and antigen in the way of interaction of molecules is promoted.It is expected that peptide mimics can allow the interaction of molecules similar to natural molecule.These principles can be used for the genetically engineered natural performance with targeting peptides described herein but with the second generation molecule of feature changing, even improveing.

Neutrokine- α and the APRIL polypeptide that can be used in the inventive method can be expressed or synthesize modified forms such as fusion protein (comprising the polypeptide that (different albumen) heterologous protein sequence is connected to through peptide bond), and secretion signal can be not only included, extra heterologous functional areas can also be included.Be connected to each other the nucleotide sequence of amino acid sequence needed for Neutrokine- α or APRIL polynucleotides and coding by using methods known in the art in suitable reading frame, and can generate this fusion protein with methods known in the art expressed fusion protein product.Or, this fusion protein for example can be generated by using peptide synthesizer with protein synthesis techniques.Thus, for example, extra amino acid region (particularly electrically charged amino acid) can be added to the N-terminal of polypeptide, to improve its stability and persistence in host cell, during purifying or during subsequent operation and storage.Peptide motif can also be added to polypeptide, to promote purifying.These regions can be removed before polypeptide preparation terminates.Peptide motif is added to polypeptide to cause secretion or discharge, improve stability and promote purifying to be all known to this area and conventional technique.

Preferred Neutrokine- α or the APRIL fusion protein that can be used in the inventive method includes can be used in the heterologous region from immunoglobulin of stable and purifying protein.For example, EP-A-O464533 (corresponding to Canada 2045869) and WO00/024782 elaborate to include the fusion protein of the different piece of the constant region of immunoglobulin molecules and another human protein or part thereof.For example in Yu, et al., (2000) Nat Immunol 1:Have been able to describe Neutrokine- alpha immunization Immunoglobulin fusion albumen in 252-256, be herein incorporated herein entire contents by quoting.APRIL domain-immunoglobulin fusion proteins for example have been described in PCT publication WO01/087977, herein entire contents are incorporated herein by quoting.In many situations, the Fc parts in fusion protein are highly suitable for treating and diagnosed, and therefore cause improved pharmacokinetics performance (EP-A 0232262).On the other hand, it is necessary to which Fc parts can be lacked after expressing, detecting and being purified into fusion protein in favourable mode described herein for some applications.When Fc part be proved its to for treat and diagnosis be hinder when it is particularly the case, such as when fusion protein is used as the antigen of immunity inoculation.In medicament research and development, such as human protein such as hIL-5 with Fc partial fusions, to identify hIL-5 antagonist with high flux screening detection method.See D.Bennett et al., J.Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J.Biol.Chem.270:9459-9471(1995).

Those skilled in the art know and as discussed above, Neutrokine- α and APRIL polypeptide can be merged with other peptide sequences.For example, the Neutrokine- α polypeptides that can be used in the inventive method can with the constant region of immunoglobulin (IgA, IgE, IgG, IgM) or part thereof (CH1, CH2, CH3, or any combination thereof and part) or albumin (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) fusion, form chimeric polyeptides.

These fusion proteins can promote purifying, can extend storage time and can increase Half-life in vivo.The chimeric protein being made up of first two domains and the heavy chain of mammalian immunoglobulin or the different zones of constant region of light chain of people's CD4 polypeptides has shown that this point.See such as EP394,827, Traunecker et al., Nature, 331:84-86(1988).The ability of immune system can be transported to enhancement antigen across epithelial barrier by being had confirmed for the antigen (such as insulin) being conjugated with FcRn binding partners such as IgG or Fc fragments (see such as PCT publication WO 96/22024 and WO99/04813).Having also been discovered that the IgG fusion proteins of the dimeric structure with disulfide bond caused by the disulfide bond of IgG parts individually itself can more effectively combine than single homodimeric polypeptide or its fragment and neutralize other molecules.See such as Fountoulakis et al., J.Biochem., 270:3958-3964(1995).

The mature form of human serum albumins (HAS or HA) is albumen (the SEQ ID NO of 585 amino acid:11), it is the major part for causing Blood osmotic pressure, and also effect can be endogenous and the carrier of exogenous part.Now, HA of the generation for clinical practice is extracted by human blood.EP330451 and EP 361991 have elaborated the generation restructuring HA (rHA) in microorganism.

Effect and its inertia performance of the albumin as carrier molecule are all carriers and the required performance of transhipment in vivo as polypeptide.The purposes that albumin is used as the carrier of various albumen as the component of albumin fusion protein has been proposed in WO 93/15199, WO 93/15200 and EP 413622.HA be also been proposed as the purposes (EP 399666) with the N-terminal fragment of the fusions of polypeptide.Merging for albumin and human cytokines can be realized by genetic manipulation, for example, the DAN for encoding HA or its fragment is added in the DNA of coding human cytokines.Then converted with the nucleotide sequence for the fusion being arranged on suitable plasmids or transfected suitable host, with expressed fusion protein.The expression can be realized from transgenic organism from protokaryon or eukaryotic or in vivo in vitro.

The albumin fusion protein that can be used in the inventive method includes the fragment of at least one Neutrokine- α polypeptide or the fragment or variant of variant and at least one human serum albumins, it is connected to each other, albumin fusion protein (is preferably generated by translating nucleic acid by genetic fusion, wherein in the nucleic acid, the all or part of polynucleotides skeleton of all or part of polynucleotides and encoding albumin that encode Neutrokine- α is connected), or chemically conjugated be connected to each other.Once as a part for albumin fusion protein, Neutrokine- α polypeptides and albumin protein can be referred to as " part ", " region " or " motif " (such as " Neutrokine- α parts " or " Albumin in Partial ") of albumin fusion protein.

In one embodiment, the albumin fusion protein that can be used in the inventive method includes Neutrokine- α polypeptides and seralbumin is either made up of them.In other embodiments, the fragment and seralbumin that the albumin fusion protein that can be used in the inventive method includes Neutrokine- α polypeptides are either made up of them.In other embodiments, the variant and seralbumin that the albumin fusion protein that can be used in the inventive method includes Neutrokine- α polypeptides are either made up of them.In a preferred embodiment, the seralbumin protein component of albumin fusion protein is sero-abluminous maturing part.

In further embodiment, the albumin fusion protein that can be used in the inventive method include Neutrokine- α polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity fragment either be made up of them.In further embodiment, the albumin fusion protein that can be used in the inventive method include Neutrokine- α polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity variant either be made up of them.In a preferred embodiment, the Neutrokine- α parts of albumin fusion protein are total length Neutrokine- α polypeptides.In a further preferred embodiment, the Neutrokine- α protein parts of albumin fusion protein are ripe, the soluble regions of Neutrokine- α polypeptides.

In further embodiment, the albumin fusion protein that can be used in the inventive method include Neutrokine- α polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity fragment or variant either be made up of them.In a preferred embodiment, the present invention, which is provided, to be included or the maturing part by Neutrokine- α polypeptides and sero-abluminous maturing part (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) albumin fusion protein of composition.In one preferred embodiment, Neutrokine- α polypeptides are merged with the human serum albumins (the 1 to 585th amino acids of the human serum albumins i.e. as shown in Fig. 1 and 2 of European patent 0322094) of mature form, are herein incorporated herein entire contents by quoting.Another preferred embodiment in, the antibody (including its fragment or variant) of the present invention is merged with the polypeptide fragment for including or being made up of the 1st to x residue of human serum albumins, wherein x is the integer from 1 to 585, and the albumin fragment has human serum albumins activity.Another preferred embodiment in, Neutrokine- α polypeptides (including its fragment or variant) are with including or being merged by the 1st polypeptide fragment constituted to z amino acids of human serum albumins, wherein z is the integer from 369 to 419, such as United States Patent (USP) 5,766, described in 883, entire contents are incorporated herein by quoting herein.Neutrokine- α polypeptides (including its fragment or variant) can be merged with the N-terminal or C-terminal of heterologous protein (such as immunoglobulin Fc polypeptide or human serum albumins polypeptide).

In a preferred embodiment, human serum albumins used in the albumin fusion protein in can be used for the inventive method contains the reference SEQ ID NO below one group or two groups:11 point mutation group：Leu-407 sports Ala, Leu-408 and sports that Val, Val-409 sport Ala and Arg-410 sports Ala；Or Arg-410 sports A, Lys-413 and sports Gln and Lys-414 and sports Gln (see such as international application WO95/23857, being herein incorporated herein entire contents by quoting).In even more preferred embodiment, the albumin fusion protein that can be used in the inventive method containing one group or two groups point mutation group recited above has improved stability/tolerance to yeast Yap3p proteolytic degradations, allows the yield for the recombinant albumin fusion protein that increase is expressed in yeast host cell.

Preferably, the albumin fusion protein that can be used in the inventive method includes the HA as N-terminal part and the Neutrokine- α polypeptides as C-terminal part.Or, the albumin fusion protein of the Neutrokine- α polypeptides including the HA as its C-terminal part and as its N-terminal part can also be used.

In other embodiments, the albumin fusion protein that can be used in the inventive method has the Neutrokine- α polypeptides merged with the N-terminal and C-terminal of albumin.In a specific embodiment, the Neutrokine- α polypeptides merged in N-terminal and C-terminal are identicals.In another embodiment, the Neutrokine- α polypeptides merged in N-terminal and C-terminal are different Neutrokine- α polypeptides.In another embodiment, Neutrokine- α polypeptides are blended in the N or C-terminal of albumin, and heterologous polypeptide is blended in remaining end.

Furthermore it is possible to can be comprising the attachment peptide being fused between part, to provide the bigger physical separation between the part for the albumin fusion protein in the inventive method.Attachment peptide can be made up of amino acid so that it is flexible or more rigid.

In general, the albumin fusion protein that can be used in the inventive method can have a HA to derive area and a Neutrokine- α area.But, multiple regions in each albumen can be used for preparing the albumin fusion protein that can be used in the inventive method.Equally, more than one albumen can be used to prepare the albumin fusion protein that can be used in the inventive method.For example, albumen can be blended in HA N-terminal and C-terminal.In this configuration, the protein part can be same or different protein molecular.The structure of the albumin fusion protein of difunctionality can be expressed as X-HA-Y or Y-HA-X.

The Neutrokine- α albumen that can be used in a specific embodiment in the inventive method or its fragment or variant can be conjugated with cytotoxin (such as cytostatic agent or cytocide).Cytotoxin or cell toxicity medicament can include any medicine being harmful to cell.Example include taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, emetine, mitomycin, etoposide, Teniposide, vincristine, vinblastine, colchicin, adriamycin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- boldenones, glucocorticoid, procaine, dicaine, lidocaine, Propranolol and puromycin with and the like or homologue.

In another embodiment, the Neutrokine- α that can be used in the inventive method or its fragment or variant can be conjugated with toxin." toxin " represent one or more can combine and activate the compounds of endogenous cytotoxicity effector system, radio isotope, holotoxin, modification toxin, the catalytic subunit of toxin or it is given cause will not normally be expressed in any molecule or the enzyme in cell surface or on surface under conditions of cell death.The toxin that can be used includes but is not limited to radio isotope known in the art, compound is for example with reference to the antibody (or its part containing complement fixation) of inherent or induction endogenous cytotoxicity effector system, thymidine kinase, endonuclease, RNase, alpha toxin, ricin (WA), abrin, ETA, diphtheria toxin, saporin (saporin), momordin (momordin), gelonin (gelonin), America pokeweed antiviral albumen, α-broom aspergillin (α-Sarcin), and cholera toxin." toxin " also includes cytostatic agent or cytocide, medicine or radioactive metal ion such as α particle emissions ion, for example²¹³Bi or other radio isotopes are such as¹⁰³Pd、¹³³Xe、¹³¹I、⁶⁸Ge、⁵⁷Co、⁶⁵Zn、⁸⁵Sr、³²P、³⁵S、⁹⁰Y、¹⁵³Sm、¹⁵³Gd、¹⁶⁹Yb、⁵¹Cr、⁵⁴Mn、⁷⁵Se、¹¹³Sn、⁹⁰Yttrium (⁹⁰Yttrium)、¹¹⁷Tin (¹¹⁷Tin)、¹⁸⁶Rhenium (¹⁸⁶Rhenium)、¹⁶⁶Holmium (¹⁶⁶Holmium) and¹⁸⁸Rhenium.

In another example, the APRIL polypeptides that can be used in the inventive method can be with immunoglobulin (IgA, IgE, IgG, IgM constant region) or part thereof (CH1, CH2, CH3, or any combination thereof and part), or albumin (includes but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1, 876, 969, European patent 0413622, the United States Patent (USP) 5 submitted with June 16th, 1998, 766, 883, entire contents are incorporated herein by quoting herein) fusion, form chimeric polyeptides.

The albumin fusion protein that can be used in the inventive method includes the fragment of at least one APRIL polypeptide or the fragment or variant of variant and at least one human serum albumins, it is connected to each other, albumin fusion protein (is preferably generated by translating nucleic acid by genetic fusion, wherein in the nucleic acid, the all or part of polynucleotides skeleton of all or part of polynucleotides and encoding albumin that encode APRIL is connected), or chemically conjugated be connected to each other.Once as a part for albumin fusion protein, APRIL polypeptides and albumin protein can be referred to as " part ", " region " or " motif " (such as " APRIL parts " or " Albumin in Partial ") of albumin fusion protein.

In one embodiment, the albumin fusion protein that can be used in the inventive method includes APRIL polypeptides and seralbumin is either made up of them.In other embodiments, the fragment and seralbumin that the albumin fusion protein that can be used in the inventive method includes APRIL polypeptides are either made up of them.In other embodiments, the variant and seralbumin that the albumin fusion protein that can be used in the inventive method includes APRIL polypeptides are either made up of them.In a preferred embodiment, the seralbumin protein component of albumin fusion protein is sero-abluminous maturing part.

In further embodiment, the albumin fusion protein that can be used in the inventive method include APRIL polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity fragment either be made up of them.In further embodiment, the albumin fusion protein that can be used in the inventive method include APRIL polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity variant either be made up of them.In a preferred embodiment, the APRIL parts of albumin fusion protein are total length APRIL polypeptides.In a further preferred embodiment, the APRIL protein parts of albumin fusion protein are ripe, the soluble regions of APRIL polypeptides.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including APRIL polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment of therapeutic activity them.In further embodiment, the present invention provides the albumin fusion protein for including the maturing part and sero-abluminous maturing part of APRIL polypeptides or being made up of them.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including APRIL polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the present invention, which is provided, to be included or the maturing part by APRIL polypeptides and sero-abluminous maturing part (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) albumin fusion protein of composition.In one preferred embodiment, APRIL polypeptides are merged with the human serum albumins (the 1 to 585th amino acids of the human serum albumins i.e. as shown in Fig. 1 and 2 of European patent 0322094) of mature form, are herein incorporated herein entire contents by quoting.Another preferred embodiment in, the antibody (including its fragment or variant) of the present invention is merged with the polypeptide fragment for including or being made up of the 1st to x residue of human serum albumins, wherein x is the integer from 1 to 585, and the albumin fragment has human serum albumins activity.Another preferred embodiment in, APRIL polypeptides (including its fragment or variant) are with including or being merged by the 1st polypeptide fragment constituted to z amino acids of human serum albumins, wherein z is the integer from 369 to 419, such as United States Patent (USP) 5,766, described in 883, entire contents are incorporated herein by quoting herein.APRIL polypeptides (including its fragment or variant) can be merged with the N-terminal or C-terminal of heterologous protein (such as immunoglobulin Fc polypeptide or human serum albumins polypeptide).

Preferably, the albumin fusion protein that can be used in the inventive method includes the HA as N-terminal part and the APRIL polypeptides as C-terminal part.Or, the albumin fusion protein of the APRIL polypeptides including the HA as its C-terminal part and as its N-terminal part can also be used.

In other embodiments, the albumin fusion protein that can be used in the inventive method has the APRIL polypeptides merged with the N-terminal and C-terminal of albumin.In a specific embodiment, the APRIL polypeptides merged in N-terminal and C-terminal are identicals.In another embodiment, the APRIL polypeptides merged in N-terminal and C-terminal are different APRIL polypeptides.In another embodiment, APRIL polypeptides are blended in the N or C-terminal of albumin, and heterologous polypeptide is blended in remaining end.

In general, the albumin fusion protein that can be used in the inventive method can have a HA to derive area and an APRIL area.But, multiple regions in each albumen can be used for preparing the albumin fusion protein that can be used in the inventive method.Equally, more than one albumen can be used to prepare the albumin fusion protein that can be used in the inventive method.For example, albumen can be blended in HA N-terminal and C-terminal.In this configuration, the protein part can be same or different protein molecular.The structure of the albumin fusion protein of difunctionality can be expressed as X-HA-Y or Y-HA-X.

The APRIL albumen that can be used in a specific embodiment in the inventive method or its fragment or variant can be conjugated with cytotoxin (such as cytostatic agent or cytocide).Cytotoxin or cell toxicity medicament can include any medicine being harmful to cell.Example include taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, emetine, mitomycin, etoposide, Teniposide, vincristine, vinblastine, colchicin, adriamycin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- boldenones, glucocorticoid, procaine, dicaine, lidocaine, Propranolol and puromycin with and the like or homologue.

In another embodiment, the APRIL that can be used in the inventive method or its fragment or variant can be conjugated with toxin.

" toxin " represent one or more can combine and activate the compounds of endogenous cytotoxicity effector system, radio isotope, holotoxin, modification toxin, the catalytic subunit of toxin or it is given cause will not normally be expressed in any molecule or the enzyme in cell surface or on surface under conditions of cell death.The toxin that can be used includes but is not limited to radio isotope known in the art, compound for example with reference to antibody (or its part containing complement fixation), thymidine kinase, endonuclease, RNase, alpha toxin, ricin (WA), abrin, ETA, diphtheria toxin, saporin, momordin, gelonin, America pokeweed antiviral albumen, α-broom aspergillin and the cholera toxin of inherent or induction endogenous cytotoxicity effector system." toxin " also includes cytostatic agent or cytocide, medicine or radioactive metal ion such as α particle emissions ion, for example²¹³Bi or other radio isotopes are such as¹⁰³Pd、¹³³Xe、¹³¹I、⁶⁸Ge、⁵⁷Co、⁶⁵Zn、⁸⁵Sr、³²P、³⁵S、⁹⁰Y、¹⁵³Sm、¹⁵³Gd、¹⁶⁹Yb、⁵¹Cr、⁵⁴Mn、⁷⁵Se、¹¹³Sn、⁹⁰Yttrium,¹¹⁷Tin,¹⁸⁶Rhenium,¹⁶⁶Holmium and¹⁸⁸Rhenium.

The feature of Neutrokine- α and APRIL polypeptides can be changed using genetically engineered, to generate the polypeptide that can be used in the inventive method.Single or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing, addition or fusion protein can be included with the new mutain of recombinant DNA technology generation known to those skilled in the art or " mutein ".These modified polypeptides can show for example to strengthen activity, reduction activity or increase stability.In addition, at least under specific purifying and condition of storage, they can be purified than corresponding natural polypeptides by higher yield, and show more preferable stability.For example, for the albumen of a variety of cell outskirts including secretory protein or mature form, it is known in the art to lack one or more amino acid from N-terminal or C-terminal, and biological function is not lost substantially.For example, Ron et al., J.Biol.Chem., 268:2984-2988 (1993), which reports the KGF albumen through modification, has heparin binding activity, even if having lacked 3, the amino acid residue of 8 or 27 amino terminals.

It can be monomer or polymer (i.e. dimer, tripolymer, the tetramer and more high polymer) that Neutrokine- α and the APRIL polypeptide in the inventive method, which can be used for, in a particular embodiment, it can be homopolymer or different aggressiveness that can be used for Neutrokine- α and the APRIL polypeptide in the inventive method.Neutrokine- α homopolymers refer to comprising only the polymer of Neutrokine- α polypeptides (including Neutrokine- α fragments, variant and fusion protein described herein).These homopolymers can contain the Neutrokine- α polypeptides with same or different amino acid sequence.In a particular embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are Neutrokine- α homodimers (such as containing two Neutrokine- α polypeptides with same or different amino acid sequence) or Neutrokine- α homotrimers (such as containing three Neutrokine- α polypeptides with same or different amino acid sequence).In one preferred embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are Neutrokine- α homotrimers.In other embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are at least homodimer, homotrimer or the same tetramer.APRIL homopolymers refer to comprising only the polymer of APRIL polypeptides (including APRIL fragments, variant and fusion protein described herein).These homopolymers can contain the APRIL polypeptides with same or different amino acid sequence.In a particular embodiment, the APRIL polypeptides that can be used in the inventive method are APRIL homodimers (such as containing two APRIL polypeptides with same or different amino acid sequence) or APRIL homotrimers (such as containing three APRIL polypeptides with same or different amino acid sequence).In one preferred embodiment, the APRIL polypeptides that can be used in the inventive method are APRIL homotrimers.In other embodiment, the APRIL polypeptides that can be used in the inventive method are at least homodimer, homotrimer or the same tetramer.

The Neutrokine- α of different aggressiveness refer to also containing in addition to Neutrokine- α polypeptides the polymer of heterologous polypeptide (polypeptides of i.e. different albumen).In a specific embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are heterodimer, heterotrimer or the different tetramer.In other embodiments, the Neutrokine- α polypeptides that can be used in the inventive method are polymers, and wherein at least is heterodimer, heterotrimer or the different tetramer.The APRIL of different aggressiveness refers to also containing in addition to APRIL polypeptides the polymer of heterologous polypeptide (polypeptides of i.e. different albumen).In a specific embodiment, the APRIL polypeptides that can be used in the inventive method are heterodimer, heterotrimer or the different tetramer.In other embodiments, the APRIL polypeptides that can be used in the inventive method are polymers, and wherein at least is heterodimer, heterotrimer or the different tetramer.In other embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are to include the heterotrimer of Neutrokine- α polypeptides and APRIL polypeptides or its fragment or variant.In other embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are to include the heterotrimer of a Neutrokine- α polypeptide (including fragment or variant) and two APRIL polypeptides (including fragment or variant).In other embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are to include the heterotrimer of two Neutrokine- α polypeptides (including fragment or variant) and an APRIL polypeptide (including fragment or variant).

In other embodiment, the Neutrokine- α polypeptides that can be used in the inventive method are the Neutrokine- α polypeptides of homopolymer, particularly homotrimer, and the wherein single protein component of polymer includes Neutrokine- α mature form (such as SEQ ID NO:2 the 134 to 285th amino acids) or its fragment or variant be either made up of them.In other specific embodiments, the Neutrokine- α polypeptides that can be used in the inventive method are heterotrimer of the Neutrokine- α polypeptides for example containing two Neutrokine- α polypeptides and APRIL polypeptide or the heterotrimer containing a Neutrokine- α polypeptide and two APRIL polypeptides of different aggressiveness, particularly heterotrimer, and the wherein single protein component of the different aggressiveness of Neutrokine- α includes Neutrokine- α ripe extracellular soluble part (such as SEQ ID NO:2 the 134 to 285th amino acids) or its fragment or variant or APRIL ripe extracellular soluble part (such as SEQ ID NO:4 the 105 to 250th amino acids) or its fragment or variant be either made from it.

In other embodiment, the APRIL polypeptides that can be used in the inventive method are the APRIL polypeptides of homopolymer, particularly homotrimer, and the wherein single protein component of polymer includes APRIL mature form (such as SEQ ID NO:4 the 105 to 250th amino acids) or its fragment or variant be either made from it.In other specific embodiments, the APRIL polypeptides that can be used in the inventive method are heterotrimer of the APRIL polypeptides for example containing two APRIL polypeptides and Neutrokine- α polypeptide or the heterotrimer containing an APRIL polypeptide and two Neutrokine- α polypeptides of different aggressiveness, particularly heterotrimer, and the single protein component of the different aggressiveness of wherein APRIL includes APRIL ripe extracellular soluble part (such as SEQ ID NO:4 the 105 to 250th amino acids) or its fragment or variant or Neutrokine- α ripe extracellular soluble part (such as SEQ ID NO:2 the 134 to 285th amino acids) or its fragment or variant be either made from it.

The polymer that can be used in the inventive method can be hydrophobic connection, hydrophilic connection, ion connection and/or be covalently attached connected result, and/or can be indirectly connected to, for example, formed by liposome.Therefore, in one embodiment, when polypeptide is in contact with each other in the solution, it is formed same polymer such as homodimer or homotrimer.In another embodiment, when polypeptide is in contact with each other in the solution, heterodimer such as heterotrimer or the different tetramer are formed.In other embodiments, polymer is formed by the covalent attachment between the covalent attachment with Neutrokine- α polypeptides or Neutrokine- α.In other embodiments, polymer is formed by the covalent attachment between the covalent attachment with APRIL polypeptides or APRIL.These covalent attachments may relate to peptide sequence (such as Neutrokine- α SEQ ID NO:2 or APRIL SEQ ID NO:4) one or more amino acid residues contained by.In oneainstance, covalent attachment is the crosslinking between the cysteine residues being located in peptide sequence, and the cysteine residues also interact in natural (i.e. naturally occurring) polypeptide.In another scenario, covalent attachment is the result after chemistry or reorganization.Or, this covalent attachment can be related to one or more contained amino acid residues in the heterologous polypeptide sequence of Neutrokine- α or APRIL fusion proteins (see United States Patent (USP) 5,478,925).In a specific example, covalent attachment is the covalent attachment between the heterologous sequence contained by Neutrokine- α-Fc fusion proteins (as described herein).In a specific example, covalent attachment is the covalent attachment between the heterologous sequence contained by APRIL-Fc fusion proteins (as described herein).In another particular embodiment of the invention, the covalent attachment for the fusion protein that can be used in the inventive method be from another can be formed covalent attachment polymer TNF families ligand/receptor member such as OPG (oseteoprotegerin) (see such as international publication WO 98/49305, being herein incorporated herein entire contents by quoting) heterologous polypeptide sequence between covalent attachment.In another embodiment, the attachment (such as peptide, carbohydrate or the polymer attachment of solubility) of two or more Neutrokine- α polypeptides and/or APRIL polypeptides through synthesis links together.Example is included in those peptide linkers (being incorporated into the application by quoting herein) described in United States Patent (USP) 5,073,627.The albumen including multiple Neutrokine- α polypeptides separated through peptide linker and/or APRIL polypeptides can be generated using traditional recombinant DNA technology.

In a specific embodiment, the Neutrokine- alpha-2 antagonists that can be used in the inventive method are Neutrokine- α and/or APRIL dominant negative regulation thing form.Specifically, Neutrokine- α and/or APRIL variant, including dominant negative regulation thing form have been described in such as International patent publications WO06/034106, WO05/113598, WO04/089982, WO04/081043 and WO03/057856 and United States patent publication US20060014248, US20050221443, US20050130892, US20050048626, US2005003480 and US20030166559.Its all the elements is incorporated herein by quoting herein.This Neutrokine- α and/or APRIL polypeptide variants for example can be by disturbing Neutrokine- α and/or APRIL to form same or heteromultimeric come antagonism Neutrokine- α functions.Or, Neutrokine- α and/or APRIL polypeptide variants can prevent to include the polypeptide and Neutrokine- α acceptors such as TACI, BCMA and BAFF-R combination and/or signal transduction of the Neutrokine- α and/or APRIL polypeptide variants.

In another embodiment, Neutrokine- alpha-2 antagonists are Gao et a.l, (2006) Biotechnol.Lett.28:Entire contents, are incorporated herein by the Neutrokine- α protein mutants described in 1649-54 by quoting herein.

In another embodiment, the Neutrokine- alpha-2 antagonists that can be used in the inventive method are Δ BAFF (SEQ ID NO:12).

B. anti-Neutrokine- Alpha antibodies

In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies or its antigen-binding fragment.For example in PCT publication WO01/087977, WO03/016468, WO01/60397, WO02/02641 and WO03/55979；US publication 2005/0070694 and 2005/0255532；With Cao et al., (2005) Immunol Lett 101:87-94；Ch’en et al.,(2005)Cell Immunol 236:78-85；Liu et al.,(2005)Acta Biochim Biophys Sin(Shanghai)37:415-420；Schneider et al.,(1999)J Exp Med 189:1747-1756；Sun et al.,(2006)Hybridoma 25:80-85；Sun et al.,(2006)Hybridoma 25:Anti- Neutrokine- Alpha antibodies and its fragment have been described in 238-242；More detailed description will be done below.Entire contents are incorporated herein by quoting herein.

The immunoactive portions of term " antibody " referred to herein as immunoglobulin molecules and immunoglobulin molecules, i.e., the molecule of the antigen binding site containing immunologic opsonin combination antigen.Term antibody not only includes whole antibody molecule, includes the variant (including derivative) of antibody fragment and antibody and antibody fragment.The example of the molecule described in term " antibody " includes but is not limited in this application：ScFv (scFv), Fab fragments, Fab ' fragments, F (ab ')₂, disulfide bond Fv (sdFv), Fv and including or by the fragment that constitutes of VL or VH domains.Term " scFv " or " scFv " referred to herein as include the polypeptide of the VL domains for the antibody being connected with the VH domains of antibody.The antibody that immunologic opsonin combines special antigen (such as Neutrokine- α) can be with the cross reactivity with other antigens.Preferably, immunologic opsonin, which combines the antibody of special antigen and other antigens, does not have cross reaction.It can for example be identified and be combined with the immunologic opsonin of special antigen by technology known to immunodetection or other those skilled in the art, the U.S. Patent application 60/834 that on July 31st, 1 submits, entire contents, are incorporated herein by the immunodetection described in 152 by quoting herein.

The antibody that can be used in the inventive method include but is not limited to monoclonal, polyspecific, the mankind or chimeric antibody, single-chain antibody, Fab fragments, F (ab ') fragment, the epitope binding fragments of antiidiotype (anti-Id) antibody and above-mentioned all antibody.The immunoglobulin molecules of the present invention can be the immunoglobulin of any kind of (such as IgG, IgE, IgM, IgD, IgA and IgY), any group (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subgroup.

The antibody that can be used in the inventive method can also include the antibody of multimeric forms.For example, the antibody that can be used in the inventive method can be the senior multimeric forms of antibody dimer, tripolymer or monomeric immunoglobulin molecule.Whole immunoglobulin molecules or F (ab ')₂The dimer of fragment is tetravalence, and the dimer of Fab fragments or scFv molecules is divalence.Single monomer in antibody multimer body can be same or different, i.e., they can be the antibody multimer of different aggressiveness or homopolymer.For example, the single antibody in polymer can have same or different binding specificity.The polymer of antibody can be completed by the natural aggregation of antibody or by chemistry known in the art or restructuring interconnection technique.For example, the antibody preparation (such as the IgG1 molecules of purifying) of part purifying can spontaneously form the protein aggregate of the antibody multimer containing antibody homodimer He other higher levels.Or, chemical linking techniques known in the art formation antibody homodimer can be passed through.For example, the crosslinking agent of Heterobifunctional includes but is not limited to SMCC (succinimido 4- (maleimidomehyl) hexamethylene -1- carboxylates) [succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (such as purchased from Pierce Bio technology, Inc. (Rockford, IL)), crosslinking agent formation antibody multimer can be used.Ghetie et al.,Proceedings of the National Academy of Sciences USA(1997)94:7509-7514 gives the exemplary scenario for forming antibody homodimer, is herein incorporated herein entire contents by quoting.The homodimers of Fab ' 2 can be changed into antibody homodimer by the digestion of pepsin.Another method for forming antibody homodimer is by using in Zhao and Kohler, The Journal of Immunology (2002) 25:Entire contents, are incorporated herein by the T15 peptides of the autophilic described in 396-404 by quoting herein.

Or, antibody multimer can be caused by recombinant DNA technology.IgM and IgA is by the interaction natural feature with J chain polypeptides into antibody multimer.Non- IgA or non-IgM molecules such as IgG molecules can be melted into the J chains interaction area containing IgA or IgM by genetic engineering, ability that non-IgA or non-IgM molecules form senior polymer is assigned accordingly (see such as Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:Entire contents, are incorporated herein by 1483-94. by quoting herein).Recombinant technique known in the art formation scFv dimers, Goel et al., (2000) CancerResearch 60 can also be passed through:6964-6971 gives the example for building scFv dimers, is herein incorporated herein entire contents by quoting.Antibody multimer can be purified into by including but is not limited to molecular exclusion chromatography using any suitable method known in the art.

Unless there is other different explanations in this manual, the specific binding of antibody or immunologic opsonin combine and all represent antibody binding target antigen, but not significantly combine other albumen (such as other TNF families parts) in for example same protein family of the albumen of (i.e. cross reaction) in addition to target antigen.It will not need not be with reference to target antigen and with the antibody of other albumen cross reactions under all conditions all without the antibody with reference to other albumen, but be compared to for its ability with reference to other albumen, target antigen specific antibody is preferably in combination with target antigen, so that its detection method or treatment suitable at least one type, that is, obtained low background level or do not resulted in irrational side effect to treatment.The protein part that antibody is combined is referred to as epitope, and this is known.Epitope can be linear (being made up of the amino acid residue of the order in protein sequence) or (discontinuous one or more amino acid residues are constituted i.e. in albumen primary structure, but are combined by two grades of albumen, three or four structure) of conformation.It is assumed that the epitope of target antigen specific antibody combination target antigen, so the antibody of specific binding target antigen can with or the fragment of target antigen and/or the variant (such as with target antigen at least 90% identical albumen) of target antigen cannot be combined, this depends on the epitope combined in target antigen fragment or variant with the presence or absence of given target antigen specific antibody.Equally, target antigen specific antibody can combine the species ortholog thing (species orthologues) of target antigen (including its fragment), and this also depends on the epitope recognized in ortholog thing with the presence or absence of antibody.In addition, target antigen specific antibody can combine the target antigen such as target antigen fusion protein of modified forms.In this case, when antibody binding target antigen fusion protein, antibody must be combined with the target antigen motif of fusion protein and contacted, so that with reference to being specific.

The antibody combined with any Special Targets antigentic specificity for example can be identified by technology known to immunodetection or other those skilled in the art, the U.S. Patent application 60/834 for example submitted by July 31st, 2006, entire contents, are incorporated herein by the immunodetection described in 152 by quoting herein.The antibody that can be used in the inventive method " can be specific to " Neutrokine- α, but this is not necessary condition.It can be described according to its cross reactivity or illustrate can be used for the anti-Neutrokine- Alpha antibodies in the inventive method.The antibody that Neutrokine- α any other analog, homologue or homologue will not be combined can be used for the method for the present invention.In a specific embodiment, the antibody that can be used in the inventive method and APRIL cross reactions.In a particular embodiment, the antibody that can be used in the inventive method and the mouse of human protein, rat and/or the homologue of rabbit or its corresponding epitope cross reaction.

In a specific embodiment, with reference to Neutrokine- α polypeptides, polypeptide fragment or SEQ ID NO:The antibody of 2 variant, and/or Neutrokine- α epitopes (by for detecting the epitope that the immunodetection well known in the art of special antibody-antigen binding is determined) can be used in the inventive method.In a specific embodiment, the antibody that can be used in the inventive method can combine the Neutrokine- α polypeptides merged with other peptide sequences.For example, Neutrokine- α polypeptides (can include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1 with the constant region of immunoglobulin (IgA, IgE, IgG, IgM) or part thereof (CH1, CH2, CH3 or its any combinations and its part) or albumin, 876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein)) fusion, form chimeric polyeptides.These fusion proteins can promote to purify and increase Half-life in vivo.The chimeric protein being made up of first two domains and the heavy chain of mammalian immunoglobulin or the different zones of constant region of light chain of people's CD4 polypeptides has shown that this point.See such as EP 394,827, Traunecker et al., Nature, 331:84-86(1988).The ability of immune system can be transported to enhancement antigen across epithelial barrier by being had confirmed for the antigen (such as insulin) being conjugated with FcRn binding partners such as IgG or Fc fragments (see such as PCT publication WO96/22024 and WO 99/04813).Having also been discovered that the IgG fusion proteins of the dimeric structure with disulfide bond caused by the disulfide bond of IgG parts individually itself can more effectively combine than single homodimeric polypeptide or its fragment and neutralize other molecules.See such as Fountoulakis et al., J.Biochem., 270:3958-3964(1995).

In another embodiment, the Neutrokine- α polypeptides for the antibody binding mutation that can be used in the inventive method, the Neutrokine- α polypeptides of the mutation are generated by the random mutation of the polynucleotides to encoding Neutrokine- α polypeptides, by the other method before error-prone PCR, random nucleotide insertion or restructuring.In another embodiment, Neutrokine- α one or more components, motif, section, part, region, the fragment of restructuring such as the antibody binding that can be used in the inventive method and one or more components, motif, section, part, region, the fragment of one or more heterologous molecules etc..In a preferred embodiment, heterologous molecule is such as TNF-α, Lymphotoxin-α (LT- α, also referred to as TNF-β), LT- β (seeing complicated heterotrimer LT- α 2- β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF- γ (international publication WO 96/14328), AIM-I (international publication WO 97/33899), AIM-II (international publication WO 97/34911), APRIL (J.Exp.Med.188 (6):1185-1190), endokine- α (international publication WO 98/07880), OPG, OX40, with nerve growth factor (NGF), with the Fas of soluble form, CD30, CD27, CD40 and 4IBB, TR2 (international publication WO96/34095), DR3 (international publication WO 97/33904), DR4 (international publication WO98/32856), TR5 (international publication WO 98/30693), TR6 (international publication WO98/30694), TR7 (international publication WO 98/41629), TRANK, TR9 (international publication WO 98/56892), TR10 (international publication WO 98/54202), 312C2 (international publication WO98/06842), TR12, CAD, and v-FLIP.In further embodiment, heterologous molecule is any member of TNF families.

In a particular embodiment, antibody binding homopolymer, particularly homotrimer the Neutrokine- α polypeptides that can be used in the inventive method.Heterotrimer of the different aggressiveness of antibody binding, particularly heterotrimer the Neutrokine- α polypeptides for example containing two Neutrokine- α polypeptides and APRIL polypeptide or the heterotrimer containing a Neutrokine- α polypeptide and two APRIL polypeptides that can be used in other specific embodiments in the inventive method.In a specific embodiment, antibody binding homopolymer, particularly homotrimer the Neutrokine- α polypeptides that can be used in the inventive method, the single protein component of wherein polymer is Neutrokine- α (such as SEQ ID NO by mature form:2 the 134 to 285th amino acids residue) composition.In other specific embodiments, heterotrimer of the different aggressiveness of antibody binding, particularly heterotrimer the Neutrokine- α polypeptides for example containing two Neutrokine- α polypeptides and APRIL polypeptide that can be used in the inventive method or the heterotrimer containing a Neutrokine- α polypeptide and two APRIL polypeptides, the protein component of the wherein different aggressiveness of Neutrokine- α is ripe extracellular soluble part (such as SEQ ID NO by Neutrokine- α:2 the 134 to 285th amino acids) or APRIL ripe extracellular soluble part (such as SEQ ID NO:4 the 105 to 250th amino acids) composition.

In a particular embodiment, the comformational epitope for the antibody binding Neutrokine- α monomeric proteins that can be used in the inventive method.In a particular embodiment, the comformational epitope for antibody binding Neutrokine- α polymers, particularly tripolymer the albumen that can be used in the inventive method.In other embodiments, it can be used for the antibody binding in the inventive method as the comformational epitope produced by the juxtaposition of Neutrokine- α and heterologous polypeptide, when the comformational epitope can occur in Neutrokine- α formation heterotrimers (such as and APRIL polypeptides) or fusion protein between Neutrokine- α and heterologous polypeptide.

The antibody that can be used in the inventive method includes but is not limited to the epitope binding fragments of polyclonal, monoclonal, polyspecific, people, humanization or chimeric antibody, single-chain antibody, Fab fragments, F (ab) ' fragment, the fragment of Fab expression libraries generation, antiidiotype (anti-Id) antibody (the anti-id antibody for including anti-Neutrokine- Alpha antibodies) and all above antibody.In a preferred embodiment, immunoglobulin is IgG1 or IgG4 isotypes.Immunoglobulin can have heavy chain and light chain.IgG, IgE, IgM, IgD, IgA and IgY arrangement can match with the light chain of κ or λ forms.

In a specific embodiment, the antibody that can be used in the inventive method is the antibody fragment with reference to Neutrokine- α, including but not limited to Fab, Fab ' and F (ab ') 2, Fd, scFv (scFv), single-chain antibody, the Fv (sdFv) of disulfide bond and include the fragment in VL or VH areas.The antibody fragment of combination Neutrokine- α including single-chain antibody can include single variable region or with all or part of following variable region combined：Hinge area, CH1, CH2 and CH3 area.In a specific embodiment, the Neutrokine- α binding fragments that can be used in the inventive method include variable region and hinge area, any combination in CH1, CH2 and CH3 area.The antibody that can be used in the inventive method can come from the antibody of any animal origin, including birds and mammal.Preferably, antibody is people, mouse (such as mouse and rat), donkey, ship rabbit, goat, cavy, camel, horse or chicken source.The antibody that " people " antibody includes at this antibody of amino acid sequence with human immunoglobulin(HIg) and is separated to including one or more human immunoglobulin(HIg)s from human immunoglobulin(HIg) library or from transgenosis and in not expressing the transgenic animals of endogenous immunoglobulin, such as in Kucherlapati United States Patent (USP) 5,939, as described in 598, entire contents are incorporated herein by quoting herein.

The antibody that can be used in the inventive method can be single special, double special, three special or higher polyspecifics.Multi-specificity antibody can be specific to the different epitopes of Neutrokine- α polypeptides or can be specific to Neutrokine- α polypeptides and heterologous epitope such as heterologous polypeptide or solid support material.See such as PCT publication WO 93/17715, WO 92/08802, WO91/00360, WO 92/05793, Tutt, et al., J.Immunol.147:60-69(1991)；United States Patent (USP) 4,474,893,4,714,681,4,925,648,5,573,920,5,601,819, Kostelnyet al., J.Immunol.148:1547-1553(1992).

The antibody that can be used in the inventive method can be described or illustrated with the binding affinity of antibody and Neutrokine- α polypeptides.In a particular embodiment, antibody binding Neutrokine- α polypeptides or its fragment or the variant that can be used in the inventive method, its dissociation constant or K_DLess than or equal to 5x10^-9M、l0^-9M、5x 10^-10M、10^-10M、5x 10^-11M、10^-11M、5x 10^-12M or 10^- ¹²M.In a particular embodiment, the dissociation constant or K available for the antibody binding Neutrokine- α polypeptides in the inventive method_DIt is in each single any scope provided between value.

The Neutrokine- Alpha antibodies for blocking the receptor/ligand with Neutrokine- α polypeptides to interact partially or completely can be used for the inventive method.Also include that ligand binding will not be prevented but the Neutrokine- alpha specific antibodies of receptor activation can be prevented.Receptor activation (i.e. signal transduction) can be determined with described herein or techniques known in the art.For example, detecting transcription factor NF-AT, AP-1, MAPK8/JNK and/or NF- κ B (including non-classical NF- κ B signals pathway) activation by using techniques known in the art and/or detecting that the phosphorylation (such as tyrosine or serine/threonine) of acceptor or its substrate can determine receptor activation by immunoprecipitation and subsequent western engram analysis.

Above-mentioned Neutrokine- Alpha antibodies can be prepared using methods known in the art.See such as PCT publication WO 96/40281；United States Patent (USP) 5,811,097；Deng et al.,Blood92(6):1981-1988(1998)；Chen et al.,Cancer Res.58(16):3668-3678(1998)；Harrop et al.,J.Immunol.161(4):1786-1794(1998)；Zhu et al.,Cancer Res.58(15):3209-3214(1998)；Yoon et al.,J.Immunol.160(7):3170-3179(1998)；Prat et al.,J.Cell.Sci.111(Pt2):237-247(1998)；Pitard et al.,J.Immunol.Methods 205(2):177-190(1997)；Liautard et al.,Cytokine 9(4):233-241(1997)；Carlson et al.,J.Biol.Chem.272(17):11295-11301(1997)；Taryman et al.,Neuron 14(4):755-762(1995)；Muller et al.,Structure 6(9):1153-1167(1998)；Bartunek et al.,Cytokine 8(1):14-20 (1996) (being herein incorporated herein entire contents by quoting).

In a specific embodiment, the Neutrokine- Alpha antibodies that can be used in the inventive method (including comprising or by the molecule that constitutes of antibody fragment or its variant) are specifically with reference to Neutrokine- α or Neutrokine- α fragment or variant.Specifically, antibody is for example with pointed SEQ ID NO in table 1:The scFv (scFv) of any amino acid sequence in 13-18 may be used to the inventive method.

Table 1：Immunologic opsonin combination Neutrokine- α scFv.

In an embodiment of the invention, the antibody binding Neutrokine- α that can be used in the inventive method and including the polypeptide with any VH areas pointed by table 1 and/or the amino acid sequence in any VL areas pointed by table 1.In a preferred embodiment, the antibody that can be used in the inventive method includes the amino acid sequence in VH areas and VL areas from the same scFv pointed by table 1.In another embodiment, the antibody that can be used in the inventive method includes the amino acid sequence in VH areas and VL areas from the different scFv pointed by table 1.In another embodiment, the antibody specificity combination Neutrokine- α that can be used in the inventive method and including the polypeptide with any one pointed by any one pointed by table 1, two, three or more VH CDR and/or table 1, two, three or more VL CDR amino acid sequence.In a preferred embodiment, the antibody that can be used in the inventive method includes the amino acid sequence of VH CDR and the VL CDR from the same scFv pointed by table 1.In another embodiment, the antibody that can be used in the inventive method includes the amino acid sequence of VH CDR and VLCDR from the different scFv pointed by table 1.The molecule that the scFv that table 1 including or by immunologic opsonin combination Neutrokine- α is pointed out antibody fragment or variant are constituted can be used for the inventive method.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:13 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:13 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:14 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:13 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:13 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:14 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:15 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:15 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:16 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:16 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:17 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:17 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include SEQ ID NO as described in Table 1:18 VH and VL areas.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes SEQ ID NO as described in Table 1:18 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include 15C10 VH and VL areas, and 15C10 is the anti-Neutrokine- Alpha antibodies of a kind of neutrality for example described in U.S. Patent application 20050186637.The amino acid sequence in 15C10 VH areas is SEQ ID NO:19.The amino acid sequence in 15C10 VL areas is SEQ ID NO:20.In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method are the antigen-binding fragments of 15C10 variants.In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method are the 15C10 of humanization.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes 15C10 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method include 4A5-3.1.1-B4 VH and VL areas, 4A5-3.1.1-B4 is the anti-Neutrokine- Alpha antibodies described in International patent publications WO03/0164468, is herein incorporated herein entire contents by quoting.In WO03/0164468, Neutrokine- α are referred to as hTNFSF13b.The amino acid sequence in 4A5-3.1.1-B4 VH areas is SEQ ID NO:21.The amino acid sequence in 4A5-3.1.1-B4 VL areas is SEQ ID NO:22.In a specific embodiment, the anti-Neutrokine- Alpha antibodies that can be used in the inventive method are the antigen-binding fragments of 4A5-3.1.1-B4 variants.In another particular embodiment of the invention, the antibody that can be used in the inventive method includes 4A5-3.1.1-B4 VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 areas.

In a specific embodiment, it can be used for the natural Neutrokine- α polypeptides of the Neutrokine- Alpha antibodies of the present invention specifically with reference to expressed by cell.

C.Neutrokine- α Binding peptides

In a specific embodiment, Neutrokine- alpha-2 antagonists are Neutrokine- alpha bindings or polypeptide.Neutrokine- alpha bindings or polypeptide for example has been described in International patent publications WO05/005462, WO05/000351, WO02/092620, WO02/16412, WO02/02641 and WO02/16411 and United States patent publication US2006135430, US2006084608, US2003194743, US20030195156 and US2003091565, herein entire contents are incorporated herein by quoting.For example in Sun et al., (2006) Biochem.Biophys.Res.Commun.346:Neutrokine- alpha bindings or polypeptide have been described in 1158-1162, herein entire contents are incorporated herein by quoting.Can be used for the Neutrokine- alpha bindings in the inventive method include from through with filobactivirus coating protein merge shown Random peptide sequences in the short polypeptide that identifies.Such as Scott et al. (1990) Science 249 is shown in discussion for phage display peptide library technology:386；Devlin et al.(1990),Science 249:404；The United States Patent (USP) 5,223,409 that on June 29th, 1993 submits；The United States Patent (USP) 5,733,731 that on March 31st, 1998 submits；The United States Patent (USP) 5,498,530 that on March 12nd, 1996 submits；The United States Patent (USP) 5,432,018 that July 11 nineteen ninety-five submits；The United States Patent (USP) 5,338,665 that August in 1994 is submitted on the 16th；The United States Patent (USP) 5,922,545 that on July 13rd, 1999 submits；The WO 96/40987 that on December 19th, 1996 publishes；The WO 98/15833 published with April 16th, 1998, is incorporated into the application by quoting herein.The bacteriophage of expression of peptides can be isolated by the affinity purification and subsequent breeding again of the Neutrokine- α targeting peptides for immobilization of continuous many wheels.To with being sequenced with the candidate bacteriophage of Neutrokine- α highest adhesion, to determine the identity of each binding peptide.Then all Neutrokine- alpha bindings identified are all connected on " carrier ", to generate the further Neutrokine- alpha bindings being used in the inventive method.Term " carrier " refers to preventing degraded and/or extension half-life period, reduction toxicity, the molecule for reducing immunogenicity or increase biological activity of Neutrokine- alpha bindings.Example carrier includes Fc areas and its variant (" peptide antibody " is preferred)；Linear polymer (such as polyethylene glycol (PEG), including 5kD, 20kD and 30kDPEG, polylysine, glucan etc.)；Branch polymer is (see the United States Patent (USP) 4,289,872 such as the Denkenwalter that September in 1981 is submitted on the 15th；The 5,229,490 of the Tam that on July 20th, 1993 submits；The Frechet that on October 28th, 1993 publishes etc. WO 93/21259)；Lipid；Cholesterol group (such as steroids)；Carbohydrate or oligosaccharides (such as glucan)；Albumen, polypeptide or the peptide of any natural or synthesis combined with salvage receptor；Albumin, including but not limited to rHA or its fragment or the variant (United States Patent (USP) 5,876,969 submitted see on March 2nd, 1999；European patent 0413622；The United States Patent (USP) 5 submitted with June 16th, 1998,766,883, be herein incorporated herein entire contents by quoting)；Leucine zipper region；With other such albumen and protein fragments.There is the carrier that at least one N-terminal through one of amino acid residue, C-terminal or side chain are connected with peptide in the Neutrokine- alpha bindings requirement that can be used in the inventive method.Multiple carriers can also be used, such as each end is that Fc or end is Fc, another end or side chain are PEG group.For Neutrokine- alpha bindings, Fc areas are preferred carriers.Fc areas can merge or be blended in two N and C-terminal of peptide with the N or C-terminal of peptide.It is preferred with merging for N-terminal.

As described above, Fc variants can be used for the suitable carrier of the Neutrokine- alpha bindings in the inventive method.Natural Fc can be modified widely, and to form Fc variants, condition has been to maintain the binding ability with salvage receptor；See such as WO 97/34631 and WO 96/32478.In these Fc variants, can remove provided in natural Fc the Neutrokine- alpha bindings that can be used in the inventive method unwanted structural behaviour or functional activity one or more sites.Residue is for example inserted by substitution or deleting residues, into site or the part containing the site is blocked can remove these sites.Residue inserted or substituted can also be the amino acid changed such as peptide mimics or D- amino acid.Fc variants are required for based on many reasons, some reasons are described below.Example Fc variants include molecule and sequence, wherein：

1. eliminate the site for being related to disulfide formation.This removal can be avoided with being used to generate other reactions containing cysteine protein having on the host cell of molecule of the present invention.Therefore, the fragment containing cysteine of N-terminal can be blocked or cysteine residues can be lacked or replace cysteine with other amino acid (such as alanine, serine).Even when eliminating cysteine residues, single-stranded Fc areas remain to be formed the Fc areas of the dimer of Non-covalent binding together.

2. the natural Fc of modification so that it is more compatible with selected host cell.For example, the PA sequences of the natural Fc of neighbouring classics N-terminal can be removed, the sequence can be recognized by such as proline imido grpup peptase of the digestive ferment in Escherichia coli.The methionine residues of N-terminal can also be added, particularly when recombinantly expressing the molecule in bacterial cell such as Escherichia coli.

3. a part for natural Fc N-terminal is removed, to avoid the heterogeneity of N-terminal when in selected host cell inner expression.Therefore, can be with any residue in first 20 amino acid of missing N-terminal.

4. remove one or more glycosylation sites.The residue (such as asparagine) being generally glycosylated can assign cell cracking effect.It can lack or replace these residues with nonglycosylated residue (such as alanine).

5. remove the site such as C1q binding sites for being related to and being interacted with complement.For example, the EKK sequences of human IgG1 can be lacked or replaced.Complement recruitment is probably unfavorable for the molecule that can be used in the inventive method, therefore can avoid this point with this Fc variants.

6. remove the site of influence and the combination of Fc acceptors rather than salvage receptor.Natural Fc can have the site interacted with some leucocytes, and these are unwanted for the Neutrokine- alpha binding fusion molecules that can be used in the inventive method, therefore can be removed it.

7. remove ADCC sites.ADCC sites are known in the art, and see such as Molec.Immunol.29 (5):633-9 (1992) elaborates IgG1 ADCC sites.These are unwanted for the fusion molecule that can be used in the inventive method, therefore can be removed it.

8. when natural Fc is derived from non-human antibody, natural Fc can be by humanization.Generally for the natural Fc of humanization, the residue in selected inhuman natural Fc can be replaced with the residue being just common in naive Fc.Technology for antibody humanization is well known in the art.

Another carrier for the Neutrokine- alpha bindings that can be used in the inventive method is can to combine albumen, polypeptide, peptide, antibody, antibody fragment or the small molecule (such as peptide simulated compound) for remedying antibody.It is, for example, possible to use in United States Patent (USP) 5, the peptide described in 739,277 is used as carrier.Peptide can also be selected with phage display or for combining the RNA- peptide screenings of salvage receptor.These salvage receptor binding compounds are also contained in the implication of " carrier ", and can be used in the Neutrokine- alpha bindings in the inventive method.These carriers should be selected according to extension half-life period (such as the sequence by avoiding protease from being recognized) and reduction immunogenicity (such as by preferential non-immunogenic sequence, as seen in antibody humanization).

As described above, polymer support can be used for can be used for the Neutrokine- alpha bindings in the inventive method.Be currently available it is various be used for connect can as the chemical part of carrier method, see such as Patent Cooperation Treaty (" PCT ") international publication WO 96/11953, be herein incorporated herein entire contents by quoting.The PCT application elaborates that water-soluble polymer is connected with the selectivity of protein N terminal.

In one preferred embodiment, polymer support preferably is polyethylene glycol (PEG).PEG group can be any easily molecular weight, can be linear or side chain.PEG average molecular weight range is preferably from about 2 kilodaltons (" kD ") to about 100kD, more preferably from about 5kD to about 10kD, most preferably from about 5kD to about 10kD.For can be used for the Neutrokine- alpha bindings in the inventive method, PEG group is typically all connected on the reactive group of invention compound (such as aldehyde radical, amino or ester group) by acylation or the reductive alkylation effect of the reactive group in PEG group sequence.

Useful strategy for the PEGylation of synthetic peptide is included by forming conjugate connection in the solution come combined peptide and PEG group sequence, and each component has the specific functionality reacted each other.Peptide can be easily prepared with traditional solid-phase synthesis.With suitable functional group on special site " pre-activate " peptide.Before being reacted with PEG group sequence, purify and simultaneously symbolize precursor completely.Peptide and PEG connection generally occur in the liquid phase, this process can be easily monitored with reverse analysis HPLC.The peptide of PEGylation can be easily purified into the HPLC of preparation scale, is inhaled with analysis level HPLC, amino acid analysis and laser and subtracts the peptide that mass spectrography symbolizes the PEGylation.

Polysaccharide polymer is the water-soluble polymer in the another type of Neutrokine- alpha bindings that can be used in the inventive method.Glucan through α 1-6 by mainly connecting the polysaccharide polymer that the single glucose subunit that key links together is constituted.Glucan in itself can be by various molecular weights scope, and the molecular weight ranges being readily obtained are from about 1kD to about 70kD.Glucan is suitable water-soluble polymer, another carrier (such as Fc) of itself or joint can as the Neutrokine- alpha bindings that can be used in the inventive method carrier.See such as WO 96/11953 and WO96/05309.It has been reported that the glucan purposes conjugated with therapeutic or diagnostic immunoglobulin；See such as European Patent publications 0315456, be herein incorporated herein entire contents by quoting.When carrier of the glucan as the present invention, about 1kD to about 20kD glucan are preferred.

In a specific embodiment, the Neutrokine- alpha bindings that can be used in the inventive method optionally include " attachment ".When there is attachment, its chemical constitution is unimportant, because it is mainly as a sept.Attachment is preferably what is be made up of the amino acid linked together through peptide bond.Therefore, in a preferred embodiment, attachment is made up of 1 to 30 amino acid linked together through peptide bond, wherein the amino acid is selected from 20 naturally occurring amino acid.Some amino acid in these amino acid can be glycosylated, and those skilled in the art are well understood by this point.In preferred embodiment, 1 to 20 amino acid are to be selected from glycine, alanine, proline, asparagine, glutamine and lysine.Even further preferably, what attachment was made up of most of spatially unimpeded amino acid such as glycine and alanine.(it is particularly (Gly) accordingly, it is preferred that attachment is polyglycine₄、(Gly)₅), poly- (Gly-Ala) and polyalanine.It is preferred that attachment be to include the amino acid linker more than 5 amino acid, suitable attachment can have most 500 amino acid in glycine, alanine, proline, asparagine, glutamine, lysine, threonine, serine or aspartic acid.The attachment of about 20 to 50 amino acid is most preferred.

Non-peptide attachment can be used for can be used for the Neutrokine- alpha bindings in the inventive method.It is, for example, possible to use alkyl linker is for example -- NH-- (CH₂)_n-- C (O) --, wherein n is 2 to 20.Group such as lower alkyl (such as C can be hindered with any non-space₁--C₆), lower alkyl, halogen (such as Cl, Br), CN, NH₂, phenyl etc. replace these alkyl linkers.

In a preferred embodiment, the Neutrokine- alpha bindings that can be used in the inventive method include amino acid sequence SEQ ID NO:23rd, amino acid sequence SEQ ID NO:24 or amino acid sequence SEQ ID NO:25.In particularly preferred embodiments, the Neutrokine- alpha bindings that can be used in the inventive method are amino acid sequence SEQ ID NO:23(AMG 623；AGP3 peptide antibodies).

Neutrokine- α acceptors

In a specific embodiment, Neutrokine- alpha-2 antagonists are Neutrokine- α receptor proteins or its fragment or variant.Neutrokine- α acceptors include such as transmembrane activator and CAML interaction agent (TACI, GenBank numbering AAC51790, SEQ ID NO:6), B cell activating factor receptor (BAFF-R, GenBank numbering NP_443177, SEQ ID NO:10), ripe antigen (BCMA, GenBank numbering NP_001183, SEQ the ID NO of B cell:8).Neutrokine- α receptor proteins, its fragment and variant and antibody for example has been described in PCT publication WO03/014294, WO02/066516, WO02/024909, WO03/014294, WO03/024991, WO02/094852 and WO04/011611 and United States patent publication US20030148445, U S20030099990, US2005070689, US2005043516 and US2003012783.This will be described in more detail below.Entire contents are incorporated herein by quoting herein.

D.Neutrokine- α acceptors, TACI

TACI polypeptides, such as those polypeptides described below can be used for the inventive method as Neutrokine- α and/or APRIL antagonists.TACI is also referred to as TR17, is a kind of albumen (the SEQ ID NO of 293 amino acid residues:6), it speculates that molecular weight is about 31.8kDa.The cDNA nucleotide sequences for encoding TACI are SEQ ID NO:5.From about 1 to about 165 amino acids of presumption constitute cell outskirt (SEQ ID NO:6), from about 166 to about 186 amino acids constitute transmembrane region (SEQ ID NO:6)；And from about 187 to about 293 amino acids constitute intracellular region (SEQID NO:6).

Therefore, in one embodiment, the TACI albumen that can be used in the inventive method is included or by amino acid sequence SEQ ID NO:The polypeptide of the separation of 6 compositions, or including or by a part of SEQ ID NO:The 6 polypeptide such as TACI cells outskirts constituted (including SEQ ID NO:6 the 1 to 165th amino acids) and/or the rich cysteine areas of TACI (including SEQ ID NO:6 the 33 to 104th amino acids)；And with the identical of polypeptide recited above at least 80% identical, more preferably at least 90% or 95%, the identical polypeptide of at least 96%, 97%, 98%, 99% or 100% still more preferably.

In another embodiment, the TACI albumen that can be used in the inventive method is to include SEQ ID NO:The polypeptide of the separation of 6 the 1 to 154th amino acids and with the identical of polypeptide recited above at least 80% identical, more preferably at least 90% or 95%, the identical polypeptide of at least 96%, 97%, 98%, 99% or 100% still more preferably.

Represent that the amino acid sequence of polypeptide is identical with canonical sequence with the polypeptide with reference to amino acid sequence at least such as 95% " identical " amino acid sequence with TACI polypeptides, except the peptide sequence can include every 100 taci receptors reference amino acid sequence amino acid in most 5 amino acid changes.In other words, in order to obtain having and reference amino acid sequence at least 95% identical polypeptide, can lack or with another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor canonical sequence most 5% amino acid residue, or the amino acid residue of for up to total amino acid residue number 5% of canonical sequence can be inserted into canonical sequence.These changes of canonical sequence can betide the amino terminal or carboxy terminal positions with reference to amino acid sequence, or any position of the generation between these terminal positions, it can be dispersed on the single residue betided in canonical sequence or betide in one or more consecutive residues in canonical sequence.

TACI polypeptide fragment is included comprising or by SEQ ID NO:The polypeptide of amino acid sequence composition contained by 6.Polypeptide fragment can be " freestanding " or in bigger polypeptide, and the fragment forms a part or the region of bigger polypeptide, most preferably as single continuum.In other embodiment, polypeptide fragment includes one or more TACI areas and is either made from it.It is preferred that polypeptide fragment include member in group：(a) include or (estimated it by TACI cells outskirt and constitute SEQ ID NO:The 1st of 6 arrive about 165 amino acids residues) composition polypeptide；(b) include or (estimated it by the rich cysteine areas of TACI and constitute SEQ ID NO:6 the 33rd to the 104th amino acids residue) composition polypeptide；(c) include or (estimated it by TACI transmembrane regions and constitute SEQ ID NO:6 the 166th to the 186th amino acids residue) composition polypeptide；(d) include or (estimated it by TACI intracellular regions and constitute SEQ ID NO:6 the 187th to the 293rd amino acids residue) composition polypeptide；Or any combination of the polypeptide in (e) (a)-(d).

The extracellular rich cystine motif for believing TACI is important for the interaction between TACI and its part (Neutrokine- α and APRIL).Therefore, in a preferred embodiment, the TACI polypeptide fragments that can be used in the inventive method include SEQ ID NO:The 33 to 66th of 6 and/or the 70 to 104th amino acids residue are either made up of them.In a specific embodiment, the TACI polypeptide fragments that can be used in the inventive method include one or two extracellular rich cystine motif (SEQ ID NO:6 the 33 to 66th and the 70 to 104th amino acids residue) or be made from it.The albumen that peptide sequence including these rich cystine motifs with one or two at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical peptide sequence are either made up of the peptide sequence is also preferred.

Other fragments for the TACI albumen that can be used in the inventive method are the fragments that the structure or function attribute through TACI is characterized.These fragments are included comprising complete (total length) TACI (SEQ IDNO:6) α spirals or α spiralizations area (" α areas "), β-pleated sheet and β-pleated sheet formation area (" β areas "), corner structure and corner structure formation area (" corner structure area "), crimp and curl into area's (" crimp zone "), hydrophilic area, hydrophobic region, α amphiphilics area, β amphiphilics area, surface formation area and high antigenic index area (have the continuous amino acid that antigenic index is more than or equal to 1.5 containing four or more, identified using the default parameters of Jameson-Wolf programs) amino acid residue, such as in United States Patent (USP) 6, 969, as discussed in 519.Being estimated such as the default parameters using these computer programs, some preferred regions include but is not limited to α areas, β areas, corner structure area and the crimp zone of Garnier-Robson presumptions；α areas, β areas and the corner structure area of Chou-Fasman presumptions；The hydrophilic area of Kyte-Doolittle presumptions；The hydrophobic region of Hopp-Woods presumptions；Eisenberg α and β amphiphilics area；Emini surfaces formation area；And Jameson-Wolf high antigenic index area.

The TACI polypeptides such as fusion protein (including the polypeptide connected through peptide bond from heterologous protein sequence (sequences of different albumen)) that can be used in the inventive method of modified forms can be expressed, secretion signal can not only be included, extra heterologous fuctional regions can also be included.Or this fusion protein for example can be prepared by using peptide synthesizer by synthetic technology.It therefore, it can add extra amino acid area, particularly electrically charged amino acid, with the stability and persistence during improving in host cell, purifying or during subsequent processing and storage to the N-terminal of polypeptide.Peptide motif can also be added in polypeptide, to promote purifying.These regions can be removed before finally polypeptide is prepared.Peptide motif is added into polypeptide to cause secretion or discharge, improve stability and promote purifying etc. to be all well known in the art and conventional technique.

The TACI fusion proteins that preferably can be used in the inventive method include the heterologous area from immunoglobulin, and it is used for solubilized protein.Such as EP-A-O 464533 (corresponding to Canada 2045869) and WO00/024782, PCT publication WO01/60397, WO01/81417, WO01/087977 and WO02/94852；US publication US2003103986 and US2006034852 and Gross, et al., (2000) Nature 404:995-999 and Yu, et al., (2000) Nat Immunol 1:252-256 is elaborated to include the fusion protein of the various pieces of the constant region of immunoglobulin molecules and another people's albumen or part thereof, is herein incorporated herein its content by quoting.In many situations, the Fc parts in fusion protein are highly suitable for treating and diagnosed, and therefore cause improved pharmacokinetics performance (EP-A 0232262).On the other hand, it is necessary to which Fc parts can be lacked after expressing, detecting and being purified into fusion protein in favourable mode described herein for some applications.When Fc part be proved its to for treat and diagnosis be hinder when it is particularly the case, such as when fusion protein is used as the antigen of immunity inoculation.In medicament research and development, such as human protein such as hIL-5 with Fc partial fusions, to identify hIL-5 antagonist with high flux screening detection method.See D.Bennett et al., J.Molecular Recognition8:52-58 (1995) and K.Johanson et al., J.Biol.Chem.270:9459-9471(1995).In a specific embodiment, the TACI-Fc fusion proteins that can be used in the inventive method are Atacicept (TACI-Ig).

Those skilled in the art know and as discussed above, TACI polypeptides can be merged with other peptide sequences.For example, the TACI polypeptides that can be used in the inventive method can with the constant region of immunoglobulin (IgA, IgE, IgG, IgM) or part thereof (CH1, CH2, CH3, or any combination thereof and part) or albumin (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) fusion, form chimeric polyeptides.

The albumin fusion protein that can be used in the inventive method includes the fragment of at least one TACI polypeptide or the fragment or variant of variant and at least one human serum albumins, it is connected to each other, albumin fusion protein (is preferably generated by translating nucleic acid by genetic fusion, wherein in the nucleic acid, the all or part of polynucleotides skeleton of all or part of polynucleotides and encoding albumin that encode TACI is connected), or chemically conjugated be connected to each other.Once as a part for albumin fusion protein, TACI polypeptides and albumin protein can be referred to as " part ", " region " or " motif " (such as " TACI parts " or " Albumin in Partial ") of albumin fusion protein.

In one embodiment, the albumin fusion protein that can be used in the inventive method includes TACI polypeptides and seralbumin is either made up of them.In other embodiments, the fragment and seralbumin that the albumin fusion protein that can be used in the inventive method includes TACI polypeptides are either made up of them.In other embodiments, the variant and seralbumin that the albumin fusion protein that can be used in the inventive method includes TACI polypeptides are either made up of them.In a preferred embodiment, the seralbumin protein component of albumin fusion protein is sero-abluminous maturing part.

In further embodiment, the albumin fusion protein that can be used in the inventive method include TACI polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity fragment either be made up of them.In further embodiment, the albumin fusion protein that can be used in the inventive method include TACI polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity variant either be made up of them.In a preferred embodiment, the TACI parts of albumin fusion protein are total length TACI polypeptides.In a further preferred embodiment, the TACI protein parts of albumin fusion protein are ripe, the soluble regions of TACI polypeptides.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including TACI polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the albumin fusion protein that present invention offer includes or the maturing part by TACI polypeptides and sero-abluminous maturing part are constituted.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including TACI polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the present invention, which is provided, to be included or the maturing part by TACI polypeptides and sero-abluminous maturing part (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) albumin fusion protein of composition.In one preferred embodiment, TACI polypeptides (including its fragment or variant) are merged with the human serum albumins (the 1 to 585th amino acids of the human serum albumins i.e. as shown in Fig. 1 and 2 of European patent 0322094) of mature form, are herein incorporated herein entire contents by quoting.Another preferred embodiment in, the antibody (including its fragment or variant) of the present invention is merged with the polypeptide fragment for including or being made up of the 1st to x residue of human serum albumins, wherein x is the integer from 1 to 585, and the albumin fragment has human serum albumins activity.Another preferred embodiment in, TACI polypeptides (including its fragment or variant) are with including or being merged by the 1st polypeptide fragment constituted to z amino acids of human serum albumins, wherein z is the integer from 369 to 419, such as United States Patent (USP) 5,766, described in 883, entire contents are incorporated herein by quoting herein.TACI polypeptides (including its fragment or variant) can be merged with the N-terminal or C-terminal of heterologous protein (such as immunoglobulin Fc polypeptide or human serum albumins polypeptide).

Preferably, the albumin fusion protein that can be used in the inventive method includes the HA as N-terminal part and the TACI polypeptides as C-terminal part.Or, the albumin fusion protein of the TACI polypeptides including the HA as its C-terminal part and as its N-terminal part can also be used.

In other embodiments, the albumin fusion protein that can be used in the inventive method has the TACI polypeptides merged with the N-terminal and C-terminal of albumin.In a specific embodiment, the TACI polypeptides merged in N-terminal and C-terminal are identicals.In another embodiment, the TACI polypeptides merged in N-terminal and C-terminal are different TACI polypeptides.In another embodiment, TACI polypeptides are blended in the N or C-terminal of albumin, and heterologous polypeptide is blended in remaining end.

In general, the albumin fusion protein that can be used in the inventive method can have a HA to derive area and a TACI area.But, multiple regions in each albumen can be used for preparing the albumin fusion protein that can be used in the inventive method.Equally, more than one albumen can be used to prepare the albumin fusion protein that can be used in the inventive method.For example, albumen can be blended in HA N-terminal and C-terminal.In this configuration, the protein part can be same or different protein molecular.The structure of the albumin fusion protein of difunctionality can be expressed as X-HA-Y or Y-HA-X.

The TACI albumen that can be used in a specific embodiment in the inventive method or its fragment or variant can be conjugated with cytotoxin (such as cytostatic agent or cytocide).Cytotoxin or cell toxicity medicament can include any medicine being harmful to cell.Example include taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, emetine, mitomycin, etoposide, Teniposide, vincristine, vinblastine, colchicin, adriamycin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- boldenones, glucocorticoid, procaine, dicaine, lidocaine, Propranolol and puromycin with and the like or homologue.

In another embodiment, the TACI albumen that can be used in the inventive method or its fragment or variant can be conjugated with toxin.

The feature for the TACI polypeptides that can be used in the inventive method can be improved or changed using genetically engineered, to generate the polypeptide that can be used in the inventive method.Single or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing, addition or fusion protein can be included with the new mutain of recombinant DNA technology generation known to those skilled in the art or " mutein ".These modified polypeptides can show for example to strengthen activity or increase stability.In addition, at least under specific purifying and condition of storage, they can be purified than corresponding natural polypeptides by higher yield, and show more preferable stability.

It can be monomer or polymer (i.e. dimer, tripolymer, the tetramer and more high polymer) that the TACI polypeptides in the inventive method, which can be used for,.In a particular embodiment, polypeptide of the invention is monomer, dimer, tripolymer or the tetramer.In other embodiment, polymer of the invention is at least dimer, tripolymer or the tetramer.It is (Beutler the and Huffel, Science 264 existed in the form of tripolymer to believe some TNF family proteins members:667,1994;Banner et al.,Cell73:431,1993).Therefore, the TACI of tripolymer can provide the advantage of enhancing biological activity.

In a particular embodiment, polymer can be homopolymer or different aggressiveness.Term " homopolymer " referred to herein as comprises only the polymer of TACI polypeptides (including TACI fragments, variant and fusion protein described herein).These homopolymers can contain the TACI polypeptides with same or different amino acid sequence.In a particular embodiment, homopolymer is the polymer for comprising only the TACI albumen with identical peptide sequence.In another particular embodiment of the invention, homopolymer is the polymer containing the TACI albumen with different peptide sequences.

Term " different aggressiveness " also contains the polymer of heterologous polypeptide (comprising only non-corresponding in the albumen of the peptide sequence of the peptide sequence of TACI coded by said gene) referred to herein as in addition to TACI polypeptides.The polymer that can be used in the inventive method can be hydrophobic connection, hydrophilic connection, ion connection and/or be covalently attached connected result, and/or can be indirectly connected to, for example, formed by liposome.Therefore, in one embodiment, when albumen is in contact with each other in the solution, it is formed same polymer such as homodimer, homotrimer, the different tetramer or the different tetramer.In other embodiments, polymer is formed by the covalent attachment between the covalent attachment with TACI albumen or TACI albumen.These covalent attachments may relate to peptide sequence (contained one or more amino acid residues of albumen.In oneainstance, covalent attachment is the crosslinking between the cysteine residues in the peptide sequence of albumen, and the cysteine residues also interact in natural (i.e. naturally occurring) polypeptide.In another scenario, covalent attachment is the result after chemistry or reorganization.

Or, this covalent attachment can be related to one or more contained amino acid residues in the heterologous polypeptide sequence of TACI fusion proteins.In an example, covalent attachment is the covalent attachment (see U.S. Patent application 5,478,925, be herein incorporated herein its content by quoting) between the heterologous sequence contained by fusion protein.In a specific example, covalent attachment is the covalent attachment between the heterologous sequence contained by TACI-Fc fusion proteins (as described herein).In another particular embodiment of the invention, the covalent attachment for the fusion protein that can be used in the inventive method be from another can be formed covalent attachment polymer TNF families ligand/receptor member such as OPG (see such as international publication WO 98/49305, being herein incorporated herein entire contents by quoting) heterologous polypeptide sequence between covalent attachment.In another embodiment, attachment (such as peptide, carbohydrate or the polymer attachment of solubility) of two or more TACI polypeptides through synthesis links together.Example is included in those peptide linkers (being incorporated into the application by quoting herein) described in United States Patent (USP) 5,073,627.Can be generated using traditional recombinant DNA technology includes the albumen of multiple TACI polypeptides separated through peptide linker.

In a specific embodiment, with reference to TACI polypeptides, polypeptide fragment or SEQ IDNO:The antibody of 6 variant, and/or TACI polypeptide epitopes (by for detecting the epitope that the immunodetection well known in the art of special antibody-antigen binding is determined) can be used in the inventive method.For example in PCT publication WO04/011611, WO01/087977, WO01/60397 and WO02/66516；United States patent publication US2005043516 and US2003012783；And Ch ' en, et al., (2005) Cell Immunol 236:78-85 and Liu, et al., (2003) XiBao Yu Fen Zi Mian Yi Xue Za Zhi 19:Have been able to describe anti-TACI antibody and its fragment in 168-169.Entire contents are incorporated herein by quoting herein.Antibody includes but is not limited to the epitope binding fragments of monoclonal, polyspecific, people, humanization or chimeric antibody, single-chain antibody, Fab fragments, F (ab ') fragment, the fragment of Fab expression libraries generation, antiidiotype (anti-Id) antibody (the anti-Id antibody for including for example anti-TACI antibody) and above-mentioned all antibody.The immunoactive portions of term " antibody " referred to herein as immunoglobulin molecules and immunoglobulin molecules, i.e., the molecule of the antigen binding site containing immunologic opsonin combination antigen.The immunoglobulin molecules of the present invention can be the immunoglobulin of any kind of (such as IgG, IgE, IgM, IgD, IgA and IgY), any group (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subgroup.In a particular embodiment, immunoglobulin molecules are IgG1.In other specific embodiments, immunoglobulin molecules are IgG4.

The antibody fragment for the combination TACI that can be used in the inventive method includes but is not limited to Fab, Fab ' and F (ab ') 2, Fd, scFv (scFv), single-chain antibody, the Fv (sdFv) of disulfide bond and include the fragment in VL or VH areas.The antibody fragment of combination TACI including single-chain antibody can include single variable region or with all or part of following variable region combined：Hinge area, CH1, CH2 and CH3 area.In a specific embodiment, the TACI binding fragments that can be used in the inventive method include variable region and hinge area, any combination in CH1, CH2 and CH3 area.The antibody that can be used in the inventive method can come from the antibody of any animal origin, including birds and mammal.Preferably, antibody is people, mouse (such as mouse and rat), donkey, shiprabbit, goat, cavy, camel, horse or chicken source.The antibody that " people " antibody includes at this antibody of amino acid sequence with human immunoglobulin(HIg) and is separated to including one or more human immunoglobulin(HIg)s from human immunoglobulin(HIg) library or from transgenosis and in not expressing the transgenic animals of endogenous immunoglobulin, such as in Kucherlapati United States Patent (USP) 5,939, as described in 598, entire contents are incorporated herein by quoting herein.

The anti-TACI antibody that can be used in the inventive method can be single special, double special, three special or higher polyspecifics.Multi-specificity antibody can be specific to the different epitopes of TACI polypeptides or can be specific to TACI polypeptides and heterologous epitope such as heterologous polypeptide or solid support material.See such as PCT publication WO 93/17715, WO 92/08802, WO91/00360, WO 92/05793, Tutt, et al., J.Immunol.147:60-69(1991)；United States Patent (USP) 4,474,893,4,714,681,4,925,648,5,573,920,5,601,819, Kostelny et al., J.Immunol.148:1547-1553(1992)；Entire contents are incorporated herein by quoting herein.

It can be described according to its cross reactivity or illustrate can be used for the TACI antibody in the inventive method.The antibody that any other analog, homologue or homologue of TACI polypeptides will not be combined can be used for the method for the present invention.In a particular embodiment, TACI antibody and mouse, rat and/or the homologue of rabbit of people's TACI albumen or its corresponding epitope cross reaction.In a specific embodiment, the anti-TACI antibody that can be used in the inventive method is not only in conjunction with TACI, also in relation with BCMA and BAFF-R.

Also it can be described according to it with the binding affinity of TACI polypeptides or illustrate can be used for the antibody in the inventive method.It is preferred that binding affinity include dissociation constant or K_DLess than or equal to 5x 10^- ⁹M、l0^-9M、5x 10^-10M、10^-10M、5x 10^-11M、10^-11M、5x 10^-12M or 10^-12M。

The TACI antibody that can be used in the inventive method can be the activator or antagonist of TACI polypeptides.For example including blocking the TACI antibody with the receptor/ligand interaction of TACI polypeptides partially or completely.Also include that ligand binding will not be prevented but the receptor specific antibody of receptor activation can be prevented.Receptor activation (i.e. signal transduction) can be determined with described herein or techniques known in the art.For example, detecting that the phosphorylation (such as tyrosine or serine/threonine) of acceptor or its substrate can determine receptor activation by using techniques known in the art detection transcription factor NF-AT, AP-1, and/or NF- κ B activation and/or by immunoprecipitation and subsequent western engram analysis.

In a specific embodiment, prevent the receptor-specific TACI antibody and identification receptor-ligand complex of ligand binding and receptor activation and may be used to the inventive method preferably without the TACI antibody for specifically recognizing uncombined acceptor or uncombined part.Above-mentioned TACI antibody can be prepared using techniques known in the art.See such as PCT publication WO 96/40281；United States Patent (USP) 5,811,097；Deng et al.,Blood 92(6):1981-1988(1998)；Chen et al.,Cancer Res.58(16):3668-3678(1998)；Harrop et al.,J.Immunol.161(4):1786-1794(1998)；Zhu et al.,Cancer Res.58(15):3209-3214(1998)；Yoon et al.,J.Immunol.160(7):3170-3179(1998)；Prat et al.,J.Cell.Sci.111(Pt2):237-247(1998)；Pitard et al.,J.Immunol.Methods 205(2):177-190(1997)；Liautard etal.,Cytokine 9(4):233-241(1997)；Carlson et al.,J.Biol.Chem.272(17):11295-11301(1997)；Taryman et al.,Neuron 14(4):755-762(1995)；Muller et al.,Structure 6(9):1153-1167(1998)；Bartunek et al.,Cytokine8(1):14-20 (1996) (being herein incorporated herein entire contents by quoting).

E.Neutrokine- α acceptors, BCMA

BCMA polypeptides, such as those BCMA polypeptides described below, can as Neutrokine- α and/or APRIL antagonist, and can be used for the inventive method.BCMA is also referred to as TR18, is albumen (the SEQ ID NO of 184 amino acid residues:8), infer that molecular weight is about 20.1kDa.Coding BCMA cDNA nucleotides sequences are classified as SEQ ID NO:7.From about 1 to about 45 amino acids of presumption constitute cell outskirt (SEQ ID NO:8)；From about 55 to about 80 amino acids constitute transmembrane region (SEQ ID NO:8)；And from about 81 to about 184 amino acids constitute intracellular region (SEQ ID NO:8).

Therefore, in one embodiment, the BCMA albumen that can be used in the inventive method is included or by amino acid sequence SEQ ID NO:The polypeptide of the separation of 8 compositions, or including or by a part of SEQ ID NO:The 8 polypeptide such as BCMA cells outskirts constituted (including SEQ ID NO:8 the 1 to 54th amino acids) and/or the rich cysteine areas of BCMA (including SEQ ID NO:8 the 8 to 41st amino acids)；And with the identical of polypeptide recited above at least 80% identical, more preferably at least 90% or 95%, the identical polypeptide of at least 96%, 97%, 98%, 99% or 100% still more preferably.

Represent that the amino acid sequence of polypeptide is identical with canonical sequence with the polypeptide with reference to amino acid sequence at least such as 95% " identical " amino acid sequence with BCMA polypeptides, except the peptide sequence can include every 100 BCMA acceptors reference amino acid sequence amino acid in most 5 amino acid changes.In other words, in order to obtain having and reference amino acid sequence at least 95% identical polypeptide, can lack or with another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor canonical sequence most 5% amino acid residue, or the amino acid residue of for up to total amino acid residue number 5% of canonical sequence can be inserted into canonical sequence.These changes of canonical sequence can betide the amino terminal or carboxy terminal positions with reference to amino acid sequence, or any position of the generation between these terminal positions, it can be dispersed on the single residue betided in canonical sequence or betide in one or more consecutive residues in canonical sequence.

BCMA polypeptide fragment is included comprising or by SEQ ID NO:The polypeptide of amino acid sequence composition contained by 8.Polypeptide fragment can be " freestanding " or in bigger polypeptide, and the fragment forms a part or the region of bigger polypeptide, most preferably as single continuum.In other embodiment, polypeptide fragment includes one or more BCMA areas and is either made from it.It is preferred that polypeptide fragment include member in group：(a) include or (estimated it by BCMA cells outskirt and constitute SEQ ID NO:The 1st of 8 arrive about 54 amino acids residues) composition polypeptide；(b) include or (estimated it by the rich cysteine areas of BCMA and constitute SEQ ID NO:8 the 8th to the 41st amino acids residue) composition polypeptide；(c) include or (estimated it by BCMA transmembrane regions and constitute SEQ ID NO:8 the 55th to the 80th amino acids residue) composition polypeptide；(d) include or (estimated it by BCMA intracellular regions and constitute SEQ ID NO:8 the 81st to the 184th amino acids residue) composition polypeptide；Or any combination of the polypeptide in (e) (a)-(d).

The extracellular rich cystine motif for believing BCMA is important for the interaction between BCMA and its part (Neutrokine- α and APRIL).Therefore, in a preferred embodiment, the BCMA polypeptide fragments that can be used in the inventive method include SEQ ID NO:8 the 8 to 41st amino acids residue is either made from it.The albumen constituted including the peptide sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical peptide sequence with the rich cystine motif or by the peptide sequence is also preferred.

Other fragments for the BCMA albumen that can be used in the inventive method are the fragments that the structure or function attribute through BCMA is characterized.These fragments are included comprising complete (total length) BCMA (SEQ IDNO:8) α spirals or α spiralizations area (" α areas "), β-pleated sheet and β-pleated sheet formation area (" β areas "), corner structure and corner structure formation area (" corner structure area "), crimp and curl into area's (" crimp zone "), hydrophilic area, hydrophobic region, α amphiphilics area, β amphiphilics area, surface formation area and high antigenic index area (have the continuous amino acid that antigenic index is more than or equal to 1.5 containing four or more, identified using the default parameters of Jameson-Wolf programs) amino acid residue, such as in United States Patent (USP) 6, 969, as discussed in 519.Being estimated such as the default parameters using these computer programs, some preferred regions include but is not limited to α areas, β areas, corner structure area and the crimp zone of Garnier-Robson presumptions；α areas, β areas and the corner structure area of Chou-Fasman presumptions；The hydrophilic area of Kyte-Doolittle presumptions；The hydrophobic region of Hopp-Woods presumptions；Eisenberg α and β amphiphilics area；Emini surfaces formation area；And Jameson-Wolf high antigenic index area.

The BCMA polypeptides such as fusion protein (including the polypeptide connected through peptide bond from heterologous protein sequence (sequences of different albumen)) that can be used in the inventive method of modified forms can be expressed, secretion signal can not only be included, extra heterologous fuctional regions can also be included.Or this fusion protein for example can be prepared by using peptide synthesizer by synthetic technology.It therefore, it can add extra amino acid area, particularly electrically charged amino acid, with the stability and persistence during improving in host cell, purifying or during subsequent processing and storage to the N-terminal of polypeptide.Peptide motif can also be added in polypeptide, to promote purifying.These regions can be removed before finally polypeptide is prepared.Peptide motif is added into polypeptide to cause secretion or discharge, improve stability and promote purifying etc. to be all well known in the art and conventional technique.

The BCMA fusion proteins that preferably can be used in the inventive method include the heterologous area from immunoglobulin, and it is used for solubilized protein.Such as EP-A-O 464533 (corresponding to Canada 2045869) and WO00/024782 elaborate to include the fusion protein of the various pieces of the constant region of immunoglobulin molecules and another people's albumen or part thereof.Such as PCT publication WO01/087977, WO01/60397 and WO01/24811 and Gross, et al., (2000) Nature 404:995-999；Thompson,et al.,(2000)J Exp Med 192:129-135；And Yu, et al., (2000) Nat Immunol 1:BCMA domain-immunoglobulin fusion proteins have been described in 252-256, are herein incorporated herein its content by quoting.In many situations, the Fc parts in fusion protein are highly suitable for treating and diagnosed, and therefore cause improved pharmacokinetics performance (EP-A 0232262).On the other hand, it is necessary to which Fc parts can be lacked after expressing, detecting and being purified into fusion protein in favourable mode described herein for some applications.When Fc part be proved its to for treat and diagnosis be hinder when it is particularly the case, such as when fusion protein is used as the antigen of immunity inoculation.In medicament research and development, such as human protein such as hIL-5 with Fc partial fusions, to identify hIL-5 antagonist with high flux screening detection method.See D.Bennett et al., J.Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J.Biol.Chem.270:9459-9471(1995).

Those skilled in the art know and as discussed above, BCMA polypeptides can be merged with other peptide sequences.For example, the BCMA polypeptides that can be used in the inventive method can with the constant region of immunoglobulin (IgA, IgE, IgG, IgM) or part thereof (CH1, CH2, CH3, or any combination thereof and part) or albumin (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) fusion, form chimeric polyeptides.

The albumin fusion protein that can be used in the inventive method includes the fragment of at least one BCMA polypeptide or the fragment or variant of variant and at least one human serum albumins, it is connected to each other, albumin fusion protein (is preferably generated by translating nucleic acid by genetic fusion, wherein in the nucleic acid, the all or part of polynucleotides skeleton of all or part of polynucleotides and encoding albumin that encode BCMA is connected), or chemically conjugated be connected to each other.Once as a part for albumin fusion protein, BCMA polypeptides and albumin protein can be referred to as " part ", " region " or " motif " (such as " BCMA parts " or " Albumin in Partial ") of albumin fusion protein.

In one embodiment, the albumin fusion protein that can be used in the inventive method includes BCMA polypeptides and seralbumin is either made up of them.In other embodiments, the fragment and seralbumin that the albumin fusion protein that can be used in the inventive method includes BCMA polypeptides are either made up of them.In other embodiments, the variant and seralbumin that the albumin fusion protein that can be used in the inventive method includes BCMA polypeptides are either made up of them.In a preferred embodiment, the seralbumin protein component of albumin fusion protein is sero-abluminous maturing part.

In further embodiment, the albumin fusion protein that can be used in the inventive method include BCMA polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity fragment either be made up of them.In further embodiment, the albumin fusion protein that can be used in the inventive method include BCMA polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity variant either be made up of them.In a preferred embodiment, the BCMA parts of albumin fusion protein are total length BCMA polypeptides.In a further preferred embodiment, the BCMA protein parts of albumin fusion protein are ripe, the soluble regions of BCMA polypeptides.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including BCMA polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the albumin fusion protein that present invention offer includes or the maturing part by BCMA polypeptides and sero-abluminous maturing part are constituted.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including BCMA polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the present invention, which is provided, to be included or the maturing part by BCMA polypeptides and sero-abluminous maturing part (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) albumin fusion protein of composition.In one preferred embodiment, BCMA polypeptides (including its fragment or variant) are merged with the human serum albumins (the 1 to 585th amino acids of the human serum albumins i.e. as shown in Fig. 1 and 2 of European patent 0322094) of mature form, are herein incorporated herein entire contents by quoting.Another preferred embodiment in, the antibody (including its fragment or variant) of the present invention is merged with the polypeptide fragment for including or being made up of the 1st to x residue of human serum albumins, wherein x is the integer from 1 to 585, and the albumin fragment has human serum albumins activity.Another preferred embodiment in, BCMA polypeptides (including its fragment or variant) are with including or being merged by the 1st polypeptide fragment constituted to z amino acids of human serum albumins, wherein z is the integer from 369 to 419, such as United States Patent (USP) 5,766, described in 883, entire contents are incorporated herein by quoting herein.BCMA polypeptides (including its fragment or variant) can be merged with the N-terminal or C-terminal of heterologous protein (such as immunoglobulin Fc polypeptide or human serum albumins polypeptide).

Preferably, the albumin fusion protein that can be used in the inventive method includes the HA as N-terminal part and the BCMA polypeptides as C-terminal part.Or, the albumin fusion protein of the BCMA polypeptides including the HA as its C-terminal part and as its N-terminal part can also be used.

In other embodiments, the albumin fusion protein that can be used in the inventive method has the BCMA polypeptides merged with the N-terminal and C-terminal of albumin.In a specific embodiment, the BCMA polypeptides merged in N-terminal and C-terminal are identicals.In another embodiment, the BCMA polypeptides merged in N-terminal and C-terminal are different BCMA polypeptides.In another embodiment, BCMA polypeptides are blended in the N or C-terminal of albumin, and heterologous polypeptide is blended in remaining end.

In general, the albumin fusion protein that can be used in the inventive method can have a HA to derive area and a BCMA area.But, multiple regions in each albumen can be used for preparing the albumin fusion protein that can be used in the inventive method.Equally, more than one albumen can be used to prepare the albumin fusion protein that can be used in the inventive method.For example, albumen can be blended in HA N-terminal and C-terminal.In this configuration, the protein part can be same or different protein molecular.The structure of the albumin fusion protein of difunctionality can be expressed as X-HA-Y or Y-HA-X.

The BCMA albumen that can be used in a specific embodiment in the inventive method or its fragment or variant can be conjugated with cytotoxin (such as cytostatic agent or cytocide).Cytotoxin or cell toxicity medicament can include any medicine being harmful to cell.Example include taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, emetine, mitomycin, etoposide, Teniposide, vincristine, vinblastine, colchicin, adriamycin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- boldenones, glucocorticoid, procaine, dicaine, lidocaine, Propranolol and puromycin with and the like or homologue.

In another embodiment, the BCMA albumen that can be used in the inventive method or its fragment or variant can be conjugated with toxin.

The feature for the BCMA polypeptides that can be used in the inventive method can be improved or changed using genetically engineered, to generate the polypeptide that can be used in the inventive method.Single or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing, addition or fusion protein can be included with the new mutain of recombinant DNA technology generation known to those skilled in the art or " mutein ".These modified polypeptides can show for example to strengthen activity or increase stability.In addition, at least under specific purifying and condition of storage, they can be purified than corresponding natural polypeptides by higher yield, and show more preferable stability.Still in yet another embodiment of the present invention, BCMA polypeptide mutants can be " dominant negative regulation thing ".Therefore, the mutant that can for example lack TNF conserved regions all or in part with deficiency BCMA polypeptides eliminates BCMA activity.Non-functional BCMA polypeptides, which can assemble to be formed, can combine but be unable to the acceptor (such as polymer) of inducement signal conduction.

It can be monomer or polymer (i.e. dimer, tripolymer, the tetramer and more high polymer) that the BCMA polypeptides in the inventive method, which can be used for,.In a particular embodiment, polypeptide of the invention is monomer, dimer, tripolymer or the tetramer.In other embodiment, polymer of the invention is at least dimer, tripolymer or the tetramer.It is (Beutler the and Huffel, Science 264 existed in the form of tripolymer to believe some TNF family proteins members:667,1994;Banner et al.,Cell73:431,1993).Therefore, the BCMA of tripolymer can provide the advantage of enhancing biological activity.

In a particular embodiment, polymer can be homopolymer or different aggressiveness.Term " homopolymer " referred to herein as comprises only the polymer of BCMA polypeptides (including BCMA fragments, variant and fusion protein described herein).These homopolymers can contain the BCMA polypeptides with same or different amino acid sequence.In a particular embodiment, homopolymer is the polymer for comprising only the BCMA albumen with identical peptide sequence.In another particular embodiment of the invention, homopolymer is the polymer containing the BCMA albumen with different peptide sequences.

Term " different aggressiveness " also contains the polymer of heterologous polypeptide (comprising only non-corresponding in the albumen of the peptide sequence of the peptide sequence of BCMA coded by said gene) referred to herein as in addition to BCMA polypeptides.The polymer that can be used in the inventive method can be hydrophobic connection, hydrophilic connection, ion connection and/or be covalently attached connected result, and/or can be indirectly connected to, for example, formed by liposome.Therefore, in one embodiment, when albumen is in contact with each other in the solution, it is formed same polymer such as homodimer, homotrimer, the different tetramer or the different tetramer.In other embodiments, polymer is formed by the covalent attachment between the covalent attachment with BCMA albumen or BCMA albumen.These covalent attachments may relate to peptide sequence (contained one or more amino acid residues of albumen.In oneainstance, covalent attachment is the crosslinking between the cysteine residues in the peptide sequence of albumen, and the cysteine residues also interact in natural (i.e. naturally occurring) polypeptide.In another scenario, covalent attachment is the result after chemistry or reorganization.

Or, this covalent attachment can be related to one or more contained amino acid residues in the heterologous polypeptide sequence of BCMA fusion proteins.In an example, covalent attachment is the covalent attachment (see U.S. Patent application 5,478,925, be herein incorporated herein its content by quoting) between the heterologous sequence contained by fusion protein.In a specific example, covalent attachment is the covalent attachment between the heterologous sequence contained by BCMA-Fc fusion proteins (as described herein).In another particular embodiment of the invention, the covalent attachment for the fusion protein that can be used in the inventive method be from another can be formed covalent attachment polymer TNF families ligand/receptor member such as OPG (see such as international publication WO 98/49305, being herein incorporated herein entire contents by quoting) heterologous polypeptide sequence between covalent attachment.In another embodiment, attachment (such as peptide, carbohydrate or the polymer attachment of solubility) of two or more BCMA polypeptides through synthesis links together.Example is included in those peptide linkers (being incorporated into the application by quoting herein) described in United States Patent (USP) 5,073,627.Can be generated using traditional recombinant DNA technology includes the albumen of multiple BCMA polypeptides separated through peptide linker.

In a specific embodiment, with reference to BCMA polypeptides, polypeptide fragment or SEQ IDNO:The antibody of 8 variant, and/or BCMA polypeptide epitopes (by for detecting the epitope that the immunodetection well known in the art of special antibody-antigen binding is determined) can be used in the inventive method.For example in PCT publication WO01/087977, WO01/60397 and WO02/66516 and Ch ' en, et al., (2005) Cell Immunol 236:Anti- BCMA antibody and its fragment have been described in 78-85.Entire contents are incorporated herein by quoting herein.Antibody includes but is not limited to the epitope binding fragments of monoclonal, polyspecific, people, humanization or chimeric antibody, single-chain antibody, Fab fragments, F (ab ') fragment, the fragment of Fab expression libraries generation, antiidiotype (anti-Id) antibody (the anti-Id antibody for including for example anti-BCMA antibody) and above-mentioned all antibody.The immunoactive portions of term " antibody " referred to herein as immunoglobulin molecules and immunoglobulin molecules, i.e., the molecule of the antigen binding site containing immunologic opsonin combination antigen.The immunoglobulin molecules of the present invention can be the immunoglobulin of any kind of (such as IgG, IgE, IgM, IgD, IgA and IgY), any group (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subgroup.In a particular embodiment, immunoglobulin molecules are IgG1.In other specific embodiments, immunoglobulin molecules are IgG4.

The antibody fragment for the combination BCMA that can be used in the inventive method includes but is not limited to Fab, Fab ' and F (ab ') 2, Fd, scFv (scFv), single-chain antibody, the Fv (sdFv) of disulfide bond and include the fragment in VL or VH areas.The antibody fragment of combination BCMA including single-chain antibody can include single variable region or with all or part of following variable region combined：Hinge area, CH1, CH2 and CH3 area.In a specific embodiment, the BCMA binding fragments that can be used in the inventive method include variable region and hinge area, any combination in CH1, CH2 and CH3 area.The antibody that can be used in the inventive method can come from the antibody of any animal origin, including birds and mammal.Preferably, antibody is people, mouse (such as mouse and rat), donkey, shiprabbit, goat, cavy, camel, horse or chicken source.The antibody that " people " antibody includes at this antibody of amino acid sequence with human immunoglobulin(HIg) and is separated to including one or more human immunoglobulin(HIg)s from human immunoglobulin(HIg) library or from transgenosis and in not expressing the transgenic animals of endogenous immunoglobulin, such as in Kucherlapati United States Patent (USP) 5,939, as described in 598, entire contents are incorporated herein by quoting herein.

The anti-BCMA antibody that can be used in the inventive method can be single special, double special, three special or higher polyspecifics.Multi-specificity antibody can be specific to the different epitopes of BCMA polypeptides or can be specific to BCMA polypeptides and heterologous epitope such as heterologous polypeptide or solid support material.See such as PCT publication WO 93/17715, WO 92/08802, WO91/00360, WO 92/05793, Tutt, et al., J.Immunol.147:60-69(1991)；United States Patent (USP) 4,474,893,4,714,681,4,925,648,5,573,920,5,601,819, Kostelny et al., J.Immunol.148:1547-1553(1992)；Entire contents are incorporated herein by quoting herein.

It can be described according to its cross reactivity or illustrate can be used for the BCMA antibody in the inventive method.The antibody that any other analog, homologue or homologue of BCMA polypeptides will not be combined can be used for the method for the present invention.In a particular embodiment, BCMA antibody and mouse, rat and/or the homologue of rabbit of people's BCMA albumen or its corresponding epitope cross reaction.In a specific embodiment, the anti-BCMA antibody that can be used in the inventive method is not only in conjunction with BCMA, also in relation with TACI and BAFF-R.

Also it can be described according to it with the binding affinity of BCMA polypeptides or illustrate can be used for the antibody in the inventive method.It is preferred that binding affinity include dissociation constant or K_DLess than or equal to 5x 10^- ⁹M、l0^-9M、5x 10^-10M、10^-10M、5x 10^-11M、10^-11M、5x 10^-12M or 10^-12M。

The BCMA antibody that can be used in the inventive method can be the activator or antagonist of BCMA polypeptides.For example including blocking the BCMA antibody with the receptor/ligand interaction of BCMA polypeptides partially or completely.Also include that ligand binding will not be prevented but the receptor specific antibody of receptor activation can be prevented.Receptor activation (i.e. signal transduction) can be determined with described herein or techniques known in the art.For example, detecting that the phosphorylation (such as tyrosine or serine/threonine) of acceptor or its substrate can determine receptor activation by using techniques known in the art detection transcription factor NF-AT, AP-1, and/or NF- κ B activation and/or by immunoprecipitation and subsequent western engram analysis.

In a specific embodiment, prevent the receptor-specific BCMA antibody and identification receptor-ligand complex of ligand binding and receptor activation and may be used to the inventive method preferably without the BCMA antibody for specifically recognizing uncombined acceptor or uncombined part.Above-mentioned BCMA antibody can be prepared using techniques known in the art.See such as PCT publication WO 96/40281；United States Patent (USP) 5,811,097；Deng et al.,Blood 92(6):1981-1988(1998)；Chen et al.,Cancer Res.58(16):3668-3678(1998)；Harrop et al.,J.Immunol.161(4):1786-1794(1998)；Zhu et al.,Cancer Res.58(15):3209-3214(1998)；Yoon et al.,J.Immunol.160(7):3170-3179(1998)；Prat et al.,J.Cell.Sci.111(Pt2):237-247(1998)；Pitard et al.,J.Immunol.Methods 205(2):177-190(1997)；Liautard etal.,Cytokine 9(4):233-241(1997)；Carlson et al.,J.Biol.Chem.272(17):11295-11301(1997)；Taryman et al.,Neuron 14(4):755-762(1995)；Muller et al.,Structure 6(9):1153-1167(1998)；Bartunek et al.,Cytokine8(1):14-20 (1996) (being herein incorporated herein entire contents by quoting).

F.Neutrokine- α acceptors, BAFF-R

BAFF-R polypeptides, such as those BAFF-R polypeptides described below, can as Neutrokine- α and/or APRIL antagonist, and can be used for the inventive method.BAFF-R is also referred to as TR21, is albumen (the SEQ ID NO of 184 amino acid residues:10), infer that molecular weight is about 18.9kDa.Coding BAFF-R cDNA nucleotides sequences are classified as SEQ ID NO:9.From about 1 to about 81 amino acids of presumption constitute cell outskirt (SEQ ID NO:10)；From about 82 to about 101 amino acids constitute transmembrane region (SEQ ID NO:10)；And from about 102 to about 184 amino acids constitute intracellular region (SEQ ID NO:10).

Therefore, in one embodiment, the BAFF-R albumen that can be used in the inventive method is to include amino acid sequence SEQ ID NO:10 or the polypeptide for the separation being made from it, or including a part of SEQ ID NO:10 or the polypeptide that is made from it, such as BAFF-R cells outskirt (including SEQ ID NO:10 the 1 to 81st amino acids) and/or the rich cysteine areas of BAFF-R (including SEQ ID NO:10 the 19 to 35th amino acids)；And with the identical of polypeptide recited above at least 80% identical, more preferably at least 90% or 95%, the identical polypeptide of at least 96%, 97%, 98%, 99% or 100% still more preferably.

In another embodiment, the BAFF-R albumen that can be used in the inventive method is to include SEQ ID NO:10 the 1 to 70th amino acids and/or amino acid sequence SEQ ID NO:The polypeptide of 26 separation.SEQ ID NO:26 show the 20th amino acids (valine) in BAFF-R the 1 to 70th amino acids, wherein BAFF-R by asparagine replace and BAFF-R in the 27th amino acids (leucine) replaced by proline.In another embodiment, the BAFF-R albumen that can be used in the inventive method is to include SEQ ID NO:The polypeptide of the separation of 26 the 2 to 70th amino acids.The inventive method is can also be used for the identical of polypeptide recited above at least 80% identical, more preferably at least 90% or 95%, the identical polypeptide of at least 96%, 97%, 98%, 99% or 100% still more preferably.

Represent that the amino acid sequence of polypeptide is identical with canonical sequence with the polypeptide with reference to amino acid sequence at least such as 95% " identical " amino acid sequence with BAFF-R polypeptides, except the peptide sequence can include every 100 BAFF-R acceptors reference amino acid sequence amino acid in most 5 amino acid changes.In other words, in order to obtain having and reference amino acid sequence at least 95% identical polypeptide, can lack or with another 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor canonical sequence most 5% amino acid residue, or the amino acid residue of for up to total amino acid residue number 5% of canonical sequence can be inserted into canonical sequence.These changes of canonical sequence can betide the amino terminal or carboxy terminal positions with reference to amino acid sequence, or any position of the generation between these terminal positions, it can be dispersed on the single residue betided in canonical sequence or betide in one or more consecutive residues in canonical sequence.

BAFF-R polypeptide fragment includes including SEQ ID NO:Amino acid sequence contained by 10 or the polypeptide being made from it.Polypeptide fragment can be " freestanding " or in bigger polypeptide, and the fragment forms a part or the region of bigger polypeptide, most preferably as single continuum.In other embodiment, polypeptide fragment includes one or more BAFF-R areas and is either made from it.It is preferred that polypeptide fragment include member in group：(a) include or (estimated it by BAFF-R cells outskirt and constitute SEQ ID NO:The 1st of 10 arrive about 81 amino acids residues) composition polypeptide；(b) include or (estimated it by the rich cysteine areas of BAFF-R and constitute SEQ ID NO:10 the 19th to the 35th amino acids residue) composition polypeptide；(c) include or (estimated it by BAFF-R transmembrane regions and constitute SEQ ID NO:10 the 82nd to the 101st amino acids residue) composition polypeptide；(d) include or (estimated it by BAFF-R intracellular regions and constitute SEQ ID NO:10 the 102nd to the 184th amino acids residue) composition polypeptide；Or any combination of the polypeptide in (e) (a)-(d).

The extracellular rich cystine motif for believing BAFF-R is important for the interaction between BAFF-R and its part (Neutrokine- α and APRIL).Therefore, in a preferred embodiment, the BAFF-R polypeptide fragments that can be used in the inventive method include SEQ ID NO:10 the 19 to 35th amino acids residue is either made from it.The albumen constituted including the peptide sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical peptide sequence with the rich cystine motif or by the peptide sequence is also preferred.

Other fragments for the BAFF-R albumen that can be used in the inventive method are the fragments that the structure or function attribute through BAFF-R is characterized.These fragments are included comprising complete (total length) BAFF-R (SEQID NO:10) α spirals or α spiralizations area (" α areas "), β-pleated sheet and β-pleated sheet formation area (" β areas "), corner structure and corner structure formation area (" corner structure area "), crimp and curl into area's (" crimp zone "), hydrophilic area, hydrophobic region, α amphiphilics area, β amphiphilics area, surface formation area and high antigenic index area (have the continuous amino acid that antigenic index is more than or equal to 1.5 containing four or more, identified using the default parameters of Jameson-Wolf programs) amino acid residue, such as in United States Patent (USP) 6, 969, as discussed in 519.Being estimated such as the default parameters using these computer programs, some preferred regions include but is not limited to α areas, β areas, corner structure area and the crimp zone of Garnier-Robson presumptions；α areas, β areas and the corner structure area of Chou-Fasman presumptions；The hydrophilic area of Kyte-Doolittle presumptions；The hydrophobic region of Hopp-Woods presumptions；Eisenberg α and β amphiphilics area；Emini surfaces formation area；And Jameson-Wolf high antigenic index area.

The BAFF-R polypeptides such as fusion protein (including the polypeptide connected through peptide bond from heterologous protein sequence (sequences of different albumen)) that can be used in the inventive method of modified forms can be expressed, secretion signal can not only be included, extra heterologous fuctional regions can also be included.Or this fusion protein for example can be prepared by using peptide synthesizer by synthetic technology.It therefore, it can add extra amino acid area, particularly electrically charged amino acid, with the stability and persistence during improving in host cell, purifying or during subsequent processing and storage to the N-terminal of polypeptide.Peptide motif can also be added in polypeptide, to promote purifying.These regions can be removed before finally polypeptide is prepared.Peptide motif is added into polypeptide to cause secretion or discharge, improve stability and promote purifying etc. to be all well known in the art and conventional technique.

The BAFF-R fusion proteins that preferably can be used in the inventive method include the heterologous area from immunoglobulin, and it is used for solubilized protein.Such as EP-A-O 464533 (corresponding to Canada 2045869) and WO00/024782 elaborate to include the fusion protein of the various pieces of the constant region of immunoglobulin molecules and another people's albumen or part thereof.Such as Pelletier, et al., (2003) J Biol Chem 278:33127-33133 and Carter, et al., (2005) Arthritis Rheum 52:BAFF-R domain-immunoglobulin fusion proteins have been described in 3943-3954, are herein incorporated herein its content by quoting.In many situations, the Fc parts in fusion protein are highly suitable for treating and diagnosed, and therefore cause improved pharmacokinetics performance (EP-A 0232262).On the other hand, it is necessary to which Fc parts can be lacked after expressing, detecting and being purified into fusion protein in favourable mode described herein for some applications.When Fc part be proved its to for treat and diagnosis be hinder when it is particularly the case, such as when fusion protein is used as the antigen of immunity inoculation.In medicament research and development, such as human protein such as hIL-5 with Fc partial fusions, to identify hIL-5 antagonist with high flux screening detection method.See D.Bennett et al., J.Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J.Biol.Chem.270:9459-9471(1995).In a specific embodiment, the BAFF-R-Fc fusion proteins that can be used in the inventive method are BR3-Fc.

Those skilled in the art know and as discussed above, BAFF-R polypeptides can be merged with other peptide sequences.For example, the BAFF-R polypeptides that can be used in the inventive method can with the constant region of immunoglobulin (IgA, IgE, IgG, IgM) or part thereof (CH1, CH2, CH3, or any combination thereof and part) or albumin (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) fusion, form chimeric polyeptides.

One example of BAFF-R-Fc albumen is the SEQ ID NO blended with the Fc areas of IgG1 immunoglobulin molecules:10 the 1 to 70th amino acids.Optionally, the 20th amino acids (valine) in BAFF-R by asparagine replace and BAFF-R in the 27th amino acids (leucine) replaced by proline.SEQ ID NO:26 show the BAFF-R changed with the two amino acid the 1 to 70th amino acids.

The albumin fusion protein that can be used in the inventive method includes the fragment of at least one BAFF-R polypeptide or the fragment or variant of variant and at least one human serum albumins, it is connected to each other, albumin fusion protein (is preferably generated by translating nucleic acid by genetic fusion, wherein in the nucleic acid, the all or part of polynucleotides skeleton of all or part of polynucleotides and encoding albumin that encode BAFF-R is connected), or chemically conjugated be connected to each other.Once as a part for albumin fusion protein, BAFF-R polypeptides and albumin protein can be referred to as " part ", " region " or " motif " (such as " BAFF-R parts " or " Albumin in Partial ") of albumin fusion protein.

In one embodiment, the albumin fusion protein that can be used in the inventive method includes BAFF-R polypeptides and seralbumin is either made up of them.In other embodiments, the fragment and seralbumin that the albumin fusion protein that can be used in the inventive method includes BAFF-R polypeptides are either made up of them.In other embodiments, the variant and seralbumin that the albumin fusion protein that can be used in the inventive method includes BAFF-R polypeptides are either made up of them.In a preferred embodiment, the seralbumin protein component of albumin fusion protein is sero-abluminous maturing part.

In further embodiment, the albumin fusion protein that can be used in the inventive method include BAFF-R polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity fragment either be made up of them.In further embodiment, the albumin fusion protein that can be used in the inventive method include BAFF-R polypeptides and it is sero-abluminous with biological activity and/or by therapeutic activity variant either be made up of them.In a preferred embodiment, the BAFF-R parts of albumin fusion protein are total length BAFF-R polypeptides.In further preferred embodiment, the BAFF-R protein parts of albumin fusion protein are ripe, the soluble regions of BAFF-R polypeptides.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including BAFF-R polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the present invention provides the albumin fusion protein for including the maturing part and sero-abluminous maturing part of BAFF-R polypeptides or being made up of them.

In further embodiment, the fragment or variant of the albumin fusion protein that can be used in the inventive method including BAFF-R polypeptides and sero-abluminous with biological activity and/or be either made up of the fragment or variant of therapeutic activity them.In a preferred embodiment, the present invention provides the maturing part for including BAFF-R polypeptides and sero-abluminous maturing part (includes but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) or the albumin fusion protein that is made up of them.In one preferred embodiment, BAFF-R polypeptides (including its fragment or variant) are merged with the human serum albumins (the 1 to 585th amino acids of the human serum albumins i.e. as shown in Fig. 1 and 2 of European patent 0322094) of mature form, are herein incorporated herein entire contents by quoting.Another preferred embodiment in, the antibody (including its fragment or variant) of the present invention is merged with the polypeptide fragment for including or being made up of the 1st to x residue of human serum albumins, wherein x is the integer from 1 to 585, and the albumin fragment has human serum albumins activity.Another preferred embodiment in, BAFF-R polypeptides (including its fragment or variant) are with including or being merged by the 1st polypeptide fragment constituted to z amino acids of human serum albumins, wherein z is the integer from 369 to 419, such as United States Patent (USP) 5,766, described in 883, entire contents are incorporated herein by quoting herein.BAFF-R polypeptides (including its fragment or variant) can be merged with the N-terminal or C-terminal of heterologous protein (such as immunoglobulin Fc polypeptide or human serum albumins polypeptide).

Preferably, the albumin fusion protein that can be used in the inventive method includes the HA as N-terminal part and the BAFF-R polypeptides as C-terminal part.Or, the albumin fusion protein of the BAFF-R polypeptides including the HA as its C-terminal part and as its N-terminal part can also be used.

In other embodiments, the albumin fusion protein that can be used in the inventive method has the BAFF-R polypeptides merged with the N-terminal and C-terminal of albumin.In a specific embodiment, the BAFF-R polypeptides merged in N-terminal and C-terminal are identicals.In another embodiment, the BAFF-R polypeptides merged in N-terminal and C-terminal are different BAFF-R polypeptides.In another embodiment, BAFF-R polypeptides are blended in the N or C-terminal of albumin, and heterologous polypeptide is blended in remaining end.

In general, the albumin fusion protein that can be used in the inventive method can have a HA to derive area and a BAFF-R area.But, multiple regions in each albumen can be used for preparing the albumin fusion protein that can be used in the inventive method.Equally, more than one albumen can be used to prepare the albumin fusion protein that can be used in the inventive method.For example, albumen can be blended in HA N-terminal and C-terminal.In this configuration, the protein part can be same or different protein molecular.The structure of the albumin fusion protein of difunctionality can be expressed as X-HA-Y or Y-HA-X.

The BAFF-R albumen that can be used in a specific embodiment in the inventive method or its fragment or variant can be conjugated with cytotoxin (such as cytostatic agent or cytocide).Cytotoxin or cell toxicity medicament can include any medicine being harmful to cell.Example include taxol, cytochalasin B, Gramicidin D, Ethidum Eremide, emetine, mitomycin, etoposide, Teniposide, vincristine, vinblastine, colchicin, adriamycin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- boldenones, glucocorticoid, procaine, dicaine, lidocaine, Propranolol and puromycin with and the like or homologue.

In another embodiment, the BAFF-R albumen that can be used in the inventive method or its fragment or variant can be conjugated with toxin.

The feature for the BAFF-R polypeptides that can be used in the inventive method can be improved or changed using genetically engineered, to generate the polypeptide that can be used in the inventive method.Single or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing, addition or fusion protein can be included with the new mutain of recombinant DNA technology generation known to those skilled in the art or " mutein ".These modified polypeptides can show for example to strengthen activity or increase stability.In addition, at least under specific purifying and condition of storage, they can be purified than corresponding natural polypeptides by higher yield, and show more preferable stability.Still in yet another embodiment of the present invention, BAFF-R polypeptide mutants can be " dominant negative regulation thing ".Therefore, the mutant that can for example lack TNF conserved regions all or in part with deficiency BAFF-R polypeptides eliminates BAFF-R activity.Non-functional BAFF-R polypeptides, which can assemble to be formed, can combine but be unable to the acceptor (such as polymer) of inducement signal conduction.

It can be monomer or polymer (i.e. dimer, tripolymer, the tetramer and more high polymer) that the BAFF-R polypeptides in the inventive method, which can be used for,.In a particular embodiment, polypeptide of the invention is monomer, dimer, tripolymer or the tetramer.In other embodiment, polymer of the invention is at least dimer, tripolymer or the tetramer.It is (Beutler the and Huffel, Science 264 existed in the form of tripolymer to believe some TNF family proteins members:667,1994;Banner et al.,Cell 73:431,1993).Therefore, the BCMA of tripolymer can provide the advantage of enhancing biological activity.

In a particular embodiment, polymer can be homopolymer or different aggressiveness.Term " homopolymer " referred to herein as comprises only the polymer of BAFF-R polypeptides (including BAFF-R fragments, variant and fusion protein described herein).These homopolymers can contain the BAFF-R polypeptides with same or different amino acid sequence.In a particular embodiment, homopolymer is the polymer for comprising only the BAFF-R albumen with identical peptide sequence.In another particular embodiment of the invention, homopolymer is the polymer containing the BAFF-R albumen with different peptide sequences.

Term " different aggressiveness " also contains the polymer of heterologous polypeptide (comprising only non-corresponding in the albumen of the peptide sequence of the peptide sequence of BAFF-R coded by said gene) referred to herein as in addition to BAFF-R polypeptides.The polymer that can be used in the inventive method can be hydrophobic connection, hydrophilic connection, ion connection and/or be covalently attached connected result, and/or can be indirectly connected to, for example, formed by liposome.Therefore, in one embodiment, when albumen is in contact with each other in the solution, it is formed same polymer such as homodimer, homotrimer, the different tetramer or the different tetramer.In other embodiments, polymer is formed by the covalent attachment between the covalent attachment with BAFF-R albumen or BAFF-R albumen.These covalent attachments may relate to peptide sequence (contained one or more amino acid residues of albumen.In oneainstance, covalent attachment is the crosslinking between the cysteine residues in the peptide sequence of albumen, and the cysteine residues also interact in natural (i.e. naturally occurring) polypeptide.In another scenario, covalent attachment is the result after chemistry or reorganization.

Or, this covalent attachment can be related to one or more contained amino acid residues in the heterologous polypeptide sequence of BAFF-R fusion proteins.In an example, covalent attachment is the covalent attachment (see U.S. Patent application 5,478,925, be herein incorporated herein its content by quoting) between the heterologous sequence contained by fusion protein.In a specific example, covalent attachment is the covalent attachment between the heterologous sequence contained by BAFF-R-Fc fusion proteins (as described herein).In another particular embodiment of the invention, the covalent attachment for the fusion protein that can be used in the inventive method be from another can be formed covalent attachment polymer TNF families ligand/receptor member such as OPG (see such as international publication WO 98/49305, being herein incorporated herein entire contents by quoting) heterologous polypeptide sequence between covalent attachment.In another embodiment, attachment (such as peptide, carbohydrate or the polymer attachment of solubility) of two or more BAFF-R polypeptides through synthesis links together.Example is included in those peptide linkers (being incorporated into the application by quoting herein) described in United States Patent (USP) 5,073,627.Can be generated using traditional recombinant DNA technology includes the albumen of multiple BAFF-R polypeptides separated through peptide linker.

In a specific embodiment, with reference to BAFF-R polypeptides, polypeptide fragment or SEQ IDNO:The antibody of 10 variant, and/or BAFF-R polypeptide epitopes (by for detecting the epitope that the immunodetection well known in the art of special antibody-antigen binding is determined) can be used in the inventive method.For example in Lee, et al., (2006)Synthetic anti-BR3antibodies that mimic BAFF binding and target both human and murine B cells Blood(Blood First Edition Paper,prepublished online July 13,2006)Vol.0,No.2006,pp.200603011；Ch’en,et al.,(2005)Cell Immunol 236:78-85；Nakamura,et al.,(2005)Virchows Arch 447:53-60；And Carter, et al., (2005) Arthritis Rheum 52:Anti- BAFF-R antibody and its fragment have been described in 3943-3954.Entire contents are incorporated herein by quoting herein.Antibody includes but is not limited to the epitope binding fragments of monoclonal, polyspecific, people, humanization or chimeric antibody, single-chain antibody, Fab fragments, F (ab ') fragment, the fragment of Fab expression libraries generation, antiidiotype (anti-Id) antibody (the anti-Id antibody for including for example anti-BAFF-R antibody) and above-mentioned all antibody.The immunoactive portions of term " antibody " referred to herein as immunoglobulin molecules and immunoglobulin molecules, i.e., the molecule of the antigen binding site containing immunologic opsonin combination antigen.The immunoglobulin molecules of the present invention can be the immunoglobulin of any kind of (such as IgG, IgE, IgM, IgD, IgA and IgY), any group (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subgroup.In a particular embodiment, immunoglobulin molecules are IgG1.In other specific embodiments, immunoglobulin molecules are IgG4.

The antibody fragment for the combination BAFF-R that can be used in the inventive method includes but is not limited to Fab, Fab ' and F (ab ') 2, Fd, scFv (scFv), single-chain antibody, the Fv (sdFv) of disulfide bond and include the fragment in VL or VH areas.The antibody fragment of combination BAFF-R including single-chain antibody can include single variable region or with all or part of following variable region combined：Hinge area, CH1, CH2 and CH3 area.In a specific embodiment, the BAFF-R binding fragments that can be used in the inventive method include variable region and hinge area, any combination in CH1, CH2 and CH3 area.The antibody that can be used in the inventive method can come from the antibody of any animal origin, including birds and mammal.Preferably, antibody is people, mouse (such as mouse and rat), donkey, ship rabbit, goat, cavy, camel, horse or chicken source.The antibody that " people " antibody includes at this antibody of amino acid sequence with human immunoglobulin(HIg) and is separated to including one or more human immunoglobulin(HIg)s from human immunoglobulin(HIg) library or from transgenosis and in not expressing the transgenic animals of endogenous immunoglobulin, such as in Kucherlapati United States Patent (USP) 5,939, as described in 598, entire contents are incorporated herein by quoting herein.

The anti-BAFF-R antibody that can be used in the inventive method can be single special, double special, three special or higher polyspecifics.Multi-specificity antibody can be specific to the different epitopes of BAFF-R polypeptides or can be specific to BAFF-R polypeptides and heterologous epitope such as heterologous polypeptide or solid support material.See such as PCT publication WO 93/17715, WO 92/08802, WO91/00360, WO 92/05793, Tutt, et al., J.Immunol.147:60-69(1991)；United States Patent (USP) 4,474,893,4,714,681,4,925,648,5,573,920,5,601,819, Kostelny et al., J.Immunol.148:1547-1553(1992)；Entire contents are incorporated herein by quoting herein.

It can be described according to its cross reactivity or illustrate can be used for the BAFF-R antibody in the inventive method.The antibody that any other analog, homologue or homologue of BAFF-R polypeptides will not be combined can be used for the method for the present invention.In a particular embodiment, BAFF-R antibody and the mouse of human BAFF-R albumen, rat and/or the homologue of rabbit or its corresponding epitope cross reaction.In a specific embodiment, the anti-BAFF-R antibody that can be used in the inventive method is not only in conjunction with BAFF-R, also in relation with TACI and BCMA.

Also it can be described according to it with the binding affinity of BAFF-R polypeptides or illustrate can be used for the BAFF-R antibody in the inventive method.It is preferred that binding affinity include dissociation constant or K_DLess than or equal to 5x 10^-9M、l0^-9M、5x 10^-10M、10^-10M、5x 10^-11M、10^-11M、5x 10^- ¹²M or 10^-12M。

The BAFF-R antibody that can be used in the inventive method can be the activator or antagonist of BAFF-R polypeptides.For example including blocking the BAFF-R antibody with the receptor/ligand interaction of BAFF-R polypeptides partially or completely.Also include that ligand binding will not be prevented but the receptor specific antibody of receptor activation can be prevented.Receptor activation (i.e. signal transduction) can be determined with described herein or techniques known in the art.For example, detecting that the phosphorylation (such as tyrosine or serine/threonine) of acceptor or its substrate can determine receptor activation by using techniques known in the art detection transcription factor NF-AT, AP-1, and/or NF- κ B activation and/or by immunoprecipitation and subsequent western engram analysis.

In a specific embodiment, prevent the receptor-specific BAFF-R antibody and identification receptor-ligand complex of ligand binding and receptor activation and may be used to the inventive method preferably without the BAFF-R antibody for specifically recognizing uncombined acceptor or uncombined part.Above-mentioned BAFF-R antibody can be prepared using techniques known in the art.See such as PCT publication WO96/40281；United States Patent (USP) 5,811,097；Deng et al.,Blood 92(6):1981-1988(1998)；Chen et al.,Cancer Res.58(16):3668-3678(1998)；Harrop et al.,J.Immunol.161(4):1786-1794(1998)；Zhu et al.,Cancer Res.58(15):3209-3214(1998)；Yoon et al.,J.Immunol.160(7):3170-3179(1998)；Prat et al.,J.Cell.Sci.111(Pt2):237-247(1998)；Pitard et al.,J.Immunol.Methods 205(2):177-190(1997)；Liautard et al.,Cytokine 9(4):233-241(1997)；Carlson et al.,J.Biol.Chem.272(17):11295-11301(1997)；Taryman et al.,Neuron 14(4):755-762(1995)；Muller et al.,Structure 6(9):1153-1167(1998)；Bartunek et al.,Cytokine 8(1):14-20 (1996) (being herein incorporated herein entire contents by quoting).

G. anti-APRIL antibody

In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-APRIL antibody or its antigen-binding fragment.For example in PCT publication WO01/087977, WO99/12965, WO01/60397 and WO02/094192；United States Patent (USP) 6,506,882；The United States patent publication 2003/0166864 that on October 11st, 2002 submits；And Ch ' en, et al., (2005) Cell Immunol236:Anti- APRIL antibody and its fragment have been described in 78-85.Entire contents are incorporated herein by quoting herein.

In a specific embodiment, with APRIL polypeptides, polypeptide fragment or SEQ IDNO:The antibody that 4 variant, and/or APRIL epitopes (being used to detect determined by the immunodetection of special antibody-antigen binding as known in the art) is combined can be used for the inventive method.In a specific embodiment, the antibody that can be used in the inventive method can combine the APRIL polypeptides merged with other peptide sequences.For example, APRIL polypeptides can with the constant region of immunoglobulin (IgA, IgE, IgG, IgM) or part thereof (CH1, CH2, CH3, or any combination thereof and part) or albumin (include but is not limited to rHA or its fragment or the variant (United States Patent (USP) 5 submitted see on March 2nd, 1,876,969th, the United States Patent (USP) 5 that European patent 0413622 and on June 16th, 1998 submit, 766,883, entire contents are incorporated herein by quoting herein) fusion, form chimeric polyeptides.These fusion proteins can promote purifying, can extend storage time and can increase Half-life in vivo.The chimeric protein being made up of first two domains and the heavy chain of mammalian immunoglobulin or the different zones of constant region of light chain of people's CD4 polypeptides has shown that this point.See such as EP 394,827, Traunecker et al., Nature, 331:84-86(1988).The ability of immune system can be transported to enhancement antigen across epithelial barrier by being had confirmed for the antigen (such as insulin) being conjugated with FcRn binding partners such as IgG or Fc fragments (see such as PCT publication WO96/22024 and WO 99/04813).Having also been discovered that the IgG fusion proteins of the dimeric structure with disulfide bond caused by the disulfide bond of IgG parts individually itself can more effectively combine than single homodimeric polypeptide or its fragment and neutralize other molecules.See such as Fountoulakis et al., J.Biochem., 270:3958-3964(1995).

In a particular embodiment, antibody binding homopolymer, particularly homotrimer the APRIL polypeptides that can be used in the inventive method.Heterotrimer of the different aggressiveness of antibody binding, particularly heterotrimer the APRIL polypeptides for example containing two APRIL polypeptides and Neutrokine- α polypeptide or the heterotrimer containing an APRIL polypeptide and two Neutrokine- α polypeptides that can be used in other specific embodiments in the inventive method.In a specific embodiment, antibody binding homopolymer, particularly homotrimer the APRIL polypeptides that can be used in the inventive method, the single protein component of wherein polymer is APRIL (such as SEQ ID NO by mature form:4 the 105 to 250th amino acids residue) composition.In other specific embodiments, heterotrimer of the different aggressiveness of antibody binding, particularly heterotrimer the APRIL polypeptides for example containing two APRIL polypeptides and Neutrokine- α polypeptide that can be used in the inventive method or the heterotrimer containing an APRIL polypeptide and two Neutrokine- α polypeptides, the protein component of the different aggressiveness of wherein APRIL is ripe extracellular soluble part (such as SEQ ID NO by APRIL:4 the 105 to 250th amino acids) or Neutrokine- α ripe extracellular soluble part (such as SEQ ID NO:2 the 134 to 285th amino acids) composition.

In a particular embodiment, the comformational epitope for the antibody binding APRIL monomeric proteins that can be used in the inventive method.In a particular embodiment, the comformational epitope for antibody binding APRIL polymers, particularly tripolymer the albumen that can be used in the inventive method.In other embodiments, it can be used for the antibody binding in the inventive method as the comformational epitope produced by the juxtaposition of APRIL and heterologous polypeptide, when the comformational epitope can occur in APRIL formation heterotrimers (such as and Neutrokine- α polypeptides) or fusion protein between APRIL and heterologous polypeptide.

The antibody that can be used in the inventive method includes but is not limited to the epitope binding fragments of polyclonal, monoclonal, polyspecific, people, humanization or chimeric antibody, single-chain antibody, Fab fragments, F (ab) ' fragment, the fragment of Fab expression libraries generation, antiidiotype (anti-Id) antibody (the anti-id antibody for including anti-APRIL antibody) and all above antibody.The immunologic competence part of term " antibody " referred to herein as immunoglobulin molecules and immunoglobulin molecules, i.e., the molecule of the antigen binding site containing immunologic opsonin combination antigen.The immunoglobulin molecules of the present invention can be any kind of (IgG, IgE, IgM, IgD, IgA and IgY), any group (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subgroup immunoglobulin molecules.In a preferred embodiment, immunoglobulin is IgG1 or IgG4 isotypes.Immunoglobulin can have heavy chain and light chain.IgG, IgE, IgM, IgD, IgA and IgY arrangement can match with the light chain of κ or λ forms.

In a specific embodiment, the antibody that can be used in the inventive method is the antibody fragment with reference to APRIL, including but not limited to Fab, Fab ' and F (ab ') 2, Fd, scFv (scFv), single-chain antibody, the Fv (sdFv) of disulfide bond and include the fragment in VL or VH areas.The antibody fragment of combination APRIL including single-chain antibody can include single variable region or with all or part of following variable region combined：Hinge area, CH1, CH2 and CH3 area.In a specific embodiment, the Neutrokine- α binding fragments that can be used in the inventive method include variable region and hinge area, any combination in CH1, CH2 and CH3 area.The antibody that can be used in the inventive method can come from the antibody of any animal origin, including birds and mammal.Preferably, antibody is people, mouse (such as mouse and rat), donkey, ship rabbit, goat, cavy, camel, horse or chicken source.The antibody that " people " antibody includes at this antibody of amino acid sequence with human immunoglobulin(HIg) and is separated to including one or more human immunoglobulin(HIg)s from human immunoglobulin(HIg) library or from transgenosis and in not expressing the transgenic animals of endogenous immunoglobulin, such as in Kucherlapati United States Patent (USP) 5,939, as described in 598, entire contents are incorporated herein by quoting herein.

The antibody that can be used in the inventive method can be single special, double special, three special or higher polyspecifics.Multi-specificity antibody can be specific to the different epitopes of APRIL polypeptides or can be specific to APRIL polypeptides and heterologous epitope such as heterologous polypeptide or solid support material.See such as PCT publication WO 93/17715, WO 92/08802, WO91/00360, WO92/05793, Tutt, et al., J.Immunol.147:60-69(1991)；United States Patent (USP) 4,474,893,4,714,681,4,925,648,5,573,920,5,601,819, Kostelny et al., J.Immunol.148:1547-1553(1992).

It can be described according to its cross reactivity or illustrate can be used for the anti-APRIL antibody in the inventive method.The antibody that any other analog, homologue or homologue of APRIL polypeptides will not be combined can be used for the method for the present invention.In a specific embodiment, the antibody that can be used in the inventive method and Neutrokine- α cross reactions.In a particular embodiment, antibody and the mouse of human protein, rat and/or the homologue of rabbit or its corresponding epitope cross reaction.

Also it can be described according to it with the binding affinity of APRIL polypeptides or illustrate can be used for the antibody in the inventive method.In a particular embodiment, the antibody binding APRIL polypeptides or its fragment or variant that can be used in the inventive method, its dissociation constant or K_DLess than or equal to 5x 10^-9M、l0^- ⁹M、5x 10^-10M、10^-10M、5x 10^-11M、10^-11M、5x 10^-12M or 10^-12M.In a specific embodiment, the dissociation constant or K of the antibody binding APRIL polypeptides that can be used in the inventive method_DIt is in each single any scope provided between value.

The APRIL antibody for e.g., including blocking the receptor/ligand with APRIL polypeptides to interact partially or completely.Also include that ligand binding will not be prevented but the APRIL specific antibodies of receptor activation can be prevented.Receptor activation (i.e. signal transduction) can be determined with described herein or techniques known in the art.For example, detecting that the phosphorylation (such as tyrosine or serine/threonine) of acceptor or its substrate can determine receptor activation by using techniques known in the art detection transcription factor NF-AT, AP-1, and/or NF- κ B activation and/or by immunoprecipitation and subsequent western engram analysis.

In a specific embodiment, prevent the receptor-specific APRIL antibody and identification receptor-ligand complex of ligand binding and receptor activation and may be used to the inventive method preferably without the APRIL antibody for specifically recognizing uncombined acceptor or uncombined part.In a specific embodiment, binding partner simultaneously prevents the neutralizing antibody and binding partner of part and acceptor combination from preventing receptor activation accordingly but not prevent the antibody of ligand binding receptor from may be used to the inventive method.Above-mentioned APRIL antibody can be prepared using techniques known in the art.Such as PCT publication WO96/40281；United States Patent (USP) 5,811,097；Deng et al.,Blood 92(6):1981-1988(1998)；Chen et al.,Cancer Res.58(16):3668-3678(1998)；Harrop et al.,J.Immunol.161(4):1786-1794(1998)；Zhu et al.,Cancer Res.58(15):3209-3214(1998)；Yoon et al.,J.Immunol.160(7):3170-3179(1998)；Prat et al.,J.Cell.Sci.111(Pt2):237-247(1998)；Pitard et al.,J.Immunol.Methods 205(2):177-190(1997)；Liautard et al.,Cytokine 9(4):233-241(1997)；Carlson et al.,J.Biol.Chem.272(17):11295-11301(1997)；Taryman et al.,Neuron 14(4):755-762(1995)；Muller et al.,Structure 6(9):1153-1167(1998)；Bartunek et al.,Cytokine 8(1):14-20 (1996) (being herein incorporated herein entire contents by quoting).

H.APRIL Binding peptides

In a specific embodiment, Neutrokine- alpha-2 antagonists are APRIL binding peptides or polypeptide.APRIL binding peptides or polypeptide for example has been described in International patent publications WO01/87977, WO01/87979 and United States patent publication US2002081296 and US2002086018, herein entire contents are incorporated herein by quoting.Can be used for the APRIL binding peptides in the inventive method include from through with filobactivirus coating protein merge shown Random peptide sequences in the short polypeptide that identifies.Such as Scott et al. (1990) Science 249 is shown in discussion for phage display peptide library technology:386；Devlin et al.(1990),Science 249:404；The United States Patent (USP) 5,223,409 that on June 29th, 1993 submits；The United States Patent (USP) 5,733,731 that on March 31st, 1998 submits；The United States Patent (USP) 5,498,530 that on March 12nd, 1996 submits；The United States Patent (USP) 5,432,018 that July 11 nineteen ninety-five submits；The United States Patent (USP) 5,338,665 that August in 1994 is submitted on the 16th；The United States Patent (USP) 5,922,545 that on July 13rd, 1999 submits；The WO 96/40987 that on December 19th, 1996 publishes；The WO 98/15833 published with April 16th, 1998, is incorporated into the application by quoting herein.The bacteriophage of expression of peptides can be isolated by the affinity purification and subsequent breeding again of the APRIL targeting peptides for immobilization of continuous many wheels.To with being sequenced with the candidate bacteriophage of APRIL highest adhesion, to determine the identity of each binding peptide.Then all APRIL binding peptides identified are all connected on " carrier ", to generate the further APRIL binding peptides being used in the inventive method.Term " carrier " refers to preventing degraded and/or extension half-life period, reduction toxicity, the molecule for reducing immunogenicity or increase biological activity of APRIL binding peptides.Example carrier includes Fc areas and its variant (" peptide antibody " is preferred)；Linear polymer (such as polyethylene glycol (PEG), including 5kD, 20kD and 30kD PEG, polylysine, glucan etc.)；Branch polymer is (see the United States Patent (USP) 4,289,872 such as the Denkenwalter that September in 1981 is submitted on the 15th；The 5,229,490 of the Tam that on July 20th, 1993 submits；The Frechet that on October 28th, 1993 publishes etc. WO 93/21259)；Lipid；Cholesterol group (such as steroids)；Carbohydrate or oligosaccharides (such as glucan)；Albumen, polypeptide or the peptide of any natural or synthesis combined with salvage receptor；Albumin, including but not limited to rHA or its fragment or the variant (United States Patent (USP) 5,876,969 submitted see on March 2nd, 1999；European patent 0413622；The United States Patent (USP) 5 submitted with June 16th, 1998,766,883, be herein incorporated herein entire contents by quoting)；Leucine zipper region；With other such albumen and protein fragments.There is the carrier that at least one N-terminal through one of amino acid residue, C-terminal or side chain are connected with peptide in the APRIL binding peptides requirement that can be used in the inventive method.Multiple carriers can also be used, such as each end is that Fc or end is Fc, another end or side chain are PEG group.For APRIL binding peptides, Fc areas are preferred carriers.Fc areas can merge or be blended in two N and C-terminal of peptide with the N or C-terminal of peptide.It is preferred with merging for N-terminal.

As described above, Fc variants can be used for the suitable carrier of the APRIL binding peptides in the inventive method.Natural Fc can be modified widely, and to form Fc variants, condition has been to maintain the binding ability with salvage receptor；See such as WO 97/34631 and WO 96/32478.In these Fc variants, can remove the APRIL binding peptides that offer can be used in the inventive method in natural Fc unwanted structural behaviour or functional activity one or more sites.Residue is for example inserted by substitution or deleting residues, into site or the part containing the site is blocked can remove these sites.Residue inserted or substituted can also be the amino acid changed such as peptide mimics or D- amino acid.Fc variants are required for based on many reasons, some reasons are described below.Example Fc variants include molecule and sequence, wherein：

Another carrier for the APRIL binding peptides that can be used in the inventive method is can to combine albumen, polypeptide, peptide, antibody, antibody fragment or the small molecule (such as peptide simulated compound) for remedying antibody.It is, for example, possible to use in United States Patent (USP) 5, the peptide described in 739,277 is used as carrier.Peptide can also be selected with phage display or for combining the RNA- peptide screenings of salvage receptor.These salvage receptor binding compounds are also contained in the implication of " carrier ", and can be used in the Neutrokine- alpha bindings in the inventive method.These carriers should be selected according to extension half-life period (such as the sequence by avoiding protease from being recognized) and reduction immunogenicity (such as by preferential non-immunogenic sequence, as seen in antibody humanization).

As described above, polymer support can be used for can be used for the APRIL binding peptides in the inventive method.Be currently available it is various be used for connect can as the chemical part of carrier method, see such as Patent Cooperation Treaty (" PCT ") international publication WO 96/11953, be herein incorporated herein entire contents by quoting.The PCT application elaborates that water-soluble polymer is connected with the selectivity of protein N terminal.

In one preferred embodiment, polymer support preferably is polyethylene glycol (PEG).PEG group can be any easily molecular weight, can be linear or side chain.PEG average molecular weight range is preferably from about 2 kilodaltons (" kD ") to about 100kD, more preferably from about 5kD to about 10kD, most preferably from about 5kD to about 10kD.For can be used for the APRIL binding peptides in the inventive method, PEG group is typically all connected on the reactive group of invention compound (such as aldehyde radical, amino or ester group) by acylation or the reductive alkylation effect of the reactive group in PEG group sequence.

Polysaccharide polymer is the water-soluble polymer in the another type of APRIL binding peptides that can be used in the inventive method.Glucan through α 1-6 by mainly connecting the polysaccharide polymer that the single glucose subunit that key links together is constituted.Glucan in itself can be by various molecular weights scope, and the molecular weight ranges being readily obtained are from about 1kD to about 70kD.Glucan is suitable water-soluble polymer, another carrier (such as Fc) of itself or joint can as the APRIL binding peptides that can be used in the inventive method carrier.See such as WO 96/11953 and WO 96/05309.It has been reported that the glucan purposes conjugated with therapeutic or diagnostic immunoglobulin；See such as European Patent publications 0315456, be herein incorporated herein entire contents by quoting.When carrier of the glucan as the present invention, about 1kD to about 20kD glucan are preferred.

In a specific embodiment, the APRIL binding peptides that can be used in the inventive method optionally include " attachment ".When there is attachment, its chemical constitution is unimportant, because it is mainly as a sept.Attachment is preferably what is be made up of the amino acid linked together through peptide bond.Therefore, in a preferred embodiment, attachment is made up of 1 to 30 amino acid linked together through peptide bond, wherein the amino acid is selected from 20 naturally occurring amino acid.Some amino acid in these amino acid can be glycosylated, and those skilled in the art are well understood by this point.In preferred embodiment, 1 to 20 amino acid are to be selected from glycine, alanine, proline, asparagine, glutamine and lysine.Even further preferably, what attachment was made up of most of spatially unimpeded amino acid such as glycine and alanine.Accordingly, it is preferred that attachment is polyglycine (particularly (Gly) 4, (Gly) 5), poly- (Gly-Ala) and polyalanine.It is preferred that attachment be to include the amino acid linker more than 5 amino acid, suitable attachment can have most 500 amino acid in glycine, alanine, proline, asparagine, glutamine, lysine, threonine, serine or aspartic acid.The attachment of about 20 to 50 amino acid is most preferred.

Non-peptide attachment can be used for can be used for the APRIL binding peptides in the inventive method.It is, for example, possible to use alkyl linker is for example -- NH-- (CH₂)_n-- C (O) --, wherein n is 2 to 20.Group such as lower alkyl (such as C can be hindered with any non-space₁--C₆), lower alkyl, halogen (such as Cl, Br), CN, NH₂, phenyl etc. replace these alkyl linkers.

I. antisense RNA and siRNA

In a specific embodiment, Neutrokine- alpha-2 antagonists are the antisense RNA, catalysis RNA (ribozyme) or short interfering rna (siRNA) for the acceptor (such as TACI, BCMA and BAFF-R) for targeting Neutrokine- α, APRIL or Neutrokine- α.In a specific embodiment, the antisense RNA for being directed to Neutrokine- α, APRIL, TACI, BCMA or BAFF-R can be used for the inventive method.By antisense DNA or RNA or by three spiralizations gene expression can be controlled with antisense technology.For example, in Okano, J.Neurochem.56:560(1991);" discuss antisense technology in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).For example, in Lee et al., Nucleic Acids Research 6:3073(1979)；Cooney et al.,Science 241:456(1988)；With Dervan et al., Science 251:Three spiralizations are discussed in 1360 (1991).Methods described is to be dependent on the combination of polynucleotides and complementary DNA or RNA.For example, 5 ' coded portions of the polynucleotides of the cell outskirt of coding polypeptide of the present invention can be used for the antisense rna oligonucleotide for designing the base-pair of length about 10 to 40.The gene region that DNA oligonucleotides can be designed to and be related to transcription is complementary, and Neutrokine- α, APRIL, TACI, BCMA or BAFF-R transcription and generation are prevented accordingly.Antisense rna oligonucleotide hybridizes with mRNA in vivo, and blocks mRNA molecules to translate into Neutrokine- α, APRIL, TACI, BCMA or BAFF-R polypeptides.Oligonucleotides recited above can also be transported to cell so that can antisence RNA or DNA in vivo, to suppress Neutrokine- α, APRIL, TACI, BCMA or BAFF-R generation.

In one embodiment, by generating Neutrokine- α, APRIL, TACI, BCMA or BAFF-R antisensenucleic acidses in can be used for the inventive method from the transcription of exogenous array.For example, carrier or one part are transcribed, the antisensenucleic acids (RNA) that can be used in the inventive method is generated.This carrier can the sequence containing coding Neutrokine- α, APRIL, TACI, BCMA or BAFF-R antisensenucleic acidses.This carrier can be still free gene or can be dyeing interbody fusion, and condition is the antisense RNA that they can be transcribed needed for generation.These carriers can be constructed with the DNA technique method of this area standard.Carrier can be plasmid, virus or the other known carrier for replicating and expressing in vertebrate cells in this area.The change for encoding Neutrokine- α, APRIL, TACI, BCMA or BAFF-R sequence can be the promoter known in the art for being preferably and being played a role in human cell in vertebrate cells greatly.These promoters can be induction type or composing type.These promoters include but is not limited to SV40 early promoters area (Bernoist and Chambon, Nature 29:304-310 (1981)), the long ends of Rous sarcoma viral 3 ' repeat contained promoter (Yamamoto et al., Cell 22:787-797 (1980)), simplex thymidine promoter (Wagner et al., Proc.Natl.Acad.Sci.U.S.A.78:1441-1445 (1981)), regulatory sequence (Brinster, the et al., Nature 296 of metallothionein gene:39-42 (1982)) etc..

The antisensenucleic acids that can be used in the inventive method includes the sequence complementary with the rna transcription body of at least a portion Neutrokine- α, APRIL, TACI, BCMA or BAFF-R genes.But, Absolute complementarity is although preferred, be not required.Referred to herein as there is " with least a portion RNA complementations " sequence enough complementarity to enable to hybridize the sequence to form stable duplex with RNA；For double-strand Neutrokine- α, APRIL, TACI, BCMA or BAFF-R antisensenucleic acidses, therefore the single-stranded of double-stranded DNA can be detected or the formation of triplex can be detected.Hybridization ability depends on the length of complementarity and antisensenucleic acids.In general, hybrid nucleic acid is bigger, then it contains more with the base of Neutrokine- α, APRIL, TACI, BCMA or BAFF-R mispairing, and can still form stable duplex (or triplex).Those skilled in the art can determine acceptable extent of mismatch with standard method, to determine the melting temperature of hybridization complex.

With the 5 ' ends such as 5 ' of mRNA until and including AUG initiation codons the complementary oligonucleotides of non-translated sequence should to suppress translation play the role of it is maximally efficient.But, complementary sequence, which has shown that, with mRNA 3 ' non-translated sequences also can effectively suppress mRNA translation.It is generally found from Wagner, R., 1994, Nature 372:333-335.Therefore, with Neutrokine- α (SEQID NO:1)、APRIL(SEQ ID NO:3)、TACI(SEQ ID NO:5)、BCMA(SEQ IDNO:Or BAFF-R (SEQ ID NO 7):9) oligonucleotides 5 ' or 3 ' untranslateds, without code area complementation can be used for antisense approach, to suppress endogenous Neutrokine- α, APRIL, TACI, BCMA or BAFF-R mRNA translation.Complementary oligonucleotides should include the complement of AUG initiation codons with mRNA 5 ' non-translational regions.Complementary ASON is the more invalid inhibitor of translation with mRNA code areas, but the method that can be used for the present invention.Regardless of whether being designed to 5 ', 3 ' or the code area hybridization with Neutrokine- α, APRIL, TACI, BCMA or BAFF-RmRNA, antisensenucleic acids should be at least the oligonucleotides of 6 length of nucleotides, preferably from 6 to about 50 nucleotides of length.Special in terms of, oligonucleotides is at least ten nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides that can be used in the inventive method can be single-stranded or double-strand DNA or RNA or its chimeric mixtures or derivative or modified forms.Can on base portion, sugar moieties or phosphate backbones modified oligonucleotide, so as to such as improve molecule hybridization stability.Oligonucleotides can include other extra group such as peptides (such as targeting host cell receptor in vivo) or promote preparation of the transhipment through cell membrane (see such as Letsinger et al., 1989, Proc.Natl.Acad.Sci.U.S.A.86:6553-6556；Lemaitre et al.,Proc.Natl.Acad.Sci.84:648-652(1987)；The PCT publication WO88/09810 that on December 15th, 1988 publishes) or the preparation (the PCT publication WO89/10134 published see on April 25th, 1) for passing through blood-brain barrier, hybridization targeted cleavage agent are transported (see such as Krol et al., BioTechniques 6:958-976 (1988)) or intercalator (see such as Zon, Pharm.Res.5:539-549(1988)).Therefore, oligonucleotides can be conjugated with another molecule such as peptide, hybridization targeting crosslinking agent, transport agents, hybridization targeted cleavage agent.

The ASON that can be used in the inventive method can include ating least one the base portion through modification,It is selected from including but not limited to 5 FU 5 fluorouracil,5-bromouracil,5- chlorouracils,5-iodouracil,Hypoxanthine,Xanthine,4- acetylcytosines,5- (carboxylic methylol) uracil,5- carboxymethyl aminomethyl -2- thiocarbamide glycosides,5- carboxymethyl aminomethyl uracils,Dihydrouracil,β-D- galactosyl pigtails glycosides (β-D-galactosylqueosine),Inosine,N6- isopentennyladenines,1- methyl guanines,M1I,2,2- dimethylguanines,2- methyl adenines,2- methyl guanines,3- methylcysteins,5-methylcytosine,N6- adenines,7- methyl guanines,5- methylaminomethyl uracils,5- methoxyl group aminomethyl -2- thiouracils,β-D-MANNOSE base pigtail glycosides (β-D-mannosylqueosine),5- methoxyl group carboxymethyl uracils,5- methoxyuracils,2- methyl mercapto-N6- isopentennyladenines,Uracil -5- glycolics (v),wybutoxosine,Pseudouracil,Pigtail glycosides (queosine),2- sulphur cytimidines,5-methyl-2-thiouracil,2- thiouracils,4- thiouracils,Methyl uracil,Uracil -5- fluoroacetic acid methyl esters,Uracil -5- fluoroacetic acid (v),5-methyl-2-thiouracil,3- (3- amino -3-N-2- carboxylics propyl group) uracil,(acp3)w,With 2,The group of 6- diaminopurines.

Can be used for the ASON in the inventive method can also include the modification glycosyl at least one group selected from including but not limited to arabinose, 2- fluorine arabinose, xylulose and hexose.

In still another embodiment, it can be used for the modification phosphate backbones that the ASON in the inventive method is selected from including at least one in the including but not limited to group of phosphorothioate, phosphorodithioate, thiophosphoramidates, phosphoramidate, phosphorodiamidite, methyl phosphonate (methylphosphonate), alkyl phosphotriester and formacetal or its analog.

In still another embodiment, the ASON that can be used in the inventive method is the different head oligonucleotides of α.The different head oligonucleotides of α and complementation RNA form special double-stranded hybrids, wherein from unlike common β units, the chain (Gautier et al., Nucl.Acids Res.15 parallel each other:6625-6641(1987)).Oligonucleotides is 2-0- methylribonucleotides (Inoue et al., Nucl.Acids Res.15:6131-6148 (1987)) or chimeric RNA-DNA analogs (Inoue etal., FEBS Lett.215:327-330(1997)).

For example the polynucleotides that can be used in the inventive method can be synthesized with standard method known in the art using the DNA synthesizer (such as can be purchased from Biosearch, Applied Biosystems etc.) of automation.For example, with Stein etc. method (Nucl.Acids Res.16:3209 (1988)) phosphorothioate oligonucleotide can be synthesized, support (Sarin et al., Proc.Natl.Acad.Sci.U.S.A.85 using controlled pore glass polymer:7448-7451 (1988)) methyl phosphonate oligonucleotides etc. can be prepared.

Although the GEM 132 complementary with Neutrokine- α, APRIL, TACI, BCMA or BAFF-R coding region sequence can be used, those complementary GEM 132s are for being most preferred in the inventive method with the non-translational region of transcription.

In a specific embodiment, the Neutrokine- alpha-2 antagonists that can be used in the inventive method also include catalytic RNA or ribozyme (the PCT international publications WO90/11364 published see such as October 4 nineteen ninety for Neutrokine- α, APRIL, TACI, BCMA or BAFF-R；Sarver et al,Science 247:1222-1225(1990)).Although Neutrokine- α, APRIL, TACI, BCMA or BAFF-R mRNA can be destroyed with the ribozyme that mRNA is cracked on the site of specific identification sequence, hammerhead ribozyme is preferred in the methods of the invention.Hammerhead ribozyme is cracking mRNA with the position indicated by the lateral section of said target mrna formation complementary base pair.Only requirement is that said target mrna has the sequence of two following bases：5′-UG-3′.The structure of hammerhead ribozyme and generation are it is known in the art that and in Haseloff and Gerlach, Nature 334:585-591 is preferably illustrated in (1988).There is the cracking site of multiple potential hammerhead ribozymes in Neutrokine- α, APRIL, TACI, BCMA or BAFF-R nucleotide sequence.Preferably, ribozyme is melted into the position for causing to crack the 5 ' ends that recognition site is located proximate to Neutrokine- α, APRIL, TACI, BCMA or BAFF-R mRNA by genetic engineering, that is, adds efficiency and the intracellular accumulation of non-functional mRNA transcriptions is minimized.

As antisense approach, (such as improving stability, targeting) that the ribozyme that can be used in the inventive method can be made up of modified oligonucleotide, it should be transported to the intracellular of expression Neutrokine- α, APRIL, TACI, BCMA or BAFF-R in vivo.The DNA of encoding ribozyme can be incorporated into the mode identical mode with above-mentioned introducing antisense code dna intracellular.It is preferred that conveyer method be directed to use with strong constitutive promoter such as pol III or pol II promoters control under encoding ribozyme DNA build carry, so that transfectional cell generates the ribozyme of sufficient amount, to destroy endogenous Neutrokine- α, APRIL, TACI, BCMA or BAFF-R courier and to suppress translation.Because ribozyme from unlike antisense molecule its be catalytic, therefore work only need to lower IC.

In a specific embodiment, it can be used for for Neutrokine- α, APRIL, TACI, BCMA or BAFF-R short interfering rna in the inventive method.The silencing complex (RISC) that can be induced with siRNA technologies by inducing cell RNA controls gene expression.For example in Hamilton AJ and Baulcombe DC.Science.1999 Oct 29;286(5441):950-2；Elbashir SM,et al.Nature.2001 May 24;411(6836):494-8 and Hanon, Gregory J.and Rossi, John J.Nature 431,371-378 (2004) discuss siRNA technologies.Method is dependent on to intracellular and introduces short double-stranded RNA (being usually 20 to 25 nucleotides).Double-stranded RNA is un-extended, and every chain is all separation.Then single stranded RNA will be integrated into RISC.Then RISC instructs to carry out the mRNA degradeds of sequence-specific, causes Translational repression.For example, the siRNA oligonucleotides that length is about 20 to 25 nucleotides can be designed with the coded portion of the polynucleotides of coding Neutrokine- α, APRIL, TACI, BCMA or BAFF-R polypeptides.It is related to DNA oligonucleotides with the reverse complemental thing of nearly 20 to 25 nucleotide fragments, the separator of nearly 9 nucleotides and selected oligonucleotide fragment.It is expected that the rna transcription body generated from this construct, which folds self, forms hairpin loop.Hairpin loop RNA causes the working process of RNase to intracellular transhipment, generates short double-strand siRNA.Integration of the chain of the siRNA into RISC effector complexs causes the degraded for the mRNA that siRNA is targetted, it is suppressed that Neutrokine- α, APRIL, TACI, BCMA or BAFF-R generation.

In one embodiment, Neutrokine- α, APRIL, TACI, BCMA or BAFF-R the siRNA nucleic acid that can be used in the inventive method is generated in the cell by the transcription of exogenous sequence.For example, transcribing out carrier or one part, the siRNA that can be used in the inventive method is generated.This carrier contains the sequence of coding Neutrokine- α, APRIL, TACI, BCMA or BAFF-R siRNA nucleic acid.These carriers can be constructed by the recombinant DNA technology method of this area standard.Carrier can be plasmid vector, viral vector or other carriers known in the art that can be replicated and express in vertebrate cells.Coding Neutrokine- α, APRIL, TACI, BCMA or BAFF-R siRNA transcription are generally realized using rna plymerase iii promoter (such as U6 or H1), the promoter usually instructs small nuclear rna (snRNA) transcription.

B cell conditioning agent

Except expression Neutrokine- α and APRIL are by external, bone-marrow-derived lymphocyte also expresses various kinds of cell surface molecular, the situation for acting as informing B cell extracellular microenvironment of the molecule, and it is used as positively or negatively modulating b cell functioning and the transmembrane signal of existence.In these acceptors, CD19, CD20 and CD22 have been identified as the promising target spot of Results.

CD20 is integral protein, and it plays a role in the complex as calcium channel.The inhibitor of CD20 calcium channels destroys Ca²⁺Inner equilibrium and cell cycle progression.In a specific embodiment, anti-CD 20 antibodies can be used in the inventive method.In one preferred embodiment, the anti-CD 20 antibodies that can be used in the inventive method are Mabtheras

(Rituximab).Another preferred embodiment in, the anti-CD 20 antibodies that can be used in the inventive method are TRU-015.Another preferred embodiment in, the anti-CD 20 antibodies that can be used in the inventive method are ocrelizumab (PRO70769).Another preferred embodiment in, the anti-CD 20 antibodies that can be used in the inventive method are IMMU-106.Another preferred embodiment in, the anti-CD 20 antibodies that can be used in the inventive method are HuMax-CD20.

CD22 is to see various kinds of cell to include the member of the protein-bonded siglec families of sialic acid in bone-marrow-derived lymphocyte.The interaction of CD22 and a variety of cis and trans carbohydrate ligands causes the regulation for many aspects developed, breed and activated to B cell.In a specific embodiment, anti-CD22 antibody can be used in the inventive method.In one preferred embodiment, the anti-CD22 antibody that can be used in the inventive method is epratuzumab.

Other immunomodulators

In a specific embodiment, the method that the present invention can be put into practice with one or more following medicines：MMF (mycophenolate mofetil；MMF、

(abatacept；C TLA4-Ig)、Riquent^TM(abetimus sodium；LJP 394)、Prestara^TM(praserone)、Edratide(TV-4710)、

(tocilizumab；Atlizumab), VX-702, TRX 1, IPP-201101, ABR-215757, sphingosine-1-phosphate ester -1 (sphingosine-1-phosphate-1, S1P1) activator, HuMax-Inflam^TM(MDX 018)、MEDI-545(MDX-1103/1333)、

Deoxyspergualin (Deoxyspergualin), ENBREL^TM(Etanercept), anti-TNF antibodies, IFN-α antibody.

Patients

Data described herein come from clinical test, and wherein lupus patient receives the treatment for the antibody for neutralizing Neutrokine- α albumen, and it is 1 that this, which is significantly alleviated with ANA titres,:80 or higher, and/or anti-dsDNA be more than or equal to the symptom (embodiment 1) related to lupus in 30IU/mL patient.Surprisingly, it is different from the whole patients that clinical test is selected in, only obtained in a sub-group of patients determine disease activity clinical endpoint (such as SELENA SLEDAI scoring reduction, have be explained in more detail below) significantly improve.Therefore, the present invention relates to identify most probable response in patient's subgroup of immunomodulator such as Neutrokine- alpha-2 antagonists treatment.

In addition, systemic loupus erythematosus (SLE) is a kind of disease of extremely heterogeneity herein, it is difficult to be diagnosed exactly, because patient may have the fact that a variety of symptoms and a variety of symptoms related to lupus are also seen in a large amount of other autoimmune diseases.Therefore, it is 1 that an embodiment of the invention, which provides treatment ANA titres,:80 or higher, and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient method, Neutrokine- alpha-2 antagonists including applying therapeutically effective amount are known in the art or other immunomodulators described herein, the diagnosis regardless of disease.It is 1 that another embodiment of the invention, which provides treatment ANA titres,:80 or higher, and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient method, Neutrokine- alpha-2 antagonists including applying therapeutically effective amount are known in the art or other immunomodulators described herein, the diagnosis regardless of disease；It is 1 to be additionally included in using determination ANA titres before immunomodulator:80 or higher, and/or its blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient.

In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from non-SLE autoimmunity disease.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from rheumatoid arthritis.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from Sjogren syndrome.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from chorionitis.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from Dermatomyositis.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from Felty syndromes.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from mixed connective tissue disease.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from raynaud's syndrome.In further embodiment, ANA titres are 1:80 or higher, and/or blood plasma or serum in Anti-hCG action be more than or equal to 30IU/mL patient suffer from juvenile chronic arthritis.

In addition, it is worth noting that, two with neutralize the Antybody therapy of Neutrokine- α albumen with systemic loupus erythematosus and rheumatoid arthritis patients 2 clinical trial phases (see, embodiment 1 and 3) in, find more likely to reply in treatment with the patient of baseline autoantibodies disease.Baseline ANA titres are 1:80 or higher, and/or anti-dsDNA be more than or equal to 30IU/mL SLE patient than its ANA titre less than 1:80 and its Anti-hCG action level be less than 30IU/mL SLE patient show stronger response.Equally, in rheumatoid arthritis, it was observed that the patient of baseline rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibody positive shows stronger response than the patient of baseline rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) negative antibody.

Therefore, in another aspect of the present invention, there is provided the method for the patient for the treatment of baseline rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibody positive, Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein including applying therapeutically effective amount, regardless of medical diagnosis on disease.The method that another embodiment of the invention provides the patient for the treatment of baseline rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibody positive, Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein including applying therapeutically effective amount, regardless of medical diagnosis on disease；And the patient of baseline rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibody positive is determined before immunomodulator is applied.In a specific embodiment, if rheumatoid factor >=12IU/ml in the blood plasma and/or serum of patient, then it is assumed that the rheumatoid factor positive of patient.In a specific embodiment, if the unit of antiCCP antibody in the blood plasma and/or serum of patient >=10, then it is assumed that the antiCCP antibody of patient is positive.

In the further aspect of the present invention, there is provided the method for the patient for the treatment of baseline autoantibodies, include apply therapeutically effective amount Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein, regardless of whether medical diagnosis on disease how.The method that another embodiment of the invention provides the patient for the treatment of baseline autoantibodies, include apply therapeutically effective amount Neutrokine- alpha-2 antagonists or other immunomodulators known in the art and/or described herein, regardless of whether medical diagnosis on disease how；And the patient of baseline autoantibodies is determined before immunomodulator is applied.

Prepare immunomodulator

The method for the immunomodulator that preparation and/or separation can be used for the present invention is known for the skilled person of association area.Below, the obtainable method on preparation property being the immunomodulator (such as the fragments and variant of anti-Neutrokine- Alpha antibodies, Neutrokine- alpha bindings and polypeptide, Neutrokine- α receptor proteins and above mentioned polypeptide) of protein sources has briefly been reviewed.

In one embodiment, the polynucleotides of encoding immune regulatory protein are inserted into carrier (such as cloning vector or expression vector).Carrier for example can be bacteriophage, plasmid, virus or retroviral vector.Retroviral vector can be reproducible or replication defective.In latter case, typically only can just occur virus breeding in complementary host cell.The polynucleotides of encoding immune regulatory protein can be connected with the carrier containing the selectable marker for being bred in host.Introducing of the vector construct into host cell can be realized by techniques known in the art, the technology includes but is not limited to calcium phosphate transfection, the transfection of DEAE- glucans mediation, transfection, electroporation, transduction, infection or the other method of cation lipid mediation.These methods are all described in the laboratory manual such as Davis et al., Basic Methods In Molecular Biology (1986) of multiple standards.

Recombinant expression carrier typically all includes replication orgin and allows to convert the ampicillin resistance gene and saccharomyces cerevisiae TRP1 genes of the selectable marker such as Escherichia coli of host cell, instructs the promoter from cance high-expression gene of downstream structural sequence transcription.These promoters can be derived from the operator of encoding glycolytic enzymes such as 3-PGA kinases (PGK), the α-factor, acid phosphatase or heat shock protein etc..Assemble the heterologous structural sequence and translation initiation and terminator sequence of proper states and preferred can instruct translation protein secretion to the targeting sequencing in plasm peripheral clearance or extracellular medium.Optionally, heterologous sequence can be with the identification peptide for the purifying that the recombinant products expressed by for example stablizing or simplifying are characterized needed for encoding fusion protein, including the transmission of N-terminal.

In one embodiment, the polynucleotides of encoding immune regulatory protein are operably connected with suitable heterologous regulatory element (such as promoter or enhancer) such as phage lambda Pv promoter, Escherichia coli lac, trp, phoA, tac promoter, the early and late promoters of SV40 and retrovirus LTR promoter.Other suitable promoters are known for those skilled in the art.

As described above, expression vector preferably includes at least one selectable marker.These labels include dihyrofolate reductase, G418 or the neomycin drug resistant gene of eukaryotic culture and the kanamycins or ampicillin resistance gene of Escherichia coli and other Bacteria Cultures.The representative example of suitable host includes but is not limited to bacterial cell such as Escherichia coli, streptomycete and salmonella typhimurium cell；Fungal cell's such as yeast cell (such as saccharomyces cerevisiae or Pichia pastoris (ATCC numberings 201178))；Insect cell such as Drosophila S2 and Spodoptera Sf9 cells；Zooblast such as CHO, COS 293 and Bowes melanoma cells；And plant cell.The suitable culture medium and condition of culture of above-mentioned host cell are known in the art.

Host cell can be that eukaryotic such as yeast cells or host cell of the more high eukaryotic such as mammalian cell (such as human archeocyte) or arable land can make prokaryotic such as bacterial cell.Host strain can be selected, it is regulated and controled the expression of inserted gene order by required particular form, or is modified and handled gene outcome.When there is specific derivant, the expression of specific promoter can be improved；Therefore the expression of the polypeptide of genetically engineered mistake can be controlled.In addition, different host cells has characteristic the and special mechanism of translation and post translational processing and modification (such as phosphorylation, cracking) albumen.Suitable cell line can be selected, with the modification needed for ensureing to expressed foreign protein and working process.It is known method to the suitable carrier for the expression in host cell and the selection of promoter, it is all the ordinary skill in the art that carrier and the necessary technology of the expression in host are introduced for expression vector establishment, into host.

By inserting the expression vector constructed with structural DNA sequence of the functional promoter in albumen needed for the coding that can manipulate reading phase together with suitable translation initiation and termination signal for bacterium.Carrier will include one or more phenotypic selection markers things and replication orgin, to ensure to maintain carrier, and provide amplification of the carrier in host if necessary.Suitable prokaryotic host for conversion includes the different strain in Escherichia coli, hay bacillus, salmonella typhimurium and pseudomonas, streptomyces and staphylococcus, although other host cells can also be selected.It is used as representational but unrestricted example, expression vector for bacterium can include the replication orgin for the selectable marker and bacterium being derived from plasmid obtained by commercialization, and the plasmid includes known cloning vector pBR322 (ATCC 37017) genetic elements.The carrier of these commercializations includes such as pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA).These pBR322 " skeleton " are partly combined with suitable carrier and expressed structure sequence.In the carrier of bacterium is preferred for, including pHE4-5 (ATCC encodes No.209311 and its variant), pQE70, pQE60 and pQE-9, purchased from QIAGEN, Inc., supra；PBS carriers, Phagescript carriers, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A, purchased from Stratagene；With ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, purchased from Pharmacia；PYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K and PAO815 are (all to be purchased from Invitrogen, Carlsbad, CA).It is preferred that eukaryotic vector be pWLNEO, pSV2CAT, pOG44, pXT1 and pSG, purchased from Stratagene；With pSVK3, pBPV, pMSG and pSVL (being purchased from Pharmacia).Other suitable carriers are obvious for those skilled in the art.

After having converted suitable host strain and host's strain grows into suitable cell density, with suitable mode (such as temperature change or chemical induction) induced selective promoter, cell is further cultured for for a period of time.Generally by the way that cell is collected by centrifugation, with cell is either physically or chemically destroyed, retain generated crude extract and further purified.

The microbial cell that Frozen-thawed cycled, ultrasound, mechanical damage or application cell decomposition agent destruction expressing protein are used can be included with the method for any convenient.These methods are known for those skilled in the art.

In one embodiment, Neutrokine- α albumen is expressed in eukaryotic system with saccharomycete Pichia pastoris.Pichia pastoris is a kind of thermophilic methyl yeast, and methanol can be metabolized as its unique carbon source by it.Methanol is oxidized to formaldehyde by having main steps that in methanol metabolic pathway using oxygen.This reaction is catalyzed by enzyme alcohol oxidase.In order to which methanol is metabolized as into its unique carbon source, Pichia pastoris must generate high-caliber pure zirconia enzyme, and partly cause is the relatively low affinity to oxygen because of alcohol oxidase.Therefore, in growth medium of the methanol as primary carbon source is relied on, the promoter region of one of two alcohol oxidase genes (AOX1) is overactive.When there is methanol, nearly the 30% of total soluble protein during the alcohol oxidase generated from AOX1 genes has accounted for Pichia pastoris.See, Ellis, S.B., et al., Mol.Cell.Biol.5:1111-21(1985)；Koutz,P.J,et al.,Yeast 5:167-77(1989)；Tschopp,J.F.,et al.,Nucl.Acids Res.15:3859-76(1987).Therefore, it is unusual high levels expression by the allogeneic coding sequence under the transcriptional regulatory of all or part of AOX1 regulatory sequences in Pichia pastoris in methyl alcohol is grown.

In an example, substantially such as " Pichia Protocols:Methods in MolecularBiology, " D.R.Higgins and J.Cregg, eds.The Humana Press; in Totowa; NJ, the Bichi yeast system described in 1998 express the DNA for encoding immune modulator defined herein or part thereof with plasmid vector pPIC9K.By the effect for the strong AOX1 promoters being connected with Pichia pastoris alkaline phosphatase (PHO) secreting signal peptide (i.e. targeting sequencing) positioned at multiple cloning site upstreams, the expression vector allows expression and secretory immune regulatory protein.

Those skilled in the art are readily apparent that, pPIC9K, such as pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZ α, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K and PAO815 can be replaced with various other yeast vectors；As long as the expression construct proposed provides the signal for being used for transcribing, translate, secreting the suitable location of (if desired) etc., including required inframe AUG.

In one embodiment, the high level expression of allogeneic coding sequence can be obtained without growth yeast culture under conditions of methanol by the way that the heterologous polynucleotide of the present invention to be cloned into expression vector such as pGAPZ or pGAPZ α and be allowed in.

The DNA that higher eucaryotic cells transcribe encoding immune regulatory protein is increased in carrier by the way that enhancer sequence is added to.Enhancer is DNA cis-acting elements, is usually that, from about 10 to 300bp, it acts on promoter to increase its transcription.Example includes the polyoma enhancer and adenovirus cancers on rear side of 100bp to 270bp locates on rear side of replication orgin SV40 enhancers, the sub- enhancer of cytomegalovirus early promoter, replication orgin.

Recombinant protein can also be expressed with various mammalian cell culture systems.The example of mammalian expression systems includes Gluzman (Cell 23:175 (1981)) described in the fibroblastic COS-7 cell lines of monkey kidney, and other cell line such as C127,3T3, CHO, the HeLa that can express compatible vector and bhk cell system.Mammalian expression vector includes replication orgin, suitable promoter and enhancer, also includes the non-transcribed sequences of any required ribosome bind site, polyadenylation site, shearing donor and acceptor site, transcription terminator and 5 ' sides.DNA sequence dna and polyadenylation site from SV40 spliced bodies can be used to provide the genetic elements of required non-transcribed.

In a specific embodiment, the construct of the cell outskirt for a part such as Neutrokine- α acceptors (such as TACI, BCMA and BAFF-R) for being designed to express immune modulator is used.Some skilled persons of this area can use respectively SEQ ID NO:5 and SEQ ID NO:6、SEQ ID NO:7 and SEQ ID NO:8 or respectively SEQ ID NO:9 and SEQ ID NO:10 polynucleotides or polypeptide design go out polynucleotide primers, to generate this expression construct.

Host cell is used for the polynucleotides for expressing encoding immune regulatory protein.These host cells include the host cell of primary, the secondary and immortality of vertebrate origin, particularly mammal source.In some cases, host cell is melted into by genetic engineering and lacks or instead of endogenous genetic stocks (such as Neutrokine- α coded sequences) and/or including genetic stocks (such as heterologous polynucleotides sequence).In some cases, host cell be modified to start and/or change encoding immune regulatory protein endogenous polynucleotide expression.For example, heterologous control regions (such as promoter and/or enhancer) and the endogenous polynucleotide sequence (United States Patent (USP) 5 submitted see on June 24th, 1 can be operably connected by homologous recombination with techniques known in the art, 641,670；The international publication WO 96/29411 that September in 1996 is published on the 26th；The international publication WO 94/12650 that August in 1994 is published on the 4th；Koller et al.,Proc.Natl.Acad.Sci.USA 86:8932-8935(1989)；and Zijlstra et al.,Nature 342:435-438(1989)；Entire contents are incorporated herein by quoting herein).

Using cell described herein immune modulator can be generated with traditional mode.Or, the RNA of the DNA construct from the present invention can also be utilized, immune modulator is generated using cell free translation system.

Can express or synthetic modification form required reading frame AUG, such as fusion protein (including the polypeptide being connected through peptide bond from (different albumen) heterologous protein sequence), secretion signal can be not only included, extra heterologous fuctional regions can also be included.It is connected to one another at methods known in the art by the required nucleotide sequence of the amino acid sequence needed for the polynucleotides and coding by encoding immune regulatory protein in suitable reading frame, and this fusion protein can be prepared with methods known in the art expressed fusion protein product.Or, this fusion protein for example can be prepared by using peptide synthesizer by protein synthesis techniques.Thus, for example, extra amino acid area, particularly electrically charged amino acid, with the stability and persistence during improving in host cell, purifying or during subsequent operation and storage can be added to the N-terminal of polypeptide.Peptide motif can also be added in polypeptide, to promote purifying.These regions can be removed before finally polypeptide is prepared.Peptide motif is added into polypeptide to trigger secretion or excretion, improve stability and promote purifying etc. to be all commonly used in the art and conventional technique.

In one embodiment, the polynucleotides of encoding immune regulatory protein can be merged with signal sequence, and the signal sequence will instruct immune modulator to be positioned in the special compartment of protokaryon or eukaryotic and/or instruct immune modulator to be come out from protokaryon or eukaryotic endocrine.For example, in Escherichia coli, it is desirable to instruct albumen to be secreted into all gaps of plasm.Expression of polypeptides can be instructed to include but is not limited to the signal sequence and the signal sequence of alkaline phosphatase of pelB signal sequences, maltose-binding protein (MBP) signal sequence, MBP, ompA signal sequence, the E.coli LT B subunits in plasm week to the signal sequence or the example of albumen (or its fragment) in all gaps of plasm of bacterium with peptide fusion of the invention.Can be obtained with commercialization some be used for build instruct albumen to position fusion protein carrier, such as New England Biolabs pMAL carrier families (particularly pMAL-p serial).In a specific embodiment, the polynucleotides of encoding immune regulatory protein can be merged with pelB pectase signal sequences, to increase the efficiency of expression and purifying of these polypeptides in gram-negative bacteria.See, United States Patent (USP) 5,576,195 and 5,846,818, be herein incorporated herein entire contents by quoting.

It can merge to instruct the example of the signal peptide of secretion of the albumen in mammalian cell to include but is not limited to plain (stanniocalcin) signal sequence (MLQNSAVLLLLVISASA, SEQ the ID NO of MPIF-1 signal sequences (GenBank numberings AAB51134 the 1 to 21st amino acids), this calcium with immune modulator:And shared signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO 27):28).The suitable signal sequence that can be used together with reference to Baculoviridae expression system is gp67 signal sequences (GenBank numberings AAA72759 the 1 to 19th amino acids).

It is preferred that fusion protein include the heterologous area from immunoglobulin, it is used for stable and purifying protein.For example, EP-A-O 464533 (corresponding to Canada 2045869) elaborates to include the fusion protein of the different piece of immunoglobulin molecules constant region and another human protein or part thereof.In many situations, the Fc parts in fusion protein are highly suitable for treating and diagnosed, and therefore cause improved pharmacokinetics performance (EP-A 0232262).On the other hand, it is necessary to which Fc parts can be lacked after expressing, detecting and being purified into fusion protein in favourable mode described herein for some applications.When Fc part be proved its to for treat and diagnosis be hinder when it is particularly the case, such as when fusion protein is used as the antigen of immunity inoculation.In medicament research and development, such as human protein such as hIL-5 with Fc partial fusions, to identify hIL-5 antagonist with high flux screening detection method.See D.Bennett et al., J.Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J.Biol.Chem.270:9459-9471(1995).

It can be used for the product that the immune modulator in the inventive method is generated including native purified product, the product of chemical synthesis process and through recombinant technique from protokaryon or true and host, the host includes such as bacterium, saccharomycete, higher plant, insect and mammalian cell.According to the difference of the host cell employed in recombinant method for production, immune modulator can be glycosylated or can not be glycosylated.In addition, immune modulator can also include initial modification methionine residues, in some cases this be host mediation working process result.

The immune modulator that can be can be used for using techniques known in the art with chemical synthesis in the inventive method (is for example shown in Creighton, 1983, Proteins:Structures and Molecular Principles,W.H.Freeman & Co.,N.Y.,and Hunkapiller,M.,et al.,1984,Nature 310:105-111).For example, the peptide of a fragment corresponding to immune modulator can be synthesized using peptide synthesizer.In addition, if desired, you can introducing non-conventional amino acid or chemical amino acid analogues as the amino acid of substitution or addition into the polynucleotide sequence of encoding immune regulatory protein.Non- conventional amino acid includes but is not limited to the D- isomers of conventional amino acid, 2, 4- diaminobutyric acids, a- aminoisobutyric acids, 4-Aminobutanoicacid, Abu, 2-amino-butyric acid, g-Abu, e-Ahx, 6-aminocaprolc acid, Aib, 2- aminoisobutyric acids, 3- alanines, ornithine, nor-leucine, norvaline, hydroxyproline, methyl amimoacetic acid, citrulling, Homocitrulline, cysteic acid, t-butylglycine, tert-butylalanine, phenylglycine, alanine cyclohexyl ester, b- alanine, fluorine amino acid, designer's amino acid such as b- methylamino acids, Ca- methylamino acids, Na- methylamino acids, with general amino acid analogue.In addition, amino acid can be D (dextrorotation) or L (left-handed) body.

The immune modulator that can be used in the inventive method can be differently modified during or after translation, such as the connection of glycosylation, acetylation, phosphorylation, aminoacylation, the derivatization of known protectiveness/closure group, proteolytic degradation and antibody molecule or other cell ligands.All a variety of chemical modifications can be realized with known technology, the technology includes but is not limited to cyanogen bromide, trypsase, chymotrypsin, papain, V8 protease, NaBH₄Special chemical degradation, formylation, oxidation, reduction, metabolic synthesis when there is tunicamycin.

Extra posttranslational modification includes addition or the missing of the N-terminal methionine residues caused by the chemical modification and prokaryotic host cell expression of the sugar chain of attachment, N connections or the O connections of sugar chain, the working process of N-terminal or C- ends, chemical part and the amino acid backbone of such as N- connections or O- connections.Can also with detectable label such as enzyme, fluorescence, radioisotopic or affine mark modified polypeptide, to allow detection and protein isolate.

The example of suitable enzyme includes horseradish peroxidase, alkaline phosphatase, beta galactosidase, glucose oxidase or acetylcholinesterase；The example of suitable prosthetic group complexes includes streptavidin/biotin and avidin/biotin；The example of suitable fluorescent material includes biotin, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluoresceins, dansyl chloride or phycoerythrin；The example of luminescent substance includes luminol；The example of bioluminescence material includes luciferase, luciferin and aequorin；The example of suitable radioactive substance includes radioactive metal ion such as alpha-radiation agent for example²¹³Bi or other radio isotopes such as iodine (¹³¹I、¹²⁵I、¹²³I、¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium (^115mIn、^113mIn、¹¹²In、¹¹¹In) and technetium (⁹⁹Tc、^99mTc), thallium (²⁰¹Ti), Gallium (⁶⁸Ga、⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F)、¹⁵³Sm、¹⁷⁷Lu、¹⁵⁹Gd、¹⁴⁹Pm、¹⁴⁰La、¹⁷⁵Yb、¹⁶⁶Ho、⁹⁰Y、⁴⁷Sc、¹⁸⁶Re、¹⁸⁸Re、¹⁴²Pr、¹⁰⁵Rh、⁹⁷Ru、⁶⁸Ge、⁵⁷Co、⁶⁵Zn、⁸⁵Sr、³²P、¹⁵³Gd、¹⁶⁹Yb、⁵¹Cr、⁵⁴Mn、⁷⁵Se、¹¹³Sn and¹¹⁷Tin.

In a particular embodiment, the immune modulator that can be used in the inventive method can be marked with europium.For example, according to product description, it is possible to use DELFIA Eu labelling kits (catalog number (Cat.No.) #1244-302, Perkin Elmer Life Sciences, Boston, MA) use europium labelled immune regulatory protein (such as Neutrokine- alpha-2 antagonists).

In a particular embodiment, immune modulator (such as) mutually adheres to macrocyclic chelants, and the macrocyclic chelants are used for radioactive metal ion, includes but is not limited to¹¹¹In、¹⁷⁷Lu、⁹⁰Y、¹⁶⁶Ho and¹⁵³Sm, is conjugated in polypeptide.In one preferred embodiment, the radioactive metal ion being connected with being attached to the macrocyclic chelants of immunomodulator is¹¹¹In.Another preferred embodiment in, the radioactive metal ion being connected with being attached to the macrocyclic chelants of immunomodulator is⁹⁰Y.In a particular embodiment, macrocyclic chelants are Isosorbide-5-Nitraes, 7,10- tetraazacyclododecanand-N, N ', N ", N " '-tetraacethyl (Isosorbide-5-Nitrae, 7,10-tetraazacyclododecane-N, N ', N ", N " '-tetraacetic acid, DOTA).In other specific embodiments, DOTA mutually adheres to through attachment molecule with immune modulator.Attachment molecule for DOTA and polypeptide to be conjugated is that this area is common, sees such as DeNardo et al., Clin Cancer Res.4 (10):2483-90,1998；Peterson et al.,Bioconjug.Chem.10(4):553-7,1999；With Zimmerman et al, Nucl.Med.Biol.26 (8):943-50,1999, entire contents are incorporated herein by quoting herein.In addition, United States Patent (USP) 5,652,361 and 5,756,065 elaborates the chelating agent that can be mutually conjugated with antibody and preparation and using the method for the chelating agent, its content is incorporated herein by quoting herein.Although United States Patent (USP) 5,652,361 and 5,756,065 concentrates on the conjugated effect of chelating agent and antibody, chelating agent and other polypeptides is easily conjugated in those skilled in the art using method described herein.

In one embodiment, the immune modulator that can be can be used for biotin labeling in the inventive method.

Solubility, stability and inner or in vitro circulation time or reduction immunogenicity that attendant advantages for example increase polypeptide can be provided (see United States Patent (USP) 4,179,337) the chemical modification derivative of immune modulator can be used in the inventive method.Chemical part for derivatization can be selected from water-soluble polymer such as polyethylene glycol, ethylene glycol/propyleneglycoles EVA, carboxymethyl cellulose, glucan, polyvinyl alcohol.Random site that can be in intramolecular or the precalculated position modified polypeptide in intramolecular, the polypeptide can include 1,2,3 or more connected chemical parts.

Polymer can be any molecular weight, can be branch or unbranched.For polyethylene glycol, for the convenience for operating and producing, it is preferred that molecular weight be (term " about " represented in polyethylene glycol preparation, and some molecules may be heavier than given molecular weight, and some molecules may be lighter) between about 1kDa and about 100kDa.Other sizes can be used, depending on required treatment spectrum (duration of example sustained release as required, effect, any effect in terms of biological activity, operation the other known effect to treatment albumen or the like of convenience, antigenic degree and shortage and polyethylene glycol).For example, the mean molecule quantity of polyethylene glycol may be about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10, 000, 10, 500, 11, 000, 11, 500, 12, 000, 12, 500, 13, 000, 13, 500, 14, 000, 14, 500, 15, 000, 15, 500, 16, 000, 16, 500, 17, 000, 17, 500, 18, 000, 18, 500, 19, 000, 19, 500, 20, 000, 25, 000, 30, 000, 35, 000, 40, 000, 50, 000, 55, 000, 60, 000, 65, 000, 70, 000, 75, 000, 80, 000, 85, 000, 90, 000, 95, 000, or 100, 000kDa.

As described above, polyethylene glycol can have branched structure.For example in United States Patent (USP) 5,643,575；Morpurgo et al.,Appl.Biochem.Biotechnol.56:59-72(1996)；Vorobjev et al.,Nucleosides Nucleotides 18:2745-2750(1999)；With Caliceti et al., Bioconjug.Chem.10:The polyethylene glycol of branch has been described in 638-646 (1999), herein entire contents are incorporated herein by quoting.

Peg molecule (or other chemical parts) and albumen should be connected according to the effect of its functional areas or antigenic region to albumen.Those skilled in the art can obtain a variety of connection methods, and such as EP 0401384 is incorporated into the application (with reference to PEG and G-CSF) by quoting herein, also sees Malik et al., Exp.Hematol.20:1028-1035 (1992) (is reported and is utilized trifluoroethyl sulfonic acid chloride (tresylchloride) by GM-CSF Pegylations).For example, by reactive group such as free amino or carboxyl, polyethylene glycol can be with amino acid residue covalent bond.Reactive group is those combinable groups of reactive polyethylene glycol molecule.Amino acid residue with free amine group can include the amino acid residue of such as lysine and N-terminal；There is the amino acid residue of free carboxy can include the amino acid residue of asparagicacid residue, glutaminic acid residue and C-terminal for those.Sulfydryl is also used as connecting the reactive group of peg molecule.Therapeutic purpose is preferably amino connection, the amino connection of such as N-terminal or lysine.

As be noted above, polyethylene glycol can be connected by the connection with any amino acid residue with albumen.For example, polyethylene glycol can be connected by the covalent bond with lysine, histidine, aspartic acid, glutamic acid or cysteine residues with albumen.The amino acid residue (such as lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the special amino acid residue (such as lysine, histidine, aspartic acid, glutamic acid or cysteine) of polyethylene glycol and albumen or more than one types of albumen can be connected using one or more reactive chemistry things.

Can distinguishingly it require in N-terminal chemically modified protein.Utilize polyethylene glycol as example, polyethylene glycol (according to molecular weight, branch etc.) can be selected from a variety of peg molecules, can be with the ratio of the peg molecule in choice reaction mixture and albumen (or peptide) molecule, the type of the pegylation reaction carried out and the method for obtaining selected N-terminal Pegylation albumen.The method (i.e. if necessary to which the motif of the motif and other single Pegylations is separated) for obtaining N-terminal polyethylene glycol preparation can be the material that N-terminal Pegylation is purified into from Pegylation protein molecular group.The selective albumen being modified by sulphation in N-terminal modification can be realized by using the different reactive standard reductive alkylations available for the different types of primary amino radical (lysine is to N-terminal amino acid) of derivatization in specific protein.Under the appropriate reaction conditions, thing containing carbonyl polymerization is realized in substantially selective derivatization of the N-terminal to albumen.

As described above, the Pegylation to albumen of the present invention can be realized with a variety of methods.For example, polyethylene glycol can be connected directly or through insertion attachment with albumen.In Delgado et al., Crit.Rev.Thera.Drug Carrier Sys.9:249-304(1992)；Francis et al.,Intern.J.of Hematol.68:1-18(1998)；United States Patent (USP) 4,002,531；United States Patent (USP) 5,349,052；WO 95/06058；With the connectionless thing system for connecting polyethylene glycol and albumen is described in WO 98/32466, its content is incorporated herein by quoting herein.

One system of the amino acid residue for being directly connected to polyethylene glycol and albumen without insertion attachment employs the MPEG of trifluoroethyl sulfonylation, and this is by using trifluoroethyl sulfonic acid chloride (tresylchloride) (ClSO₂CH₂CF₃) modify the product that mono methoxy polyethylene glycol (MPEG) is generated.After with the MPEG of trifluoroethyl sulfonylation and albumino reaction, the amino of polyethylene glycol and albumen is joined directly together.Therefore, invention includes albumen-polyethylene glycol conjugate of the reaction generation of the albumen and the peg molecule with 2,2,2- trifluoroethyl sulfonyls by the present invention.

Different insertion attachment connection polyethylene glycol and albumen can also be utilized.For example, United States Patent (USP) 5,612,460 (being herein incorporated herein its content by quoting) elaborates the carbamate attachment for connecting polyethylene glycol and albumen.By albumen and compound such as MPEG- succinimidos succinate (MPEG-succinimidylsuccinate), through 1, MPEG, MPEG-2 of 1 '-N,N'-carbonyldiimidazole activation, 4,5- trichlorophenyl carbonates (MPEG-2,4,5-trichloropenylcarbonate), the reaction of MPEG- paranitrophenols carbonic ester (MPEG-p-nitrophenolcarbonate) and various MPEG- succinic acid derivatives can also generate albumen-polyethylene glycol conjugate that polyethylene glycol and albumen are wherein connected through attachment.The a variety of extra polyethyleneglycol derivatives and reactive chemistry thing that are used to connect polyethylene glycol and albumen are described in WO 98/32466, herein entire contents are incorporated herein by quoting.The protein product of the Pegylation generated using reactive chemistry thing given herein is included within the scope of the present invention.

The number (replacing degree) for the polyethylene glycol motif being connected with every kind of albumen of the present invention can also be different.For example, the polyethylene glycol albumen of the present invention can connect average 1,2,3,4,5,6,7,8,9,10,12,15,17,20, or more a peg molecule.Equally, averagely substitution degree is within the scope of for example each protein molecular 1-3,2-4,3-5,4-6,5-7,6-8,7-9,8-10,9-11,10-12,11-13,12-14,13-15,14-16,15-17,16-18,17-19 or 18-20 polyethylene glycol motifs.In Delgado et al., Crit.Rev.Thera.DrugCarrier Sys.9:The method for determining substitution degree is discussed in 249-304 (1992).

The immune modulator that can be used in the inventive method can be reclaimed and purified with known method, and methods described includes but is not limited to ammonium sulfate or ethanol precipitation, acid extraction, cation or anion-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography method and agglutinin chromatography.Most preferably purified using high performance liquid chromatography (" HPLC ").

Preparation and administration

The present invention provides through the treatment including the immunomodulator such as pharmaceutical composition of Neutrokine- alpha-2 antagonists that therapeutically effective amount is applied to object, the method for suppressing and preventing.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- Alpha antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are TACI-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are BAFF-R-Fc albumen.In a specific embodiment, Neutrokine- alpha-2 antagonists are anti-Neutrokine- α peptide antibodies.In a specific embodiment, Neutrokine- alpha-2 antagonists are the Neutrokine- α protein fragments or variant for playing the effect of dominant negative regulation thing.In one preferred embodiment, immunomodulator is (there is no the material for limiting its effect or generating unnecessary side effect) substantially purified.Object is preferably animal, and including but not limited to animal is such as ox, pig, horse, chicken, cat, dog, preferably mammal, most preferably people.

Clinical condition (side effect that particularly single immunomodulator is treated) with reference to individual patient, the transport sites of the composition containing immunomodulator, application process, factor known to using plan and other medical personnels, come preparation and quantitative immunological conditioning agent in the way of being consistent with good medical practice.Therefore, " therapeutically effective amount " of the immunomodulator herein proposed is determined according to these considerations.

Various movement systems are all known, and it can be used for applying and include the pharmaceutical composition of immunomodulator, such as liposome-encapsulated, particulate, microcapsules, the recombinant cell that can express compound, receptor-mediated endocytosis are (see such as Wu and Wu, J.Biol.Chem.262:4429-4432 (1987)), by part that nucleic acid construct is retrovirus or other carriers etc..Medication is including but not limited to intracutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, Epidural cavity and oral route.Can be by any convenient way such as including the pharmaceutical composition of immunomodulator by infusion or bolus in ection, by the absorption administration of epithelium or mucous layer (such as oral mucosa, rectum and intestinal mucosa), can be applied together with other biological active medicine include the pharmaceutical composition of immunomodulator.Using can be whole body or part.In addition, it is probably needs to include the pharmaceutical composition including immunomodulator is incorporated into central nervous system with intrathecal injection in the ventricles of the brain by suitable approach；For example, by potentially contributing to intraventricular injection with the storage capsule intraventricular catheter that for example Ommaya capsules are connected.Pulmonary administration for example can also be used by using the preparation of inhalator or atomizer and Alevaire.

In a specific embodiment, the present invention relates to the pharmaceutical preparation of medicine (such as immunomodulator known in the art and/or described herein).In particular it relates to be essentially the pharmaceutical preparation of the medicine (such as albumen and antibody) of protein sources.The pharmaceutical preparation of the present invention contains pharmaceutically acceptable excipient.In general, the pharmaceutical preparation of the present invention is formulated such that medicine remains the activity of its physics, chemistry and biology.The pharmaceutical preparation of the present invention can be stored at a suitable temperature.For example, the pharmaceutical preparation of the present invention can be stored under 2-8 °C, -40 °C or -80 °C.In a specific embodiment, stabilization formulations are to observe that aggegation occurs less than about 1%, observes that oxidation occurs less than about 1%, and/or the preparation for occurring desamidation less than about 4% medicine was observed in 2 years for medicine in 2 years for medicine wherein in 2 years.Amount for example by combining the medicine in the pharmaceutical preparation of required dosage volume and method of application to determine the present invention.In an embodiment of the invention, the concentration of the medicine in pharmaceutical preparation of the invention is about 1-160mg/ml, about 0-155mg/ml, about 20-150mg/ml, about 30-145mg/ml, about 40-140mg/ml, about 50-135mg/ml, about 60-130mg/ml, about 70-125mg/ml, about 80-120mg/ml, about 90-115mg/ml, about about 95-110mg/ml, 100-105mg/ml or about 100mg/ml.Concentration range e.g., from about 11-154mg/ml in the middle of above-mentioned concentration range is expected to be also a part of the invention.For example, it is contemplated that using number range of any value recited above as the combination of the upper limit and/or lower limit." about " specific described scope and (5,4,3,2 or 1) mg/ml bigger or smaller than the upper limit and/or lower limit of scope scope are included herein.

The aqueous pharmaceutical preparations of the present invention include pH buffer solutions.In an embodiment of the invention, the pH value range of the buffer solution used in pharmaceutical preparation of the invention is from about 5 to about 7.In one preferred embodiment, the pH value range of the buffer solution used in pharmaceutical preparation of the invention is from about 5.5 to about 6.5.Another preferred embodiment in, the pH value range of the buffer solution used in pharmaceutical preparation of the invention is from about 5.8 to about 6.2.Another preferred embodiment in, the pH value range of the buffer solution used in pharmaceutical preparation of the invention is about 6.0.PH value range in the middle of pH value range described above is expected to be also a part of the invention.For example, it is contemplated that using number range of any value recited above as the combination of the upper limit and/or lower limit." about " include herein specific described scope and it is bigger than the upper limit and/or lower limit of the scope or small by 0.5,0.4,0.3,0.2 or 0.1pH value units scope.The buffer example in preferred scope is controlled to include acetate (such as sodium acetate), succinate (such as sodium succinate), gluconate, histidine salt, citrate, Tris salt, phosphate, glycylglycine and other organic acid buffer agent pH value.Extra exemplary buffer agent is those buffers that are pharmaceutically useful and being generated from suitable acid, alkali and its salt, such as those buffers defined below.

Pharmaceutically useful acid is nontoxic inorganic acid and organic acid in terms of being included in the concentration and mode that it is formulated.For example, suitable inorganic acid includes hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfinic acid, p-aminobenzene sulfonic acid, phosphoric acid, carbonic acid etc..Suitable organic acid includes straight chain and straight chained alkyl acid,Aromatic radical acid,Naphthenic acid,Cycloaliphatic acids,Aromatic series aliphatic acid,Different naphthenic acid,Saturated acid,Unsaturated acids,It is single,Double or tricarboxylic acids,Including such as formic acid,Acetic acid,2- glycolics,Trifluoroacetic acid,Phenylacetic acid,Trimethylace tonitric,Tertiary fourth acetic acid,Ortho-aminobenzoic acid,Propionic acid,2 hydroxy propanoic acid,2- oxygen propionic acid,Malonic acid,Pentamethylene propionic acid,Pentamethylene propionic acid,3- benzenpropanoic acids,Butyric acid,Succinic acid,Benzoic acid,3- (4- hydroxyphenyls) benzoic acid,2- acetic acid-benzoic acid,Ascorbic acid,Cinnamic acid,Bay sulfuric acid,Stearic acid,Muconic acid,Mandelic acid,Butanedioic acid,Pamoic acid,Fumaric acid,Malic acid,Maleic acid,Hydroxymaleic acid,Malonic acid,Lactic acid,Citric acid,Tartaric acid,Glycolic,Gluconic acid,Gluconic acid,Pyruvic acid,Glyoxalic acid,Oxalic acid,mesylate,Butanedioic acid,Salicylic acid,Phthalic acid,palmoic,palmeic,Thiocyanic acid,Loprazolam,Ethyl sulfonic acid,1,2- ethionic acids,2- ethylenehydrinsulfonic acids,Benzene sulfonic acid,4- chlorobenzenesulfonic acids,Naphthalene-2-sulfonic acid (napthalene-2-sulphonic),To toluene thiosulfonic acid (p-toluenesulphonic),Camphorsulfonic acid,- 2- alkene -1- the carboxylic acids of 4- methyl bicycles [2.2.2]-eight (4-methylbicyclo [2.2.2]-oct-2-ene-1-carboxylic),Glucoheptonic acid,4,4 '-di-2-ethylhexylphosphine oxide -3- (hydroxyl -2- alkene -1- carboxylic acids) (4,4′-methylenebis-3-(hydroxy-2-ene-1-carboxylic acid)),Carbonaphthoic acid (hydroxynapthoic) etc..

Pharmaceutically useful alkali is nontoxic inorganic base and organic base in terms of being included in the concentration and mode that it is formulated.For example, suitable alkali includes those from inorganic base formation metal such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminium, N- meglumins, morpholine, the alkali of piperidines and organically nontoxic alkali, including primary ammonium, secondary ammonium and tertiary amine, substitution ammonium, cycloaminium and deacidite, [such as N (R ')₄ ⁺(wherein R is independently H or C_1-4Alkyl such as ammonium, Tris)], such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropyl amine (TPA), monoethanolamine, 2-diethylaminoethanol, tromethamine, dicyclohexyl amine, lysine, arginine, histidine, caffeine, procaine, Hai Baming (hydrabamine), choline, glycine betaine, aminophylline, aminoglucose, methylglucosamine, theobromine, purine, piperazine, piperidines, N-ethylpiperidine, polyamino resin.Particularly preferred organically nontoxic alkali is isopropylamine, diethylamine, monoethanolamine, trimethylamine, two hexamethylene ammoniums, choline and caffeine.

The extra pharmaceutically useful bronsted lowry acids and bases bronsted lowry that can be used in the present invention includes those bronsted lowry acids and bases bronsted lowries for being derived from amino acid, including histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine and asparagine.

Pharmaceutically useful buffer solution includes the acidity and the buffer solution of the addition salts of alkalescence that those are derived from bronsted lowry acids and bases bronsted lowry recited above.In an embodiment of the invention, the buffer solution of pharmaceutical preparation of the invention is succinate, histidine salt, citrate and/or phosphate.In one preferred embodiment, the buffer of pharmaceutical preparation of the invention is histidine salt.Another preferred embodiment in, the buffer of pharmaceutical preparation of the invention is citrate.

In yet another embodiment of the present invention, the buffer concentration in pharmaceutical preparation of the invention is about 5-50mM, about 5-20mM, about 5-15mM or about 10mM.Concentration range e.g., from about 6-48mM in the middle of above-mentioned concentration range is expected to be also a part of the invention.For example, it is contemplated that using number range of any value recited above as the combination of the upper limit and/or lower limit." about " specific described scope and (5,4,3,2 or 1) mM bigger or smaller than the upper limit and/or lower limit of scope scope are included herein.

Surfactant can also be added into the pharmaceutical preparation of the present invention.The surfactant of example includes nonionic surface active agent such as polysorbate (such as polysorbate 20 or 80) or poloxalkol (such as poloxamer 188).Other pharmaceutically useful surfactants are known to the present invention, also within the scope of the invention.In a specific embodiment, the amount of the surfactant added is enough to reduce the aggegation (such as in the aggegation occurred after rocking or transporting) of medicine, causes the particle formation in pharmaceutical preparation of the invention to minimize, and/or reduce the non-specific adsorption of medicine.In one preferred embodiment, pharmaceutical preparation of the invention includes surfactant, and the surfactant is polysorbate.

In one embodiment, pharmaceutical preparation of the invention contains surfactant Polysorbate 20.In one preferred embodiment, pharmaceutical preparation of the invention contains the polysorbate 20 between at least 0.007% and about 0.07%.Another preferred embodiment in, pharmaceutical preparation of the invention contains the polysorbate 20 between at least 0.01% and about 0.05%.Another preferred embodiment in, pharmaceutical preparation of the invention contains the polysorbate 20 between at least 0.01% and about 0.03%.Another preferred embodiment in, pharmaceutical preparation of the invention contains about 0.01% polysorbate 20." about " specific described scope and the scope of (0.01%, 0.009%, 0.008%, 0.007%, 0.006% or 0.005%) bigger or smaller than the upper limit and/or lower limit of the scope are included herein, condition is that the percentage of polysorbate 20 is not less than 0.007%.

Another preferred embodiment in, pharmaceutical preparation of the invention contains surfactant Polysorbate 80.In one preferred embodiment, pharmaceutical preparation of the invention contains the polyoxyethylene sorbitan monoleate between at least 0.0015% and about 0.07%.Another preferred embodiment in, pharmaceutical preparation of the invention contains the polyoxyethylene sorbitan monoleate between at least 0.003% and about 0.05%.Another preferred embodiment in, pharmaceutical preparation of the invention contains the polyoxyethylene sorbitan monoleate between at least 0.005% and about 0.03%.Another preferred embodiment in, pharmaceutical preparation of the invention contains the polyoxyethylene sorbitan monoleate between at least 0.01% and about 0.03%.Another preferred embodiment in, pharmaceutical preparation of the invention contains about 0.01% polyoxyethylene sorbitan monoleate." about " specific described scope and the scope of (0.01%, 0.009%, 0.008%, 0.007%, 0.006% or 0.005%) bigger or smaller than the upper limit and/or lower limit of the scope are included herein, condition is that the percentage of polyoxyethylene sorbitan monoleate is not less than 0.0015%.

Tension regulator can also be added into the pharmaceutical preparation of the present invention.Useful tension regulator includes salt and amino acid.The salt of pharmaceutical preparation pharmaceutically useful and suitable for the present invention includes sodium chloride, sodium succinate, sodium sulphate, potassium chloride, magnesium chloride, magnesium sulfate and calcium chloride.The salt of pharmaceutical preparation preferred for the present invention is NaCl and MgCl₂.NaCl can improve the stability of medicine by protecting medicine to avoid desamidation and aggegation.In one preferred embodiment, pharmaceutical preparation of the invention contains NaCl.Another preferred embodiment in, pharmaceutical preparation of the invention contains the NaCl between about 150 and about 500mM.Another preferred embodiment in, pharmaceutical preparation of the invention contains about 150mM NaCl." about " specific described scope and the scope of (1,2,3,4,5,10,25 or 50mM) bigger or smaller than the upper limit and/or lower limit of the scope are included herein.In one preferred embodiment, pharmaceutical preparation of the invention is isotonic.The isotonic pharmaceutical preparation for representing the present invention has the osmotic pressure substantially the same with blood of human body.Isotonic preparation typically has the osmotic pressure from about 250 to about 350mOsm, preferably from about 290 to about 310mOsm." about " specific described scope and (5,4,3,2 or 1) mOsm bigger or smaller than the upper limit and/or lower limit of scope scope are included herein.For example isotonicty can be determined using steam die mould or frost type osmometer.

In one embodiment, the pharmaceutical preparation of the present invention contains medicine as defined above (i.e. medicine, buffer, surfactant and tension regulator), and there is no one or more preservatives such as phenmethylol, phenol, m- cresols, Acetochlorone and phenyl ethylamine chlorine.In another embodiment, pharmaceutical preparation of the invention can include preservative, when particularly wherein described preparation is multi-dose formulation.The pharmaceutical preparation of the present invention can include one or more other pharmaceutical acceptable carrier, excipient or stabilizer, for example those are in Remington ' s Pharmaceutical Sciences 16thedition, Osol, A.Ed. carrier, excipient or stabilizer described in (1980), condition are the characteristics that they will not significantly needed for negative sense influence preparation.Carrier, excipient or stabilizer used is all nontoxic for acceptor in the dosage and concentration used, and including：Extra buffer, cosolvent, antioxidant include ascorbic acid and methionine, chelating agent such as EDTA, metal composite (such as Zn albumen compositions), biodegradable polymer such as polyester, preservative, antifreeze, anti-decomposition agent, filler.Suitable antifreeze example includes polyalcohol, polyethylene glycol (PEG), bovine serum albumin(BSA) (BSA), glutamic acid, other amino acid etc..Other suitable antifreezes include sugar and sugar alcohol such as sucrose, mannose, trehalose, glucose, sorbierite and mannitol.Suitable anti-decomposition agent can include sugar such as sucrose, trehalose, lactose and maltose.Suitable filler includes mannitol, glycine and D-sorbite etc..

In one preferred embodiment, pharmaceutical preparation of the invention does not include antifreeze.In further embodiment, pharmaceutical preparation of the invention does not include sucrose.

The pharmaceutical preparation of the present invention can also include the EDTA for being usually used in stablizing protein formulation.EDTA can suppress the sulfhydryl oxidase of metal catalytic as chelating agent, and the formation of the aggregation of disulfide bond is reduced accordingly.EDTA preferred concentration is from about 0.01% to about 0.2%.

For the special indication treated, pharmaceutical preparation of the invention can also be combined with one or more other medicines on demand, preferably those medicines with the medicine in complementary activity and the not pharmaceutical preparation of the negative sense influence present invention.The combination with the mixture of immunomodulator is formulated into the present invention relates to extra other medicines.In addition, by independent formulations but being intended for use the combination of synchronous with immunomodulator or overlapping administration the present invention also relates to wherein extra medicine.In combination, these extra medicines, which have, can effectively realize the amount of planned purpose.Further describing herein can be by the extra medicine with the formulation compositions of the present invention.

In one preferred embodiment, the present invention provides the pharmaceutical preparation for including or being made up of 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5).Another preferred embodiment in, the pharmaceutical preparation of the present invention includes amount from about 1mg/ml to about 160mg/ml antibody, is preferably from about 80mg/ml to about 120mg/ml antibody, 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, pharmaceutical preparation of the invention for intravenous administration includes amount from about 1mg/ml to about 160mg/ml antibody, is preferably from about 80mg/ml to about 120mg/ml antibody, 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, pharmaceutical preparation of the invention for subcutaneous administration includes amount from about 1mg/ml to about 160mg/ml antibody, is preferably from about 80mg/ml to about 120mg/ml antibody, 10mM histidine buffering liquids, 150mMNaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it." about " specific described scope and (5,4,3,2 or 1) mg/ml bigger or smaller than the upper limit and/or lower limit of scope scope are included herein.

In one preferred embodiment, pharmaceutical preparation of the invention includes 100mg/ml antibody, 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, the pharmaceutical preparation of the invention for intravenous administration includes 100mg/ml antibody, 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or being made from it.Another preferred embodiment in, the pharmaceutical preparation of the invention for subcutaneous administration includes 100mg/ml antibody, 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or being made from it.

In one preferred embodiment, the present invention provides the pharmaceutical preparation for including or being made up of 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5).Another preferred embodiment in, the pharmaceutical preparation of the present invention includes amount from about 1mg/ml to about 160mg/ml antibody, is preferably from about 80mg/ml to about 120mg/ml antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, pharmaceutical preparation of the invention for intravenous administration includes amount from about 1mg/ml to about 160mg/ml antibody, is preferably from about 80mg/ml to about 120mg/ml antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, pharmaceutical preparation of the invention for subcutaneous administration includes amount from about 1mg/ml to about 160mg/ml antibody, is preferably from about 80mg/ml to about 120mg/ml antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it." about " specific described scope and (5,4,3,2 or 1) mg/ml bigger or smaller than the upper limit and/or lower limit of scope scope are included herein.

In one preferred embodiment, pharmaceutical preparation of the invention includes 100mg/ml antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, the pharmaceutical preparation of the invention for intravenous administration includes 100mg/ml antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.Another preferred embodiment in, the pharmaceutical preparation of the invention for subcutaneous administration includes 100mg/ml antibody, 0.74mg/ml L-Histidines, the hydrochloride of 1.1mg/ml L-Histidines one, 8.8mg/ml NaCl and 0.1mg/ml polyoxyethylene sorbitan monoleate (pH 6.0 ± 0.5) or is made from it.

In one preferred embodiment, the antibody in pharmaceutical preparation of the invention is monoclonal antibody.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention is IgG antibody.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention is IgG1 antibody.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention is IgG1/ λ antibody.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention is the antibody of people or humanization.

In one preferred embodiment, pharmaceutical preparation of the invention stable at least six month under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention stable at least nine month under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention is stable at least 1 year under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention is stable at least 1.5 years under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention is stable at least 2 years under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention is stable at least 3 years under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention is stable at least 4 years under 2-8 °C.Another preferred embodiment in, pharmaceutical preparation of the invention is stable at least 5 years under 2-8 °C.

In one preferred embodiment, the antibody in pharmaceutical preparation of the invention can show significant stability in the freezing and thawing cycle repeated, and after these processing, still keep stable after thawing.In general, the preparation being frozen is rapidly frozen, for example, it is frozen in liquid nitrogen.It can be melted within the scope of certain temperature, be, for example, that, from about 0 °C to about 25 °C, this is slow melts；Either from about 26 °C to 40 °C, this is fast melt." about " scope of specific described scope and (5,4,3,2 or 1) bigger or smaller than the upper limit and/or lower limit of the scope degree Celsius is included herein.The example of fast melt is the pharmaceutical preparation for melting the present invention in 37 °C of water tank.In one preferred embodiment, the antibody in pharmaceutical preparation of the invention can keep stablizing at least one freezing and thawing cycle.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention can keep stablizing at least two freezing and thawing cycle.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention can keep stablizing at least three freezing and thawing cycle.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention can keep stablizing at least four freezing and thawing cycle.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention can keep stablizing at least five freezing and thawing cycle.Another preferred embodiment in, the antibody in pharmaceutical preparation of the invention can keep stablizing at least ten freezing and thawing cycle.

In order to keep stability, it may be necessary to determine the preferred plan of the pharmaceutical preparation of the freeze thawing present invention, or in order to provide maximum stability to by the antibody for carrying out special freezing and thawing cycle, it may be necessary to the pharmaceutical preparation of the identification present invention.Therefore, in an embodiment of the invention, it have rated this parameter.For example, the stability of the pharmaceutical preparation of the present invention can be detected under the various Freezing-Melting Conditions such as snap frozen of various combination, slow freezing, fast melt, slow thawing, to determine the scheme for generating minimum catabolite (i.e. with maximum stability).

Concentration research has shown that the IgG/ λ antibody in the pharmaceutical preparation of the invention including 10mM histidine buffering liquids, 150mM NaCl and 0.01% polyoxyethylene sorbitan monoleate (pH 6.0) can be concentrated at least 160mg/ml, and purity (being determined through SEC-HPLC) and aggregation (particle is not observed) are not adversely influenced (data are not shown).Further, it was observed that concentration adds viscosity.Due to the increase of viscosity, therefore add using difficult possibility.Research is had been shown in less than in 10 seconds, easily injection viscosity can be less than 7.75cP sample through 30G1/2 inch needles.As shown in Table X, or even when the concentration of IgG1/ λ antibody is 160mg/ml, the viscosity of pharmaceutical preparation is still below 7.75cP, therefore remains to easily inject through syringe.

Table X：It is used as the viscosity of the function of IgG1/ λ antibody concentrations

Concentration (mg/ml)	Viscosity (cP)
		0.0	0.93
18.9	0.97
		35.5	1.13
65.7	1.48
		79.2	1.80
89.4	1.96
		104.0	2.38
113.2	2.68
		122.2	3.15
128.0	3.43
		136.5	3.89
144.1	4.58
		161.7	6.74

The preparation that can be used for applying in vivo is most preferably sterile.By before or after prepared by preparation the filtering through sterilised membrane filter can be easily accomplished this point.

In one preferred embodiment, antibody of the invention is formulated in 10mM sodium citrates, 1.9% glycine, 0.5% sucrose, 0.01% polyoxyethylene sorbitan monoleate (pH 6.5 ± 0.3).Another preferred embodiment in, the antibody of the invention for intravenous administration is formulated in 10mM sodium citrates, 1.9% glycine, 0.5% sucrose, 0.01% polyoxyethylene sorbitan monoleate (pH 6.5 ± 0.3).

In one preferred embodiment, antibody of the invention is formulated in 10mM sodium citrates, 8% sucrose, 0.04% (w/v) polyoxyethylene sorbitan monoleate (pH 6.5 ± 0.3).Another preferred embodiment in, the antibody of the invention for intravenous administration is formulated in 10mM sodium citrates, 8% sucrose, 0.04% (w/v) polyoxyethylene sorbitan monoleate (pH 6.5 ± 0.3).Another preferred embodiment in, the antibody of the invention for subcutaneous administration is formulated in 10mM sodium citrates, 8% sucrose, 0.04% (w/v) polyoxyethylene sorbitan monoleate (pH 6.5 ± 0.3).

In general, equably and being closely contacted by Neutrokine- alpha-2 antagonists or other immunomodulators and liquid-carrier known in the art and/or described herein or the solid carrier finely separated or both come preparation of preparation.Then, if it is desired, product is shaped as required formulation.Preferably, carrier is parenteral carrier；The more preferably solution isotonic with the blood of acceptor.The example of these carriers includes water, salt solution, Ringer's solution and glucose solution.Non-aqueous carrier such as expressed oi and ethyl oleate and liposome are also useful carrier herein.

Carrier suitably contains less amount of additive, for example, strengthen the material of isotonicty and chemical stability.These materials are all nontoxic for acceptor in the dosage and concentration that it is used, and including buffer such as phosphoric acid, citric acid, butanedioic acid, acetic acid and other organic acids or their salt；Antioxidant such as ascorbic acid；The polypeptide of low molecule amount (less than about 10 residues), such as poly arginine or tripeptides；Albumen such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinyl pyrrolidone；Amino acid such as glycine, glutamic acid, aspartic acid or arginine；Monosaccharide and disaccharide and other carbohydrate include cellulose or derivatives thereof, glucose, maltose, sucrose or dextrin；Counter ion counterionsl gegenions such as sodium；Preservative such as cresols, phenol, Acetochlorone, phenmethylol and p-hydroxybenzoate；And/or nonionic surface active agent such as polysorbate, poloxalkol or PEG.

In these carriers, including to be usually formulated as concentration be about 0.001mg/ml to 100mg/ml to the composition of immunoloregulation polypeptide, or 0.1mg/ml to 100mg/ml, preferably about 1-10mg/ml or 1-10mg/ml, pH value is about 3 to 10 or 3 to 8, more preferably 5-8, most preferably 6-7.It will be apparent to apply the formation that some above mentioned excipient, carrier or stabilizer will cause polypeptide salt.

The composition that can be used for therapeutic administration is most preferably sterile.This point can be easily accomplished by the filtering through sterilised membrane filter (for example, 0.2 micron membranes) before or after prepared by preparation.Therapeutic composition is usually placed in the container with sterile access port, such as intravenous solution bag or bottle with the plug that can be penetrated by hypodermic needle.

Including can be used for the pharmaceutical composition of the immunomodulator in the inventive method often as the aqueous solution or as rebuild lyophilized formulation be stored in unit dose or multi-dose container, such as sealed ampoule or bottle.It is used as the example of lyophilized formulation, the aqueous Neutrokine- α polypeptide solutions filling 10ml bottles of 1% (w/v) being sterile filtered with 5ml, by the mixture lyophilized formed.The Neutrokine- α polypeptides for rebuilding lyophilized by using antibacterial water for injection prepare infusion solution.

Or, including can be used for the pharmaceutical composition of the immunomodulator in the inventive method can be to be stored in the form of lyophilized in single-dose containers.Infusion solution is rebuild using sterile injection carrier.

In a particular embodiment, the immunomodulator that can be used for the present invention is the Neutrokine- α or anti-CD 20 antibodies of radiolabeled polypeptide such as radio-labeled forms.These pharmaceutical compositions for including radio-labelled molecule can also include radiation protective and extender,plasma such as sodium ascorbate, dextran-40 and glycerine.In a particular embodiment, the composition that can be used in the inventive method includes the Neutrokine- α polypeptides or its fragment or variant of iodate form, and it is formulated in 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40 (Genetran-40).

In a particular embodiment, the composition that can be used in the inventive method includes the SEQ ID NO of at least 1mg/mL iodate form:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, the composition that can be used in the inventive method includes the SEQ ID NO of at least 2mg/mL iodate form:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, the composition that can be used in the inventive method includes the SEQ ID NO of at least 3mg/mL iodate form:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, the composition that can be used in the inventive method includes the SEQ ID NO of at least 4mg/mL iodate form:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mMHEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, the composition that can be used in the inventive method includes the SEQID NO of about 4.6mg/mL iodate forms:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.

In a particular embodiment, can be used for the composition in the inventive method includes the SEQ ID NO of the iodate form between about 0.1mg/mL and 20mg/mL:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, can be used for the composition in the inventive method includes the SEQID NO of the iodate form between about 1mg/mL and 10mg/mL:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, can be used for the composition in the inventive method includes the SEQ ID NO of the iodate form between about 2mg/mL and 8mg/mL:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.In a particular embodiment, can be used for the composition in the inventive method includes the SEQ ID NO of the iodate form between about 3mg/mL and 6mg/mL:2 134-285 amino acids residue, 10.0mM sodium citrates, 140mM sodium chloride, 8.7mM HEPES, 4% (w/v) sodium ascorbate, 3.3% (w/v) dextran-40.

In a preferred embodiment, the composition that can be used in the inventive method includes anti-Neutrokine- Alpha antibodies.In other embodiments, the composition that can be used in the inventive method includes specific binding Neutrokine- α antibody.In other embodiments, the composition that can be used in the inventive method includes the anti-Neutrokine- Alpha antibodies of Antagonism.In other embodiments, the composition that can be used in the inventive method includes specific binding Neutrokine- α and neutralizes the antibody of Neutrokine- α biological activity.In other embodiments, the composition that can be used in the inventive method includes the antibody for combining the restructuring Neutrokine- α albumen being purified into from cell culture, wherein the restructuring Neutrokine- α albumen is by least encoding SEQ ID NO:The polynucleotide encoding of 2 the 134 to 285th amino acids.In other embodiments, the composition that can be used in the inventive method includes specific binding Neutrokine- α antibody, the restructuring Neutrokine- α albumen that wherein described antibody binding is purified into from cell culture, wherein the restructuring Neutrokine- α albumen is by least encoding SEQ ID NO:The polynucleotide encoding of 2 the 134 to 285th amino acids.

Can orally, rectum, in parenteral, subcutaneous, brain pond, intravaginal, intraperitoneal, local (such as through powder, cartilage, drops or transdermal skin patches), buccal portion (bucally) or oral cavity or nasal spray (such as through sucking inhalant or powder) apply the pharmaceutical composition containing immunomodulator.

Term " parenteral " referred to herein as mode of administration, it include intravenous, intramuscular, in intraperitoneal, breastbone, subcutaneous and intra-articular injection and infusion.

In one preferred embodiment, subcutaneous administration contains the composition of immunomodulator.

In one preferred embodiment, it is intravenous to apply the composition containing immunomodulator.

The composition containing immunomodulator can also be applied with sustained release system.Continue whether the suitable example of composition includes suitable polymeric material (for example, semi permeable polymer substrate of formed article form such as film or microcapsules), suitable hydrophobic material (can for example use oily emulsion) or ion exchange resin and slightly soluble derivatives (for example omiting soluble).

Sustained-release matrix includes copolymer (Sidman, the U.et al., Biopolymers22 of polylactide (United States Patent (USP) 3,773,919, EP 58,481), Pidolidone and γ-ethyl-L-glutamate salt:547-556 (1983)), poly- (2- ethoxys methacrylate) (R.Langer et al., J.Biomed.Mater.Res.15:167-277(1981)；And R.Langer, Chem.Tech.12:98-105 (1982)) ethylene vinyl acetate (R.Langer et al., ibid) or poly- D- (-) -3- hydroxybutyric acids (EP 133,988).

In a specific embodiment, the composition containing immunomodulator is formulated in biodegradable, polymer drug delivery system, such as United States Patent (USP) 4,938,763,5,278,201st, 5,278,202,5,324,519,5,340,849 and 5,487,897 and international publication WO01/35929, WO00/24374 and WO00/06117 described in those movement systems, entire contents are incorporated herein by quoting herein.In a particular embodiment, utilize

Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colorado) preparation contains the composition of immunomodulator.In other specific embodiments, using purchased from Alkermes, Inc.'s (Cambridge, MA)

Sustained release system preparation contains the composition of immunomodulator.

The example for the biodegradable polymer that can be used in pharmaceutical preparation including but not limited to polylactic acid compounds, polyglycolide, PCL, polyanhydride, polyamide, polyurethanes, polyesteramide, polyorthoesters, polydioxanone, acetal resin, poly- acetyl, polycarbonate, gather positive carbonate, polyphosphazenes, polyhydroxybutyrate valerate, poly- hydroxypentanoic acid, poly- alkylene oxalic acid, poly- alkylene butanedioic acid, polymalic acid, polyaminoacid, poly- ethylene methacrylic ether, polymalic acid acid anhydride, polyvinylpyrrolidone, polyethylene glycol, poly- hydroxylated cellulose, chitin, the copolymer of chitosan and above-mentioned substance, trimer or composition or mixture.It is preferred that polymer be that those have the polymer of relatively low crystallinity and more high hydrophobicity.These polymer and copolymer have higher solubility than the polymer such as polyglycolide and chitin of high-crystallinity in bio-compatibility solvent, and the polymer also has high-caliber hydrogen bond.Preferably the material with required solubility parameter is the copolymer of polylactic acid compounds, PCL and these polymer and glycolide, wherein having more amorphous regions, to strengthen solubility.In particularly preferred embodiments, the biodegradable polymer that can be used for preparing the composition containing immunomodulator is PLG.It can be composed with modified polymer performance such as molecular weight, hydrophobicity and lactide/glycolides ratio with the insoluble drug release needed for obtaining (see such as Ravivarapu et al., Journal of Pharmaceutical Sciences89:732-741(2000)；Its content is incorporated herein by quoting herein).

It is also preferred that the solvent for biodegradable polymer is nontoxic, miscible and bio-compatible the solvent of water.The example of this solvent includes but is not limited to N- methyl -2- pyrrolones, 2- pyrrolones, C2 to C6 alkenols, C1 to C15 alcohol, glycol, triol and tetrol such as ethanol, glycerine, propane diols, butanol；C3 to C15 alkyl ketones such as acetone, diethyl ketone and MEK；C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate；Alkyl ketone such as MEK；C1 to C15 acid amides such as dimethylformamide, acetic acid dimethylamide and caprolactam；C3 to C20 ethers such as tetrahydrofuran or glyceryl alcohol contracting acetone (solketal)；Tween, glyceryl triacetate, propylene carbonate, Decylmethyl Sulphoxide, dimethyl sulfoxide (DMSO), oleic acid, Azone (1-dodecylazacycloheptan-2-one).Other preferred solvents are benzyl alcohol, Ergol, dipropylene glycol, glycerine, tributyrin, ethyl oleate, glycerine, glycogen (glycofural), isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate and triethyl citrate.According to solvability and their compatibility, most preferred solvent is N- methyl -2- pyrrolones, 2- pyrrolones, dimethyl sulfoxide (DMSO), glyceryl triacetate and propylene carbonate.

In addition, the preparation containing immunomodulator and biodegradable polymer can also contain release rate modifier and/or pore former.The example of release rate modifier includes but is not limited to aliphatic acid, triglycerides, other similar hydrophobic compound, organic solvent, plastic molding compound and hydrophilic compounds.Suitable release rate modifier include it is for example single-, the ester such as 2- N-P-ethoxyethyl acetates of double and tricarboxylic acids, methyl acetate, ethyl acetate, diethyl phthalate, repefral, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, lemon dimethyl phthalate, triethyl citrate, acetyltributyl citrate, citric acid acetyl triethyl, glycerol triacetate, two (normal-butyl) sebacates (di (n-butyl) sebecate) etc.；Polyhydroxy-alcohol is such as propane diols, polyethylene glycol, glycerine, D-sorbite；Aliphatic acid；Three esters of glycerine such as triglycerides, epoxidised soybean oil and other epoxidized vegetable oils；Alcohol such as cholesterol；Alcohol is such as C6-C12 alkenols, cellosolvo.Can individually or these other substance migration release rate modifiers of joint.The appropriate combination of release rate modifier includes but is not limited to glycerine/propyleneglycoles, D-sorbite/glycerine, ethylene oxide/trimethylene oxide, butylene glycol/adipic acid etc..It is preferred that release rate modifier include but is not limited to dimethyl citric acid, triethyl citrate, cognac oil, glycerine and hexylene glycol.The suitable pore former that can be used in polymer composition includes but is not limited to sugared such as sucrose and glucose, salt such as sodium chloride and sodium carbonate, polymer such as hydroxypropyl cellulose, carboxymethyl cellulose, polyethylene glycol and polyvinyl pyrrolidone.It is preferred that the solid crystal such as salt or sugar of set aperture, which can be supplied to,.

In a particular embodiment, BEMA is utilized^TM BioErodible Mucoadhesive System、MCA^TM MucoCutaneous Absorption System、SMP^TMSolvent MicroParticle System or BCP^TMBioCompatible Polymer System of Atrix Laboratories, Inc. (Fort Collins, Colorado) can make the composition containing immunomodulator.

The composition of sustained release also (is commonly found in Langer, Science 249 including liposome embedded composition:1527-1533(1990)；Treat et al.,in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler(eds.),Liss,New York,pp.317-327and 353-365(1989)).With known method such as DE 3,218,121；Epstein et al.,Proc.Natl.Acad.Sci.(USA)82:3688-3692(1985)；Hwang et al.,Proc.Natl.Acad.Sci.(USA)77:4030-4034(1980)；EP 52,322；EP 36,676；EP 88,046；EP 143,949；EP 142,641；Japanese patent application 83-118008；United States Patent (USP) 4,485,045 and 4,544,545；And EP 102,324 can prepare liposome.Liposome is typically all the liposome of small (about 200-800 Ethylmercurichlorendimides) single layer type, and wherein fat content is more than about 30 molar percentage cholesterol, the selected ratio of adjustment, to carry out optimal immune modulating treatment.

In another embodiment, the composition of sustained release includes crystal preparation known in the art.

In still one other embodiments, being transported with pump includes the composition of immunomodulator (see Langer, ibid；Sefton,CRC Crit.Ref.Biomed.Eng.14:201(1987)；Buchwald et al.,Surgery 88:507(1980)；Saudek et al.,N.Engl.J.Med.321:574(1989)).

Other controlled release durg delivery systems (Science 249 is discussed in Langer summary:1527-1533(1990)).

For parenteral administration, in one embodiment, immunomodulator is prepared typically by the way that the immunomodulator and pharmaceutical acceptable carrier of purity, unit dose injectable forms (solution, suspension or emulsion) needed for mixing are the carrier nontoxic to acceptor and mutually compatible with the other components of preparation in the dosage and concentration used.For example, when active component is immune modulator, preparation does not include oxidant and known other compounds being harmful to polypeptide preferably.

In a specific embodiment, it may be necessary to the region for the treatment of needed for locally applying to medical compounds or composition；For example, by but the perioperative local infusion of non-limited way, topical application for example after surgery the application of joint wound dressing, this point can be realized by injection, by conduit mode, by suppository routes or by implant mode (implant can be porous, non-porous or gel material, including film such as sialate film or filter membrane).Preferably, when applying antibody of the albumen including the present invention, it is necessary to the material that will not be carefully adsorbed using albumen.

In another embodiment, immunomodulator can be transported with carrier particularly liposome (see Langer, Science 249:1527-1533(1990)；Treat et al.,in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler(eds.),Liss,New York,pp.353-365(1989)；Lopez-Berestein, ibid, pp.317-327).

Still in another embodiment, immunomodulator can be transported with controlled release durg delivery system.In one embodiment, pump can be applied (see Langer, ibid；Sefton,CRC Crit.Ref.Biomed.Eng.14:201(1987)；Buchwald et al.,Surgery 88:507(1980)；Saudek et al.,N.Engl.J.Med.321:574(1989)).In another embodiment, polymeric material can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Florida (1974)；Controlled Drug Bioavailability,Drug Product Design and Performance,Smolen and Ball(eds.),Wiley,New York(1984)；Ranger and Peppas,J.,Macromol.Sci.Rev.Macromol.Chem.23:61(1983)；Also Levy et al., Science 228 is seen:190(1985)；During et al.,Ann.Neurol.25:351(1989)；Howard et al.,J.Neurosurg.71:105(1989)).Still in another embodiment, controlled release durg delivery system can be positioned over to the therapy target i.e. near-end of brain, therefore a part for whole-body dose is only needed to (see such as Goodson, in Medical Applications of Controlled Release, ibid, vol.2, pp.115-138 (1984)).

In a specific embodiment, wherein compound of the invention is the nucleic acid of encoding proteins, can administration of nucleic acid in vivo, to promote the expression of its coded albumen, by building it as a part for suitable nucleic acid expression vector and by using retroviral vector (see United States Patent (USP) 4, 980, 286) or direct injection is passed through, or by using microparticle bombardment (such as particle gun, Biolisitic, Dupont), or pass through lipid coatings or cell surface receptor or transfection agents, or by using the homeotic box sample peptide with being known to enter in core (see such as Joliot et al., Proc.Natl.Acad.Sci.USA 88:1864-1868 (1991)) connected nucleic acid etc. can use the compound so that and it turns into intracellular portion.Or, can be with intracellular introducing nucleic acid by homologous recombination, and be integrated in host cell DNA and expressed.

Present invention provides pharmaceutical composition.These compositions include the compound and pharmaceutical acceptable carrier of therapeutically effective amount.In a specific embodiment, term is " pharmaceutically acceptable " to represent listed in the authentic supervisory organ through federal or state government or American Pharmacopeia or other universally recognized animal applications and the particularly pharmacopeia of mankind's application.Term " carrier " refers to diluent, adjuvant, excipient or the carrier applied together with medicine.These pharmaceutical carriers can be sterile liquid such as water and oil, including those oil, animal, vegetables or synthesis source oil such as peanut oil, soybean oil, mineral oil, sesame oil.When intravenous administration pharmaceutical composition, water is preferred carrier.Salting liquid and aqueous glucose and glycerite can also be adopted to liquid-carrier, particularly Injectable solution.Suitable drug excipient includes starch, glucose, lactose, sucrose, gelatin, malt, paddy rice, flour, chalk, silica gel, odium stearate, glycerin monostearate, talcum, sodium chloride, skimmed milk power, glycerine, propylene, ethylene glycol, water, ethanol etc..If desired, composition can also contain a small amount of wetting agent or emulsifying agent or pH buffer.These compositions can use the form of solution, suspension, emulsion, tablet, pill, capsule, powder, extended release preparation etc..With traditional bonding agent and carrier such as triglycerides together, composition can be formulated into suppository.Oral formulations can include standard vector such as the mannitol of pharmaceutical grade, lactose, starch, magnesium stearate, saccharin sodium, cellulose magnesium carbonate." example of suitable pharmaceutical carrier is described in Remington ' s Pharmaceutical Sciences " in E.W.Martin.These compositions contain the compound of therapeutically effective amount, the preferably compound of purified form and proper amount of carrier, and the form for being administered to patient is suitable for provide.Preparation should meet mode of administration.

In one preferred embodiment, composition is configured to suitable for pharmaceutical composition of the intravenous administration to human body according to conventional methods.Composition for intravenous administration is typically the solution of sterile isotonic aqueous buffer solution.If desired, composition can also include solubilizer and local anaesthetics such as lidocaine, to mitigate the pain of injection site.In general, can dividually or be mixed provide unit dosage form component, such as in the form of the drying lyophilized powder or without the form of aqueous concentrate being labeled with the hermetically sealed container of amount of activating agent such as ampoule or Sachette provide., can be with the infusion bottle containing sterile pharmaceutical grade water or salt solution come dispensing composition if applying composition by infusion.If applying composition by injection, the sterile water for injection or salt solution of an ampoule can be provided so that can blending ingredients before administration.

Immunomodulator can be formulated into neutrality or salt form.Pharmaceutically useful salt includes those salt for being formed of anion with anion such as those are derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and those salt for being formed with cation of the cation such as those are derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2- ethylaminoethanols, histidine, procaine.

The amount of the effective immunomodulator of energy in method described herein can be determined with the clinical technology of standard.Furthermore it is possible to optionally with vitro detection method, help to identify optimal dose scope.Exact dose employed in preparation also depends on the severity of route of administration and disease or lesion, should determine the dosage according to the judgement of medical personnel and the situation of each patient.Effective dose can be extrapolated from from external or animal model test system dose-effect curve.

For antibody, the dosage for being administered to patient is typically 0.1mg/kg to 100mg/kg weight in patients.Preferably, it is between 0.1mg/kg to 20mg/kg weight in patients, more preferably between 0.1mg/kg to 10mg/kg weight in patients to be administered to the dosage of patient.In a preferred embodiment, give patient the intravenous dosage for applying 1,4,10 or 20mgkg.In general, human antibodies have longer half-life period than the antibody from other species in human body, because human body is to the immune response of extraneous polypeptide.Therefore, the administration of the human antibodies of lower dosage and less frequency is often possible.In addition, by the way that the application dosage and frequency of antibody of the invention can be reduced through modifying for example lipidization intake and tissue infiltration (such as entering intracerebral) to strengthen antibody.

As general suggestion, per dosage in parenteral administration polypeptide the medicinal effective dose of totality be within the scope of about 1mg/kg/ days to 10mg/kg/ days weight in patients, although as described above, this should be depending on treating judgement.It is highly preferred that this dosage is at least 0.01mg/kg/ days, for people, most preferably between about 0.01 and 1mg/kg/ days.

In another embodiment, it is that between 0.0001 and 0.045mg/kg/ days, preferably dosage is between 0.0045 and 0.045mg/kg/ days, more preferably using the dosage of the polypeptide given people, the dosage of the mankind is that the dosage of mouse was about 3mg/kg/ days in about 45 micrograms/kg/ days.

If be administered continuously, polypeptide generally is applied with the dose rates of about 1 microgram/kg/ hours to about 50 micrograms/kg/ hours, or for example polypeptide is applied using mini-pump by daily 1 to 4 times injections or by continuous h inf.The intravenous solution that is packed in bag can also be used.

The change of response generation and interval are different to observe after treatment for the length of required treatment, and this depends on required effect.

Can with continuous infusion, daily multiple injection (such as 3 times a day or more time, or twice daily), the form of daily single or intermittent infusion (for example twice daily, once a day, once every other day, twice a week, once in a week, monthly, monthly twice and quarterly once) is applied includes the composition of immunomodulator.If be administered continuously, polypeptide generally is applied with the dose rates of about 0.001 microgram/kg/ hours to about 10 micrograms/kg/ hours to about 50 micrograms/kg/ hours, or for example polypeptide is applied using mini-pump by daily 1 to 4 times injections or by continuous h inf.

The effective dose for the composition including immunomodulator that can determine to be applied by method known to those skilled in the art, wherein involved parameter is biological half life, bioavailability and toxicity.These determination methods are within the limit of power of those skilled in the art, especially in conjunction with elaborating that this is provided.

Organism also plays an important role to the biology of immunomodulator exposed to determination treatment and/or medicine effective dose scheme aspect.The variation of dosage for example for quite a long time in apply the immunomodulator of suitable low dosage repeatedly and can have an effect, it is described to act on and acting on of being obtained of the immunomodulator of the suitable high dose of administration is therapeutically and/or pharmaceutically differentiable repeatedly within extremely short a period of time.

Using Freireich, E.J., (Cancer Chemotherapy Reports 50 (4) are waited:219-44 (1966)) the equivalent body surface area dose conversion coefficient that is provided, those skilled in the art can advantageously by the data conversion obtained from the pilot system in given application immunomodulator into the medicine effective quantity for the immunomodulator that each dosage is applied in another pilot system accurate estimate.Using the conversion coefficient of the offers such as Freireich, the test data obtained by applying immunomodulator in mouse can for example be converted into the accurate estimate of medicine effective quantities of the Neutrokine- α in rat, monkey, dog and people.Following conversion table (Table III) is the general introduction for the data that Freireich etc. is provided.Table III is given for the equivalent body surface area dosage represented by the use mg/kg that will be converted into from the dosage represented with mg/kg in a species in table in another listed species.Table III：Equivalent body surface area dose conversion coefficient.

Thus, for example the dosage 50mg/kg in the conversion coefficient provided using Table III, mouse can be converted into the approximate dosage 12.5mg/kg in monkey, because (50mg/kg) x (1/4)=12.5mg/kg.As extra example, dosage 0.02,0.08,0.8,2 and 8mg/kg in mouse are equivalent to 1.667 micrograms of effective dose/kg, 6.67 micrograms/kg, 66.7 micrograms/kg, 166.7 micrograms/g and 0.667mg/kg in people respectively.

In some embodiments, it is related to the Neutrokine- α or anti-Neutrokine- Alpha antibodies using radio-labeled forms.The radioactive dosage applied is essentially all different.Can be about the 0.1 radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions for arriving about 100mCi/70kg body weight with application dosage.In another embodiment, it with application dosage can be about the 0.1 radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions for arriving about 50mCi/70kg body weight.In another embodiment, can be about the radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions of 0.1,0.5,1,5,10,15,20,25,30,35,40,50,60,70,80,90 or 100mCi/70kg body weight with application dosage.

Can be about the 0.1 radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions for arriving about 10mCi/kg body weight with application dosage.In another embodiment, it with application dosage can be about the 0.25 radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions for arriving about 5mCi/kg body weight.In a particular embodiment, can be about the radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions of 0.35,0.70,1.35,1.70,2.0,2.5 or 3.0mCi/kg body weight with application dosage.

Can be about 1 to about 50mCi/m with application dosage²Radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions.In another embodiment, it with application dosage can be about the 10 radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions for arriving about 30mCi/m2.In a particular embodiment, can be about 10,15,20,25 or 30mCi/m with application dosage²Radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions.

The concentration of total Neutrokine- α albumen, Neutrokine- α SV albumen, anti-Neutrokine- Alpha antibodies and/or anti-Neutrokine- α SV antibody in radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions can also be different, such as from about 1 microgram/kg to about 1mg/kg.In a particular embodiment, in radiolabeled Neutrokine- α or anti-Neutrokine- Alpha antibodies compositions Neutrokine- α albumen, Neutrokine- α SV albumen, the total concentration of anti-Neutrokine- Alpha antibodies and/or anti-Neutrokine- α SV antibody may be about 5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100 micrograms/kg.

For example, as it is known that lymthoma is radiosensible tumour.For immunodiagnostic imaging, the trace labelling of compound can be applied, generally 60mCi labelled with radioisotope 1-20mgNeutrokine- α albumen is arrived with about 1.Dosage sometimes depends on the isotope for imaging；Upper limit amount in scope is preferably 40 and arrives 60mCi, can use^99mTc；Lower limit amount in scope is preferably 1-20mCi, can use¹¹¹In.For imaging purpose, object about 1 can be given to about 30mgNeutrokine- α compounds.For radioimmunotherapy treatment purpose, enough Neutrokine- α compounds can be applied to object so that the whole-body dose received up to 1100cGy, but preferably less or equal to 500cGy.Total amount scope of the Neutrokine- α albumen including Neutrokine- α albumen, Neutrokine- alpha conjugates and Neutrokine- α compounds for being administered to object can be from 1.0 micrograms/kg to 1.0mg/kg weight in patients.In another embodiment, be administered to object Neutrokine- α albumen total amount scope can from 20 micrograms/g to 100 micrograms/g weight in patients.

Estimate about 825mCi's¹³¹I can provide the amount of approximate 500cGy radioactive intensity to whole human body.The amount for the radioactive intensity applied depends in part on selected radio isotope.For⁹⁰Y is treated, it is believed that from about 1 to the radioactive intensities of about 200mCi amounts be suitable, wherein it is preferred that amount be 1 to 150mCi, and 1 to 100mCi (such as 60mCi) is most preferred.The method for optimizing that tissue dose is estimated from the radioactive intensity applied is to be imaged with tracer dose or other pharmacokinetic regimens, to obtain the estimation to predetermined dosimetry value.It is determined that being administered in the suitable dose of the radiopharmaceutical of individual, it is necessary to the amount of the radiation received in view of the single organ of the maximal tolerance dose relative to this organ.These information are known for those skilled in the art, for example, see Emami et al., International Journal of Radiation Oncology, Biology, Physics 21:109-22(1991)；And Meredith, Cancer Biotherapy ＆Radiopharmaceuticals 17:Both full contents, are incorporated herein by 83-99 (2002) by quoting herein.

" high dose " scheme, such as systemic administration 200 arrive 600cGy (or higher) amount, it may be necessary to which marrow replaces the support of scheme, because marrow is due to toxicity and limits the tissue of dose of radiation.

In a particular embodiment, patient receives the multiple administration of composition (antibody or other immunomodulators known in the art and/or described herein that for example specifically bind Neutrokine- α).Repeatedly apply for one group and be referred to as a cycle.Signal period can include applying for such as 1,2,3,4,5,6,7,8,9,10,11,12 or more times.For any applied once, its dosage can be fixed either different, to allow to realize initial drug load and/or to compensate for patient-specific difference in terms of body weight, body surface area, disease activity, disease responsiveness, drug tolerance, recovery time, PK parameters, and/or pharmacology response.

In period demand it is any it is administered twice between time can be fixed either different, to adapt to the patient-specific difference in terms of disease activity, disease responsiveness, drug tolerance, recovery time, PK parameters and/or pharmacology response.In a particular embodiment, patient is given the loading dose just controlled, and the dosage is twice of the amount given given in subsequent apply.In other implementations, it is any it is administered twice between time can be 1,2,3,4,5,6 or 7 days it is (1 week) or longer.In a particular embodiment, it is any it is administered twice between time can be 1,2,3,4,5,6,7 or 8 weeks (or longer).Patient can receive the treatment in multiple cycles.If necessary to the cycle more than more than 1, time between any two treatment cycle can be fixed or different, to adapt to the patient-specific difference in terms of disease activity, disease responsiveness, drug tolerance, recovery time, PK parameters and/or pharmacology response.In a particular embodiment, the time between any two cycle can be 1,2,3,4,5 or 6 weeks or longer.In a particular embodiment, the time between any two cycle can be 1,2,3,4,5,6,7,8,9,10,11,12 months or longer.In a particular embodiment, the time between any two cycle can be 1,2,3,4,5 years or longer.In a particular embodiment, patient receives initial bullet formula and applied, and then receives the treatment in one or more cycles.

In one embodiment, initial bullet formula, which is applied, includes the antibody antagonists to the intravenous application dosage of patient for the Neutrokine- α more than or equal to 2mg/kg.In one embodiment, initial bullet formula, which is applied, includes the antibody antagonists to the intravenous application dosage of patient for the Neutrokine- α more than or equal to 5mg/kg.In a preferred embodiment, the administration of initial bullet formula is the antibody antagonists for the Neutrokine- α more than or equal to 10mg/kg to the intravenous application dosage of patient.In other implementations, the administration of initial bullet formula is the antibody antagonists for the Neutrokine- α more than or equal to 15mg/kg to the intravenous application dosage of patient.In one embodiment, initial bullet formula, which is applied, includes the antibody antagonists to the intravenous application dosage of patient for the Neutrokine- α more than or equal to 20mg/kg.

In other implementations, initial bullet formula, which is applied, includes anti-CD 20 antibodies.

In other implementations, initial bullet formula, which is applied, includes B cell depletor.

Present invention provides the medicine bag or kit of the container including one or more one or more components for filling up pharmaceutical composition.Optionally, with these containers accompany can be government supervision department defined on medicine or biological product production, using or sale in terms of notice, the notice reflect for human body apply medicine or biological product production, using or sale superintendent office certification.

Can individually or the other medicines of joint apply immunomodulator together, other medicines include but is not limited to one or more other immunomodulators, chemotherapeutics, antibiotic, antiviral agent, steroid or non-steroid anti-inflammatory drug, traditional immunotherapeutic agent and cell factor.Can concomitantly for example as mixture, discretely but simultaneously or synchronously or order apply the combination.This includes wherein composition of medicine by as treatment mixture situation about being applied together, and be also separated including wherein composition of medicine ground but simultaneously enters the internal process of same individual using for example separated venous channel." combination ", which is applied, also to be included discretely first applying one of which compound or medicine, then applies second of compound or medicine.

In subsequent paragraph, elaborate that can combine other compounds applies immunomodulator.In some cases, other compounds are also a kind of immunomodulator in itself.Elaboration in those paragraphs is intended to pass on a kind of viewpoint, i.e., two or more different immunomodulators can be administered in combination in the method being specifically related to reference to the present invention.Anti- Neutrokine- Alpha antibodies are used in combination for example, being specifically related to and can combine anti-CD 20 antibodies with reference to method of the invention.

The traditional nonspecific immunosuppressive agent that can be applied with combined immunization conditioning agent includes but is not limited to steroids, cyclosporin, cyclosporin analog, endoxan, endoxan IV, methylprednisolone, prednisolone, imuran, FK506,15- deoxyspergualin and other immunodepressant played a role by the function of depression effect T cell.Other immunodepressant that can be applied with combined immunization conditioning agent include but is not limited to prednisolone, methopterin, Thalidomide, Methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ^TM), cloth quinoline that, deoxyspergualin and Azaspirane (SKF 105685).

In a particular embodiment, immunomodulator can be applied with combined immunization inhibitor.The immunosuppressive agents that can be applied together with immunomodulator include but is not limited to ORTHOCLONE

3 (muromonab-CD3s), SANDIMMUNE^TM、NEORAL^TM、SANGDYA^TM(cyclosporin),(FK506, tacrolimus),(mycophenolate, its active metabolite is mycophenolic acid), IMURAN^TM(imuran), glucocorticoid, adrenocorticotro such as DELTASONE^TM(metacortandracin) and HYDELTRASOL^TM(prednisolone), FOLEX^TMAnd MEXATE^TM(amethopterin), OXSORALEN-ULTRA^TM(Methoxsalen) and RAPAMUNE^TM(sirolimus).In a specific embodiment, it can be prevented with immunodepressant to organ or the rejection of bone marrow suppression.

In another embodiment, joint Patients Treated with Steroid applies immunomodulator.The steroids that can be applied with combined immunization conditioning agent includes but is not limited to oral glucocorticoid, metacortandracin and methylprednisolone (such as IV methylprednisolones).In a specific embodiment, joint metacortandracin applies immunomodulator.In a further specific embodiment, joint metacortandracin and immunodepressant apply immunomodulator.The immunodepressant that can be applied together with immunomodulator and metacortandracin is those immunodepressant described herein, including but not limited to imuran, endoxan and endoxan IV.In another particular embodiment of the invention, joint methylprednisolone applies immunomodulator.In a further specific embodiment, joint methylprednisolone and immunodepressant apply immunomodulator.The immunodepressant that can be applied together with immunomodulator and methylprednisolone is those immunodepressant described herein, including but not limited to imuran, endoxan and endoxan IV.

In one preferred embodiment, joint antimalarial applies immunomodulator.The antimalarial that can be applied together with immunomodulator includes but is not limited to HCQ (such as PLAQUENIL^TM), chloroquine, and/or acrinamin.

In one preferred embodiment, joint NSAID applies immunomodulator.

In a non-exclusive embodiment, joint 1,2,3,4,5,10, or more plant following medicament administration immunomodulator：NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), this uh potassium propionate (Monsanto), Mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), Ilyushin is replaced sour (Byk Gulden), Campath, AGM-1470 (Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra gene therapies (Valentis), JTE-522 (Japan Tobacco), taxol (Angiotech), DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), (the synergen of soluble TNF acceptor 1；Amgen), IPR-6001 (Institute for Pharmaceutical Research), Trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), with butixocort propionate (WarnerLambert).

In one embodiment, one or more following medicament administration immunomodulators are combined：Infliximab (also referred to as Remicade^TMCentocor, Inc.), Trocade (Roche, RO-32-3555), leflunomide (also referred to as Arava^TM, from Hoechst Marion Roussel), Kineret^TM(IL-1 receptor antagonists, also referred to as anakinra.From Amgen, Inc.), SCIO-469 (p38 kinase inhibitors, from Scios, Inc),

(adalimumab from Abbott laboratories), and/or ASLERA^TM(Astenile, dehydrobenzene, GL701, from Genelabs Technologies Inc).

Collect in another embodiment, can combine 1,2,3,4,5, or more plant following medicament administration immunomodulator：Amethopterin, SASP, Sanocrysin, Anranofin, cyclosporin, penicillamine, imuran, antimalarial (as described herein), endoxan, knurl can so, gold preparation, ENBREL^TM(Etanercept), anti-TNF antibodies, LJP394 (La Jolla Pharmaceutical Company, San Diego, California) and prednisolone.

In another embodiment, joint antimalarial, amethopterin, anti-TNF antibodies, ENBREL^TM, and/or SASP applies immunomodulator.In one embodiment, joint methopterin applies immunomodulator.In another embodiment, joint anti-TNF antibodies apply immunomodulator.In another embodiment, joint methopterin and anti-TNF antibodies apply immunomodulator.In another embodiment, joint SASP applies immunomodulator.In the embodiment of another special number, joint methopterin, anti-TNF antibodies and SASP apply immunomodulator.In another embodiment, ENBREL is combined^TMUsing immunomodulator.In another embodiment, ENBREL is combined^TMImmunomodulator is applied with methopterin.In another embodiment, ENBREL is combined^TM, methopterin and SASP apply immunomodulator.In another embodiment, ENBREL is combined^TMAnd SASP applies immunomodulator.In other implementations, one or more antimalarials are administered in combination together with one of which combinations of the above.In a specific embodiment, joint antimalarial (such as HCQ), ENBREL^TM, methopterin and SASP apply immunomodulator.In another particular embodiment of the invention, joint antimalarial (such as HCQ), ENBREL^TM, SASP, anti-TNF antibodies and methopterin apply immunomodulator.

In an other embodiment, individually or the one or more IVIG products of joint apply immunomodulator.The IVIG product that can be applied with immunomodulator includes but is not limited to GAMMAR^TM、IVEEGAM^TM、SANDOGLOBULIN^TM、GAMMAGARD S/D^TMAnd GAMIMUNE^TM.In a specific embodiment, in transplantation treatment (such as bone-marrow transplantation), joint IVIG product applies immunomodulator.

In an other embodiment, individually or joint anti-inflammatory agent apply immunomodulator.The anti-inflammatory agent that can be applied with immunomodulator includes but is not limited to glucocorticoid and non-steroid anti-inflammatory drug, amino aromatic carboxylic acid derivatives, fragrant acetogenin, fragrant butanoic acid derivative, aromatic carboxylic acid derivatives, fragrant propanoic derivatives, pyrazoles, pyrazolone, salicyclic acid derivatives, Thiazinecarboxamides, e- acetylamino caproic acids, S-adenosylmethionine, 3- amino -4- hydroxybutyric acids, Amixetrine, Bendazac, benzydamine, bucolome, Difenpiramide, ditazole, Emorfazone, guaiazulene, Nabumetone, aulin, orgotein, Oxaceprol, paranyline, Perisoxal, pifoxime, proquazone, aerbron, and Tenidap.

In a particular embodiment, individually or joint anti-CD 4 antibodies apply immunomodulator.In one embodiment, the co-application of immunomodulator and anti-CD 4 antibodies be used to treat rheumatoid arthritis.In one embodiment, the co-application of immunomodulator and anti-CD 4 antibodies is used for systemic lupus erythematosus.

In a particular embodiment, individually or combine anti-IL-15 antibody and apply immunomodulator.In one embodiment, the co-application of immunomodulator and anti-IL-15 antibody be used to treat rheumatoid arthritis.In one embodiment, the co-application of immunomodulator and anti-IL-15 antibody is used for systemic lupus erythematosus.

In a particular embodiment, individually or joint CTLA4-Ig and LEA29Y apply immunomodulator.In one embodiment, immunomodulator and CTLA4-Ig and LEA29Y co-application be used to treat rheumatoid arthritis.In one embodiment, the co-application of immunomodulator and CTLA4-Ig and LEA29Y is used for systemic lupus erythematosus.

In a particular embodiment, individually or combine anti-IL-6 receptor antibodies and apply immunomodulator.In one embodiment, the co-application of immunomodulator and anti-IL-6 receptor antibodies be used to treat rheumatoid arthritis.In one embodiment, the co-application of immunomodulator and anti-IL-6 receptor antibodies is used for systemic lupus erythematosus.

In a particular embodiment, individually or combine anti-C5 (complement component) antibody and apply immunomodulator.In one embodiment, the co-application of immunomodulator and anti-C5 antibody be used to treat rheumatoid arthritis.In one embodiment, the co-application of immunomodulator and anti-C5 antibody is used for systemic lupus erythematosus.

In a particular embodiment, individually or joint complement cascade inhibitors apply immunomodulator.Complement cascade inhibitors include but is not limited to anti-properdin antibody (Gliatech), TP-10, recombinant soluble I types complement receptors (AVANT Immunotheragenetics Inc.)；Pexelizmab, complement C5 inhibitor (Alexion Pharmaceuticals Inc.)；And 5G1.1, tissue complement part C5 are cracked into its monoclonal antibody for promoting inflammatory components.In one embodiment, the co-application of immunomodulator and complement cascade inhibitors is used for treatment inflammation, rheumatoid arthritis and/or systemic loupus erythematosus.

In another embodiment, combined chemotherapy medicament administration immunomodulator.The chemotherapeutics that can be applied together with immunomodulator includes but is not limited to antibiotic derivatives (such as adriamycin, bleomycin, daunorubicin and dactinomycin D)；Antiestrogen (such as TAM)；Antimetabolite (such as fluorouracil, 5-FU, methopterin, Floxuridine, Interferon Alpha-2b, glutamic acid, mithramycin, purinethol and 6-thioguanine)；Cell toxicity medicament (such as carmustine, BCNU, lomustine, CCNU, cytarabine, endoxan, estramustine, hydroxycarbamide, procarbazine, mitomycin, busulfan, cis-platinum and vincristine sulphate)；Hormone (such as Medroxyprogesterone, estramustine phosphate sodium, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, stilphostrol, Chlorotrianisene and Testolactone)；Nitrogen mustard derivatives (such as melphalan, knurl can so, chlormethine (mustargen) and phosphinothioylidynetrisaziridine)；Steroids and composition (such as betamethasone sodium phosphate)；With other drugs (such as Dacarbazine, asparaginase, vincristine sulphate, Vinblastine Sulfate and etoposide).

In a specific embodiment, joint CHOP (endoxan, adriamycin, vincristine and metacortandracin) or the component combined in one or more CHOP apply immunomodulator.In one embodiment, joint anti-CD 20 antibodies such as human monoclonal anti-CD 20 antibodies apply immunomodulator.In another embodiment, joint anti-CD 20 antibodies and CHOP, or any combination (particularly endoxan and/or metacortandracin) of anti-CD 20 antibodies and one or more CHOP components apply immunomodulator.In a specific embodiment, joint Rituximab applies immunomodulator.In a further embodiment, immunomodulator is applied together with any combination (particularly endoxan and/or metacortandracin) of Rituximab and CHOP, or Rituximab and one or more CHOP components.In a specific embodiment, joint tositumomab (anti-CD 20 antibodies for coming from Coulter Pharmaceuticals, San Francisco, CA) applies immunomodulator.In a further embodiment, immunomodulator is applied together with any combination (particularly endoxan and/or metacortandracin) of tositumomab and CHOP, or tositumomab and one or more CHOP components.Tositumomab can optionally with¹³¹I is connected.Anti-CD 20 antibodies optionally can be connected with radio isotope, toxin or cell toxicant pro-drug.

In another particular embodiment of the invention, Zevalin is combined^TMUsing immunomodulator.In a further embodiment, with Zevalin^TMAnd CHOP, or Zevalin^TMImmunomodulator is applied together with any combination (particularly endoxan and/or metacortandracin) of one or more CHOP components.Zevalin^TMIt can be connected with one or more radio isotopes.Particularly preferred isotope is⁹⁰Y and¹¹¹In。

In other embodiment, combine Rituximab (Rituxan^TM) and/or Ibritumomab Tiuxetan (Zevalin^TM, such as (In-111) Ibritumomab Tiuxetan or (Y-90) Ibritumomab Tiuxetan) and apply immunomodulator.In a specific embodiment, Rituximab and/or Ibritumomab Tiuxetan and immunomodulator is administered in combination to treat NHL.

In a particular embodiment, the long-term treatment that anti-CD20 is applied is supplemented as after seizure of disease such as lupus breaking-out using immunomodulator.

In other embodiment, combine imatinib mesylate (Gleevec

4- [(4-Methyl-1-piperazinyl) methyl]-N- [4-methyl-3- [[4- (3-pyridinyl) -2-pyrimidinyl] amino]-phenyl] benzamide methanesulfonate) apply immunomodulator.

In other embodiment, combine bortezomib (Bortezomib^TM[(1R) -3-methyl-1- [[(2S) -1-oxo-3-phenyl-2- [(pyrazinylcarbonyl) amino] propyl] amino] butyl] boronic acid) apply immunomodulator.

In other embodiment, combine Allan pearl monoclonal antibody

Using immunomodulator.

In other embodiment, joint fludarabine phosphate (Fuda China

9H-Purin-6-amine, 2-fluoro-9- (5-O-phosphono- β-D-arabinofuranosyl) (2-fluoro-ara-AMP)) apply immunomodulator.

One or more medicines for being used to treat Huppert's disease can be combined and apply immunomodulator, the medicine includes but is not limited to alkylating agent, anthracycline, carmustine (DTI-015, BCNU, BiCNU, Gliadel

), endoxan (

CTX), dexamethasone

Adriamycin

Melphalan (L-PAM,

Phenyalamine mustard), metacortandracin, Thalidomide and vincristine (OncoVCR、

)。

The preferred compositions for the medicine for treating Huppert's disease that can be applied with combined immunization conditioning agent include but is not limited to endoxan+metacortandracin, melphalan+metacortandracin (MP), vincristine+adriamycin

The alternate scheme (VMCP/VBAP) of+dexamethasone (VAD), vincristine+carmustine+melphalan+endoxan+metacortandracin (VBMCP, M2 scheme) and vincristine+melphalan+endoxan+metacortandracin and vincristine+carmustine+adriamycin+metacortandracin.

Can with combined immunization conditioning agent apply one or more be used for treat NHL medicine include but is not limited to 2-chlorodeoxyadenosine, Amifostine (

WR-272), bexarotene (

Targretin

Targretin

LGD1069), bleomycin

Busulfan

Carboplatin (

CBDCA), carmustine (DTI-015, BCNU, BiCNU, Gliadel

), knurl can be right

Cis-platinum (

CDDP), Cladribine (2-CdA,

), endoxan (

CTX), cytarabine (

Ara-C, Ara-C,

), Dacarbazine (DTIC), daunorubicin (Daunomycin,

Daunorubicin

)、Denileukin diftitox

Dexamethasone

Dolasetron mesilate

Adriamycin

Hematopoietin

Phosphoric acid etoposideEtoposide (VP-16,

), fludarabine (

FAMP)、Granisetron

Hydrocortisone, demethyl daunorubicin (

DMDR, IDA), ifosfamide

Interferon-' alpha '

Interferon a2a (Intron

), mustargen (Nitrogen Mustard, HN₂、

), melphalan (L-PAM,

Phenyalamine mustard), methopterin

(MTX、

), methylprednisolone

Mitoxantrone (

DHAD)、Ondansetron

Pentostatin (

2- deoxycoformycins), peroxophosphoric acid amine (4-hydroperoxycyclophosphamide, 4-HC), metacortandracin, procarbazineRituximab

(

Anti-CD20 monoclonal antibody), phosphinothioylidynetrisaziridine (Sanya and vulcanization phosphamide,), TPT (

SK&F-104864、NSC-609699、

), vinblastine (

VLB), vincristine (

Onco

VCR、

) and eldisine

The preferred compositions for the medicine for treating NHL that can be applied with combined immunization conditioning agent include but is not limited to：Adriamycin

+ bleomycin+vinblastine+Dacarbazine (ABVD), anti-idiotype treats (BsAb)+interferon Alpha, anti-idiotype treatment (BsAb)+knurl can be right, anti-idiotype treats (BsAb)+proleulzin, BCNU (carmustine)+etoposide+Ara-C (cytarabine)+melphalan (BEAM), bleomycin+etoposide+adriamycin+endoxan+vincristine+procarbazine+metacortandracin (BEACOPP), bryostatin+vincristine, endoxan+BCNU (carmustine)+VP-16 (etoposide) (CBV), endoxan+vincristine+metacortandracin (CVP), endoxan+adriamycin(Hydroxydaunomycin)+vincristine (Oncovorin)+metacortandracin (CHOP), endoxan+Novantrone

(mitoxantrone)+vincristine (Oncovorin)+metacortandracin (CNOP), endoxan+adriamycin+VM-26+metacortandracin, endoxan+adriamycin

(Hydroxydaunomycin)+vincristine (Oncovorin)+metacortandracin+Rituximab (CHOP+ Rituximabs), endoxan+adriamycin+VM-26+metacortandracin+interferon Alpha, cytarabine+bleomycin+vincristine+mitoxantrone (CytaBOM), dexamethasone+cytarabine+cis-platinum (DHAP), dexamethasone+ifosfamide+cis-platinum+etoposide (DICE), adriamycin+vinblastine+mustargen+vincristine+bleomycin+etoposide+metacortandracin (Stanford V), etoposide+vinblastine+adriamycin (EVA), etoposide+Methyllprednisolone+cytarabine+cis-platinum (ESHAP), etoposide+metacortandracin+ifosfamide+cis-platinum (EPIC), fludarabine+mitoxantrone+dexamethasone (FMD), fludarabine+dexamethasone+cytarabine (ara-C)+cis-platinum

(FluDAP), ifosfamide+cis-platinum+etoposide (ICE), mustargen+

(vincristine)+procarbazine+metacortandracin (MOPP), Mei Sina+ifosfamide+demethyl daunorubicin+etoposide (MIZE), methopterin+Leucovorin rescue+bleomycin+adriamycin+endoxan+Oncovorin+ dexamethasone (m-BACOD), metacortandracin+methopterin+adriamycin+endoxan+etoposide (ProMACE), phosphinothioylidynetrisaziridine+busulfan+endoxan, phosphinothioylidynetrisaziridine+busulfan+melphalan, TPT+taxol, and vincristine

+ adriamycin

+ dexamethasone (VAD).

Can with combined immunization inhibitor apply the medicine for treating NHL more examples include but is not limited to A007 (4-4 '-dihydroxy benaophenonel -2,4- dinitrophenylhydrazones), AG-2034 (AG-2024, AG-2032, GARFT [glycinamide ribonucleotide transformylase] inhibitor), Aldesleukin (IL-2,

), Allan pearl monoclonal antibody

Alitretinoin (

LGN-1057), hemel (Hexamethyl melamine,

), amino camptothecin (9-AC, 9-aminocamptothecin, NSC 603071), anti-CD19/CD3 monoclonal antibodies (anti-CD19/CD3scFv, anti-NHL monoclonal antibodies), anti-idiotype treatment (BsAb), arabinose guanine (Ara-G, GW506U78), arsenic trioxide (ATO), B43- genisteins (anti-19 antibody of CD/genistein conjugate), B7 antibody conjugates, Betathine (Beta-LT), BlyS antagonists, Bryostatin-1 (BMY-45618, NSC-339555), CHML (Cytotropic Heterogeneous Molecular Lipids), clofarabine (chlorine-fluorine-araA), daclizumab

Depsipeptide (FR901228, FK228), dolastatin -10 (DOLA-10, NSC-376128), epirubicin (

EPI, 4 ' tables-adriamycin), Epratuzumab (

Humanization anti-CD22, HAT), Fly3/flk2 parts

G3139(

Antisense Bcl-2), Hu1D10 (anti-HLA-DR monoclonal antibodies, SMART1D 10), HumaLYM (anti-CD20 monoclonal antibody), Ibritumomab tiuxetan

Interferon gamma (gamma interferon, Gamma

γ-IF), Irinotecan (CPT-11、

CaptoCPT-1), ISIS-2053, ISIS-3521 (antisense PKC- α), Lmb-2 immunotoxins (anti-CD25 recombinant immunotoxins, anti-Tac (Fv)-PE38),

(cytofectin+IL-2 genes, IL-2 gene therapies), Lym-1 (¹³¹I LYM-1), lymphoma vaccine (Genitope), naphthalene draw shore (compound 506, U78), Neugene compounds (

Antisense myc), NovoMAb-G2scFv (NovoMAb-G2IgM), O6-benzylguanine (BG,

), oxalic acid platinum

TaxolTaxol-DHA

Peldesine (BCX-34, PNP inhibitor), Rebeccamycin and Rebeccamycin analogs, SCH-66336, Sobuzoxane (MST-16,

)、SU5416(

VEGF inhibitor), TER-286, Thalidomide, TNP-470 (AGM-1470), tositumomab

Valspodar (PSC 833), Vaxid (B cell lymphoma DNA vaccination), vinorelbine

WF10 (macrophage conditioning agent) and XR-9576 (XR-9351, P- glycoprotein/MDR inhibitor).

Can with combined immunization conditioning agent apply one or more be used for treat ALL medicine include but is not limited to amsacrine, carboplatin (

CBDCA), carmustine (DTI-015, BCNU, BiCNU, Gliadel), Cholecaliferol, endoxan (

CTX), cytarabine (

Ara-C, cytarabine,), daunorubicin (Daunomycin,Daunorubicin

), dexamethasone

Adriamycin

Etoposide (VP-16,

), Filgrastim(

G-CSF、

), fludarabine (

FAMP), demethyl daunorubicin (

DMDR, IDA), ifosfamide

Imatinib mesylate (STI-571,

Abl tyrosine kinase inhibitors), interferon gamma (Gamma- interferon, Gamma

Gamma-IF), L-ASP (

Asparaginase

), purinethol (Ismipur, 6-MP), methopterin

(MTX、

), mitoxantrone (DHAD)、

Metacortandracin, vitamin A acid, VM-26 (VM-26,

), thioguanine (6- thioguanines, 6-TG), TPT (

SK&F-104864、NSC-609699、

), vitamin A acid (

ATRA、

) and vincristine (

Onco

VCR、)。

Can with combined immunization conditioning agent apply the medicine for treating ALL more examples include but is not limited to amino camptothecin (9-AC, 9-aminocamptothecin, NSC603071), aminopterin, Annamycin (AR-522, annamycin LF,

), arabinose guanine (Ara-G, GW506U78,

), arsenic trioxide (

ATO、

), B43- genisteins (anti-CD 19 antibodies/genistein conjugate), B43-PAP (anti-CD19Ab/ pokeweed antiviral proteins conjugate), Cordycepin, CS-682, Decitabine (the miscellaneous nitrogen -2 of 5- '-deoxycytidine), dolastatin -10 (DOLA-10, NSC-376128), G3139 (

Antisense Bcl-2), irofulven (MGI-114, Ivofulvan, acyl group fulvene analog), MS-209, phenyl butyrate, quinine, TNP-470 (AGM-1470, Fumngillin), Trimetrexate

Troxacitabine (BCH-204、BCH-4556、

), UCN-01 (7- hydroxyls staurosporin), WHI-P131 and WT1 vaccines.

The preferred compositions for the medicine for treating ALL that can be applied with combined immunization conditioning agent include but is not limited to carboplatin+mitoxantrone,Carmustine+endoxan+etoposide,Cytarabine+daunorubicin,Cytarabine+adriamycin,Cytarabine+demethyl daunorubicin,Cytarabine+interferon gamma,Cytarabine+L-ASP,Cytarabine+mitoxantrone,Cytarabine+fludarabine and mitoxantrone,Etoposide+cytarabine,Etoposide+ifosfamide,Etoposide+mitoxantrone,Ifosfamide+etoposide+mitoxantrone,Ifosfamide+VM-26,Methopterin+purinethol,Methopterin+purinethol+vincristine+metacortandracin,Phenyl butyrate+cytarabine,Phenyl butyrate+etoposide,Phenyl butyrate+TPT,Phenyl butyrate+vitamin A acid,Quinine+adriamycin,Quinine+mitoxantrone+cytarabine,Thioguanine+cytarabine+amsacrine,Thioguanine+etoposide+demethyl daunorubicin,Thioguanine+vitamin A acid+Cholecaliferol,Vincristine+metacortandracin,Vincristine+metacortandracin and L-ASP,Vincristine+dexamethasone/metacortandracin+asparaginase+daunorubicin/adriamycin,Vincristine+dexamethasone/metacortandracin+asparaginase+daunorubicin/adriamycin+Filgrastim,Vincristine+dexamethasone/metacortandracin+asparaginase+daunorubicin/adriamycin+endoxan+methopterin,With vincristine+dexamethasone/metacortandracin+asparaginase+daunorubicin/adriamycin+endoxan+methopterin+Filgrastim.

Can with combined immunization conditioning agent apply one or more be used for treat chronic lymphocytic leukemia medicine include but is not limited to knurl can be right

Cladribine (2-CdA,

), endoxan (

CTX), cytarabine (Cytosar-Ara-C, Ara-C,

Cytarabine ocfosfate, ara-CMP), adriamycinFludarabine (

FAMP), Pentostatin (

2- deoxycoformycins), metacortandracin and vincristine (

Onco

VCR、

)。

The more examples for the medicine for treating chronic lymphocytic leukemia that can be applied with combined immunization conditioning agent include but is not limited to Allan pearl monoclonal antibody

Amino camptothecin (9-AC, 9-aminocamptothecin, NSC 603071), aminopterin, Annamycin (AR-522, annamycin LF,), arabinose guanine (Ara-G, GW506U78,

Compound 506U78), arsenic trioxide (

ATO、

), Bryostatin-1 (

BMY-45618, NSC-339555), CS-682, dolastatin -10 (DOLA-10, NSC-376128), Filgrastim (

G-CSF、Leukine)、Flavopiridol (NSC-649890、HMR-1275)、G3139(

Antisense Bcl-2), irofulven (MGI-114, Ivofulvan, acyl group fulvene analog), MS-209, phenyl butyrate, Rituximab

(

Anti-CD20 monoclonal antibody), Thalidomide, Theophylline, TNP-470 (AGM-1470, Fumngillin), UCN-01 (7- hydroxyls staurosporin) and WHI-P131.

The preferred compositions for the medicine for treating chronic lymphocytic leukemia that can be applied with combined immunization conditioning agent include but is not limited to fludarabine+metacortandracin and endoxan+adriamycin+vincristine+metacortandracin (CHOP).

In other embodiment, the associational cells factor applies immunomodulator.The cell factor that can be applied together with immunomodulator includes but is not limited to GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-α, IFN-β, IFN-γ, TNF-α and TNF-β.In another embodiment, immunomodulator can be applied together with any interleukin, including but not limited to IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 and IL-22.In a preferred embodiment, joint IL4 and IL10 applies immunomodulator.Inventor has been observed that IL4 and IL10 can strengthen the alpha mediated B cell proliferations of Neutrokine-.

In vitro, inventor has been observed that IFN γ and IL-10 add monocyte and macrophage cell surface expression Neutrokine- α (obtaining macrophage by cultivating primary monocyte and 20ng/mL M-CSF12-15 days), wherein IL-4 processing reduces the Neutrokine- α of the cell surface expression of monocyte and macrophage.Together with IL-10 the complete inhibition to the IL-10 cell surface expression Neutrokine- α induced is caused using IL-4.Cell surface expression Neutrokine- α increase is caused using IL-4 together with IFN γ.Compared with untreated macrophage, the amount for causing solubility (activity) the Neutrokine- α being discharged into culture medium with IFN-γ and IL-10 processing macrophages adds 3 times.

In other embodiment, and chemotactic factor (CF) applies immunomodulator together.In another embodiment, immunomodulator is applied together with chemokine beta -8, chemokine beta -1, and/or macrophage inflammatory protein -4.In one preferred embodiment, immunomodulator is applied together with chemokine beta -8.

In an other embodiment, joint IL-4 antagonists apply immunomodulator.The IL-4 antagonists that can be applied together with immunomodulator include but is not limited to：Soluble IL-4 receptor polypeptide, the soluble IL-4 receptor polypeptide of multimeric forms, the IL-4 of the anti-IL-4 antibody combined with reference to the anti-IL-4 receptor antibodies of IL-4 acceptors and non-conducting through the IL-4 biological signals triggered, blocking IL-4 and one or more IL-4 acceptors and combination IL-4 acceptors and non-conducting through the IL-4 biological signals triggered mutain.Preferably, antibody of the present invention is monoclonal antibody (including antibody fragment, such as those fragments described herein).

In an other embodiment, joint hemopoieticgrowth factor applies immunomodulator.The hemopoieticgrowth factor that can be applied together with immunomodulator includes but is not limited to LEUKINETM (SARGRAMOSTIM^TM) and NEUPOGEN^TM(Filgrastim^TM)。

In an other embodiment, joint fibroblast growth factor applies immunomodulator.The fibroblast growth factor that can be applied together with immunomodulator includes but is not limited to FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15.

In an other embodiment, joint antihypertensive applies immunomodulator.The antihypertensive that can be applied together with immunomodulator includes but is not limited to calcium ion antagonist such as nifedipine (ADALAT^TM、PROCARDIA^TM), peripheral vasodilation agent such as hydrolazine (APRESOLINE^TM), beta adrenergic blocking drug such as Propranolol (INDERAL^TM), α/β adrenergic blocking drug such as labetolol (NORMODYNE^TM、TRANDATE^TM), suppress Angiotensin II generation medicine such as captopril (CAPOTEN^TM), directly suppress Angiotensin II activity medicine such as Losartan (COZAAR^TM) and thiazide diuretic such as Hydrochioro (HYDRODIURIL^TM、ESIDREX^TM)。

Individually or other adjuvants can be combined apply immunomodulator.The adjuvant that can be applied with immunomodulator includes but is not limited to alum, alum plus deoxycholic acid (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG and MPL.In another particular embodiment of the invention, joint QS-21 applies immunomodulator.The more adjuvants that can be applied with immunomodulator include but is not limited to monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminium salt, MF-59 and Virosomal adjuvant technologies.The vaccine that can be applied with immunomodulator includes but is not limited to specific aim protection MMR (measles,mumps,rubella), polio, varicella, lockjaw/diphtheria, hepatitis A, hepatitis B, haemophilus influenzae B, pertussis, pneumonia, influenza, Lyme diseases, rotavirus, cholera, yellow fever, encephalitis B, polio, rabies, typhoid fever and pertussis and/or PNEUMOVAX-23^TMVaccine.In another particular embodiment of the invention, PNEUMOVAX-23 is combined^TMUse immunomodulator.

In one embodiment, another member of joint TNF families applies immunomodulator.The TNF that can be applied with immunomodulator, TNF correlation molecules or TNF sample molecules include but is not limited to the TNF-α of soluble form, Lymphotoxin-α (LT- α, also referred to as TNF-β), LT- β (see compound heterotrimer LT- α 2- β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF- γ (international publication WO 96/14328), TRAIL/AIM-I (international publication WO 97/33899), LIGHT/AIM-II (international publication WO 97/34911), APRIL (J.Exp.Med.188 (6):1185-1190), endokine- α (international publication WO 98/07880), FASTR/TR6 (international publication WO 98/30694), OPG (OPG), with Neutrokine- α (international publication WO 98/18921), OX40, with nerve growth factor (NGF), with the Fas of soluble form, CD30, CD27, CD40 and 4-IBB, TR2 (international publication WO96/34095), DR3 (international publication WO 97/33904), TRAIL-R1/DR4 (international publication WO 98/32856), TRAIL-R3, TR5 (international publication WO 98/30693), TR6 (international publication WO 98/30694), TRAIL-R2/TR7 (international publication WO 98/41629), TRANK, TR9 (international publication WO 98/56892), TRAIL-R4/TR10 (international publication WO 98/54202), 312C2 (international publication WO 98/06842), and TR12.

In another embodiment, joint one or more Neutrokine- α acceptors (such as TACI, BCMA and BAFF-R) apply immunomodulator.In a preferred embodiment, Neutrokine- α acceptors are soluble.Other preferred embodiment in, the Fc areas of the Fc areas such as IgG molecules of Neutrokine- α acceptors and immunoglobulin molecules are blended.For example, the 1 to 81st amino acids (GenBank numberings NP 443177) of TACI 1-154 amino acids residue (GenBank numbering AAC51790), BCMA 1-48 amino acids (GenBank numbering NP_001183) or BAFF-R can be merged with the Fc areas of IgG molecules, and be used in combination with immunomodulator another known in the art and/or described herein.In another embodiment, the BAFF-R-Fc albumen that can be applied with combined immunization conditioning agent is the SEQ ID NO merged with the Fc areas of IgG1 immunoglobulin molecules:10 the 1 to 70th amino acids.Optionally, the 20th amino acids (valine) in BAFF-R are replaced by asparagine, and the 27th amino acids (leucine) in BAFF-R are replaced by proline.

In one preferred embodiment, joint anti-CD40L antibodies and/or anti-CD 40 antibodies apply immunomodulator.

In an other embodiment, individually or joint anti-angiogenic agent apply immunomodulator.The anti-angiogenic agent that can be applied with immunomodulator includes but is not limited to Angiostatin (Entremed, Rockville, MD), (the Boston Life Sciences of troponin -1, Boston, MA), the anti-invasion factor, vitamin A acid and its derivative, taxol (Taxol), suramin, the Tissue Inhibitor of metalloproteinases 1, the Tissue Inhibitor of metalloproteinases -2, VEGI, plasminogen activator inhibitor -1, plasminogen activator inhibitor -2 and various forms of lighter " d groups " transition metal.

Lighter " d races " transition metal includes such as vanadium, molybdenum, tungsten, titanium, niobium and tantalum species.These kinds of transition metals can form transition metal composite.The compound of suitable above mentioned kinds of transition metals includes oxo transition metal composite.

The representative example of vanadium compound includes oxo vanadium compound such as vanadic acid and vanadyl complexes.Suitable vanadic acid compound includes metavanadic acid and positive vanadic acid compound such as ammonium metavanadate, sodium metavanadate and sodium vanadate.Suitable vanadyl complexes, which include such as vanadyl levulinic ketone ester and vanadic sulfate, includes vanadic sulfate hydrate such as vanadic sulfate list water and trihydrate.

The representative example of tungsten and molybdenum compound also includes oxo compound.Suitable oxo tungsten compound includes wolframic acid and tungsten oxide compound.Suitable wolframic acid compound includes ammonium tungstate, artificial schellite, sodium tungstate dihydrate and wolframic acid.Suitable tungsten oxide includes tungsten oxide (IV) and tungsten oxide (VI).Suitable oxo molybdenum compound includes molybdic acid, molybdenum oxide and molybdenyl compound.Suitable molybdic acid compound includes ammonium molybdate and its hydrate, sodium molybdate and its hydrate and potassium molybdate and its hydrate.Suitable molybdenum oxide includes oxidizing ferment (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdenyl compound includes such as molybdenyl acetylacetone,2,4-pentanedione.Other suitable tungsten and molybdenum compounds include the hydrogen-oxygen derivative from such as glycerine, tartaric acid and sugar.

In the present invention, various other anti-angiogenesis can also be utilized.Representational example includes but is not limited to platelet factor 4, protamine sulfate, sulfuric acid chitin derivativ (being made up of female Carapax Eriocheir sinensis) (Murata et al., Cancer Res.51:22-26, 1991), sulfated polysaccharide peptide glycan compound (SP-PG) (presence of steroids such as estrogen and tamoxifen citrate can strengthen the function of this compound), staurosporine, the conditioning agent of matrix metabolism includes such as proline analogs, cis hydroxyproline, d, L-3, 4- dehydroprolines, Thioproline, α, α-dipyridyl, aminopropionitrile fumaric acid, 4- propyl group -5- (4- pyridine radicals) -2 (3H)-azolactone, methopterin, mitoxantrone, heparin, interferon, serum beta2-microglobulin, chIMP (Pavloff et al., J.Bio.Chem.267:17321-17326,1992), (Tomkinson et al., Biochem are J.286 for chymostatin:475-480,1992), the sulfuric ester of cyclodextrin 14, Eponemycin, camptothecine, Fumngillin (Ingber et al., Nature 348:555-557,1990), disodium aurothiomalate (" GST "；Matsubara and Ziff, J.Clin.Invest.79:1440-1446,1987), serum anticollagenase, α 2- antiplasmins (Holmes et al., J.Biol.Chem.262 (4):1659-1664,1987), bisantrene (National Cancer Institute), Lobenzarit Disodium (N- (2)-carboxyphenyl-4-chloroanthronilic acid disodium or " CCA " (Takeuchi et al., Agents Actions 36:312-316,1992)) and metal protease inhibitors such as BB94.

The other anti-angiogenesis that can also be utilized in the present invention include Thalidomide (Celgene, Warren, NJ), Angiostatic steroid, AGM-1470 (H.Brem and J.Folkman J Pediatr.Surg.28:445-51 (1993)), the antagonists of beta 2 integrin alpha v β 3 (C.Storgard et al., J Clin.Invest.103:47-54(1999)),Carboxynaminolmidazole,Carboxyamidotriazole(CAI)(National Cancer Institute,Bethesda,MD),Conbretastatin A-4(CA4P)(OXiGENE,Boston,MA),Squalamine (Magainin Pharmaceuticals,Plymouth Meeting,PA),TNP-470(Tap Pharmaceuticals,Deerfield,IL),ZD-0101(AstraZeneca,London,UK),APRA(CT2584),Benfluralin,Byrostatin-1(SC339555),CGP-41251(PKC 412),CM101,Dexrazoxane (ICRF187),DMXAA,Endostatin,Flavopridiol,Genistein (Genestein),GTE,ImmTher,Iressa(ZD 1839),Octreotide (Somatostatin),Panretin,Penacillamine,Photopoint,PI-88,Prinomastat (AG-3340) Purlytin,Suradista(FCE26644),TAM (Nolvadex),Tazarotene,Tetrathiomolybdate,Xeloda (Capecitabine),And 5 FU 5 fluorouracil.

The anti-angiogenic agent that can be applied with combined immunization conditioning agent can be played a role by number of mechanisms, and the mechanism includes but is not limited to suppress the integrin receptor expressed by the protein cleavage of extracellular matrix, the function of blocking endothelial cell-extracellular matrix adhesion molecule, the function of antagonizing vessel generation derivant such as growth factor and Inhibit proliferaton endothelial cell.The example for the anti-angiogenic agent that interference cell extracellular matrix protein is cracked and can applied with combined immunization conditioning agent includes but is not limited to AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol Myers Squibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK) and Metastat (Aeterna, St-Foy, Quebec).The example for the anti-angiogenesis inhibitor that plays a role and can be applied with combined immunization conditioning agent by blocking the function of endothelial cell-extracellular matrix adhesion molecule includes but is not limited to EMD-121974 (Merck KcgaA Darmstadt,) and Vitaxin (Ixsys Germany, La Jolla, CA/Medimmune, Gaithersburg, MD).The example for the anti-angiogenic agent that is played a role and can be applied with combined immunization conditioning agent by direct antagonism or suppression angiogenesis inducer includes but is not limited to Angiozyme (Ribozyme, Boulder, CO), anti-VEGF antibody (Genentech, S.San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S.San Francisco, CA), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, NJ) and SU-6668 (Sugen).The effect of other anti-angiogenic agents is indirect suppression angiogenesis.The example of the indirect inhibitor for the angiogenesis that can be applied with combined immunization conditioning agent includes but is not limited to IM-862 (Cytran, Kirkland, WA), interferon-' alpha ', IL-12 (Roche, Nutley,) and Pentosan polysulfate (Georgetown University NJ, Washington, DC).

In a particular embodiment, the use in conjunction of immunomodulator and anti-angiogenic agent be used to treating, prevent, and/or improving autoimmune diseases autoimmunity disease as described herein.

In a specific embodiment, the use in conjunction of immunomodulator and anti-angiogenic agent be used to treating, prevent, and/or improving arthritis.In one more specifically embodiment, the use in conjunction of immunomodulator and anti-angiogenic agent be used to treating, prevent, and/or improving rheumatoid arthritis.

In another embodiment, immunomodulator is applied in United anticoagulation agent.The anti-coagulants that can be applied together with immunomodulator includes but is not limited to heparin, warfarin and aspirin.In a specific embodiment, joint heparin and/or warfarin apply immunomodulator.In another particular embodiment of the invention, joint warfarin applies immunomodulator.In another particular embodiment of the invention, joint warfarin and aspirin apply immunomodulator.In another particular embodiment of the invention, joint heparin applies immunomodulator.In another particular embodiment of the invention, joint heparin and aspirin apply immunomodulator.

In another embodiment, joint suppresses the medicament administration immunomodulator of anticardiolipin antibodies generation.In a particular embodiment, the medicament administration immunomodulator of combined occlusion and/or reduction anticardiolipin antibodies combination phospholipid-binding plasma albumen beta 2-glycoprotein I (b2GPI) ability.

In some embodiments, joint antiretroviral agent, NRTI, non-nucleoside reverse transcriptase inhibitor, and/or protease inhibitors apply immunomodulator.The NRTI that can be applied with combined immunization conditioning agent includes but is not limited to RETROVIR^TM(Zidovudine/AZT), VIDEX^TM(Didanosine/ddI), HIVID^TM(zalcitabine/ddC), ZERIT^TM(stavudine/d4T), EPIVIR^TM(Lamivudine/3TC) and COMBIVIR^TM(Zidovudine/Lamivudine).The non-nucleoside reverse transcriptase inhibitor that can be applied with combined immunization conditioning agent includes but is not limited to VIRAMUNE^TM(NVP), RESCRIPTOR^TM(delavirdine) and SUSTIVA^TM(efavirenz).The protease inhibitors that can be applied with combined immunization conditioning agent includes but is not limited to CRIXIVAN^TM(indinavir), NORVIR^TM(Ritonavir), INVIRASE^TM(inverase) and VIRACEPT^TM(nelfinavir).

In some embodiments, joint antiretroviral agent, nucleoside/nucleotide RTI (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and/or protease inhibitors (PI) apply immunomodulator.The NRTIs that can be applied with combined immunization conditioning agent includes but is not limited to RETROVIR^TM(Zidovudine/AZT), VIDEX^TM(Didanosine/ddI), HIVID^TM(zalcitabine/ddC), ZERIT^TM(stavudine/d4T), EPIVIR^TM(Lamivudine/3TC) and COMBIVIR^TM(Zidovudine/Lamivudine).The NNRTIs that can be applied with combined immunization conditioning agent includes but is not limited to VIRAMUNE^TM(NVP), RESCRIPTOR^TM(delavirdine) and SUSTIVA^TM(efavirenz).The PIs that can be applied with combined immunization conditioning agent includes but is not limited to CRIXIVAN^TM(indinavir), NORVIR^TM(Ritonavir), INVIRASE^TM(inverase) and VIRACEPT^TM(nelfinavir).

Other NRTI include LODENOSINE^TM(F-ddA；The stable adenosine NRTI of acid；Triangle/Abbott)、COVIRACIL^TM(emtricitabine/FTC；Similar in construction to Lamivudine (3TC), but external activity is 3 to 10 times of Lamivudine；Triangle/Abbott)、dOTC(BCH-10652；Also Lamivudine is structurally similar to, but still remains the activity of anti-most of lamivudine drug resistance separation strains；Biochem Pharma), adefovirdipivoxil (FDA refusal approval its be used for anti-hiv therapy；Gilead Sciences)、

(two good fortune esters of adefovirdipivoxil；The active precursor medicine of adefovirdipivoxil；Its activity form be PMEA-pp), TENOFOVIR^TM(bis-POCPMPA, PMPA pro-drug；Gilead), DAPD/DXG (DAPD active metabolites；Triangle/Abbott), D-D4FC (being similar to 3TC, the activity with anti-AZT/3TC drug-resistant virus), GW420867X (Glaxo Wellcome), ZIAGEN^TM(Abacavir/159U89；Glaxo Wellcome Inc.), CS-87 (3 ' azidos -2 ', 3 '-dideoxyuridine；WO 99/66936) and β-L-FD4C and β-L-FddC carrying S- acyl group -2- sulphur ethyls (S-acyl-2-thioethyl, SATE) prodrug form (WO 98/17281).

Other NNRTI include COACTINON^TM(the strong NNRTI of emivirine/MKC-442, HEPT type；Triangle/Abbott)、CAPRAVIRINE^TM(AG-1549/S-1153, it is follow-on that there is the NNRTI for resisting the virus activity being mutated containing K103N；Agouron), PNU-142721 (has the activity than its high 20 to 50 times of predecessor's delavirdine, and is resistant to K104N mutant；Pharmacia ＆ Upjohn), DPC-961 and DPC-963 (be designed to the anti-second generation derivative with the K103N viral efavirenzs being mutated；DuPont), GW-420867X (has 25 times higher than HBY097 of activity, and is resistant to K103N mutant；Glaxo Wellcome), the CALANOLIDE A (naturally occurring materials from rubber tree；It is resistant to the virus of the mutation containing Y181C and K103N)；With Propolis (WO 99/49830).

Other protease inhibitors include LOPINAVIR^TM(ABT378/r；Abbott Laboratories), a kind of BMS-232632 (azepine peptides；Bristol-Myres Squibb)、TIPRANAVIR^TM(PNU-140690, non-peptide pyrrolin ketone；Pharmacia ＆Upjohn), PD-178390 (non-peptide pyrrolin ketone；Parke-Davis a kind of), (azepine peptides of BMS 232632；Bristol-Myers Squibb), L-756,423 (indinavir analogs；Merck), DMP-450 (cyclic urea compounds；Avid ＆ DuPont), AG-1776 (have external antiprotease inhibitor drug-resistant virus activity peptide mimics；Agouron), VX-175/GW-433908 (phosphate prodrugs of amprenavir；Vertex ＆ Glaxo Welcome), CGP61755 (Ciba) and AGENERASE^TM(amprenavir；Glaxo Wellcome Inc.).

Other antiretroviral agents include fusion inhibitor/gp41 bonding agents.Fusion inhibitor/gp41 bonding agents include T-20, and (peptide of the 643 to 678th amino acids from HIV gp41 transmembrane protein ectodomains, it is combined with the gp41 of inactive state, and prevents it from changing into Fusion Strain；) and T-1249 (second generation fusion inhibitors Trimeris；Trimeris).

Other antiretroviral agents include fusion inhibitor/chemokine receptor anagonists.Fusion inhibitor/chemokine receptor anagonists include CXCR4 antagonists such as AMD3100 (bicyclam), SDF-1 and the like and ALX40-4C (cationic peptide), the T22 (peptides of 18 amino acid；) and T22 analogs T134 and T140 Trimeris；CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES and TAK-779；With CCR5/CXCR4 antagonists such as NSC 651016 (distamycin analog).Also CCR2B, CCR3 and CCR6 antagonist are included.Chemokine receptor agonists also suppress fusion such as RANTES, SDF-1, MIP-1 α, MIP-1 β.

Other antiretroviral agents include integrase inhibitor.Integrase inhibitor includes anthraquinone, the ZINTEVIR of dicaffeoylquinic acid (DFQA), L- Cichoric acids (a kind of chicoric acid (DCTA)), quinazoline (QLC) and correlation^TM(AR 177, oligonucleotides may act on cell surface rather than real integrase inhibitor；Arondex), those naphthols of naphthols for example described in WO 98/50347.

Other antiretroviral agents include hydroxycarbamide sample compound such as BCX-34 (purine nucleoside phosphorylase, Biocryst), ribonucleotide reductase inhibitor such as DIDOX^TM(Molecules for Health), hypoxanthine monophosphate dehydrogenase (IMPDH) inhibitor such as VX-497 (Vertex) and mycophenolic acid such as CellCept (mycophenolates；Roche).

Other antiretroviral agents include inhibitor such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, RANTES and the soluble complex and AMD-3100 of aminoglucan (GAG) that the inhibitor of viral integrase enzyme, double (MIBK) compounds of inhibitor such as arlydene of viral genome nuclear translocation, HIV enter；Nucleocapsid zinc finger inhibitor such as dithiane compound；HIV Tat and Rev target spot；And drug enhancers such as ABT-378.

Other ARTs and auxiliary treatment include cell factor and lymphokine such as MIP-1 α, MIP-1 β, SDF-1 α, IL-2, PROLEUKIN^TM(Aldesleukin/L2-7001；Chiron), IL-4, IL-8, IL-10, IL-12 and IL-13；Interferon such as IFN-α 2a；TNF, NF κ B, GM-CSF, M-CSF and IL-10 antagonist；Regulate and control the medicine such as cyclosporin and metacortandracin of immune activation；Vaccine such as Remune^TM(HIV immunogenes), APL 400-003 (Apollon), restructuring gp120 and fragment, divalence (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2rgp120, gp120/ solubility CD4 compounds, Delta JR-FL albumen, branch's synthetic peptide from discrete gp120C3/C4 areas, fusion-complementation immunogene and Gag, Pol, Nef and Tat vaccine；Gene therapy such as heredity suppresses subcomponent (GSE；WO 98/54366) and Intrakine (intrakine) (target CC chemotactic factor (CF)s (Yang et al., PNAS94 that ER blocks the genetic modification of the CCR5 surface expression newly synthesized:11567-72(1997)；Chen et al.,Nat.Med.3:1110-16(1997)))；For example anti-CXCR4 antibody 12G5 of antibody, antibodies against CCR 5 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, anti-CD 4 antibodies Q4120 and RPA-T4, anti- CCR3 antibody 7B11, anti- gp120 antibody 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti- Tat antibody, anti-TNF-α antibody, with monoclonal antibody 33A, aromatic hydrocarbon (AH) receptor stimulating agent and antagonist such as TCDD, 3, 3 ', 4, 4 ', 5- pentachlorodiphenyls, 3, 3 ', 4, 4 '-tetrachloroethanes, with α-naphthoflavene (WO 98/30213), with antioxidant such as gamma-L-glutamine-cysteine ethyl ester (γ-GCE；WO99/56764).

In other embodiments, opportunistic infection treatment medicament administration immunomodulator can be combined.TRIMETHOPRIM-SULFAMETHOXAZOLE can be included but is not limited to the opportunistic infection treatment medicine of combined immunization conditioning agent^TM、DAPSONE^TM、PENTAMIDINE^TM、ATOVAQUONE^TM、ISONIAZID^TM、RIFAMPIN^TM、PYRAZINAMIDE^TM、ETHAMBUTOL^TM、RIFABUTIN^TM、CLARITHROMYCIN^TM、AZITHROMYCIN^TM、GANCICLOVIR^TM、FOSCARNET^TM、CIDOFOVIR^TM、FLUCONAZOLE^TM,ITRACONAZOLE^TM、KETOCONAZOLE^TM、ACYCLOVIR^TM、FAMCICOLVIR^TM、PYRIMETHAMINE^TM、LEUCOVORIN^TM、NEUPOGEN^TM(Filgrastim/G-CSF) and LEUKINE^TM(sargramostim/GM-CSF).In a specific embodiment, with immunomodulator and TRIMETHOPRIM-SULFAMETHOXAZOLE^TM、DAPSONE^TM、PENTAMIDINE^TM, and/or ATOVAQUONE^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic Pneumocystis carinii pneumonia infection.In another particular embodiment of the invention, with immunomodulator and ISONIAZID^TM、RIFAMPIN^TM、PYRAZINAMIDE^TM, and/or ETHAMBUTOL^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic mycobacterium avium compound infection.In another particular embodiment of the invention, with immunomodulator and RIFABUTIN^TM、CLARITHROMYCIN^TM, and/or AZITHROMYCIN^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic mycobacterium tuberculosis infection.In another particular embodiment of the invention, with immunomodulator and GANCICLOVIR^TM、FOSCARNET^TM, and/or CIDOFOVIR^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic cytomegalovirus infection.In another particular embodiment of the invention, with immunomodulator and FLUCONAZOLE^TM、ITRACONAZOLE^TM, and/or KETOCONAZOLE^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic fungal infection.In another particular embodiment of the invention, with immunomodulator and ACYCLOVIR^TMAnd/or FAMCICOLVIR^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic I types and/II herpes simplex virus types infection.In another particular embodiment of the invention, with immunomodulator and PYRIMETHAMINE^TMAnd/or LEUCOVORIN^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic toxoplasma gondii infection.In another particular embodiment of the invention, with immunomodulator and LEUCOVORIN^TMAnd/or NEUPOGEN^TMAny combination prophylactic treatment, prevention, and/or diagnosis opportunistic bacterium infection.

In a further embodiment, immunomodulator is applied in combination antiviral agent.The antivirotic that can be applied together with immunomodulator includes but is not limited to ACV, Ribavirin, amantadine and Rimantadine (remantidine).

In a further embodiment, combine antibiotic administration immunomodulator.The antibiotic that can be applied together with immunomodulator includes but is not limited to Amoxicillin, Aminoglycoside, beta-lactam (glycopeptide), beta-lactamase, clindamycin, chloramphenicol, cynnematin, Ciprofloxacin, Ciprofloxacin, erythromycin, fluoquinolone, macrolide, metronidazole, penicillin, quinolone, rifampin, streptomysin, sulfanilamide (SN), tetracycline, trimethoprim, trimethoprim-trimethoprim sulfate and vancomycin.

Furthermore it is possible to which individually or the other therapeutic schemes of joint apply immunomodulator, the therapeutic scheme includes but is not limited to radiotherapy.Can with order and/or synchronously apply these combined therapies.Kit

Present invention provides the medicinal bag or kit of the container including one or more components for being filled with one or more pharmaceutical compositions.Optionally, with these containers accompany can be government supervision department defined on medicine or biological product production, using or sale in terms of notice, the notice reflect for human body apply medicine or biological product production, using or sale superintendent office certification.Furthermore it is possible to using the polypeptide of the invention combined with other treatment compound.In a specific embodiment, kit contain government supervision department defined on medicine or biological product production, using or sale in terms of notice, the notice reflect for human body apply medicine or biological product production, using or sale superintendent office have authenticated its ANA titres be more than or equal to 1:80 and/or the application that is more than or equal in 30IU patient of blood plasma or serum Anti-hCG action.

Embodiment

Although having broadly described the present invention, the present invention will be better understood with reference to the following examples in those skilled in the art, and the purpose that the embodiment is merely illustrative herein is not as a limitation of the invention.

Embodiment 1：Using the summary of the clinical test results of antibody (Belimumab (Baily the monoclonal antibody)) systemic lupus erythematosus (SLE) for neutralizing Neutrokine- α albumen

One perspective, random, double blinding, placebo-controlled trial are research on adding Belimumab (antibody for neutralizing Neutrokine- α) treatments SLE on the basis of SLE standard cares.It has been selected in 449 altogether and has met ACR standards (Tan et al., Arthritis Rheum.25:1271-7,(1982)；With Hochberg et al., Arthritis Rheum.40:1725, (1997)), the medical history with measurable autoantibody and during screening SELENA SLEDAI scorings >=4 SLE objects.

The 0th, 14,28 days, then every 28 days intravenous study drug-administrations (1,4,10mg/kg Belimumab) or placebo, totally 52 weeks.It is made whether to continue the selection of 24 week extended period research to the object for completing treatment in 52 weeks.Belimumab is formulated in 10mM sodium citrates, 1.9% glycerine, 0.5% sucrose, 0.01% (w/v) polyoxyethylene sorbitan monoleate (pH6.5 ± 0.3).The object for receiving placebo also receives the preparation (10mM sodium citrates, 1.9% glycerine, 0.5% sucrose, 0.01% (w/v) polyoxyethylene sorbitan monoleate (pH6.5 ± 0.3)) without Belimumab.Every 1 to 2 months curative effect was evaluated with SELENASLEDAI (SS), SLE Flare Index, doctor's overall evaluation (PGA).Also the scoring of BILAG and SF-36 disease activities is regularly evaluated.SS scores when predetermined primary efficacy endpoint is 24 weeks decline percentage and in 52 weeks as SLE breaks out defined in index to duration of seizure.Biological marker includes ANA, Anti-hCG action (Ab), C3/C4, Ig isotype and surrounding B cell FACS.Every 1 to 2 months B cell was analyzed with 4 color FACS (CD19, CD20, CD27, CD69, CD38, CD138 and CD45).Autoantibodies level is obtained in a follow-up includes anti-dsDNA Ab, Ig isotype, total protein and albumin level.With likelihood ratio chi-square analysis, Wilcoxon inspections or the change of t- check analysis biological markers.

In our current research, the average age of object is 42 years old；Average SLE sick times in these objects are 8.8 years.Baseline Disease Activity level in these objects is at a relatively high, and the SS scorings close to 67% object are 8 points or higher (average SS scorings are 9.6).93% selected object of research is all women.70% object is white people；24% object is non-descendants American；3% object is Asia descendants；And 18% object is Hispanic (overlapping classification).98% object has the ANA of historical record positive, and 71.5% object be when selected ANA+ (during screening/the 0th day ANA titre>1:80 and/or anti-dsDNA Ab >=30IU/ml).Anti-dsDNA titre >=30IU/ml of 50% object when being selected in.The medicine of SLE treatments is most commonly used for during baseline includes following medicine：Steroids (close to 70% object), aminoquinoline (such as antimalarial) (70%), cox 2 inhibitor (28%), COX-1 inhibitor (26%), imuran (20%), methopterin (16%) and mycophenolate (16%).In baseline, the whole body glucocorticoid that the test group and placebo object for having 34% and 42% respectively receive clinical meaning dosage (is defined as>The metacortandracin of 7.5mg/ days or other glucocorticoids of dose,equivalent).Baseline between test group is characterized or completion rate aspect is all not significantly different (81% completes experiment).

Although primary efficacy endpoint is not reaching to statistically-significant difference, in ANA+ objects, SS scorings have been considerably reduced 29% (Fig. 1 is seen in p=0.0435) at the 52nd week.Using 24 weeks baselines, SLE breaking-out (log rank p=0.036) of the Belimumab objects during the 24 to 52nd week is reduced.Although BILAG combinations digit score is not observed (by the way that tract staged conversion is calculated into BILAG combinations into digit score in accordance with the following methods), but in ANA+ objects, two organ areas scoring through the Belimumab objects treated has less increase (skeletal muscle, p when finding the 52nd week to the analysis that 8 single organ areas are scored<0.008；Nerve, p<0.038) and three organ areas scoring oriented less increased trend (cardiovascular and breathing, p=0.06；Whole body, p=0.15；Kidney, p<0.15).To the 16th week (p=0.016) to the 52nd week (p<0.002；Gross activity is compared with placebo) when all improve PGA scoring.Therefore there is improvement, although the metacortandracin dosage of placebo is increased (increase to more than 7.5mg/ days, 15% pair 7%) compared with Belimumab treatment groups.In ANA+ objects, metacortandracin increase was just observed when the 8th week (from low dosage≤7.5mg/ days to high dose>7.5mg/ days) being remarkably decreased of frequency (p at the 8-12 weeks and during the 32 to 40th week<0.05).There is no the curative effect of dose response, illustrate that all dosage suffer from equal activity.Include adverse events (AE), AE seriousness, infection or laboratory toxicity in terms of security, clinically significant difference is all not observed in the comparison of all Belimumab and placebo.There is pleuritic less (3.3% couple of 8%, p in Belimumab group objects<0.05), there are nettle rash more (4% pairs 0%, p<0.05).Infusion reaction is rare, only reported 1 matters of aggravation.The immunogenicity to Belimumab is observed in 1 object (1mg/kg).

As shown in Table IX, the analysis at the 52nd week to ANA+ objects is found, for placebo, and Belimumab, which is treated, causes the notable stabilization of disease, as being determined such as BILAG indexes (the 3rd row) and PGA (the 4th row).In addition, also response rate in test of cure group is analyzed with combination response terminal (the 1st row), the endpoint composite scores the measured value of the measured value for the overall disease mobility being measured to and the measured value for the patient's overall status being measured to through PGA Disease Activity Index and the special organ's system lesion being measured to through BILAG grade forms through SS.If the SELENA SLEDAI scorings of patient reduce >=4 points, its PGA scorings (are defined as PGA scoring increases without deterioration<0.3 point) and any specific organ systems do not deteriorate and (be defined as without new BILAG A organ areas scoring or without 2 new BILAG B organ areas scorings), then patient is regarded as response in the treatment in terminal is combined.Using combination terminal recited above, the notable response (p=0.0058) to Belimumab is found to the analyses of ANA+ objects.

In addition, in ANA+ objects, significantly improving for PGA and SF-36 physical efficiencys component scoring (SF-36PCS) is just observed in treatment early stage.Compared with the object of ANA+ placebo treatments, the ANA+ objects treated through Belimumab most just showed baseline PGA change average percents when the 4th week and significantly improve (p<0.05).Compared with the object of ANA+ placebo treatments, the ANA+ objects treated through Belimumab also show baseline PGA changes the significantly improving of average percent numerical value (p when the 8th, 16 weeks and 48 weeks at the 8th week, 16 weeks, 48 weeks and 52 weeks<0.05；P at the 52nd week<0.01).Compared with ANA+ object placebo treatment groups, what average SF-36PCS also showed that quality of life of the ANA+ objects treated through Belimumab at the 12nd week, 24 weeks, 48 weeks and 52 weeks significantly improves (each time point p<0.05).

Substantially reducing and (changing percentage with middle position baseline number to represent) for the B cell number in Belimumab treatment targets is observed during whole research, including (each measured value taken during the 8 to 52nd week is p to CD19+B cells<0.01), activating B cell (CD20+/CD69+；The each measured value taken during the 8 to 52nd week is p<0.01), (CD20+/CD27-, each measured value taken during the 8 to 52nd week is p to pure B cell<And Plasmacytoid B cell (CD20+/CD138+ 0.01)；The each measured value taken during the 16 to 52nd week is p<0.01).Measurement at the 24th week to B cell number is confirmed, compared with the object of placebo treatment, B cell when Belimumab (collecting for all treatments) is significantly reduced the 24th week.At the 24th week, it was observed that being remarkably decreased of cell number (changing percentage with middle position baseline number to represent), including CD19+B cells, activating B cell (CD20+/CD69+), pure B cell (CD20+/CD27-) and Plasmacytoid B cell (CD20+/CD 138+).B cell (median) when Belimumab (being combined with all treatments) is significantly reduced the 52nd week.At the 52nd week, the change percentage for combining the middle position CD20+B cells of all treatment groups was 54%*, just observed when the 8th week and is remarkably decreased (p<0.0001).At the 52nd week, the change percentage for combining the middle position Plasmacytoid B cell (CD20+/CD138+) of all treatment groups was 62%*.At the 52nd week, middle position activating B cell (CD20+/CD69+B cells) the change percentage for combining all treatment groups was that (* is p to 70%*<0.02).At the 52nd week, CD19+B cells and pure B cell (CD20+/CD27-) are significantly reduced, while remaining memory cell group.On the contrary, in Belimumab treatment targets, thick liquid cell at the 52nd week adds 72.5% than baseline (2.7%), and there was only 30.6% (p=0.02) in placebo/standard care group.In addition, the reduction of the B cell number of Belimumab inductions has been continued until the 76th week.At the 76th week, the change percentage for combining the middle position CD20+B cells of all treatment groups was 61%.At the 76th week, the change percentage for combining the middle position Plasmacytoid B cell (CD20+/CD138+) of all treatment groups was 60%.At the 76th week, middle position activating B cell (CD20+/CD69+B cells) the change percentage for combining all treatment groups was 84%.In the comparison of Belimumab treatment targets and placebo treatment object, (each measured value obtained during the 4 to 12nd week is p to the anti-dsDNA titre (changing percentage with middle position baseline to represent) that just observed when Belimumab significantly reduces selected in anti-dsDNA titre >=30IU/ml subject early in the 4th week<0.01；The each measured value obtained during the 16 to 24th week is p<0.03；The each measured value obtained during the 32 to 52nd week is p<0.01).At the 52nd week, Belimumab reduced the (p of anti-dsDNA Ab 30%<0.002, baseline is positive), and placebo is 9%.The effect is kept, because the measured value of the 76th week shows that anti-dsDNA Ab reduces 28%.Compared with the control of placebo treatment, in Belimumab treatment targets, just confirmed that the serum IgG of Belimumab inductions, the significantly reducing of IgA, IgE and IgM level (change percentage to represent) (p with middle position baseline when the 8th week<0.0001).At the 52nd week, serum IgG, IgA, IgE and IgM level (being 10%, 14%, 34% and 29% respectively) are reduced.The reduction has continueed to the 76th week (being 12%, 15%, 35% and 34% respectively).In addition, the object that Ig isotype levels increase during for those baselines, 41% object for receiving Belimumab treatments (52/128, p=0.0014) return to normal Ig isotypes level, and only 16% (7/45) control object has returned to normal level.In the patient of the low C4 level of complement of baseline in Belimumab treatment groups, dramatically increasing and (changing percentage with middle position baseline to represent) for C4 level of complement is all observed for all measured values obtained during the 4 to 52nd week.At the 52nd week, the C4 in Belimumab treatment groups added 33% (p=0.0126, low baseline C4 levels).Belimumab effects are kept again, and C4 during Belimumab treatment groups the 76th week has brought up to 46%.At the 52nd week, compared with placebo (3.5%, 2/58), anti-dsDNA+object that 14.5% (24/165) receives Belimumab was transformed to negative (p=0.012).At the 76th week, there are 3 anti-dsDNA+objects for receiving Belimumab to be transformed to feminine gender again.

Belimumab is resistant to very well, and shows significant biological activity.Belimumab improves PGA scorings, reduces B cell number, add C4, reduce anti-dsDNA, and reduction/normalization Ig isotype levels.Belimumab delays the generation of the breaking-out after 6 months.When being selected in object positive ANA, SS scorings when considerably improving the 52nd week.Finally, combination response terminal shows the notable response (being shown in Table IX) that ANA+ objects are treated to Belimumab.

Table ix：Response rate of the ANA+ objects at the 52nd week

^aThe likelihood ratio test of paired comparisons between gross activity and placebo of the P values from combination.

Embodiment 2：SELENA SLEDAI albuminuria scores

Renal insufficiency is usually related to systemic loupus erythematosus.Those skilled in the art know a variety of standard methods that can be used for evaluating renal function, for example, advance to ESRD time, serum creatinine continual multiplication time, CrCl, the sour clearance rate of opacin, the protein concentration in single urine sample and the protein concentration in twenty-four-hour urine liquid sample.

The albuminuria change calculated from " twenty-four-hour urine liquid sample " is one of project that SELENA SLEDAI are scored.Albuminuria measurement can be carried out with any known method in this area.In a specific embodiment, single urine sample is collected, and protein content and/or CrCl is determined, see such as Lemann, et al., Clin Chem., 33:297-9,1987 and Schwab, et al., Arch Intern Med., May;147(5):943-4,1987.In a specific embodiment, the urine of 24 hours is collected, and determines protein content and/or CrCl.In a specific embodiment, single urine sample is collected, the ratio of protein content and CrCl amount is determined, the protein content in twenty-four-hour urine liquid sample is estimated with the ratio, see such as Ruggenenti, et al., BMJ.316 (7130):504-9,1998.Therefore, in this embodiment, " twenty-four-hour urine liquid sample " can represent the albumen grams in the urine based on twenty-four-hour urine liquid sample or the estimate to the albumen grams in twenty-four-hour urine liquid sample.Estimation to the albumen grams in twenty-four-hour urine liquid sample can be according to the protein content in such as single urine sample and the ratio of the CrCl amount in single urine sample.

In the SELENA SLEDAI points-scoring systems of standard, show emerging albuminuria or recent albuminuria increase (cause albuminuria numeric ratio in nearest twenty-four-hour urine liquid sample close to previous twenty-four-hour urine liquid sample in the albuminuria numerical value that is measured to be higher by least 0.5g) patient be designated as 4 points of albuminuria in delivered SELENA SLEDAI grade forms, see such as Bombardier, et al., Arthritis Rheum.Jun;35(6):630-40,1992.Therefore, under the SELENASLEDAI points-scoring systems of standard, as long as the albuminuria in twenty-four-hour urine liquid sample does not continue to increase>0.5g, then SELENA SLEDAI improvement just occurs in the object that baseline is designated as 4 points of albuminuria in subsequent follow-up (i.e. even when being faced with stable albuminuria or albuminuria increase≤0.5g/24, the total score of patient can subtract 4 points).

Modification to SELENA SLEDAI albuminuria code of points is described below.As the SELENA SLEDAI points-scoring systems of standard, show emerging albuminuria or recent albuminuria increase (cause albuminuria numeric ratio in nearest twenty-four-hour urine liquid sample close to previous twenty-four-hour urine liquid sample in the albuminuria numerical value that is measured to be higher by least 0.5g) patient be designated as 4 points of albuminuria.If the albuminuria numerical value of patient does not improve (i.e. in addition, albuminuria in current twenty-four-hour urine liquid sample not than close to previous twenty-four-hour urine liquid sample in the albuminuria numerical value that is measured to reduce at least 0.5g), then patient continues to be designated as 4 points of albuminuria.But if the albuminuria numerical value of patient be improved (albuminuria in i.e. current twenty-four-hour urine liquid sample than close to previous twenty-four-hour urine liquid sample in the albuminuria numerical value that is measured to reduce at least 0.5g), then patient is designated as 0 point of albuminuria.

In a specific embodiment, close to previous albuminuria measured value be before current measurement≤26 weeks in twenty-four-hour urine liquid sample in obtained measured value.

Embodiment 3：Using the summary of the clinical test results for antibody (Belimumab) rheumatoid arthritis (SLE) for neutralizing Neutrokine- α albumen

2 phases, multicenter, random, double blinding, placebo-controlled study have been carried out in the object with RA.Object is randomly divided into 4 treatment groups (placebo, 1mg/kg, 4mg/kg and 10mg/kg).The 0th, 14,28 days, then apply within every 28 days 1,4,10mg/kgBelimumab or placebo, totally 24 weeks.It is 24 weeks optional extended peroids afterwards.Belimumab is formulated in 10mM sodium citrates, 1.9% glycerine, 0.5% sucrose, 0.01% (w/v) polyoxyethylene sorbitan monoleate (pH6.5 ± 0.3).The object for receiving placebo also receives the preparation (10mM sodium citrates, 1.9% glycerine, 0.5% sucrose, 0.01% (w/v) polyoxyethylene sorbitan monoleate (pH6.5 ± 0.3)) without Belimumab.283 objects have participated in the research altogether.1,4 or 10mg/kg Belimumab are application of to 214 objects in the research treatment phase of 24 weeks.99 objects receive placebo.

The more excellent ACR20 responses of statistics are all obtained in 1mg/kg treatment groups (p=0.0097) and all active treatment groups (p=0.0213) of combination.ACR20 is American society of rheumatism opening for evaluating index of the patient to the response for the treatment of rheumatoid arthritis.ACR20 responses are defined as in addition to 3 evaluations that 5 other symptom or disease performances are evaluated in (i.e. Patient Pain Assessment, patient's overall evaluation, the physical efficiency overall evaluation, the disabled of patient's self-assessment, acute phase reactant (ESR or CRP)) improve at least 20%, and tenderness joint number and swollen joint number at least reduce 20%.In addition, after the adjustment of the multiple comparisons carried out using Bonferroni closed methods, the result of 1mg/kg treatment groups is still statistically significant (p<0.0166).As SLE objects, Belimumab is related to the raising of the ACR20 responses in the object that the object and baseline c reactive protein (CRP) of baseline autoantibodies disease (rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP)) are positive.It observed biological activity, including statistically significant CD20+B cells, pure B cell, activating B cell and RF reduction；Memory cell increasing in treatment first month and the slow reduction during continued treatment.The Belimumab of all dosage can be resistant to well.In our current research, the dose-effect relationship of curative effect, security or biological marker effect is not obvious.The continued treatment of research extended peroid can be resistant to well.At the 48th week, ACR20 responses added nearly 40%.With continuing for treatment, increase and maintain the effect to biological marker, while memory cell number continues to be reduced to baseline values.Serum-concentration in this research is that within the scope of being arrived according to 1 issue desired by, the exposure of associated medicine (such as methopterin, leflunomide or HCQ) to Belimumab all has no significant effect.

Embodiment 4：Neutrokine- α extend the B cell life-span by two independent signal transduction paths

Neutrokine- α are also referred to as the key factor (Rolink that BLyS (bone-marrow-derived lymphocyte stimulant), BAFF, TALL-1, THANK, TNFSF13B and zTNF4 are the survivals of stagnant ambient bone-marrow-derived lymphocyte, A.G., and Melchers, F. (2002) .Curr Opin Immunol 14,266-275).The discovery that the Neutrokine- α deficient mices generated by lacking or introducing soluble decoy acceptor through target gene have the major defect of marginal belt and follicular B cells (main ripe periphery B cell group) has confirmed importance (Grosss of the Neutrokine- α in terms of pure B cell homeostasis well, J.A., et al. (2001) .Immunity 15,289-302；Schiemann,B.,et al.(2001).Science293,2111-2114).On the contrary, the ectopic expression for the Neutrokine- α that transgenosis is caused has been obviously prolonged the life-span of folliculus and marginal belt periphery B cell, and do not influence the development B cell (Mackay in T cell, B1 cells, early stage (T1) transition periphery B cell or marrow, F., et al (2003) .Annu Rev Immunol 21,231-264).For the maintenance of a variety of B cell tumours, necessary to Neutrokine- α are also, the Neutrokine- α of imbalance are stimulated and have been saved autoreactivity B cell, so that it avoids being lacked, generation (the Kalled of autoantibody is promoted accordingly, S.L. (2005) .Immunol Rev 204,43-54).Therefore, Neutrokine- α serve key effect in terms of normal B cells and the cell homeostasis of pathogenic B cell.The present embodiment describe in detail the result of carried out experiment, to understand the mechanism that Neutrokine- α promote B cell survival.

Testing program

Mouse：From the Pim-1 of New York Paul Rothman Columbia universities^+/-2^+/-Pim-1 is generated in strain^+/+2^+/+、Pim-1^-/-2^+/+、Pim-1^+/+2^-/-And Pim-1^-/-2^-/-Mouse.C57BL/6(B6)；Jackson laboratories from Bar Harbor ME or from National Cancer Institute Production Program, NCI-Fredrick, Fredrick MD.According to research institute's animal feeding and using committee's guide is by animal feeding and maintains in the University of Pennsylvania, Harvard Medical School or Massachusetts medical college.

B cell is purified：Handled with anti-thy1.2 and to the complement of splenocyte and collect cell to obtain splenic B cells then by using the gradient-purified remaining B cells of progressively percoll and at 60%-70% interfaces.In some experiments, by positive selection and with biotinylated anti-CD23 antibody (BDBiosciences-Pharmingen, San Diego CA) and microballon (Miltenyi Biotec, Auburn CA) the Magneto separate Spleen cell suspensions of streptavidin coating obtain CD23+B cells.CD23+B cells are not by Percoll size selections, because internal environment activation causes CD23 loss.It is by antibody and the Percoll B cell prepared>90%B220+, and CD23+B cells are>95% purity.

Cell culture：By the B cell of purifying or CD23+B cell culture in added with the RPMI-1640 culture mediums of 2 mercapto ethanol, MEM- nonessential amino acid, glutamine, penicillin and streptomysin (complete medium, CM).For B cell survival and other detections, the recombined human Neutrokine- α prepared using 50-100ng/ml in Human Genome Sciences, Rockville MD.Randolph Noelle doctors of the people Neutrokine- α of FLAG labels from Dartmouth Medical School.Mouse Neutrokine- α are purchased from Alexis Biochemicals, San Diego CA；Interferon-' alpha ' (IFN-α) is purchased from PBL Biomedical, Piscataway, NJ.The final concentration of 50nM rapamycin from the mother liquor for being dissolved in methanol is added into culture.In the experiment using rapamycin, control B cell is handled as control vector with methanol.For kinetic measurement, B cell is prepared and in 4 °C of cool overnights.Then each sample 5-6x 10 is pressed⁶Individual B cell adds it to anti-FLAG M2 antibody (Sigma) coating of 5 micrograms/ml monoclonals, washed through 1%BSA PBS, closing, then 1 hour is added in 24 orifice plates per the people Neutrokine- α of hole 2ug FLAG labels before washing and adding cell.The control B cell not stimulated is the B cell only treated through anti-FLAG antibody that those are added in hole.B cell has also been activated by the incubation of anti-mouse IgM (5 micrograms/ml), anti-CD40 (0.5 microgram/ml) or 100ng/ml recombined human Neutrokine- α with being added into kinetic measurement buffer solution (Hank balanced salt solutions add 2%BSA).

Antibody and western blot method：The anti-Pim2 of mouse (1D12), Pim1 (19F7), goat anti-actin (1-19), anti-mouse Ig, anti-rabbit Ig and the anti-goat Ig antibody that is combined with HRP both are from Santa Cruz Biotechnology, Santa Cruz CA.Rabbit-anti phosphoserine 473Akt, phosphothreonine 389p70S6 kinases, phosphothreonine 24/32FKHR/FKRHL1, phosphoGSK3, GSK, p70S6K, FKHR and Akt are purchased from Cell Signaling, Beverly MA.Rabbit anti-mouse Mcl-1 is purchased from Rockland, Wilmington MA.By washing B cell in ice-cold PBS and being cracked added with protease inhibitors (minitab, Roche, Indianapolis IN) and phosphatase inhibitor cocktail I and II (Sigma) RIPA (150mM NaCl, 1%NP-40,0.5% NaTDC, 0.1%SDS, 50mM Tris, pH8.0) in prepare full cell pyrolysis liquid.By 10-50ug protein dissolutions on 4-12%NuPage bis-tris polyacrylamide gels (Invitrogen, Carlsbad CA), and it is transferred into NC Nitroncellulose.Trace is closed with 3%BSA (Sigma, no IgG), 0.2%Tween-20PBS, and with being incubated overnight under 4 °C of first antibody in same buffer.Trace is washed with PBS-0.2%Tween-20, and with being conjugated with HRP secondary antibody incubation, is developed with ECLplus (Amersham Bioscience, Piscataway NJ).Probe again is carried out by being incubated to peel off trace in 20 minutes under in the PBS added with 1%SDS and 100uM mercaptoethanols 65 °C.Then as above wash and close trace.

Survival detection：By 5x 10 under 37 °C of CM⁶The culture of/ml B cells is in 24 plate culture dishes.The combination of 50-100ng/ml Neutrokine- α, 50nM rapamycin, 200U people IFN or these reagents is added into B cell.1 hour before with experiment additive culture, with 50nM rapamycins or Vehicle element B cell, fresh rapamycin is added after 48 hours are cultivated.Expect blue exclusive method counting survivaling cell to monitor survival rate by using tongue daily, it is each to determine in triplicate.

As a result

The studying for mechanism for how promoting Neutrokine- α B cell to survive finds that Neutrokine- α have activated the Akt/mTOR approach in B cell.With 100ng/ml recombined humans or mouse Neutrokine- α or anti-Ig (positive control) 37 °C, Secondary Culture base moderate stimulation purified

splenic B cell

0,5,20,60 or 120 minutes.Lysate is prepared from ice-cold sample, and is analyzed with western blot method.As the serine 473 by Akt and the increase of the phosphorylation of threonine 308 are determined, this stimulations of the restructuring Neutrokine- α to primary B cell causes the activation of Akt approach.Wherein show that Akt has been activated in itself with Neutrokine- α, the solubility Neutrokine- α (100ng/ml) of FLAG labels or the additional testing of the anti-CD40 of 0.5 microgram/ml (positive control) stimulation purified splenic B cells of hardened conjunction, as shown in Akt substrate GSK β and upstream the runner factor FOXO1 and FOXO3a phosphorylation.MTOR is the main downstream effect things of Akt.MTOR in the primary B cell of post activation of Neutrokine- α stimulations is also showed that by the phosphorylations of mTOR substrates (p70S6 kinases and Translational repression thing 4E-BP1).Western blot method have studied modified phoshorylation pattern.

Rapamycin is mTOR potent inhibitor and B cell proliferation and the high inhibition element of differentiation.The small resting B cell from Normal donor is cultivated in the case where being with or without 100ng/ml Neutrokine- α, carrier or 50nM rapamycins, the rapamycin is used for pretreatment B cell before cultivating, it is introduced directly into culture, and was added again once every 2 days after culture is started.Number of viable cells was determined at the 4th day.The co-cultivation of total B or CD23+B cells and Neutrokine- α and rapamycin is without the alpha mediated survival facilitations of Neutrokine- are prevented, as the number of viable cells occurred for the 4th day after incubation is determined.This result shows that in the B cell that Neutrokine- α are treated another survival routes may have been activated.

Pims is the kinase families of three kinds of serine/threonine; it provides the apoptosis protective effect of resistance to rapamycin; the apoptosis protective effect is that a variety of activator induce (Fox, C.J., et al. in hematopoietic cell; (2003) .Genes Dev 17; 1841-1854 and Fox, C.J., et al.; (2005) .J Exp Med 201,259-266).Western blot is shown in after 100ng/ml Neutrokine- α processing 2 days, and primary B cell has raised Pim1 and Pim2 expression.

In order to study whether Pim1 and 2 is related to the alpha mediated B cell survivals of Neutrokine-, wild type or Pim 1 will be come from^-/+2^-/+Heterozygote, Pim1^-/-2^-/-Double defect or the defects of Pim 2 (Pim 1^+/-2^-/-) CD23+B cells in donor and carrier, 100ng/ml rhuNeutrokine- α and be with or without 50nM rapamycins and cultivated in CM 4 days.All expect that blue exclusive method determines viability with tongue daily.It is interesting that when exposed to Neutrokine- α, the B cell (Pim-1 from Pim1 and Pim2 double defect mouse^-/-2^-/-B cell) survival enhancing is shown really.If mTOR and Pim1 and 2 is operated in different signal transduction paths, each approach has mediated Neutrokine- α to the facilitation of cell survival, and this can just explain the above results.In order to check whether that the approach for being related to two separation is theoretical, the rapamycin Pim-1 alpha mediated to Neutrokine- checked^-/-2^-/-The effect of B cell survival.The addition of rapamycin eliminates Neutrokine- α enhancings Pim-1^-/-2^-/-The ability of B cell survival in B cell.Further research shows Pim-1^+/-2^-/-B cell (only Pim2 functional defects) and Pim-1^-/-2^-/-B cell illustrates that Pim1 is not necessary for Neutrokine- α to the effect that B cell is survived equally to rapamycin sensitive.Sum it up, these data displays have two independent approach to mediate the existence that Neutrokine- is alpha mediated, every approach is all individually enough to mediate Neutrokine- α this activity.

Further tests showed that effective enhancing (anti-apoptotic protection induction) of B cell survival needs the expression of Mcl-1, Bcl-2 family member, and the member serves effect in terms of periphery B and T cell homeostasis is promoted (data are not shown).

It therefore, it can simulate the effect that Neutrokine- alpha-2 antagonists are induced with inhibitor (such as rapamycin) and the composition of the inhibitor of Pim2 approach including akt/mTOR approach.Including the inhibitor (such as rapamycin) of Akt/mTOR approach and the composition of inhibitor of Pim2 approach are used as suppressing B cell survival or treat the Neutrokine- alpha-2 antagonists of one or more diseases described herein or lesion therefore,.For example, the B cell life-span can be shortened with the inhibitor (such as rapamycin) and the composition of the inhibitor of Pim2 approach for including akt/mTOR approach.Furthermore it is possible to use inhibitor (such as rapamycin) and the composition treatment autoimmunity disease of the inhibitor of Pim2 approach including akt/mTOR approach.In a particular embodiment, the autoimmunity disease that can be mediated with the inhibitor (such as rapamycin) and the composition treatment B cell of the inhibitor of Pim2 approach for including akt/mTOR approach.In other specific embodiments, the popular autoimmunity disease of the composition treatment wherein autoantibody for the inhibitor (such as rapamycin) and the inhibitor of Pim2 approach for including akt/mTOR approach can be used.In a particular embodiment, the inhibitor (such as rapamycin) and the composition treatment rheumatoid arthritis of the inhibitor of Pim2 approach that include akt/mTOR approach, systemic loupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren syndrome, type 1 diabetes, ITP, actue infectious polyradiculoneuritis, Hashimoto thyroiditis or Grave diseases can be used.

Furthermore it is possible to simulate the effect that Neutrokine- alpha-2 antagonists are induced with the composition including Mcl-1 inhibitor.Including the composition of Mcl-1 inhibitor is used as suppressing B cell survival or treats the Neutrokine- alpha-2 antagonists of one or more diseases described herein or lesion therefore,.For example, the B cell life-span can be shortened with the composition for including Mcl-1 inhibitor.Furthermore it is possible to including the composition treatment autoimmunity disease of Mcl-1 inhibitor.The autoimmunity disease that can be mediated with the composition treatment B cell for including Mcl-1 inhibitor.In other specific embodiments, the popular autoimmunity disease of the composition treatment wherein autoantibody for including Mcl-1 inhibitor can be used.In a particular embodiment, can be sick with the composition treatment rheumatoid arthritis, systemic loupus erythematosus, multiple sclerosis, myasthenia gravis, Sjogren syndrome, type 1 diabetes, ITP, actue infectious polyradiculoneuritis, Hashimoto thyroiditis or the Grave that include Mcl-1 inhibitor.Embodiment 5：The sign of antibody preparation

The heat endurance of the antibody in each preparation is evaluated with the analysis of the 1mg/ml IgG/ λ antibody by differential scanning calorimetry to preparation in 10mM histidines and 10mM citrate buffer solutions.It is to be specific to Neutrokine- α and can neutralize the IgG1/ λ antibody of Neutrokine- alpha actives for the distinct antibodies in this research.Analysis finds melting temperature highest of the pH scopes for two kinds of buffer solutions in 6.0-7.5, and higher melting temperature typicallys represent higher heat endurance.Within the scope of this pH, the melting temperature of citrate buffer solution is higher than histidine buffering liquid 2 °C, illustrates that citrate buffer solution may generate more stable antibody preparation.But, the antibody in histidine buffering liquid has higher thermal reversibility than the antibody in citrate buffer solution.This shows that antibody has higher biophysics stability in histidine, although it has lower melting temperature.This point is confirmed to the stability study of antibody preparation, research finds that 10mM histidines cause less aggregation than 10mM citric acid when being stored 18 months under 2-8 °C.During stability study, the buffer capacity of two kinds of buffer solutions is evaluated with pH measure is repeated.In addition to higher biophysics stability being provided to antibody, pH6.0-6.5 histidine shows to provide bigger buffer capacity for 6.5-7.0 citric acid than pH scope.In 18 months stability studies, histidine remains stable pH value at all temperature (2-8 °C, 25 °C and 40 °C) tested.On the contrary, lemon acid supplement has wider array of variation at higher temperatures (data are not shown).

Embodiment 6：The Journal of Sex Research steady in a long-term of antibody preparation

In order to determine the shelf life of antibody preparation, the Journal of Sex Research steady in a long-term of the 100mg/ml antibody to preparation in 10mM histidines, 150mM NaCl, 0.01% (w/v) polyoxyethylene sorbitan monoleate (pH 6.0) has been carried out.It is to be specific to Neutrokine- α and can neutralize the IgG1/ λ antibody of Neutrokine- alpha actives for the distinct antibodies in this research.2ml antibody preparations in 5ml vials are uprightly deposited 24 months at -80 °C, 2-8 °C, 25 °C and 40 °C.With the sample for being stored in -80 °C as control, shelf life, and any possible degradation pathway occurred with the sample monitoring being stored under acceleration environment (25 °C and 40 °C) are determined with the sample for being stored in 2-8 °C.In 24 months, regularly analyze sample with multiple detection methods, including visually observe, pH value, concentration, SDS-PAGE, SEC-HPLC, ion exchange HPLC (IE-HPLC), bioassay method, capillary isoelectric focusing (cIEF), peptide mapping, RP-HPLC and

The result that three all methods are obtained, which is in fact suitable, to be found to the analysis of the sample in 2-8 °C and -80 °C storage 24 months through SEC-HPLC, IE-HPLC and RP-HPLC, the difference of very little has been only seen.The reduced rate of the SEC-HPLC purity of 2-8 °C of sample is every month approximate 0.03%, and the increment rate at the IE-HPLC peaks (most-likely due to desamidation) of early stage elution is every month 0.14% (data are not shown).Storage 24 months after, 2-8 °C of sample antibody aggregation (<1%), deamidation (~4%) and oxidation (1%) aspect value show seldom change.But all detections of the sample to storage under acceleration conditions all observed significant degraded.Include aggregation and fragmentation through the SEC-HPLC degradeds observed under acceleration conditions.IE-HPLC detections show the increase for accelerating the storage under price adjustment to cause early stage eluting peak.Deamidation and fragmentation when peptide mapping observed 25 °C；Deamidation, oxidation, fragmentation and aspartic acid when observed 40 °C to different aspartic acid rearrangement.Therefore, the IgG1/ λ antibody in 100mg/ml pharmaceutical preparations at least can be 24 months in 2-8 °C of storage-stable.

Embodiment 7：Determine the vitro detection method to the inhibitory action of Neutrokine- α-Neutrokine- α acceptor interactions

Describe below and can be used in determining the detection method whether compound is used as Neutrokine- alpha-2 antagonists.Specifically, it is that compound suppresses the ability that solubility Neutrokine- α are combined with the homoreceptor on its IM9 cell that this detection method, which is determined,.

Biotinylation Neutrokine- α preparation

Using slide-a-lyzer boxes (Pierce), dialyse 100 microgram people under 4 °C with 50mM sodium acid carbonates (sodium acid carbonate) or mouse Neutrokine- α are stayed overnight.Next day, NHS- biotins (Pierce) are dissolved in DMSO, concentration is 13.3mg/ml.It is then added into Neutrokine- α, biotin and Neutrokine- α mol ratio are 20：1, mix and be incubated 2 hours on ice.Then, using slide-a-lyzer frames, the biotinylated Neutrokine- α that dialysed with sterile PBS (Sigma) under 4 °C are stayed overnight.Combined using acceptor and suppress the biological activity that detection method (see below) verifies biotinylated Neutrokine- α.

The culture of IM9 cells

IM9 cells are the human B lymphocyte systems for expressing Neutrokine- α acceptors.By IM9 cell maintenances in added with 4mM Glus, 10%FCS, 10U penicillin, the RPMI-1640 of 100g/ml streptomysins (all formulations both are from Sigma).Melt from the mother liquor of freezing and obtain these cells, and when its density reaches 4-8x 10 after cultivating 5 days⁵During/ml, detection is used it for.

Acceptor, which is combined, suppresses detection method

With 100 microlitre 1 of every hole:PLL (Sigma) PBS solutions coating flat 1 hour of 96 orifice plate (Costar) at room temperature of 10 dilutions.Then plate is washed with water twice, allows that it is air-dried, and in 4 °C of left overnights.Then 100 microlitres of IM9 cells (10 are added into each hole⁶/ ml RPMI-1640 culture mediums).Then plate is centrifuged 5 minutes under 3200rpm, so as to sedimentation cell.Culture medium is carefully pumped out, 200 microlitres of MPBS (PBS of the confining liquid containing 3%Marvel) are added into each hole.Then allow that plate is closed 1 hour at room temperature.

In 96 orifice plates of a separation, 10 microlitres of biotinylated Neutrokine- α (162.5ng/ml) MPBS solution, final concentration of 25ng/ml are added into each hole.55 microlitres of various test compounds are added into each hole.Final volume in each hole is 65 microlitres.Preferably, test compound is also diluted in MPBS.Then plate is incubated at room temperature 30 minutes.

The plate that IM9 coatings are washed with PBS twice, gently beats plate, and is added immediately 50 microlitres of bacteriophages/biotinylation Neutrokine- α mixtures, and is incubated 1 hour at room temperature.Plate is washed three times in PBST, is washed three times in PBS, gently beats plate, 50 microlitres are added into each hole and presses 1:1000 are diluted in the streptavidin-Delfia (Wallac) in the detection buffer solution of business men.Then plate is incubated to 1 hour at room temperature, is washed 6 times with Delfia wash solutions (Wallac).After plate plate is gently beaten, add per 100 microlitres of hole Delfia enhancing solution (Wallac).Plate is lightly beaten, to contribute to particulate to be formed, is incubated 10 minutes at room temperature, the fluorescence at 6520nm is read with the work stations of Wallac 1420.

The appropriate control that this detection is included is to only include biotinylation Neutrokine- α to confirm the biotinylation Neutrokine- α in this detection with the sample of the maximum combined of its acceptor and not comprising biotinylation Neutrokine- α to confirm the sample of the background signal in this detection.Other useful controls are that non-Neutrokine- α are special or " unrelated " compound is (similar to test compound structure but believe the compound that it will not be with Neutrokine- α or one of which Neutrokine- α acceptor interactions.If test compound is the anti-Neutrokine- Alpha antibodies of IgG1 isotypes, suitable " unrelated control " is another non-specific in Neutrokine- α or the IgG1 antibody of one of which acceptor.

Embodiment 8：Human B cell proliferation assay for in-vitro screening Neutrokine- α antagonistic molecules

Neutrokine- α, responsive cell and the hypothesis antagonist (every kind of be all prepared to 3X mother liquor) isometric by mixing carries out the biological detection for evaluating the effect for assuming Neutrokine- alpha-2 antagonists in 96 orifice plates, and each detection is repeated 3 times.

Bone-marrow-derived lymphocyte is purified into from human tonsil with MACS (AntiCD3 McAb is deprived), washing, and it is resuspended in complete medium (CM) (RPMI1640 and penicillin containing 100U/ml, 100 μ g/ml streptomysins, 4mM glutamine, 5x 10^-5The 10%FBS of M beta -mercaptoethanols), cell concentration is every milliliter of 3x 10⁶Individual cell.To intracellular addition 3X concentration (the 1 of 3X=mother liquor:33,333 dilutions) staphylococcus aureus CowanI (SAC, CalBiochem).

Meanwhile, the dilution (3 times) of 8 serial potential antagonists is prepared with CM so that the antagonist of dilution is the 3X of test final concentration in this detection.For example, by final concentration of 10 micrograms of the starting of the antibody of conventionally test/mL, being diluted to about 1.5ng/mL.

People r Neutrokine- α are prepared with CM so that its concentration in CM is 3X (3X=300ng/mL, 30ng/mL and 3ng/mL).With the potential antagonist of Neutrokine- α conventionally tests of some concentration, to avoid due to the false negative result that unpredictable low-affinity or Antagonist concentration are caused.

Then the antagonist and 50 microlitres of Neutrokine- α diluted of 50 microlitres of dilutions are added into the hole containing 50 microlitres of cell mixtures.

Then cell is incubated to 72 hours (37 °C, 5%CO in very moist incubator₂).After 72 hours, to 0.5 μ Ci/ holes of intracellular addition³H- thymidines (6.7Ci/mmol), and 24 hours are incubated again.With Tomtec cell harvester plates, the cell number in filter is counted out with TopCount Scintillation counters (Packard).

The appropriate control that this detection is included is wherein maximum in this detection to confirm without antagonist³The sample of H- thymidines blending and without Neutrokine- α to confirm the sample of the background signal in this detection.Other useful controls are that non-Neutrokine- α are special or " unrelated " compound is (similar to test compound structure but believe the compound that it will not be with Neutrokine- α or one of which Neutrokine- α acceptor interactions.If test compound is the anti-Neutrokine- Alpha antibodies of IgG1 isotypes, suitable " unrelated control " is another non-specific in Neutrokine- α or the IgG1 antibody of one of which acceptor.

Those skilled in the art, which know, to modify this detection, for example, can be modified in terms of the order of step or the reagent of administration.As a particular embodiment, anti-IgM substitution SAC pretreatment B cells can be used.Those skilled in the art are it is also known that ability of other detection method test compounds as Neutrokine- alpha-2 antagonists can be used.

Embodiment 9：Mouse B cell for in-vitro screening Neutrokine- alpha-2 antagonists molecules breeds detection

In order to determine whether potential Neutrokine- alpha-2 antagonists can suppress the alpha mediated B cell proliferations of Neutrokine-, mouse spleen cell proliferation detection has been carried out.Briefly, by using 25g syringe needles and 10ml complete mediums (RPMI1640 and penicillin containing 100U/ml, 100 μ g/ml streptomysins, 4mM glutamine, 5x 10^-5The 10%FBS of M beta -mercaptoethanols) rinse spleen isolate mouse boosting cell.Cell flows through 100 micrometer nylon filter membranes, to remove cell mass.Then 25 minutes (1 15ml conical tubes/spleens of 400xg ficoll centrifuge cells suspension at room temperature；3ml ficol, 10ml cell suspending liquid/spleen；Ficol 1083 comes from Sigma).The cell being recovered to is washed 3 times in complete medium, and counted.Then by the cell being recovered in (3X=1 of SAC containing 3X:33,333 mother liquors dilute；Mother liquor is the suspension of 10% staphylococcus aureus (CowanI bacterial strains) from Calbiochem) complete medium in be diluted to concentration for 3x106/ml.

For each antibody, 50 microlitres of concentration are assigned in the single hole of 96 orifice plates for 30 μ g/ml, 3.0 μ g/ml and 0.3 μ g/ml antibody diluent, are repeated 3 times.Negative control is used as with the culture medium without antibody (and people's isotype controls (commercialization purchase), if desired).

It is 300ng/ml, 90ng/ml and 30ng/ml that Neutrokine- α albumen is diluted into concentration with complete medium.Then 50 microlitres of every kind of Neutrokine- α dilutions are added in the antibody diluent of plate series.Then by the plate containing antibody and Neutrokine- α dilutions in 37 °C, 5%CO₂It is lower to be incubated 30 minutes, backward each hole in 50 microlitres of Spleen cell suspensions containing SAC of addition.Then plate 72 hours (37 °C, 5%CO are incubated₂)。

After 72 hours, 50 μ l are added into each hole containing 0.5 μ Ci/ holes³The complete medium of H- thymidines (6.7Ci/mmol, Amersham), and cell is incubated to 24 hours (37 °C, 5%CO again₂).With Tomtec cell harvester plates, the cell number in filter is counted out with TopCount Scintillation counters (Packard).

Those skilled in the art, which know, to modify this detection, for example, can be modified in terms of the order of step or the reagent of administration.As specific example, anti-IgM substitution SAC pretreatment B cells can be used.Those skilled in the art are it is also known that ability of other detection method test compounds as Neutrokine- alpha-2 antagonists can be used.

It is evident that the present invention can be put into practice with the mode different from the special description in previous description and embodiment.All it is possible to a variety of modifications of the present invention and variation according to religious doctrine above, and is therefore within the scope of claims of annex.

All the elements of all publications (including patent, patent application, magazine article, laboratory manual, books or other documents) cited herein are incorporated into the application by quoting.

In addition, will be incorporated herein by quoting in the full content of the sequence table of this computer and paper form submitted.In addition, the full content (including specification, sequence table and accompanying drawing) of following each U.S.'s temporary and non-provisional patent application and international patent application is incorporated into the application by quoting herein：The U.S. Provisional Application 60/725 that on October 13rd, 2005 submits,625,60/735 submitted on November 14th, 2005,967,60/776 submitted for 27th for 2 months for 2006,664,60/781 submitted on March 13rd, 2006,387,60/787 submitted on March 31st, 2006,557,60/797 submitted on May 4th, 2006,360,60/814 submitted on June 20th, 2006,870,60/815 submitted on June 22nd, 2006,558,60/815 submitted on June 23rd, 2006,827,60/834 submitted on July 31st, 2006,150,60/725 submitted on October 13rd, 2005,626,60/735 submitted on November 14th, 2005,988,60/776 submitted for 27th for 2 months for 2006,665,60/797 submitted on May 4th, 2006,351,60/814 submitted on June 20th, 2006,869,60/815 submitted on June 22nd, 2006,559,60/834 submitted July 31 in 2006,152,60/725 submitted on October 13rd, 2005,627,60/735 submitted on November 14th, 2005,964,60/776 submitted for 27th for 2 months for 2006,658,60/725 submitted on October 13rd, 2005,629,60/735 submitted on November 14th, 2005,963,60/776 submitted for 27th for 2 months for 2006,660,60/725 submitted on October 13rd, 2005,628,60/735 submitted on November 14th, 2005,987,60/776 submitted for 27th for 2 months for 2006,659,60/543 submitted for 11st for 2 months for 2004,261,60/580 submitted on June 18th, 2004,387,60/617 submitted on October 12nd, 2004,191,60/368 submitted on April 1st, 2002,548,60/336 submitted on December 7th, 2001,726,60/331 submitted on November 16th, 2001,478,60/330 submitted on October 31st, 2001,835,60/329 submitted on October 18th, 2001,747,60/329 submitted with October 17th, 2001,508,August in 2000 submit within 15th 60/225,628,August in 2000 submit within 23rd 60/227,008,September in 2000 submit within 22nd 60/234,338,60/240 submitted on October 17th, 2000,806,60/250 submitted on November 30th, 2000,020,60/276 submitted on March 6th, 2001,248,60/293 submitted on May 25th, 2001,499,60/296 submitted on June 7th, 2001,122,60/304 submitted on July 13rd, 2001,809,60/122 submitted on March 2nd, 1999,388,60/124 submitted on March 12nd, 1999,097,60/126 submitted on March 26th, 2000,599,60/127 submitted on April 2nd, 1999,598,60/130 submitted on April 16th, 1999,412,60/130 submitted on April 23rd, 1999,696,60/131 submitted on April 27th, 1999,278,60/131 submitted on April 29th, 1999,673,60/136 submitted on May 28th, 1999,784,60/142 submitted on July 6th, 1999,659,60/145 submitted on July 27th, 1999,824,60/167 submitted on November 24th, 1999,239,60/168 submitted on December 3rd, 1999,624,60/171 submitted on December 16th, 1999,108,60/176 submitted on December 23rd, 1999,015,And on January 14th, 1997 submit 60/036,100；U.S.'s non-provisional application：11/054 submitted for 10th for 2 months for 2005, 539, 10/739 submitted on December 19th, 2003, 042, 10/735 submitted on December 16th, 2003, 865, 10/270 submitted on October 16th, 2002, 487, August in 2001 submit within 14th 09/929, 493, 09/588 submitted on June 8th, 2000, 947, 09/589 submitted on June 8th, 2000, 285, 09/589 submitted on June 8th, 2000, 286, 09/589 submitted on June 8th, 2000, 287, 09/589 submitted on June 8th, 2000, 288, 09/507 submitted for 22nd for 2 months for 2000, 968, 09/255 submitted for 23rd for 2 months for 1999, 09/005 submitted on January 12nd, 794 and 1998, 874；International patent application：The PCT/US96/17957 that PCT/US01/25549 that August in 2001 is submitted on the 15th, 25, PCT/US00/04336 and 1996 on the October submitted for 22nd for 2 months 2000 submit.

Claims

Purposes of the 1.Neutrokine- alpha-2 antagonists in the pharmaceutical composition for systemic lupus erythematosus (SLE) patient is prepared, the patient has ANA titre >=1 in its blood plasma or serum:80 or Anti-hCG action >=30IU/mL, wherein the antagonist is applied with therapeutically effective amount；

And wherein described antagonist is anti-Neutrokine- Alpha antibodies, it includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 amino acid sequence selected from the group being made up of the following：

(a)SEQ ID NO:13 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(b)SEQ ID NO:14 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(c)SEQ ID NO:15 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(d)SEQ ID NO:16 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(e)SEQ ID NO:17 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(f)SEQ ID NO:18 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(g)SEQ ID NO:19 VH CDR1, VH CDR2, VH CDR3, and SEQ IDNO:20 VL CDR1, VL CDR2 and VL CDR3；With

(h)SEQ ID NO:21VH CDR1, VH CDR2, VH CDR3, and SEQ IDNO:22 VL CDR1, VL CDR2 and VL CDR3.
Purposes of the 2.Neutrokine- alpha-2 antagonists in the pharmaceutical composition for systemic lupus erythematosus (SLE) patient is prepared, wherein it is determined that the patient has ANA titre >=1 in its blood plasma or serum:The antagonist is applied with therapeutically effective amount after 80 or Anti-hCG action >=30IU/mL；

Wherein described antagonist is anti-Neutrokine- Alpha antibodies, and it includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 amino acid sequence selected from the group being made up of the following：

(a)SEQ ID NO:13 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(b)SEQ ID NO:14 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(c)SEQ ID NO:15 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(d)SEQ ID NO:16 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(e)SEQ ID NO:17 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(f)SEQ ID NO:18 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(g)SEQ ID NO:19 VH CDR1, VH CDR2, VH CDR3, and SEQ IDNO:20 VL CDR1, VL CDR2 and VL CDR3；With

(h)SEQ ID NO:21VH CDR1, VH CDR2, VH CDR3, and SEQ IDNO:22 VL CDR1, VL CDR2 and VL CDR3.
3. the purposes of claim 2, wherein it is determined that patient there is at least one feature in the group being made up of the following after apply the Neutrokine- alpha-2 antagonists：

(a) SELENA SLEDAI scorings >=6；

(b) the C3 complement factor levels reduced in its blood plasma or serum；

(c) the C4 complement factor levels reduced in its blood plasma or serum；

(d) patient receives the metacortandracin of >=7.5 mg/days；With

(e) patient is receiving or was previously once receiving to be used to treat the related indication immunosuppressive therapy of lupus.
4.Neutrokine- alpha-2 antagonists are being prepared for reducing the purposes being administered in the pharmaceutical composition of the frequency of the glucocorticoid of Patients with SLE or amount, wherein the antagonist is applied into the patient with therapeutically effective amount.
5. the purposes of claim 4, wherein it is determined that patient there is at least one feature in the group being made up of the following after apply the Neutrokine- alpha-2 antagonists：

(a) ANA titre >=1:80；

(b) Anti-hCG action >=30IU/mL in its blood plasma or serum；

(c) SELENA SLEDAI scorings >=6；

(d) the C3 complement factor levels reduced in its blood plasma or serum；

(e) the C4 complement factor levels reduced in its blood plasma or serum；

(f) patient receives the metacortandracin of >=7.5 mg/days；With

(g) patient is receiving or was previously once receiving to be used to treat the related indication immunosuppressive therapy of lupus.
6. determine whether lupus patient replys the in-vitro method in drug therapy, including：

(a) SELENA SLEDAI, BILAG and PGA scoring of patient is determined before drug therapy is applied；

(b) drug therapy is applied；With

(c) SELENA SLEDAI, BILAG and PGA scoring of patient is determined after drug therapy is applied；

If wherein：The SELENA SLEDAI scorings determined in step (c) are scored few 4 points or more points than the SELENA SLEDAI determined in step (a)；Compared with the BILAG scorings determined in step (a), the BILAG index scores determined in step (c) do not include the new scoring of BILAG A organ areas or 2 new BILAG B organ areas scorings；And the PGA scorings determined in step (c) score high than the PGA determined in step (a)<0.3,

Then think that patient is had answered that in drug therapy.
7. the method for claim 6, wherein the drug therapy is the pharmaceutical composition for including Neutrokine- alpha-2 antagonists.
8. any one of claim 1-5 purposes, wherein the patient has ANA titre >=1 in its blood plasma or serum:80 and >=30IU/mL Anti-hCG action.
9. any one of claim 3-5 purposes, wherein applying the Neutrokine- alpha-2 antagonists after it is determined that having the C3 complement factors less than 90mg/dL in the blood plasma or serum of patient.
10. any one of claim 3-5 purposes, wherein applying the Neutrokine- alpha-2 antagonists after it is determined that having the C4 complement factors less than 16mg/dL in the blood plasma or serum of patient.
11. the purposes of any one of claim 2,3 and 5, wherein carrying out described determination according to the medical history record or test in laboratory of patient.
12. the purposes of claim 4, wherein the glucocorticoid is selected from the group being made up of metacortandracin, prednisolone, hydrocortisone, methylprednisolone and dexamethasone.
13. the purposes of claim 12, wherein glucocorticoid are metacortandracins, and it is administered to the amount of the metacortandracin of patient and is reduced by least 25% to≤7.5mg/ days.
14. the purposes or method of claim 4 or 7, wherein the antagonist is protein, it is included：TACI Neutrokine- α binding structural domains (SEQ ID NO:6), BCMA Neutrokine- α binding structural domains (SEQ ID NO:8), BAFF-R Neutrokine- α binding structural domains (SEQ ID NO:10) or with SEQ ID NO:The variant of the BAFF-R Neutrokine-a binding structural domains of the amino acid sequence of 26 the 2 to 70th amino acids residue.
15. the purposes or method of claim 4 or 7, wherein the antagonist is Neutrokine- alpha bindings, peptide antibody, Neutrokine- α protein variants or anti-Neutrokine- α receptor antibodies.
16. the purposes or method of claim 15, wherein Neutrokine- α protein variants work as dominant negative regulation thing.
17. the purposes or method of claim 4 or 7, wherein the antagonist is anti-Neutrokine- Alpha antibodies.
18. the purposes or method of claim 17, wherein the antibody includes the amino acid sequence of one group of VH and VL domain in the group being made up of the following：

(a)SEQ ID NO:13 VH domains and VL domains；

(b)SEQ ID NO:14 VH domains and VL domains；

(c)SEQ ID NO:15 VH domains and VL domains；

(d)SEQ ID NO:16 VH domains and VL domains；

(e)SEQ ID NO:17 VH domains and VL domains；

(f)SEQ ID NO:18 VH domains and VL domains；

(g)SEQ ID NO:19 VH domains and SEQ ID NO:20 VL domains；With

(h)SEQ ID NO:21 VH domains and SEQ ID NO:22 VL domains.
19. the purposes or method of claim 18, wherein the antibody includes SEQ ID NO:17 VH and VL domains.
20. the purposes or method of claim 17, wherein the antibody includes VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 amino acid sequence selected from the group being made up of the following：

(a)SEQ ID NO:13 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(b)SEQ ID NO:14 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(c)SEQ ID NO:15 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(d)SEQ ID NO:16 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(e)SEQ ID NO:17 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(f)SEQ ID NO:18 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3；

(g)SEQ ID NO:19 VH CDR1, VH CDR2, VH CDR3, and SEQ IDNO:20 VL CDR1, VL CDR2 and VL CDR3；With

(h)SEQ ID NO:21VH CDR1, VH CDR2, VH CDR3, and SEQ IDNO:22 VL CDR1, VL CDR2 and VL CDR3.
21. the purposes or method of claim 17, wherein in the antibody and Neutrokine- α.
22. the purposes or method of claim 17, wherein the antibody, which suppresses Neutrokine- α, combines its acceptor.
23. the purposes or method of claim 17, wherein the antibody suppresses B cell proliferation.
24. the purposes or method of claim 17, wherein the antibody suppresses B cell survival.
25. the purposes or method of claim 17, are produced wherein the antibody suppresses immunoglobulin.