CN1856325A

CN1856325A - Treatment of respiratory diseases with anti-IL-2 receptor antibodies

Info

Publication number: CN1856325A
Application number: CN 200480027204
Authority: CN
Inventors: R·S·沙梅斯
Original assignee: PDL Biopharma Inc
Current assignee: PDL Biopharma Inc
Priority date: 2003-09-23
Filing date: 2004-09-21
Publication date: 2006-11-01

Abstract

The present invention provides a method of treating respiratory diseases. In particular, it provides a method for the treatment of asthma comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation comprising an antibody, wherein said antibody binds to IL-2 receptor.

Description

Antybody therapy respiratory tract disease with anti-IL-2 receptor

Invention field

The present invention generally relates to the Antybody therapy field, it specifically is anti--IL-2 receptor antibody, also relating to cell-mediated respiratory tract disease and the anaphylactic disease with these antibody therapies treatment T-, specifically is cell-mediated anaphylactic disease of Th1 cell and Th2 and/or symptom, the most preferably method of asthma.

Background of invention

The activation of T cell and cytokine secretion be in many respiratory tract diseases and anaphylactic disease, comprises in the asthma of interest playing pivotal role.Asthma is a kind of disease of complexity, it is characterized in that airway inflammation is with intermittence, reversibility airway obstruction and air flue allergy.Though its cause of disease the unknown, the airway inflammation that comprises lymphocyte, mastocyte, eosinophilic granulocyte and neutrophil cell are chronic all patients' of persistence asthma common traits.Major part causes and has kept the inflammatory process (Drazen J.M. etc., J Exp.Med.183:1-5 (1996)) of air flue by the cytokine that activating T cell synthesizes and discharges.CD4 ⁺/ CD25 ⁺The secreted various cytokines of T cell have participated in the chronic asthma inflammation, comprise IL-3, IL-4, IL-5 and granulocyte-macrophage colony stimutaing factor (Kon O.M. etc., Inflamm.Res.48:516-23 (1999)).In causing the Fibrotic asthma of air flue, the activated T cell may be the key link that causes and regulate the air flue repair process.Therefore, the therapeutic scheme of specificity guiding inhibition activating T cell may be useful to asthmatic patient.

Obduction research and bronchial biopsy specimen data confirm that the T cell quantity increases in the asthma air flue.Autopsy research to 15 routine asthmatic patients discloses, and the T cell quantity approximately is the individual twice (Azzawi M. etc., Am.Rev.Respir.Dis.145:1477-82 (1992)) that was complementary at 10 no asthma ages of example in the asthmatic patient air flue.These cells are activatory, as indicated in expressing interleukin-2 (IL-2) receptor (CD25), human leucocyte antigen (HLA)-DR, very late antigen-1 and IL-5mRNA.Serious asthmatic patient peripheral blood studies have shown that CD4 ⁺/ CD25 ⁺The T cell quantity increases (Corrigan C.J. etc., Lancet 1:1129-32 (1988)).The research of asthmatic patient bronchoalveolar lavage fluid also proves activatory CD25 ⁺The T cell quantity increases, and the rising of the level of IL-2 and solubility IL-2 receptor (Alexander A.G. etc., J.Eur.Respir.8:574-8 (1995); Park C.S. etc., Chest.106:400-6 (1994); Walker C. etc., J.Allergy Clin.Immunol.88:935-42 (1991)).

Daclizumab (Daclizumab) is a kind of immunosuppressant, and it is the Humanized immunoglobulin IgGl monoclonal antibody of producing with recombinant DNA technology.Daclizumab can combine with α subunit (p55 α, CD25 or the Tac subunit) specificity of people's high-affinity IL-2 receptor of activated lymphocyte surface expression.The Tac subunit is only just expressed after interacting with exogenous antigen or with IL-2.Because daclizumab by 90% human normal immunoglobulin's sequence and only 10% Mus sequence constitute, so its immunogenicity is very low.U.S. Patent number 5,530 discloses the aminoacid and the nucleotide sequence of daclizumab in 101 and 5,693,761, includes it in this paper in full as a reference separately.

U.S. food and drug control management board have ratified daclizumab to be used to accept ciclosporin and steroid and or discord imuran or mycophenolate (ZENEPAX ^, Package Insert, RocheLaboratories (2000)) and treatment and repelling with prevention kidney allograft among the immunosuppressant patient.Adopt placebo to compare in the double blind controling test of accepting for the first time corpse kidney allograft patient with 2, the patient who accepts 5 dosage (1mg/kg) daclizumab acute cellular rejection incidence rate in back 6 months of transplanting is reduced to 40%.Adopt daclizumab without other toxicity, generator opportunistic infections or lymphoma do not increase (Vincenti F. etc., J.Med.N.Engl.338:161-5 (1998) yet; Nashan B. etc., Transplantation 67:110-5 (1999)).

Also estimated daclizumab in immunosuppressant autoimmunity uveitis patient accepting ciclosporin and/or steroid.Allow patient's general immunosuppressant of stopping using, final per 4 weeks are accepted a daclizumab transfusion.It seems treatment during 12 months among 10 patients daclizumab prevented 8 intraocular inflammation diseases that serious threat vision do not occur, vision does not have and increases the weight of.Toleration good (Nussenblatt R.B. etc., Proc.Nat ' l.Acad Sci U.S.A.96:7462-6 (1999)) to this treatment patient.

Daclizumab I phase multiple dose research in that 19 routine moderates are carried out in the severe psoriatic shows that this Drug tolerance is good, does not follow the specific side reaction of its administration.Give patient infusion daclizumab (2mg/kg loads dosage, is 1mg/kg then) in the 2nd, 4,8,12 and 16 weeks.This studies show that, during the preceding 4 weeks treatment of per 2 all administrations, has all blocked the CD25 in peripheral blood and the tissue.Seriousness has on average reduced by 30% (P=0.02) when the patient P ASI of treatment in advance scoring showed for 8 weeks less than 36.Begin to make in varying degrees the receptor desaturation after 4 weeks, this reverses relevant with amelioration of disease.During the treatment of 16 weeks, the expression of IL-2 receptor α-subunit has reduced by 44.8%.T cell absolute counting does not show obviously change in this research process.Daclizumab does not produce tangible side reaction (Krueger J.G. etc., J.Am.Acad.Dermatol.43:448-58 (2000)) during this research.

Because disease that respiratory tract disease, especially T are cell-mediated such as asthma is popular, and lacks the effective ways for the treatment of respiratory tract disease, so be starved of more effective Therapeutic Method provided by the present invention and preparation.Need new Therapeutic Method and preparation especially, treat conventional nonspecific immunity is suppressed unresponsive more serious obstinate asthma of therapy and cell-mediated respiratory tract disease and the anaphylactic disease of other T.The present invention includes cell-mediated respiratory tract disease and anaphylactic disease, the especially respiratory tract disease of treatment T such as the method for asthma, also comprise the anaphylactic disease that many Th1 cells of treatment and Th2 are cell-mediated and/or the method for symptom.This method comprises the antibody that gives anti-IL-2 receptor, preferred humanized antibody, and daclizumab can be in conjunction with the antibody of IL-2 receptor epi-position with identical with daclizumab.As II disclosed herein phase clinical research result proves, to compare with existing Therapeutic Method, daclizumab provides excellent clinical efficacy and secular beneficial effect for the treatment moderate to severe asthma.

Summary of the invention

The invention provides treatment or preventative smelting and treat the cell-mediated disease of T, especially respiratory tract disease and/or the anaphylactic disease that is caused or increased the weight of by IL-2 receptor-mediated (T cell) activation is as the cell-mediated anaphylactic disease of Th1 cell or Th2 or the method for symptom.The method that respiratory tract disease and/or anaphylactic disease and/or symptom are treated in treatment or preventative smelting comprises and will treat or prevent the patient that can need this treatment with the pharmaceutical preparation of the bonded antibody of IL-2 receptor-specific of containing of effective dose.In another embodiment, this Therapeutic Method comprises that also the coupling medicine with the described disease of treatment gives patient.

In a preferred implementation, available the inventive method is treated and is selected from following disease: asthma, allergic rhinitis, atopic dermatitis, nasal polyp, Churg-Strauss syndrome (allergic granuloma vasculitis), sinusitis and chronic obstructive pulmonary disease (COPD).

In other preferred implementation, the disease of available Therapeutic Method treatment of the present invention is: anaphylactic disease that Th2-is cell-mediated and/or symptom are selected from asthma, atopic dermatitis, anaphylaxis, rubella (urticaria), allergic rhinitis, nasal polyp, sinusitis, allergic conjunctivitis, skin allergy, eczema, pollinosis, irritable bowel gastritis or Churg-Strauss syndrome.

In another embodiment, the disease of available Therapeutic Method treatment of the present invention is cell-mediated disease of Th1 and/or symptom, is selected from: interstitial lung disease (ILD) (as spy's property sent out pulmonary fibrosis), hypersensitivity pneumonopathy and hypersensitivity pneumonia.In a preferred embodiment, this Therapeutic Method can be used for caused slight, moderate of the cause of disease of any kind or pathogenesis or severe asthma patient.In particularly preferred embodiments, this method is used for chronic persistence asthmatic patient or moderate to the severe asthma patient.Specifically, this method can be used for the not good asthmatic patient of corticosteroid control.In other specific implementations, available the inventive method treatment atopy or ergotropy asthmatic patient, atopy or ergotropy asthma include but not limited to: the asthma that the asthma that allergic asthma, bronchial asthma, exercise cause, occupational asthma, bacterial infection cause and " baby pant (wheezy-infant) syndrome ".In one embodiment, the method for treatment asthma comprises that also the asthmatic medicament with coupling gives the patient.In a preferred embodiment, described coupling asthmatic medicament is selected from and sucks or oral steroid, and the leukotriene modifying agent sucks or oral β 2-agonist and imbedibility ipratropium.In a preferred implementation, the coupling asthmatic medicament is an inhaled steroid, is selected from beclometasone, budesonide, flunisolide, fluticasone, triamcinolone, mometasone and Nai De (acetonide).

In a preferred embodiment, using monoclonal antibody, specifically is that chimeric humanized antibody or people's antibody is implemented the inventive method.In some embodiments of the present invention, in this antibody capable and one or more biologic activity of IL-2 receptor.

In especially preferred embodiment, implementing in the Therapeutic Method of the present invention with the bonded antibody of IL-2 receptor-specific is daclizumab, or is incorporated into the antibody of identical epi-position with daclizumab.In another embodiment, can adopt and contain the antibody identical and implement this Therapeutic Method with the cdr amino acid sequence at least 60% of daclizumab.In other embodiments, implement among the CDR of the used anti--IL2 receptor antibody of this method at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% aminoacid sequence identical with daclizumab CDR.In a preferred embodiment, implement the used antibody of the inventive method and the binding affinity of described human IL-2's receptor and be at least 10 ⁸M ^-1, more preferably at least 10 ⁹M ^-1

In a preferred embodiment, implementing the inventive method can be outer by gastrointestinal tract, intravenous, intramuscular or the subcutaneous pharmaceutical preparation that contains anti-IL2 receptor antibody.In a preferred embodiment, described preparation contains daclizumab.In a preferred embodiment, implementing the used pharmaceutical preparation of this method is to contain the 100mg/ml daclizumab of having an appointment, about 20-60mM succinic acid buffer (or 20-70mM histidine buffering liquid), pH is about 5.5-6.5, contains 0.01%-0.1% polysorbate and the tension force buffer agent (for example about 75-150mM NaCl or the about 1-100mM MgCl that keep isotonicity approximately ₂) liquid.

In other embodiments, the treatment effective dose of implementing the pharmaceutical preparation of the inventive method is 0.001mg/kg to 10mg/kg, preferably about 0.5mg/kg to 4.0mg/kg.In some embodiments, the treatment effective dose is the fixed dosage between about 100mg and the 200mg.

Brief Description Of Drawings

Fig. 1 has described the research approach that embodiment 1 described moderate was studied to the daclizumab II phase of severe chronic persistence asthmatic patient.

Fig. 2 carries out the titrating scheme of imbedibility corticosteroid during having described the preparation (run-in) that embodiment 1 described moderate studies to the daclizumab II phase of severe chronic persistence asthmatic patient.

Fig. 3 studies the timetable that the patient is estimated the 1 described II phase of embodiment.

Detailed Description Of The Invention

Definition

" antibody " used herein refer to be combined with certain concrete antigentic specificity, or produce immunoreactive immunoglobulin molecules with certain concrete antigen, comprises polyclone and monoclonal antibody. This term also comprises genetically engineered form or the modified forms of immunoglobulin (Ig), such as the Fab of chimeric antibody, humanized antibody, heterogeneous coupling antibody (such as bispecific antibody, two antibody, three antibody and four antibody) and antibody, for example comprise Fab ', F (ab ')₂, Fab, Fv, rIgG and scFv fragment. Term " scFv " refers to single-chain Fv antibody, and the variable region of the heavy chain of traditional double-chain antibody and light chain connects into a chain in this antibody. Usually, the joint peptide is inserted between two chains, make it suitably folding and produce active binding site. In addition, the meaning of term used herein " antibody " also comprises mixture to more than one antibody of responding property of the specific antigen mixture of the dissimilar monoclonal antibodies of IL-2 receptor response (if with).

Term used herein " specific binding ", " selective binding ", " specific reaction " or " specific immune response " refer to be used in determines the association reaction that antibody exists in heterologous albumen and other biomolecule colony. In other words, refer to antibody basically not with specific antigen beyond the association reaction of any antigen generation cross reaction. Therefore, for example, the affinity of being combined with required antigen when antibody is at least than the high twice of background (namely non-specific/cross reaction is in conjunction with level), generation specific binding when more generally doubly above than the high 10-100 of background. The specific binding of antibody and required antigen need to be selected antibody for concrete antigen usually. For example, can select to produce the polyclonal antibody that to modify variant, splice variant specific binding with certain concrete albumen or its polymorphism variant, allele, straight homologues, conservative, only obtain with selected protein (such as the IL-2 acceptor) not the antibody with other oroteins generation specific immune response. Can realize this kind selection by removing with the antibody of other molecule generation cross reaction. Can adopt various forms of immunoassays to select antibody with certain concrete albumen generation specific reaction. For example, normal adopt solid phase ELISA immunoassays to select to play the antibody of specific reaction (referring to for example with certain albumen, Harlow and Lane, " antibody, laboratory manual " in (Antibodies, A Laboratory Manual) (1988) to can be used for determining the immunoassays form of specific immune response and the description of condition).

" IgG antibody-like " used herein refers to IgGl, IgG2, IgG3 and IgG4 antibody. Amino acid residue numbering in heavy chain and the light chain is EU index number (Kabat etc., " sequence of immunology proteins of interest matter " (sequence of Proteins of Immunological Interest), the 5th edition, NIH, Bethesda, MD (1991); This paper has adopted the EU numbering).

" epi-position " or " antigenic determinant " refers to the antibody combining site on the antigen. The contiguous amino acid of protein or non-contiguous amino acid can form epi-position side by side by three dimensional fold. Usually the epi-position of contiguous amino acid formation can preserve when the contact metamorphism solvent, and is often lost with the processing of sex change solvent by the epi-position that three dimensional fold forms. An epi-position generally comprises at least three with unique space conformation, more normal at least 5 or 6-10 amino acid. The method of determining the space conformation of epi-position comprises, for example, and X-ray crystallography and two dimensional NMR. (Glenn E.Morris compiles, (1996) for Epitope Mapping Protocols in Methods in Molecular Biology, the 66th volume referring to the drawing of the epi-position in the molecular biology method for example scheme. If the amino acid mutation in certain albumen reduces or the combination of having eliminated a kind of antibody also can reduce or eliminate the combination of another kind of antibody, if and/or two kinds of antibody competitions are in conjunction with this albumen, be a kind of antibody and this albumen in conjunction with reducing or eliminate the combination of another kind of antibody, then claim these two kinds of antibody capables to be combined with the same epi-position of this albumen.

" V used herein_H" or " VH " refer to the variable region of heavy chain of antibody immunoglobulin, comprise the Fab of heavy chain of antibody, for example Fv, scFv or Fab. Mention " V_L" or " VL " refer to the variable region of light chain of immunoglobulin (Ig), comprise the Fab of light chain of antibody, for example Fv, scFv, dsFv or Fab.

The light chain of antibody and variable region of heavy chain contain four " framework " district that is separated by three hypervariable regions, and three hypervariable regions are also referred to as " complementary determining region " or " CDR ". The implication of framework region and CDR is that those of ordinary skills know (referring to such as Kabat etc., " the interested protein sequence of immunology " (Sequences of Proteins of Immunological Interest), the 5th edition, NIH, Bethesda, MD (1991)). The framework region sequence of different light chains or heavy chain is relatively conservative in a kind of animal. The framework region of antibody, the combined frame work district effect that namely forms light chain and heavy chain are with three-dimensional fix and arrange CDR. CDR mainly is responsible for the conjugated antigen epi-position. The CDR of each chain is commonly referred to as CDR1, CDR2 and CDR3, from the terminal beginning of N number consecutively, generally also identifies with the chain at concrete CDR place. Therefore, V_HCDR3 is arranged in the heavy chain of antibody variable domains, and V_LCDR1 is the CDR1 of light chain of antibody variable domains.

Term used herein " monoclonal antibody " is not limited to the antibody by the hybridoma technology generation, also refers to by monoclonal, comprises the antibody that eucaryon, protokaryon or phage clone are derived, rather than produces its method. Available various techniques known in the art comprise that hybridoma, restructuring and phage display techniques or its combination come for the preparation of monoclonal antibody of the present invention. For example, available hybridoma technology, comprise technology known in the art and, for example, Harlow and Lane, " antibody: laboratory manual " (Antibodies:A Laboratory Manual), Cold Spring Harbor Laboratory Press, New York (1988); Hammerling etc., " monoclonal antibody and T quadroma " (Monoclonal Antibodies and T-cell Hybridomas), Elsevier, New York (1981), the technology described in the 563-681 page or leaf (including in full it in this paper as a reference) is come the production monoclonal antibody. Can be by selecting bacteriophage or the similar substrates library of recombinant antibodies, referring to for example, Huse etc., Science 246:1275-1281 (1989); Ward etc., Nature 341:544-546 (1989) and Vaughan etc., Nature Biotech.14:309-314 (1996) or by with antigen or with the coding this antigen the dna immunization animal produce antibody.

Term " antibody that genetic method changes " refers to that its aminoacid sequence is different from the antibody of parental generation (promptly not changing) antibody.Therefore, the aminoacid sequence of the antibody of the used anti-IL2 receptor of the inventive method is not only limited to the sequence of finding in the natural antibody; The available recombinant DNA technology redesign antibody of knowing makes it obtain desirable characteristics.Possible variation is from only changing one or several aminoacid to redesign, for example variable region or constant region fully.Can in constant region, introduce change by positional mutation, improving or to change the functional characteristic of therapeutic antibodies, as immunogenicity, pharmacokinetic properties (as serum half-life), complement in conjunction with, with interaction and other effector functions of film.Usually, but the antagonist variable region change to improve the antigen binding characteristic.

" essentially identical constant region " refers in the antibody constant region at least about 85-90%, and preferred at least 95% aminoacid sequence is with natural or not change antibody constant region identical.

Term used herein " chimeric antibody " refers to a kind of immunoglobulin molecules: (a) its constant region or its part are changed, replace or exchange, make its antigen binding site (variable region) be connected in dissimilar or type, effector function constant region that change and/or another kind of animal, or be connected in the diverse molecule that to give these chimeric antibody new features, for example enzyme, toxin, hormone, somatomedin, medicine etc.; Or (b) its variable region or its part variable region different by antigenic specificity or that change changes, replaces or exchange.Those of ordinary skills know the method that produces chimeric antibody.Referring to for example Morrison etc., Science 229:1202-1207 (1985); Oi etc., BioTechniques 4:214-221 (1986); Gillies etc., J.Immunol.Methods 125:191-202 (1989); With U.S. Patent number 5,807,715; 4,816,567 and 4,816,397, include this paper in full in as a reference separately.

At least one CDR that term " humanized antibody " refers to comprise people's framework and non-human antibody is the immunoglobulin of all CDR preferably, and its constant region and human normal immunoglobulin's constant region are basic identical, and promptly at least about 85-90%, preferably at least 95% is identical.Therefore, may be except that CDR, all parts of Humanized immunoglobulin are all basic identical with the appropriate section of one or more natural human immunoglobulin sequences.Therefore, this humanized antibody is a chimeric antibody, and whole basically people variable region is nearly all replaced by non-human animal's corresponding sequence in this antibody.Framework residue in people's framework region can be replaced by the corresponding residue of CDR donor antibody, to change, preferably improves its antigen-binding energy.Available well known method, for example, can be by the interaction of simulation CDR and framework residue, identify antigen is identified relatively that in conjunction with very important framework residue with by sequence uncommon framework residue on the particular location identifies the replacement of these frameworks.Referring to for example, Queen etc., U.S. Patent number: 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (including this paper in as a reference in full separately).Available various techniques known in the art for example comprise, (EP 239,400 in the CDR-transplanting; PCT announces WO 91/09967; U.S. Patent number 5,225,539; 5,530,101 and 5,585,089), surperficial frosting or upgrade the surface (EP 592,106; EP519,596; Padlan, Mol.Immunol., 28:489-498 (1991); Studnicka etc., Prot.Eng.7:805-814 (1994); Roguska etc., Proc.Natl.Acad.Sci.91:969-973 (1994) and chain reorganization (U.S. Patent number 5,565,332) make the antibody humanization, include them in this paper in full as a reference.

Term " people's antibody " refers to contain the antibody of people variable region and constant region.With the inventive method treatment human patients the time, may need people's antibody.Available the whole bag of tricks known in the art comprises that above-mentioned phage presents method, with the antibody library generation or the acquisition people antibody of derived from human immunoglobulin sequences.Referring to U.S. Patent number 4,444,887 and 4,716,111; With PCT bulletin WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735 and WO 91/10741 include this paper in as a reference separately in full.Also available can not the expressive function endogenous immunoglobulin, but but the transgenic mice of expressing human immunoglobulin gene is produced people's antibody.The summary of producing people's antibody technique is referring to Lonberg and Huszar, Int.Rev.Immunol.13:65-93 (1995).The method of producing the technology of people's antibody and human monoclonal antibodies and producing this antibody goes through referring to for example, and PCT announces WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European patent number 0 598 877; U.S. Patent number 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 and 5,939,598, include it in this paper in full as a reference.In addition, some companies such as Abgenix, Inc. (Fremont, CA) and Medarex (Princeton NJ) engages in the similar technology of above-mentioned technology and provides at selected antigenic antibody.The technology generation of also available being called " guiding is selected " can be discerned the fully human antibodies of selected epi-position.In the method, utilize selected non-human monoclonal antibodies, instruct selection (Jespers etc., the Biotechnology 12:899-903 (1988) of the fully human antibodies of the identical epi-position of identification as mouse antibodies.

Term " primatesization antibody " refers to contain the antibody of monkey variable region and human constant region.The method of producing primatesization antibody is known in the art.Referring to for example, U.S. Patent number 5,658,570; 5,681,722 and 5,693,780, include it in this paper in full as a reference.

Term " aminoacid " refers to the aminoacid and the synthetic aminoacid of natural generation, and with similar amino acid analogue of the aminoacid functional of natural generation and amino acid analog thing.The one-letter symbol represented amino acid that trigram symbol known to using usually herein or IUPAC-IUB biochemical nomenclature commission are recommended.Equally, the available single-letter code of usually being accepted is represented nucleotide.

" aminoacid of natural generation " refers to by the genetic code amino acids coding, and adorned afterwards aminoacid, as hydroxyproline, Gla and neighbour-phosphoserine.

" amino acid analogue " refers to the chemical compound identical with the amino acid whose basic chemical structure of natural generation, for example, its α carbon is incorporated into the aminoacid of hydrogen, carboxyl, amino and R group, as homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.These analog can have the R group (as nor-leucine) of modification or the amide groups of modifying, but have kept the basic chemical structure identical with the aminoacid of natural generation." amino acid analog thing " refers to its structure and the different chemical compound of amino acid whose common chemical constitution, but the amino acids of its function and natural generation seemingly.

Term " polypeptide ", " peptide " and " protein " use interchangeably herein, refer to the amino acid residue polymer that connects by peptide bond.This term application is amino acid polymers of the amino acid whose artificial chemical simulation thing of corresponding natural generation in wherein one or more amino acid residues, also is used for the amino acid polymer that amino acid polymer that containing of natural generation modify residue and non-natural produce.

In two or more aminoacid or nucleotide sequence, term " identical " or " homogeny " percent refer to two or more sequences or subsequence is identical or the amino acid residue of certain percent or nucleotide are identical (promptly relatively and the comparison window zone of comparison or appointed area when at utmost corresponding, on the appointed area, have 60% identical approximately, preferred 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homogeny), as measuring with following default parameters with BLAST or BLAST 2.0 sequence comparison algorithms, or (, be positioned at referring to for example by manual arrangement comparison and visual observations Www.ncbi.nlm.nih.govBLAST explanation on the NCBI website).Claim these sequences " basic identical " then.This definition also refers to, or can be applicable to the complement of cycle tests.This definition also comprises the sequence that contains disappearance and/or add, and the sequence that contains replacement, and the sequence of natural generation, for example, and polypeptide variant or allele variant and artificial variant.The well-known algorithms of measuring the sequence homogeny can be regarded as out space etc.Preferably, on long zone, or more preferably on the zone of a long 50-100 aminoacid or nucleotide, there is homogeny at least about 25 aminoacid or nucleotide.

" variant that conservative is modified " used herein can be applicable to aminoacid or nucleotide sequence variant.For aminoacid sequence, the variant sequence that conservative is modified comprises and contains the aminoacid that adds or lack one or little percent, or with the sequence of amino acid whose replacement, disappearance or the adding of one of chemically similar aminoacid replacement or little percent.Conservative replacement table provides similar aminoacid on the function, and it is well known in the art.In general, a seed amino acid and another kind of amino acid whose conservative replacement comprise: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) agedoite (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); With 8) cysteine (C), methionine (M) (referring to for example, ThomasE.Creighton, " protein: structure and molecular characterization " (Proteins:Structures and MolecularProperties) (ISBN 071677030X, W.H.Freeman, 1992)).The conservative of these aminoacid sequences modification variant does not comprise congener and allele between polypeptide variant of the present invention, kind in addition.

For nucleotide sequence, the variant that conservative is modified refer to the to encode sequence (modifying the nucleotide sequence of variant aminoacid sequence as the coding conservative) of identical or essentially identical aminoacid sequence.When nucleotide sequence not during encoding amino acid sequence, basic identical or relevant, for example, natural sequence of adjoining.Since the degeneracy of genetic code, the most of protein of the variant nucleic acid sequences codified that many conservatives are modified.For example, codon GCA, GCC, GCG and GCU coded amino acid alanine all.Therefore, at determined each the position alanine of codon, this codon can be changed into another described corresponding codon, and can not change encoded polypeptides.This nucleic acid variation is " silent variant ", and it is that conservative is modified a kind of of variation.The silent variant of this nucleic acid herein each nucleic acid encoding sequence has also been described.One skilled in the art will appreciate that in some content, but each codon (except AUG and TGG, the former is the unique codon of methionine normally, and the latter is the unique codon of tryptophan normally) in the modification of nucleic acids, and produce the identical molecule of function.Therefore, often there is silent variant to lie in the described sequence of expression product in the nucleic acid of certain polypeptide of encoding, but can not implying aspect the actual probes sequence.

Term " separation ", " purification " or " biological pure " refer to substantially or do not contain in fact the material of the composition that accompanies when seeing its native state usually.Generally measure purity and homogeneity with technique of analytical chemistry such as polyacrylamide gel electrophoresis or high performance liquid chromatography.Basically purification is that the main matter that exists in the goods is protein or nucleic acid.Specifically, isolating nucleic acid from some open reading frame, the natural side joint of these open reading frame has gene, and the albumen of this gene code is different from reads the coded albumen of frame.In some embodiments, term " purification " nucleic acid or albumen refer to that it only produces a band basically in gel electrophoresis.Preferably, it is 85% pure to refer to that nucleic acid or albumen are at least, and more preferably at least 95% is pure, and most preferably at least 99% is pure.In other embodiments, " purification " refers to remove at least a pollutant in the compositions to be purified.In this case, it is homogeneous that purification does not need the chemical compound of purification, and for example 100% is pure.

" carrier " used herein comprises pharmaceutically acceptable carrier, excipient or stabilizing agent, and they are nontoxic to the cell or the mammal that contact during with concentration at the dosage that is adopted.Physiologically acceptable carrier usually is the pH aqueous buffer solution.The example of physiologically acceptable carrier comprises buffer such as phosphate, citrate and other organic acid, and antioxidant comprises ascorbic acid; Low-molecular-weight (being less than 10 residues approximately) polypeptide; Albumen is as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone; Aminoacid such as glycine, glutamine, agedoite, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate comprise glucose, mannose or dextran; Chelating agen such as EDTA; Sugar alcohol such as mannitol or sorbitol; Salify counter ion such as sodium; And/or non-ionic surface active agent such as tween, Polyethylene Glycol (PEG) and PLURONICS ^TM

" treatment effective dose " used herein refers to be enough to cure, alleviate, weaken or suppress to small part the amount of medicine, pharmaceutically active substance, pharmaceutical preparation or the compositions of disease and/or its symptom and/or complication.

" treatment " refers to therapeutic treatment and preventive measure.Need the object of treatment to comprise that object and the needs of suffering from this disease prevent the object of this disease.

Use interchangeably herein " object " or " patient ", they refer to vertebrates, preferred mammal, more preferably people.

Term used herein " derived from " refer to " available from " or " by ... produce " or " from ... hand down ".

Invention is described

The disease indication

The invention provides the method for in the object of needs treatment or prevention, treating or preventing respiratory tract disease.This Therapeutic Method comprises the antibody for the treatment of effective dose, and this antibody can specific inhibition of IL-6-2 and the lymphocyte activation that combines and/or suppress the IL-2 mediation of IL-2 receptor.In a preferred implementation, the respiratory tract disease of targeting is an asthma.This method can be used for treating any kind, the cause of disease or pathogenetic slight, moderate or severe asthma.Prove that as data disclosed herein this method is effective especially to chronic persistence asthma, especially to the not good asthmatic patient of corticosteroid control.And, can adopt the inventive method treatment atopy or ergotropy asthma, comprise asthma that asthma, occupational asthma, bacterial infection that allergic asthma, bronchial asthma, exercise cause cause, " baby pant syndrome " (promptly also can be accredited as initial stage or early stage 4 years old or 5 years old following asthma infant asthma symptom at observed night) and other non-allergic asthma.

Available well known method is weighed the curative effect of asthma.Found that method of treatment for asthma of the present invention can produce following one or more results that show its curative effect: pulmonary function improves (spirometry), asthma increases the weight of to slow down, morning, the peak value exhalation flow rate improved, the rescue drug use reduces, daytime and symptoms of asthma minimizing at night, no asthma natural law increases, and is developed to time lengthening that asthma increases the weight of and forced expiratory volume (FEV in a second ₁) increase.

This Therapeutic Method can comprise that also the asthmatic medicament (as inhaled steroid) with coupling gives the patient.Preferably, in the steroid therapy respiratory tract disease, as a kind of medicine that adopts in the asthma.More preferably, this steroid is selected from beclometasone, budesonide, flunisolide, one or more of fluticasone and triamcinolone.In one embodiment, steroid can be formulated in the same dosage form of antibody of anti-IL-2 receptor.In another embodiment, give patient's steroid and the antibody that gives anti-IL-2 receptor respectively.In one embodiment, when the antibody of not anti-IL-2 receptor, the quantity not sufficient that gives steroid is with treatment or prevention respiratory tract disease, as asthma.

In one embodiment, give the quantity not sufficient of steroid so that any side reaction or flushing take place the patient.Preferably giving the highest of steroid may dosage be that deficiency is so that the dosage of any side reaction or flushing takes place the patient.

According to the curative effect that antibody proved of anti-IL2 receptor, daclizumab can reduce the eosinophil levels of serious asthmatic patient, can estimate reasonably that the inventive method can be used in other respiratory tract of treatment or anaphylactic disease and/or symptom.It is the feature of the cell-mediated anaphylactic disease of many T that eosinophil levels improves.Know that also daclizumab can reduce the generation of T cell relevant cell factor.Therefore, according to the inventive method, relevant disease or the symptom of inflammatory reaction that available daclizumab (or antibody of other anti-IL2 receptor) treatment T is cell-mediated.

Respiratory tract that medicable T is cell-mediated and/or anaphylactic disease and/or symptom comprise Th1 cell and the cell-mediated disease of Th2.For example, can include but not limited to according to specificity T h2 cell-mediated anaphylactic disease and/or the symptom of the inventive method: asthma, atopic dermatitis, anaphylaxis, rubella (urticaria), allergic rhinitis, nasal polyp, sinusitis, allergic conjunctivitis, skin allergy, eczema, pollinosis, irritable bowel gastritis, Churg-Strauss syndrome with the daclizumab treatment.Respiratory tract disease and/or symptom that the Th1 of available daclizumab therapy for treating of the present invention is cell-mediated include but not limited to: interstitial lung disease (ILD) (for example, the atopy pulmonary fibrosis), hypersensitivity pneumonopathy and hypersensitivity pneumonia.

In addition, interstitial lung disease (ILD) often with various general autoimmune diseases, includes but not limited to: rheumatoid arthritis, systemic lupus erythematosus (sle), ankylosing spondylitis, systemic sclerosis, Sjogren syndrome, pollinosis, scleroderma, sarcoidosis, polymyositis or dermatomyositis.As a result, the antibody therapy of anti-IL2 receptor of the present invention can be separately or with other treatment be used for the treatment of in combination these general autoimmune diseases with disease and/or symptom.

The disease and/or the symptom of eosinophilic granulocyte's mediation include but not limited to: lung eosinophilia, eosinophilic granulocyte's property muscle pain syndrome, tropical eosinophilia, high eosinophilic granulocyte's syndrome and parasitic infection include but not limited to schistosomicide.At present, adopt the disease of many these eosinophilic granulocyte's mediations of IL-5 therapy for treating.Reduce the effect of eosinophil levels in the T cell mediated diseases (as asthma) according to it, the antibody therapy of anti-IL2 receptor of the present invention also can be used for the treatment of the disease that these eosinophilic granulocytes mediate separately or with other treatment in combination.

Chronic obstructive lung (or respiratory tract) disease (COPD) is the disease that is defined as airway obstruction on the physiology, and this peripheral airways obstruction that is caused by emphysema and chronic bronchitis usually mixes mutually and causes.COPD is the 5th a major causes of death in the world, and the utmost point needs effective medicine and therapy.COPD is a subgroup of chronic lung disease, also comprises asthma, it is characterized in that airway tissue chronic inflammatory disease and/or fibrosis.These diseases have many common pathological characteristicses, therefore, can estimate reasonably that the antibody therapy of anti-IL2 receptor of the present invention can be used for treating COPD.

The antibody of anti-IL-2 receptor

The antibody that is used for anti-IL-2 receptor of the present invention comprises can be in conjunction with the antibody of any epi-position of IL-2 receptor.Preferred this epi-position is found on the α subunit (p55 α, CD25 or Tac subunit) of IL-2 receptor.These antibody comprise the antibody (antibody that is produced by host animal) of natural anti-IL-2 receptor and the antibody of the anti-IL-2 receptor of recombinating.The antibody that comprises the anti-IL-2 receptor of all animal origins.The antibody of the natural anti-IL-2 receptor of non-restrictive illustrative comprises what people, chicken, goat and rodent (for example rat, mice, hamster and rabbit) produced, comprise through genetically engineered transgenic rodent produced (referring to for example, U.S. Patent number 6,300,129 B1 (Lonberg etc.) and U.S. Patent number 6,114,598 (Kucherlapati etc.) include this paper in as a reference separately in full) people's antibody of anti-IL-2 receptor.The manufacturing of also available phage rendering method is used for antibody of the present invention (referring to for example, U.S. Patent number 5,427,908 (Dower etc.) and U.S. Patent number 5,969,108 (Bonnert etc.) are included this paper in as a reference separately in full).In order to be used for human patients, the necessary energy of this antibody specificity is in conjunction with human IL-2's receptor.The binding affinity of this antibody and IL-2 receptor should be at least 10 ⁷M ^-1, but preferably at least 10 ⁸M ^-1, more preferably at least 10 ⁸M ^-1, most preferably 10 ⁹M ^-1, ideal is 10 ¹⁰M ^-1Or it is higher.Present the affinity that method or other method in vitro mutagenesis can improve antibody (referring to for example, Co etc., U.S. Patent number 5,714,350 is included it in this paper as a reference in full) with phage.

Preferred this antibody capable specificity is in conjunction with the α subunit (p55 α, CD25 or Tac subunit) of IL-2 receptor.More preferably the IL-2 receptor is the IL-2 receptor of expressing on the activated lymphocyte surface.The preferred T cell of lymphocyte.

In preferred this antibody capable and at least a biological characteristics of IL-2 receptor, but all biological characteristicses that most preferably can neutralize, for example lymphocyte activation of IL-2 mediation.This antibody can suppress or block combining of IL-2 receptor and IL-2 usually.This antibody should be able to suppress the propagation and the activation of activating T cell, or induces the activated T natural death of cerebral cells.

Preferred this antibody can not specificity in conjunction with Fc γ receptor, therefore in great majority or all patients, this antibody is the mitogenic response of activated T cell not basically.Preferred this antibody has following good characteristic as immunosuppressant: can suppress the immune response of great majority (for example at least 67%, 75%, 90% or 95%) patient's T cell at least, can not cause the mitogenic activity that discharges harmful cytokine and do not induce.

Can be by produce the polyclonal antibody of anti-IL-2 receptor with the inhuman host animal of human IL-2's receptor immunity.Available immunity well known in the art and hybridoma method produce this monoclonal antibody (referring to for example, Harlow and Lane, " antibody: laboratory manual " (Antibodies:A Laboratory Manual), Cold Spring HarborLaboratory Press, New York (1988); Hammering etc., " monoclonal antibody and T quadroma " (Monoclonal Antibodies And T-Cell Hybridomas), Elsevier, New York (1981), 563-681 page or leaf).For example, can be as Uchiyama etc., J.Immunol.126 (4), 1393 (1981) described methods produce and the Preliminary screening monoclonal antibodies, with the specific antibody of acquisition IL-2 receptor.The another kind of monoclonal antibody that is fit to is the M7/20 monoclonal antibody, as described in (Clin.Immunol. and Immunopath (1985)) such as Gaulton, it is that specific monoclonal rat anti-mouse κ, u, the Ig antibody of L-2 receptor is seen U.S. Patent number 5,916,559, include it in this paper in full as a reference).Another suitable monoclonal antibody is the 2A3 monoclonal antibody that hybridoma ATCCHB-8555 produces, and is disclosed in U.S. Patent number 4,845,198, includes it in this paper in full as a reference.U.S. Patent number 4,411 has been described other suitable anti-IL-2 antibody in 993 and 4,473,493, includes this paper in full in as a reference separately.

Also can adopt recombinant DNA technology to produce the antibody of the anti-IL-2 receptor of reorganization, be used for the present invention.The variable region of this recombinant antibodies and/or constant region aminoacid sequence do not need genetically engineered, but can be identical with the sequence of natural antibody.The antibody that is used for the anti-IL-2 receptor of reorganization of the present invention comprises that any expression system comprises the antibody that protokaryon and eukaryotic expression system produce.

Exemplary prokaryotic system generally is the bacterial system that can express the outside nucleotide sequence of introducing.Exemplary eukaryotic expression system comprises fungal expression system, virus expression systems comprises eukaryotic cell such as insect cell, plant cell, especially mammalian cell (as Chinese hamster ovary celI and myeloma cell such as NSO and SP2/0), and these are well known to those of ordinary skill in the art.Referring to for example, Morrison etc., Science 229:1202-1207 (1985); Oi etc., BioTechniques 4:214-221 (1986); Gillies etc., J.Immunol.Methods 125:191-202 (1989) and U.S. Patent number 5,807,715; 4,816,567 and 4,816,397, include this paper in full in as a reference separately.Also can produce this antibody by chemosynthesis.In any case production antibody, available well known method is as the antibody of filtration, chromatography (as affinity chromatograph, as passing through protein A, cation-exchange chromatography, anion-exchange chromatography and gel filtration) the anti-IL-2 receptor of purification.Usually, it is 90% that the I that is used for the antibody of pharmaceutical preparation is accepted purity, preferred 95%, more preferably 98%, most preferably 99% or higher.

Perhaps, can change the variable region and/or the constant region sequence of this recombination to construct thing by genetic method.The antibody that is preferred for the anti-IL-2 receptor that genetic method of the present invention changes comprise can in conjunction with and in the mosaic or the humanized antibody of IL-2 receptor.The antibody of the anti-IL-2 receptor of exemplary preferred humanization is daclizumab.The aminoacid of daclizumab and nucleotide sequence are disclosed in U.S. Patent number 5,530, in 101 and 5,693,761, include this paper in full in as a reference separately.The ripe light chain of daclizumab and the aminoacid sequence of heavy chain show below:

The sophisticated κ light chain of daclizumab (SEQ ID NO:1) DIQMTQSPSTLSASVGDRVTITCSASSSISYMHWYQQKPGKAPKLLIYTTSNLASG VPARF SGSGSGTEFTLTISSLQPDDFATYYCHQRSTYPLTFGQGTKVEVKRTVAAPSVFIF PPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Ripe γ-1 heavy chain (SEQ ID NO:2) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGYINPSTG YTEYN QKFKDKATITADESTNTAYMELSSLRSEDTAVYYCARGGGVFDYWGQGTLVTVSSA STKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVKGVEVHNAKTKPREEQYNSTYR VVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVM HEALHNHYTQKSLSLSPGK

Daclizumab (trade mark ZENAPAX ^) be the Humanized monoclonal antibodies of energy specificity in conjunction with people's high-affinity IL-2 receptor alpha subunit (p55 α, CD25 or Tac subunit), this receptor is expressed in the activated lymphocytes surface.ZENAPAX ^By Protein Design Labs, Inc. is (hereinafter referred to as " PDL "; Fremont CA) foundes, and by RocheLaboratories (Hoffmann-La Roche Inc., Nutley, NJ) exploitation and sale.In current clinical implementation mode, daclizumab is the IgGl isotype antibody, yet also can produce the IgG2M3 isotype of the daclizumab with similar treatment feature.

Other preferred antibody comprise identical with daclizumab can be in conjunction with the antibody of IL-2 receptor epi-position.Preferably, identical with daclizumab can be identical in conjunction with the aminoacid sequence at least 60% of the aminoacid sequence of this antibody of IL-2 receptor epi-position and daclizumab.In other preferred implementation, the sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98% of its aminoacid sequence and daclizumab, 99% or 100% identical.

In other preferred implementation, identical with daclizumab can be identical in conjunction with the aminoacid sequence at least 60% of the cdr amino acid sequence of the antibody of IL-2 receptor epi-position and daclizumab CDR.In other preferred implementation, its cdr amino acid sequence is identical with the CDR sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of daclizumab.The antibody of anti-IL-2 receptor can be any known isotype, but preferred especially IgG2 in four kinds of IgG isotypes.

Also can adopt the antibody of the anti-IL-2 receptor that genetic method changes, comprise combination and in and the IL-2 receptor, or prevent that IL-2 receptor and the bonded chimeric antibody of IL-2 from implementing the inventive method.Preferred this chimeric antibody contains derived from the variable region of mice or rat and the constant region of derived from human, so that when the administration of human class object, this chimeric antibody has the longer half-life, and immunogenicity is littler.The method of making chimeric antibody is known in the art.

The present invention comprises that also employing can keep the antibody fragment of anti-IL-2 receptor of binding specificity of the antibody of above-mentioned complete anti-IL-2 receptor.Example includes but not limited to: heavy chain, light chain and variable region, and the Fab of antibody described herein and (Fab ') ₂

Also can adopt the antibody of anti-IL2 receptor to pass through modification (as passing through site-directed mutagenesis) but antibody enforcement the inventive method of function identical (as having suitable IL-2 receptors bind affinity).For example, but modified antibodies improves its stability (as serum half-life) and/or curative effect.The example of modified antibodies comprises the conservative replacement with amino acid residue and the antibody of one or more aminoacid deletion or adding, and these modifications can obviously not change its antigen in conjunction with purposes nocuously.

Replace to be included in to change under the prerequisite that to keep curative effect (as specific binding capacity) or modify one or more amino acid residues and extremely redesign this zone fully.In a preferred implementation, can utilize in FcRn land positional mutation to produce the daclizumab (or binding antibody of any other IL2 receptor) of serum half-life significant prolongation, as the U.S. Patent Application Serial Number of submitting on October 15th, 2,003 10/687,118 is described, includes it in this paper as a reference.Also can be by post translational modification (as acetylation and phosphorylation) or synthetic (as the linkage flag group) antibody of the present invention.Also can adopt the fragment of these modified antibodies that keep this binding specificity.

Pharmaceutical preparation or compositions

Antibody of the present invention can be made pharmaceutical compositions.Therefore, the present invention also provides the method and composition of the antibody of the anti-IL2 receptor for the treatment of effective dose.Definite dosage will depend on therapeutic purposes, those of ordinary skills can utilize and know technology (referring to for example Ansel etc., " pharmaceutical dosage form and medicine are sent " (PharmaceuticalDosage Forms and Drug Delivery) (the 6th edition, Media, Pa.:Williams and Wilkins, 1995); " pharmaceutical dosage form " (Pharmaceutical Dosage Forms) (1-3 volume, ISBN number 0824785762,082476918X, 0824712692,0824716981), Lieberman etc. compile (New York:Marcel Dekker, Inc., 1992); Loyd V.Allen, Jr., " art of drug regimen, Science and Technology " (The Art, Science and Technology of Pharmaceutical Compounding), (AmericanPharmaceutical Association, 1999) and Gloria Pickar, " Rapid Dose Calculation " be (Delmar Learning, 1999) (DosageCalculations)) determine this dosage.As known in the art, need degrade according to physiological, whole body and positioning delivery, with new protease synthesis rate, and the order of severity adjustment of age, body weight, general health, sex, diet, administration time, drug interaction and symptom, these factors are that those of ordinary skills are confirmable with normal experiment.

The antibody of the present invention that pharmaceutical preparation of the present invention or compositions comprise can be fit to dosage form and give patient.In a preferred embodiment, this pharmaceutical preparation is water-soluble form, as pharmaceutically acceptable salt, comprises that promptly acid-addition salts and base addition salts provide." pharmaceutically-acceptable acid addition " referred to keep the favourable biological action of free alkali but do not had the salt of other ill effect biology, they are the salt that is formed by mineral acid example hydrochloric acid, hydrobromic acid, sulfacid, nitric acid, phosphoric acid etc. and organic acid such as acetic acid, propanoic acid, glycolic, acetone acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethyl sulfonic acid, p-methyl benzenesulfonic acid, salicylic acid etc." pharmaceutically acceptable base addition salts " comprises the salt derived from inorganic base, as sodium salt, potassium salt, lithium salts, ammonium salt, calcium salt, magnesium salt, iron salt, zinc salt, mantoquita, manganese salt, aluminum salt etc.Especially preferred is ammonium salt, potassium salt, sodium salt, calcium salt and magnesium salt.The amine salt that comprises primary amine, secondary amine and tertiary ammonium salt and replacement derived from the salt of pharmaceutically acceptable organic nontoxic alkali, replace replacement amine, cyclammonium and deacidite that amine comprises natural generation, as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropyl amine (TPA) and ethanolamine.

This pharmaceutical preparation or compositions also can comprise one or more of following material: carrier protein such as serum albumin; Buffer; Filler such as microcrystalline Cellulose, lactose, corn and other starch; Binding agent; Sweeting agent and other flavoring agent; Coloring agent and Polyethylene Glycol.

Can give this pharmaceutical preparation with various unit dosage forms according to medication.For example, the unit dosage form that is suitable for oral administration includes but not limited to: powder, tablet, pill, capsule and lozenge.Recognize, when giving antibody, should protect it not digested when oral.Generally can realize this kind protection in the following manner: make this molecule and certain chemical compound compound, maybe this molecule is packaged into suitable toleration carrier, in liposome or protection barrier so that it can resist acid hydrolysis and enzyme hydrolysis.The method that the protection medicine is not digested is well known in the art.

This medicament that gives comprises usually and is dissolved in pharmaceutically acceptable carrier or excipient, the antibody of the present invention in the preferred aqueous carrier.Can adopt various aqueous carriers, for example, buffer saline etc.These solution should be sterilized, and do not contain undesirable substance usually.Can make these composition steriles by the sterilization technology of knowing of routine.These compositionss can contain pharmaceutically acceptable auxiliary substance, and these materials are needed near physiological condition, as pH regulator and buffer agent, toxicity regulator etc., as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate etc.The concentration of active substance can have a great difference in these preparations, main volume, viscosity, body weight etc. according to liquid, selected concrete administering mode and needs of patients are selected (referring to for example, " Lei Mingdun pharmaceutical science " (Remington ' sPharmaceutical Science) (the 15th edition, Mack Publ.Co., Easton PA, 1980); With Goodman and Gillman, " pharmacological basis of treatment " (The Pharmacologial Basis ofTherapeutics) (volume such as Hardman, TheMcGraw-Hill Companies, Inc., 1996)).

Preparation provided herein also can be according to the required active component that contains more than one of disease specific to be treated, dysgenic composition with complementary activity can not take place between preferably containing mutually.Comprise the effectively this molecule of the amount of accomplishing the end in view suitably.

The active component of said medicine preparation can be wrapped in microcapsule, colloid drug delivery system (as liposome, albumin microsphere, microemulsion, nano-particle and Nano capsule), thick emulsion or the slow releasing preparation.These technology are (referring to for example, " Lei Mingdun pharmaceutical science " (Remington ' s PharmaceuticalScience) (the 15th edition, Mack Publ.Co., Easton PA, 1980)) well known to those skilled in the art.

The pharmaceutical preparation or the compositions that can contain the antibody of the anti-IL2 receptor of the present invention are used for the treatment of or preventative smelting is treated.In treatment is used, said composition is suffered from the patient of disease (as asthma), administered dose is enough to cure or suppresses disease to small part, or alleviates its symptom and/or complication.The amount that will be enough to finish this purpose is defined as " treatment effective dose ".Used treatment effective dose depends on the type of concrete respiratory tract disease indication, pharmaceutical preparation, the order of severity of disease and patient's overall health.Can requiredly according to patient give single agent or multi-agent pharmaceutical preparation with dosage and frequency that tolerated.Under any circumstance, said preparation should provide the active component of capacity, with effective treatment patient.

Can prevent or the amount of the pharmaceutical preparation of releasing mammal disease progression is called " prevention effective dose ".Preventative smelting is treated required concrete dosage and is depended on medical condition and mammal medical history, the disease specific that is prevented and other factors such as age, body weight, sex, route of administration, drug effect etc.For example, can implement this preventative smelting to the mammal that before once suffered from disease and treat with the prevent disease recurrence, or to suspecting that the mammal that disease takes place probably implements this preventative smelting and treat.

Dosage and give preparation

Usually can be mixed with the storable antibody drug preparation of lyophilizing or aqueous solution form mutually by the antibody that will have required purity and physiologically acceptable carrier, excipient or the stabilizing agent chosen wantonly.Acceptable carrier, excipient or stabilizing agent are tackled the receiver under used dosage and concentration nontoxic, comprises buffer such as phosphate, citrate and other organic acid; Antioxidant, antiseptic, low molecular weight polypeptide, albumen, hydrophilic polymer, aminoacid, carbohydrate, chelating agen, sugar and other standard analysis well known by persons skilled in the art (above-mentioned " Lei Mingdun pharmaceutical science ").

The daclizumab preparation that is used for vivo medicine-feeding described herein is stored in 2-8 ℃ usually.Said preparation does not usually contain antiseptic, should use in 4,12 or 24 hours after taking out from bottle and being diluted to saline.Preferred intravenous or subcutaneous giving after filtration or the said preparation of filtered.In one embodiment, according to including it in this paper in full as a reference with resisting the antibody preparation of IL2 receptor to store described in the U.S. Patent Application Serial Number of submitting on July 25th, 2,002 10/206,469 with stable lyophilized form.

Preferably the antibody daclizumab with the anti-IL-2 receptor of humanization is stored in the disposable bottle, and the Sterile Saline buffer soln of 5.0mL daclizumab is housed in this bottle, and concentration is 5.0mg/mL.Yet the present invention also comprises 1-10mg/mL (as 1,2,5 or 10), the concentration of 20-50mg/mL (as 20,30,40 or 50) or 60-100mg/mL (as 60,70,80,90 or 100).At the preferred formulation that is used for storing, said preparation contains the antibody of 5mg/mL, 3.6mg/mL one hypophosphite monohydrate sodium dihydrogen, 11mg/mL seven hypophosphite monohydrate disodium hydrogens, 4.6mg/mL sodium chloride, 0.2mg/mL polysorbate.Said preparation also can comprise hydrochloric acid or sodium hydroxide, adjusts to about 6.9 with the pH with said preparation.

In a preferred implementation, daclizumab can be made into stable liquid preparation.U.S. Patent Application Serial Number 10/291,528 (the application number 2003/0138417Al that the U.S. publishes, on July 24th, 2003 published) as JIUYUE in 2002 submission on the 8th is described, includes it in this paper in full as a reference.This stable liquid preparation is particularly useful for the subcutaneous daclizumab that gives, and can be used for treating in the method for respiratory tract disease of the present invention.In a preferred embodiment, the stable liquid preparation of this daclizumab contains the 100mg/ml daclizumab of having an appointment, the succinate buffer that about 20-60mM pH is 5.5-6.5 (or about 20-70mM histidine buffering liquid), about 0.01%-0.1% polysorbate, with the tension force buffer agent of keeping the preparation isotonicity (75-150mM NaCl, or about 1-100mM MgCl according to appointment ₂).

Can be by any suitable pathways, comprise oral, rectum, nasal cavity, part (comprising transdermal, aerosol, buccal and Sublingual), gastrointestinal tract outer (comprising subcutaneous, intramuscular, intravenous and Intradermal) or prepare therapeutic antibodies in pharmaceutical preparation by anapnotherapy.In one embodiment, available needleless air pressure injection gives said preparation.Also can understand, optimization approach can be different with the age because of receiver's symptom.Preferably outside gastrointestinal tract, as this pharmaceutical preparation of injected delivery of vein giving drugs into nose group, so that absorb and the described preparation of blood circulation delivery treatments effective dose by whole body.

The treatment effective dose of said preparation depends on the order of severity (as severe chronic asthma) of concrete respiratory tract disease, patient's clinical medical history and reaction, and attending doctor's judgment.Can once or through a series of treatments give the patient with said preparation.Originally, can give the patient candidate dosage, monitor the development of this conditions of patients by the routine techniques of knowing with those of ordinary skills and determine proper dosage and therapeutic scheme.

Produce the amount of the active component of single dosage form with the carrier material mixing, depend on treatment target and concrete administering mode.Yet should understand, given dose level to certain concrete patient depends on various factors, the order of severity of the disease specific that comprises activity, age, body weight, general health, sex, diet, administration time, route of administration, discharge rate, the drug regimen of used concrete preparation and treating, those skilled in the art can determine these factors.

Specifically, the exemplary effective dose of treatment asthma is about 0.001mg/kg (i.e. the every kg body weight of milligram)-100mg/kg, preferably about 0.001mg/kg-10mg/kg, more preferably from about 0.005mg/kg-0.100mg/kg.The preferred dosage level comprises about 0.001mg/kg, about 0.005mg/kg, about 0.0075mg/kg, about 0.010mg/kg, about 0.015mg/kg, about 0.020mg/kg, about 0.030mg/kg, about 0.045mg/kg, about 0.050mg/kg, about 0.060mg/kg, about 0.070mg/kg, about 0.080mg/kg and about 0.1mg/kg.Preferred dosage can be equal to or less than about 0.5mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg or 5mg/kg.Preferred dose can be between above-mentioned any two dosage levels.

Also can prepare and give " fixed dosage " dosage form of the antibody of the anti-IL2 receptor of patient.For example, can with fill in advance 100 or the 1ml syringe of 200mg/ml daclizumab preparation give all asthmatic patients, no matter weight in patients what.Suppose typical adult patients crowd's body weight 50 and 100kg between, send 1mg/kg-2mg/kg with the 100mg fixed dosage.The fixed dosage dosage form can make contingent dose error minimum in administration, especially be preferred for the treating asthma of patient's self-administer.

Usually, can adopt the therapeutic antibodies of higher dosage (as each patient 0.1mg-every day up to about 100mg), when especially not entering blood flow when delivering medicine to concealment part, as when entering body cavity or entering organ lumen.Topical can adopt quite high dosage.Those skilled in the art understand or understand the practical methods of preparation through the compositions of gastrointestinal tract external administration, referring to for example " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Science) and Goodman and Gillman, " pharmacological basis of treatment " (The Pharmacologial Basis ofTherapeutics) is on seeing.

Therapeutic scheme

According to treatment process and patient's body situation, the treatment of asthma scheme can be obviously different.Usually, any single-dose thing preparation that contains antibody described herein to the major general gives the patient, is referred to as " initial dose " or " initial administration " or " loading dosage " if there is any additional dose (" maintenance dose ") back.Of course, for example every day, weekly, per two weeks, per 6 weeks or every month, perhaps per 2,3 or gave time antibody drug 1,2,3 or 4 times or repeatedly in 6 months.The treatment time of a course of treatment should continue at least one day or two days, as one to number (2,3,4,5 or 6) sky, several weeks, several months or several years, or unlimited time, this depends on the character and the seriousness of disease.Treatment time is calculated as the time that this antibody once gives this antibody to the end that gives for the first time.The patient can accept 2,3,4 or more a plurality of course of treatment.Can adjust administration frequency according to patient's improvement process.Preferred loading dosage is 2mg/kg approximately.After loading dosage, preferred maintenance dose is 1mg/kg approximately.In the preferred dosage scheme, loaded dosage at 30 minutes in the clock time, gave each maintenance dose at 15 minutes in the clock time.

For reducing the transfusion related symptoms, the antibody drug preparation that contains anti-IL-2 receptor also can give respectively with the other medicines of coupling.These medicines generally include methylprednisolone, hydrocortisone, ondansetron, acetaminophen and have many other reagent of identity function, and this is well known to those skilled in the art.Can comprise oral, rectum, nasal cavity, part, gastrointestinal tract outer (comprising subcutaneous, intramuscular, intravenous and Intradermal) by any suitable pathways, or give these other medicines by anapnotherapy.

The dosage level of these medicines also is known in the art, for example, and each patient 1mg-100g.Exemplary dosage comprises methylprednisolone, hydrocortisone and the ondansetron of 10-50mg, 60-200mg or 200-500mg; Acetaminophen with 100-500mg, 600-1000mg, 1-5g.For example, give in initial or/and each time before the pharmaceutical preparation of antibody of anti-IL-2 receptor or/and afterwards at least about 1,2,3,4,5,6,7,8,10,12,14,20,24,36 hour or 2,3,4,5,7,10,20,40 or 60 days, but single or multiple gives patient's immunomodulator extraly.

In one embodiment, Therapeutic Method of the present invention also comprises the coupling medicine that gives target disease.For example, the coupling asthmatic medicament (being used for chronic and acute) that can be used for the inventive method includes but not limited to: suck and oral steroid (as beclometasone, budesonide, flunisolide, fluticasone, triamcinolone, mometasone and Nai De); Systematicness corticosteroid (as methylprednisolone, prednisolone, prednisone, dexamethasone and deflazacort); Suck or oral β2Ji Dongji (as salmaterol, formoterol, bitolterol, pirbuterol, terbutaline, bambuterol and albuterol); Cromoglicic acid and nedocromil; Antiallergic ophthalmic medicine (as dexamethasone); Methylxanthine (as theophylline and pyrilamine-acetic acid theophylline); Leukotriene modification property medicine (as zafirlukast, zileuton, montelukast (montekulast) and pranlukast); Anticholinergic agents (as ipratropium bromide); Other therapeutic antibodies (as the antibody of adhesion molecule or IgE in the anti-born of the same parents); The coagulation dioxane A2 synthase inhibitor; The thromboxan prostaglandin receptor antagonist; Other quasi-eicosane alcohol dressing agent is (as Alprostadil and PGE1, dinoprostone and PGE2, epoprostenol and prostacyclin and PGI2 analog (as the PG12 Beraprost), seratrodast, ozagrel, phosphodiesterase 4 isozyme inhibitor, coagulation dioxane A2 synthase inhibitor (as nitrogen  Si spit of fland); Ditec (low dosage disodium cromoglycate and fenoterol); The platelet activating factor receptor antagonist; Hydryllin; Kang coagulation dioxane A2 medicine; Anti-Kallidin I medicine (as icatibant); The medicine (as ketotifen) that suppresses activatory eosinophilic granulocyte and T cell aggregation, IL-13 blocker (as solubility IL-13 receptor fragments), IL-4 blocker (as solubility IL-4 receptor fragments); In conjunction with and the active part of blocking-up IL-13 or IL-4, and xanthine derivative (as pentoxifylline (pentoxifyolline)).

Non-limited way provides following examples in the explanation mode.Include the disclosure of all references document in the description in this paper as a reference especially, be used for all purposes.

Embodiment

Embodiment 1: the daclizumab II phase of chronic persistence asthmatic patient, at random, double blinding, placebo, parallel group of research

Present embodiment has been described design, enforcement and the result of the Pilot Study that II phase, dosage progressively improves, and has proved the effectiveness for the treatment of chronic persistence asthmatic patient method with daclizumab.

A. research overview

1. purpose

The main purpose of this research is to estimate safety, the toleration and preliminary active of daclizumab in the not good chronic persistence asthma adult patients of imbedibility corticosteroid treatment control.

2. total research design

This research is with randomization, multicenter, double blinding, placebo, parallel group of research in daclizumab treatment imbedibility corticosteroid control not good (being equivalent to every day＞1200 microgram imbedibility triamcinolone) chronic persistence asthmatic patient.The overall design sketch map that has shown this research among Fig. 1.

After reaching between the warming up period in 5 weeks, screen visit, on baseline, active medicine or placebo (3: 1) are carried out the randomization grouping.The research visit occurs in screening (visit 1), between warming up period (nearly 4 visits), 1 once (0-84 days per 2 weeks of treatment phase, 6 visits), 2 per 2 weeks of treatment phase are (85-140 days, steroid reduces gradually, 4 visits) once, then in the every 2-4 of follow-up period week once (141-239 days, 5 times visit).Carried out all research visit, dosage and evaluations on the 3rd in the front and back of specifying research day.

3. the selection of patient population

The competent person of registration is age 18-70 year non-smoking masculinity and femininity asthmatic patient in this research, the FEC (FEV in its 1 second ₁) be the 50%-80% of predicted value, use β ₂The reversibility of agonist treatment is 12%, although the triamcinolone that needs inhalation dose every day＞1200 micrograms before the registration how moral (triamcinolone) or its equivalent more than 3 months.For the randomization grouping, between warming up period, the patient must prove needs to suck corticosteroid treatment, (absolute value) FEV during examination ₁＞90%, and FEV ₁＞predicted value is greater than 50%.Especially, absolute FEV ₁Value raising 〉=12% and 200mL suck salbutamol MDI 2-4 time then, prove between screening or warming up period, need carry out qualification to the patient and judge to carry out the randomization grouping.Used sample size is 120 patients's (90 of active medicines, 30 of placebo) at random.

Meet the listed standard of all tables 1 and do not meet wherein the patient of listed any exclusion standard and think to register and enter this research.

Table 1: comprise standard and exclusion standard

Comprise standard
Comprise standard	18-70 year sex patient
Asthma medical history 〉=6 month	18-70 year sex patient
Asthma medical history 〉=6 month	Chronic persistence asthma medical history daily requirement sucks 〉=1200 microgram triamcinolones, and the perhaps suitable corticosteroid of inhalation dose is before registration medication 〉=3 month (seeing table 2)
FEV when registration ₁Be the 50%-80% of predicted value.This value is appointed as the preceding baseline of research.Absolute FEV ₁Value raising 〉=12% and 200mL, suck salbutamol MDI then 2-4 time, proof need be carried out qualification to the patient and judge to carry out randomization between screening or warming up period.
	Women that might be conceived is serum pregnancy tests feminine gender when screening, urine pregnancy test feminine gender in 24 hours before first dosage of double-blind study medicine.Masculinity and femininity all need proof during studying and last medication infusion study and adopted both sides' contraceptives back 4 middle of the month.
The patient of Informed Consent Form is provided
The patient of Informed Consent Form is provided	Exclusion standard
Body weight is less than 70% of the ideal body weight that calculates by height and sex	Exclusion standard
	Register the patient who received any experimental drug treatment in 30 days in research
Register the patient who received any Mus, mosaic, humanized antibody or quiet notes IG treatment in 90 days in research
	Register the patient who received cyclosporin, methotrexate or triacetyloleandomycin/methylprednisolone treatment in 60 days in research
In preceding 8 weeks of research registration, donate blood＞500 milliliters patient
	The patient of smoking history 〉=10 year or smoking in research registration 12 months
Register the patient who suffers from upper respiratory tract or lower respiratory infection in 14 days in research
	Vaccinated patient in 6 weeks of research beginning (patient of inoculation influenza vaccines can enter experiment in 2 weeks after inoculation)
The patient of or chronic sinusitis acute with antibiotic therapy
	Suffer from tangible organ dysfunction, comprise the patient of lung (except that asthma), heart, liver, kidney, central nervous system, blood vessel, gastrointestinal tract, endocrine or metabolic dysfunction
Use kreatinin 〉=1.6mg/dL, the patient that alanine aminotransferase (ALT) or aspartate aminotransferase (AST) 〉=the normal value upper limit are 2.5 times
	≤ 6 patients that myocardial infarction, congestive heart failure or arrhythmia medical history are arranged the middle of the month of research registration
Accept the patient of Beta receptor blockers treatment
Accept the patient of Beta receptor blockers treatment	The sign of preexist shows that it has infected the human immunodeficiency virus or has had hbs antigen or the patient of hepatitis C serum tests positive

Hemoglobin＜11g/dL, platelet＜100,000/mm ³Or neutrophilic granulocyte＜1500 cells/mm ³
	Register the patient who did major operation in 30 days in research
In research registration 30 days, used the patient of corticosteroid outside the oral or gastrointestinal tract
	Studying the patient who registers hospitalization asthma in 60 days
Accept the anaphylactogen immunization therapy or accepted the immunization therapy of inferior maintenance anaphylactogen in 6 months or accepted the patient of maintenance anaphylactogen immunization therapy in 2 years in registration in registration
	Pregnancy or breast-feeding female
The super quick patient of heparin	Pregnancy or breast-feeding female
The super quick patient of heparin	To the super quick patient of any excipient in daclizumab or the said preparation
Register the severe infections that needs to carry out intravenous antibiotic therapy or hospitalization in 60 days in research
	Pro-suffers from malignant tumor or concurrent malignant tumor (except plain tumor skin carcinoma of non-black or the carcinoma in situs of cervix through fully treating) in 5 years
Can not abide by this research approach or obvious deformity be arranged or unable patient
	The patient who in research 6 weeks of registration, suffers from chickenpox, herpes zoster or other serious viral infection
Studying the patient who registers contacted chickenpox in 21 days

Table 2: the steroid every day inhalation dose of equal value that comprises standard-required

Medicine	Dosage
Medicine	Dosage	Fluticasone MDI	〉=330 micrograms (〉=3110 microgram MDI air blowing)
Beclometasone	〉=588 micrograms (air blowing of 〉=1442 micrograms)	Fluticasone MDI	〉=330 micrograms (〉=3110 microgram MDI air blowing)
Beclometasone	〉=588 micrograms (air blowing of 〉=1442 micrograms)	Budesonide	〉=400 micrograms (＞2 suctions)
Flunisolide	〉=1200 micrograms (〉=5 air blowings)	Budesonide	〉=400 micrograms (＞2 suctions)
Flunisolide	〉=1200 micrograms (〉=5 air blowings)	Triamcinolone	〉=1200 micrograms (〉=12 air blowings)
Fluticasone DPI (comprising Advair disckus)	Suck for 〉=4 times 100 microgram DPI or 〉=suck for 2 times 250 microgram DPI or 〉=suck 500 microgram DPI 1 time	Triamcinolone	〉=1200 micrograms (〉=12 air blowings)

Think that the patient with following feature can be registered:

A. register the patient of nasal cavity corticosteroid, nasal cavity cromoglicic acid, local antianaphylaxis ophthalmic remedy or corticosteroid emulsifiable paste or the Ointment in Treatment of once using consistent dose in preceding 30 days

B. once used patient's (these medicines of when registration, must stopping using) of theophylline, salmaterol, oral salbutamol, Advair (the oral inhalant of fluticasone and salmaterol), cromoglicic acid, nedocromil or the treatment of leukotriene dressing agent.

C. use fugitive imbedibility or sprayability β as required ₂The patient of agonist treatment.

Filter out the patient who has signed Informed Consent Form.The patient that will meet these standards is assigned randomly to one of two researchs (daclizumab or placebo).The NRS center sends patient's randomization numbering to specified research center pharmacists.First day of blind method treatment (daclizumab or placebo) is appointed as the 0th day.

4. medication preparation, dosage and send

The daclizumab that is used for this research is the daclizumab preparation ZENEPAX of the FDA approval of buying from the market ^Placebo is a drugs compared.Provide daclizumab with small bottle packing, the 5ml solution of 25 milligrams of daclizumabs is housed in the bottle.Every ml soln contains 5mg daclizumab and 3.6mg one hypophosphite monohydrate sodium dihydrogen, 11mg seven hypophosphite monohydrate disodium hydrogen e, and 4.6mg sodium chloride, the 0.2mg polysorbate, and can contain hydrochloric acid or sodium hydroxide adjusts to 6.9 with pH.Do not add any antiseptic.

The all the components that is provided in this activity dosage form is provided placebo, deducts this active component (daclizumab).The fill volume of placebo bottle is 5mL.

Used dosage is to load dosage 2mg/kg and subsequent dose 1mg/kg.Dilute this dosage with 50ml normal saline (0.9%) in pouch after, intravenous gives this research medicine.The loading dosage of input 2mg/kg in 30 minutes time.All follow-up 1mg/kg dosage of input in 15 minutes time.Carry out all transfusions with infusion pump.Daclizumab is not used in direct injection, must dilute with preceding.

For the first time studying medication infusion and finish back (the 0th day) stays the patient and observed all follow-up transfusions observation afterwards at least 1 hour at least 2 hours.

Daclizumab and placebo are stored under the in check refrigerator condition 2-8 ℃.Said preparation does not contain antiseptic, takes out in back 24 hours from bottle and uses.When preparing when transfusion, should be in administration in 4 hours angular veins.If do not use said preparation immediately, should be placed in 2-8 ℃ the refrigerator 24 hours at the most, or at room temperature keep 4 hours.During this period of time, should abandon this preparation solution.

5. research spacing of doses time, dosage progressively improve and follow-up period

In the following table 3 brief summary this research therapeutic dose scheme.

Table 3: therapeutic dose scheme

	The treatment phase 1 ¹						The treatment phase 2 ²				Follow-up period
	The treatment phase 1 ¹						The treatment phase 2 ²				Follow-up period						Tx	The 0th day	The 14th day	The 28th day	The 42nd day	The 56th day	The 70th day	The 84th day	The 98th day	The 112nd day	The 126th day	The 140th day	The 154th day	The 168th day	The 182nd day	The 210th day	The 238th day
Daclizumab	x ³	x	x	x	x	x	x	x	x	x	N/A	N/A	N/A	N/A	N/A	N/A	Tx	The 0th day	The 14th day	The 28th day	The 42nd day	The 56th day	The 70th day	The 84th day	The 98th day	The 112nd day	The 126th day	The 140th day	The 154th day	The 168th day	The 182nd day	The 210th day	The 238th day
Daclizumab	x ³	x	x	x	x	x	x	x	x	x	N/A	N/A	N/A	N/A	N/A	N/A	Placebo	x ³	x	x	x	x	x	x	x	x	x	N/A	N/A	N/A	N/A	N/A	N/A

1. except that blind method treatment, all patients also will accept the imbedibility triamcinolone (0-83 days) of randomization dosage in advance.

2. except that accepting blind method treatment, since the 84th day, all patients reduced randomized in advance triamcinolone dosage 25% every 2 weeks.

The 0th day in 30 minutes infusion 2mg/kg load dosage, then in all other dosage days, infusion 1mg/kg dosage in 15 minutes.N/A=does not use-does not have the infusion studies medicine

This research comprises-warming up period of 28--1 days.The purpose of warming up period is that the needs of patients that checking is studied sucks corticosteroid.Between this warming up period, the patient is carried out the titration of imbedibility corticosteroid, as the brief summary of Fig. 2 sketch map institute.The patient is transformed into the triamcinolone that sucks equivalent, the asthma control medicine of all other couplings of stopping using when warming up period begins.Enter this warming up period after the patient of inactive leukotriene dressing agent waited for for 2 weeks, get back to visit 2A.Every other patient only waits for the visit 2A that surrounds behind all other asthma control medicines of stopping using.In order to carry out the randomization grouping, require the patient to prove that they need to suck the FEV of corticosteroid, screening (absolute value) between warming up period ₁＞90%, 50% of FEV1＞predicted value.Need the asthma of corticosteroid whole body administration to increase the weight of not random packet of patient, but stop this research.

In the table shown in Figure 3 brief summary spacing of doses and the timetable that increases progressively and relevant patient estimate.This timetable is divided into two treatment periods: (1) treatment phase 1 (blind method treatment, 0-84 days): the loading dosage 2mg/kg that gave daclizumab or placebo on the 0th day, imported in the clock time at 30 minutes, gave subsequent dose 1mg/kg then at the 14th, 28,42,56 and 70 day, imported in the clock time at 15 minutes; (2) the treatment phase 2 (blind method treatment adds steroid and successively decreases 85-140 days): 84th, gave 1mg/kg daclizumab or placebo in 98,112 and 126 days, imported in the clock time at 15 minutes.

During the treatment phase 1, make qualified patient's randomization (3: 1, active medicine: placebo), accepted daclizumab or infusion with placebo, keep baseline dosage before the randomization of imbedibility triamcinolone simultaneously at the 0th, 14,28,42,56 and 70 day.For accepting the patient of daclizumab, gave the loading dosage 2mg/kg of daclizumab on the 0th day, imported in the clock time at 30 minutes, give the dosage of 1mg/kg then in treatment day subsequently, imported in the clock time at 15 minutes.Be not later than last warming up period and visit first dosage that allowed the patient accept blind method research medicine in back 7 days.Behind the randomization, per 2 weeks are observed a patient, to estimate and to adjust dosage.

During the treatment phase 2 (being the successive treatment phase 1), make patient's imbedibility triamcinolone (TAA) dosage the dosage that the patient finishes 1 o'clock treatment phase be reduced by 25% every two weeks (the 84th, 98 and 112 day), fully phased out suction steroid (the 126th day) up to them, accept daclizumab 1mg/kg or placebo (the 84th, 98,112 and 126 day) transfusion simultaneously, input in 15 minutes.Per 2 weeks are observed a patient, to estimate and to adjust dosage.

To meet following standard, the fixed time point of research stop to treat the patient from further adjustment research drug dose, withdraw from, allow them enter follow-up period: (1) experiences once above asthma and increases the weight of, need the general steroid therapy during the treatment phase 1; (2) during the treatment phase 2, live through an asthma and increase the weight of, need the general steroid therapy; (3) during any asthma increases the weight of, need the every day＞the 60mg prednisone; (4) need during asthma increases the weight of with prednisone treatment＞14 days; Or (5) asthma increases the weight of to spend the night in hospital (promptly needing＞stayed institute to treat in 24 hours).

In follow-up period (140-238 days), the monitoring patient cancels the situation in 16 weeks behind the research medicine, estimates at the 140th, 154,168,182,210 and 238 day.

Do not understand this therapeutic scheme at locational all individualities of research.These individualities comprise researcher, research coordination person, patient and other research worker.PDL medical monitoring person and CRA do not understand therapeutic scheme yet.Do not participate in the specified PDL safety monitoring person that this research carries out and checked all 2 grades or more senior (being moderate, severe or life-threatening) symptom in every month, no matter whether it is relevant with the administration of research medicine.This individuality is to understand therapeutic scheme.

6. compliance, safety and pharmacodynamics are measured

Under research work personnel's supervision, study medicine.Guarantee compliance by allowing researcher or qualified designee carry out the blind method research of each time medication infusion.All triamcinolone MDI medicinal cuppings of specifying research worker and record to distribute to the patient for each patient are estimated the use of imbedibility triamcinolone.

The safety measurement of carrying out during the research comprises vital signs, nurse/doctor's observation, side reaction evaluation and laboratory safety implementations.

By measuring the pharmacodynamic profile that the whole blood eosinophilic granulocyte measures all research patients.

Obtained the daclizumab serum-concentration by this research, with its limited pharmacokinetics feature of passing in time as daclizumab.Collect blood serum sample from each patient's selected position, carry out then immediately with administration after the dosage of each time point (referring to table 3) medicine of studying.

Estimate immunogenicity by analyzing in each patient serum sample anti-daclizumab antibody (anti-Ab).

7. curative effect is measured

The main curative effect parameter that is used to estimate daclizumab is pulmonary function (vital capacity), by FEV during the treatment phase 1 (0-84 days) ₁Change apart from the percent of baseline and to estimate.

Second kind of curative effect parameter comprises: patient's ratio that experience asthma increases the weight of during (1) treatment phase 1 (0-84 days);

(2) percent that AM PEFR and PM PEFR change during treatment phase 1 (0-84 days) and 2 (85-140 days);

(3) save the variation of the no asthma natural law that writes down among the symptoms of asthma at operating position, daytime and night of medicine (β2Ji Dongji MDI) and the IVRS during the treatment phase 1 and 2; (4) patient's who withdraws from the research from then on during the treatment phase 1 and 2 ratio; (5) during the treatment phase 1 and 2 to time that asthma increases the weight of; (6) FEV during treatment phase 2 and the follow-up period ₁The percent that changes.

Vital capacity determination

Write down the mensuration of vital capacity according to the timetable of Fig. 3.Carry out all vital capacity determinations according to American Thoracic Society's guide.Used predicted value is consistent in whole research.Do not adjust the vital capacity value because of ethnic group.Write down FEV best in three effort ₁Vital capacity comprises vital capacity (FVC), the FEV when firmly breathing ₁And FEV ₁/ FVC, the forced expiration stream during FVC (FEF25-75) mid point.Do not give fugitive imbedibility β 2-agonist in preceding 6 hours at vital capacity determination.With at every turn the visit the vital capacity determination outcome record in patient's case replacement form.

Treatment asthma increases the weight of

Asthma increases the weight of to be defined as that cough increases the weight of, pressure in chest or stridulate, and with following 1 or multinomial: (1) in 48 hours, baseline will use rescue salbutamol per 24 hours 〉=8 times blow (suctions); (2) in 48 hours, use rescue salbutamol per 24 hours 〉=16 times blow (suctions); (3) peak value expiratory flow (PEF) is less than reference level's 65%, even rescue was treated 60 minutes; (4) even rescue treatment (be defined as air blowing in per 20 minutes and suck salbutamol 2-4 time, as many as 1 hour) still had symptom in 60 minutes; And/or (5) need whole body to give (oral or injection) corticosteroid.

Researcher has been estimated the needs that whole body given the steroid rescue.Except above-mentioned standard, researcher also can be according to his or his judgement, and prescription gives the whole body steroid and treats asthma and increase the weight of.Use the reason of corticosteroid to be recorded on patient's the case replacement form (CRF) whole body.If patient experience asthma increases the weight of (according to above-mentioned standard), the suggestion patient calls out the research center.The patient that permission need increase the weight of with the asthma of oral corticosteroid treatment uses prednisone assault treatment, 60 milligrams at the most of every days, 14 days at the most.Stop also not enter follow-up period to meeting any patient's who ends the treatment standard further dose titration.Visit 13 is as end visit eventually.Only to needing whole body to use the patient that increases the weight of of steroid rescue to determine to end treatment.

During the treatment phase 1, live through the needs of patients whole body that asthma for the first time increases the weight of and give the corticosteroid rescue, also allow dosage baseline before the randomization of its imbedibility triamcinolone improve 25%, and keep this dosage, finish up to the treatment phase 1.

Symptoms of asthma/every day medicine record

During studying, utilize the journal record (by the interactive voice response system) of each patient every day to estimate symptoms of asthma, drug use situation and peak air flow record.Think that IVRS is a kind of source file; Verified that this class diary can be used for the asthma clinical trial.

Daytime, average symptom scope provided the reaction classification of 0-6 branch scope for variety of issue, represented the slightest to the most serious symptoms of asthma.The reaction classification of scope from 0 minute (expression is not awakened because of symptoms of asthma) to 3 minutes (expression is not slept whole night) adopted in the diary at night.Mean value calculation scope on daytime every day scoring according to 4 problems in the daytime symptoms scope.Calculating the total diary scoring of this week is at least 4 days the meansigma methods that 7 day daytime every day, scope was marked.Calculate the average score weekly of diary at night scope in a similar manner.Mark weekly daytime and the reduction of night scoring represents that asthma makes moderate progress.Last week of (the-7--1 days) last all average score and treatment phase 1 (77-84 days) and last all difference calculating asthma of treatment phase 2 (134-140 days) variation of scope of marking between warming up period before treat apart from baseline.

No asthma natural law is defined as daytime and the diary at night all shows the natural law that does not have symptom.The method of analyzing this variable depends on the distribution of the journal record of losing.If obliterated data seldom, the variation of average no asthma natural law will be analyzed.If the symptoms of asthma of patient's report is inconsistent, patient's ratio of the no asthma natural law of experience in the relatively treatment group.

Adopt β with having write down in the symptom diary night to save by day ₂Agonist MDI.Instruct the patient to write down used β in the diary by day ₂The amount of agonist MDI and the sleeping back used β of record in the diary at night ₂The amount of agonist MDI.Utilize weekly journal record to calculate and use daytime and average every day at night β ₂The amount of agonist MDI.Calculate used β by the difference that mark the average every day in last week of last week of (the-7--1 days) that week and treatment phase 1 (77-84 days) and treatment phase 2 (134-140 days) between warming up period before the treatment ₂Agonist rescue medicine is apart from the variation of baseline.

The monitoring of peak value expiratory flow

In screening during the visit, instruct the patient to use small-sized Wright peak value flowmeter measuring device.Measure also 3 best PEFR of record in the diary by day before the patient sleeps at night, take any medicine after getting up morning and write down the journal record at night before.Calculate weekly grand mean night every day and morning peak air flow.Last week by last week between warming up period before treating and treatment phase 1 (77-84 days) and treatment phase 2 (134-140 days) average every day PEFR difference calculating night and morning peak air flow apart from the variation of baseline.

8. statistical method

To each being described property of treatment group statistical analysis, calculate demographic variable and baseline variables.Estimate continuous variable by t check or suitable non parametric tests, as age, disease persistent period and symptom score.Check with card side or Fisher degree of accuracy as required and estimate classified variable.

For placebo and descriptive preliminary curative effect (preliminary curative effect: the FEV that provides of daclizumab ₁Apart from the variation of baseline, second curative effect: symptom score, β ₂Use, the asthma of agonist rescue medicine increase the weight of number of times and average every day AM peak value exhalation flow rate) evidence.Check to estimate in the group by the rank of paired t-test or Wilcoxon design and change.Determine statistical significance between group by t check or Wilcoxon-Mann-Whitney check.With Kaplan-Meier method and logarithm rank method evaluation time-event variable.

B. detailed research method

Be used for method and being described in more detail of scheme that this II phase studies and be disclosed in the U.S. Provisional Application of submitting on March 12nd, 2,004 60/552,974, include it in this paper as a reference, be used for all purposes.

The complete description of II phase method sees albumen Design Laboratory scheme numbering DAC-1003 (" the daclizumab II phase of chronic persistence asthmatic patient, randomization, double blinding, placebo, parallel grouping research " (A Phase II, Randomized, Double-Blind, Placebo-Controlled, parallel-Group Study ofDaclizumab in Patient with Chronic, Persistent Asthma)), daclizumab, the date is March 27 calendar year 2001; Revise A:2001 July 16; Revise B:2001 August 24; Revise C:2002 April 15; Revise D:2002 July 8; Revise E:2002 August 28 (including it in this paper in full as a reference).

Table 4: be used for the abbreviation of II phase scheme

Absolute baseline (value)	FEV ₁Absolute value, measure in when visit screening
Absolute baseline (value)	FEV ₁Absolute value, measure in when visit screening	AE	Side reaction (experience)
FEV ₁	FEC in 1 second	AE	Side reaction (experience)
FEV ₁	FEC in 1 second	HEENT	Head, eye, ear, nose and larynx
IVIG	The intravenous injection immunoglobulin	HEENT	Head, eye, ear, nose and larynx
IVIG	The intravenous injection immunoglobulin	MDI	Gageable dose inhaler
PEF	The peak value exhaled air flow	MDI	Gageable dose inhaler
PEF	The peak value exhaled air flow	PEFR	The peak value exhalation flow rate
SAE	Serious side reaction (experience)	PEFR	The peak value exhalation flow rate
SAE	Serious side reaction (experience)	TAA	Triamcinolone acetonide (being also referred to as triamcinolone)

C. detailed result of study

1. the patient distributes and baseline characteristic

Table 5-8 has listed with patient's distribution, demographics and baseline characteristic before the daclizumab treatment.

Table 5: the patient before the randomization distributes

Describe	Total N (%)
Describe	Total N (%)	The patient of registration	208
The patient who rejects before the randomization	92	The patient of registration	208
The patient who rejects before the randomization	92	Increase the weight of	3(3.3)
Preparation (phase) failure	61(66.3)	Increase the weight of	3(3.3)
Preparation (phase) failure	61(66.3)	Be not obedient to	3(3.3)
The violation scheme	2(2.2)	Be not obedient to	3(3.3)
The violation scheme	2(2.2)	Researcher is judged	3(3.3)
Patient's decision	13(14.1)	Researcher is judged	3(3.3)
Patient's decision	13(14.1)	Side reaction	1(1.1)
Other	6(6.5)	Side reaction	1(1.1)

Treat randomized patient

116

Table 6: the patient behind the randomization distributes

Describe	Daclizumab N (%)	Placebo N (%)	Total N (%)
Describe	Daclizumab N (%)	Placebo N (%)	Total N (%)	Randomized patient, n (row %)	89(76.7)	27(23.3)	116(100)
The patient who rejects before the dosed administration	2(2.2)	0(0.0)	2(1.7)	Randomized patient, n (row %)	89(76.7)	27(23.3)	116(100)
The patient who rejects before the dosed administration	2(2.2)	0(0.0)	2(1.7)	Researcher is judged	1(1.1)	0(0.0)	1(0.9)
Patient's decision	1(1.1)	0(0.0)	1(0.9)	Researcher is judged	1(1.1)	0(0.0)	1(0.9)
Patient's decision	1(1.1)	0(0.0)	1(0.9)	The patient who rejects behind the dosed administration	11(12.4)	4(14.8)	15(12.9)
Side reaction	1(1.1)	0(0.0)	1(0.9)	The patient who rejects behind the dosed administration	11(12.4)	4(14.8)	15(12.9)
Side reaction	1(1.1)	0(0.0)	1(0.9)	Be not obedient to	1(1.1)	0(0.0)	1(0.9)
Cancel	7(7.9)	2(7.4)	9(7.8)	Be not obedient to	1(1.1)	0(0.0)	1(0.9)
Cancel	7(7.9)	2(7.4)	9(7.8)	Researcher is judged	1(1.1)	0(0.0)	1(0.9)
Other	1(1.1)	2(7.4)	3(2.6)	Researcher is judged	1(1.1)	0(0.0)	1(0.9)
Other	1(1.1)	2(7.4)	3(2.6)	Finish the patient of research	76(85.4)	23(85.2)	99(85.3)

Table 7: patient demographic feature

	Daclizumab N=88 (77%)	Placebo N=27 (23%)	The p value
	Daclizumab N=88 (77%)	Placebo N=27 (23%)	The p value	The women	56(63.6)	19(70.4)	0.68
The Caucasian	75(85.2)	20(74.1)	0.30	The women	56(63.6)	19(70.4)	0.68
The Caucasian	75(85.2)	20(74.1)	0.30	Age
Meansigma methods (SD)	43.5(12.5)	41.0(12.4)	0.42	Age
Meansigma methods (SD)	43.5(12.5)	41.0(12.4)	0.42	Median	44.2	37.6
(minima, maximum)	(18.6，73.3)	(23.4，70.9)		Median	44.2	37.6

Table 8: baseline characteristic (1)

Meansigma methods (SD)	Daclizumab N=88 (77%)	Placebo N=27 (23%)	The p value
Meansigma methods (SD)	Daclizumab N=88 (77%)	Placebo N=27 (23%)	The p value	The disease persistent period (year)	22.6(13.6)	24.9(14.0)	0.47
The use year number of imbedibility corticosteroid	5.4(6.1)	4.6(5.3)	0.53	The disease persistent period (year)	22.6(13.6)	24.9(14.0)	0.47
The use year number of imbedibility corticosteroid	5.4(6.1)	4.6(5.3)	0.53	The randomization predose (microgram) of triamcinolone	2089(769)	2111(729)	0.89
FEV1 (L/ second)	2.3(0.7)	2.2(0.5)	0.46	The randomization predose (microgram) of triamcinolone	2089(769)	2111(729)	0.89
FEV1 (L/ second)	2.3(0.7)	2.2(0.5)	0.46	The FEV1% that estimates	68.8(11.3)	68.0(11.1)	0.75
Every day symptom score	7.8(4.1)	9.4(4.2)	0.10	The FEV1% that estimates	68.8(11.3)	68.0(11.1)	0.75
Every day symptom score	7.8(4.1)	9.4(4.2)	0.10	Use B2 agonist rescue medicine every day	3.9(3.2)	4.6(3.1)	0.32
(pressure-vaccum number of times)				Use B2 agonist rescue medicine every day	3.9(3.2)	4.6(3.1)	0.32
(pressure-vaccum number of times)				The PEFR in morning every day (L/ branch)	370.4(96.1)	344.9(78.9)	0.19
Whole blood eosinophilic granulocyte (%)	2.6(2.1)	2.4(1.9)	0.69	The PEFR in morning every day (L/ branch)	370.4(96.1)	344.9(78.9)	0.19
Whole blood eosinophilic granulocyte (%)	2.6(2.1)	2.4(1.9)	0.69	Reversibility changes %	21.9(9.8)	21.0(9.4)	0.68

2. the specificity analyses of method

Table 9-17 has listed the patient data that obtains during the treatment phase 1 (0-84 days) has been carried out the method specificity analyses.

Table 9:FEV ₁(0-84 days)

	Daclizumab N=88 (77%)	Placebo N=27 (23%)
	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline (L/ second)
Meansigma methods (s.e.)	2.34(0.07)	2.25(0.1)	Baseline (L/ second)
Meansigma methods (s.e.)	2.34(0.07)	2.25(0.1)	N	88	27
The 84th day (L/ second)			N	88	27
The 84th day (L/ second)			Meansigma methods (s.e.)	2.43(0.08)	2.20(0.1)
N	76	26	Meansigma methods (s.e.)	2.43(0.08)	2.20(0.1)
N	76	26	% apart from the baseline variation
Meansigma methods (s.e.)	4.40(1.80)	-1.52(2.39)	% apart from the baseline variation
Meansigma methods (s.e.)	4.40(1.80)	-1.52(2.39)	N	76	26
P value in the group	0.02	0.53	N	76	26
P value in the group	0.02	0.53	P value between group		0.05

Table 10: the FEV of expectation ₁% (0-84 days)

	Daclizumab N=88 (77%)	Placebo N=27 (23%)
	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline (L/ second)
Meansigma methods (s.e.)	68.78(1.2)	68.00(2.14)	Baseline (L/ second)
Meansigma methods (s.e.)	68.78(1.2)	68.00(2.14)	N	88	27
The 84th day (L/ second)			N	88	27
The 84th day (L/ second)			Meansigma methods (s.e.)	71.37(1.38)	67.57(2.61)
N	76	26	Meansigma methods (s.e.)	71.37(1.38)	67.57(2.61)
N	76	26	% apart from the baseline variation
Meansigma methods (s.e.)	2.45(0.98)	-0.97(1.42)	% apart from the baseline variation
Meansigma methods (s.e.)	2.45(0.98)	-0.97(1.42)	N	76	26
P value in the group	0.02	0.50	N	76	26
P value in the group	0.02	0.50	P value between group		0.06

Table 11:FEV ₁/ FVC (0-84 days)

	Daclizumab N=88 (77%)	Placebo N=27 (23%)
	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline (L/ second)
Meansigma methods (s.e.)	0.70(0.01)	0.71(0.1)	Baseline (L/ second)
Meansigma methods (s.e.)	0.70(0.01)	0.71(0.1)	N	88	27
The 84th day (L/ second)			N	88	27
The 84th day (L/ second)			Meansigma methods (s.e.)	0.72(0.01)	0.70(0.01)
N	76	26	Meansigma methods (s.e.)	0.72(0.01)	0.70(0.01)
N	76	26	% apart from the baseline variation
Meansigma methods (s.e.)	0.02(0.01)	-0.02(0.01)	% apart from the baseline variation
Meansigma methods (s.e.)	0.02(0.01)	-0.02(0.01)	N	76	26
P value in the group	0.002	0.39	N	76	26
P value in the group	0.002	0.39	P value between group		0.01

Table 12:FEF (25-75%) (0-84 days)

	Daclizumab N=88 (77%)	Placebo N=27 (23%)
	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline (L/ second)
Meansigma methods (s.e.)	1.74(0.09)	1.64(0.1)	Baseline (L/ second)
Meansigma methods (s.e.)	1.74(0.09)	1.64(0.1)	N	88	27
The 84th day (L/ second)			N	88	27
The 84th day (L/ second)			Meansigma methods (s.e.)	1.85(0.1)	1.55(0.11)
N	76	26	Meansigma methods (s.e.)	1.85(0.1)	1.55(0.11)
N	76	26	% apart from the baseline variation
Meansigma methods (s.e.)	0.16(0.05)	-0.10(0.09)	% apart from the baseline variation
Meansigma methods (s.e.)	0.16(0.05)	-0.10(0.09)	N	76	26
P value in the group	0.002	0.63	N	76	26
P value in the group	0.002	0.63	P value between group		0.005

Four kinds of distinct methods specificity analyses of listed patient's vital capacity data among the his-and-hers watches 9-12 (FEV1, estimate FEV1%, FEV1/FVC and FEF (25-75%)) show that separately significant difference is arranged, and this helps this when studying the 84th day daclizumab is apart from the average change of baseline.

And, to observe between the 14th day and the 112nd day of this research, daclizumab treatment patient's baseline FEV1 percent measured value as one man remains on (about 2-5%) on the placebo analog value.

Table 13: symptom score (Sx) (0-84 days)

[0-24]	Daclizumab N=88 (77%)	Placebo N=27 (23%)
[0-24]	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline
Meansigma methods (s.e.)	8.0(0.5)	9.7(0.9)	Baseline
Meansigma methods (s.e.)	8.0(0.5)	9.7(0.9)	N	77	23
The 84th day			N	77	23
The 84th day			Meansigma methods (s.e.)	6.8(0.6)	9.9(1.2)
N	67	21	Meansigma methods (s.e.)	6.8(0.6)	9.9(1.2)
N	67	21	% apart from the baseline variation
Meansigma methods (s.e.)	-1.2(0.4)	0.1(0.4)	% apart from the baseline variation
Meansigma methods (s.e.)	-1.2(0.4)	0.1(0.4)	N	62	20
P value in the group	0.002	0.79	N	62	20
P value in the group	0.002	0.79	P value between group		0.018

Table 14: adopt β2Ji Dongji MDI (0-84 days)

[# pressure-vaccum/sky]	Daclizumab N=88 (77%)	Placebo N=27 (23%)
[# pressure-vaccum/sky]	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline
Meansigma methods (s.e.)	4.1(0.4)	4.5(0.6)	Baseline
Meansigma methods (s.e.)	4.1(0.4)	4.5(0.6)	N	71	20
The 84th day			N	71	20
The 84th day			Meansigma methods (s.e.)	2.9(0.4)	5.0(0.9)
N	55	18	Meansigma methods (s.e.)	2.9(0.4)	5.0(0.9)
N	55	18	% apart from the baseline variation
Meansigma methods (s.e.)	-0.9(0.4)	0.5(0.3)	% apart from the baseline variation
Meansigma methods (s.e.)	-0.9(0.4)	0.5(0.3)	N	49	16
P value in the group	0.03	0.15	N	49	16
P value in the group	0.03	0.15	P value between group		0.009

Table 15:AM PEFR (0-84 days)

[L/ branch]	Daclizumab N=88 (77%)	Placebo N=27 (23%)
[L/ branch]	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline
Meansigma methods (s.e.)	367.6(10.9)	344.6(16.8)	Baseline
Meansigma methods (s.e.)	367.6(10.9)	344.6(16.8)	N	77	23
The 84th day			N	77	23
The 84th day			Meansigma methods (s.e.)	389.3(12.2)	348.8(16.2)
N	67	21	Meansigma methods (s.e.)	389.3(12.2)	348.8(16.2)
N	67	21	% apart from the baseline variation
Meansigma methods (s.e.)	12.9(3.8)	-0.1(9.5)	% apart from the baseline variation
Meansigma methods (s.e.)	12.9(3.8)	-0.1(9.5)	N	62	20
P value in the group	0.001	0.99	N	62	20
P value in the group	0.001	0.99	P value between group		0.217

Table 16:PM PEFR (0-84 days)

[L/ branch]	Daclizumab N=88 (77%)	Placebo N=27 (23%)
[L/ branch]	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline
Meansigma methods (s.e.)	359.4(10.6)	329.6(14.0)	Baseline
Meansigma methods (s.e.)	359.4(10.6)	329.6(14.0)	N	79	23
The 84th day			N	79	23
The 84th day			Meansigma methods (s.e.)	374.5(12.2)	326.7(13.1)
N	61	21	Meansigma methods (s.e.)	374.5(12.2)	326.7(13.1)
N	61	21	% apart from the baseline variation
Meansigma methods (s.e.)	15.1(4.1)	-4.1(7.3)	% apart from the baseline variation
Meansigma methods (s.e.)	15.1(4.1)	-4.1(7.3)	N	57	19
P value in the group	＜0.001	0.77	N	57	19
P value in the group	＜0.001	0.77	P value between group		0.029

The method specificity analyses of listed patient data is also supported the curative effect of daclizumab treatment among the his-and-hers watches 13-16.Shown in the result in table 13 and 14, during the treatment phase 1, the symptom (Sx) and the β of daclizumab treatment group ₂The agonist rescue is used obviously and is reduced.Also have, shown in the result of table 16,, obviously help the patient of daclizumab treatment during the treatment phase 1 apart from the mean change of dusk peak velocity (PMPEFR) baseline with respect to the patient of placebo treatment.

Though do not obtain the significance,statistical data of other continuous variable,, with respect to placebo, help the patient that daclizumab is treated to the 84th day mean change apart from baseline to these variablees AM_PEFR, no Sx% natural law and %FEV1 separately.This general trend that helps daclizumab is further supported its curative effect in treating asthma.

Table 17: increase the weight of

	Daclizumab N=88 (77%)	Placebo N=27 (23%)	The p value
	Daclizumab N=88 (77%)	Placebo N=27 (23%)	The p value	Increase the weight of	6 people (8%) are arranged in 76	26 philtrums have 4 people (15%)	0.37

Shown in the result of table 17, the daclizumab of mensuration treatment patient organizes asthma and increases the weight of situation obviously lower (8% pair 15%).And, find that daclizumab treatment patient is developed to the response curve of the Kaplan-Meier curve of the time of increasing the weight of apparently higher than (according to the survival distribution function) placebo-treated patients.Observed survival distribution function value improves and will be developed to the time of increasing the weight of and extend to 225 days from about 14 days in daclizumab treatment patient.

Similarly, find that daclizumab treatment patient is developed to the response curve of the Kaplan-Meier curve of the time that needs use whole body corticosteroid apparently higher than (according to the survival distribution function) placebo-treated patients.In this case, observed survival distribution function value improves and will be developed to the time of increasing the weight of and extend to 250 days from about 42 days in daclizumab treatment patient.

3. pharmacodynamics terminal point

Table 18 has been listed the result according to pharmacodynamics data and terminal point acquisition.In addition, the percent of observing periphery eosinophilic granulocyte's baseline values of daclizumab treatment patient maintained the level that is lower than placebo-treated patients on all time points (the 28th, 56 and 84 day) basically in 0-84 days.

Table 18: periphery eosinophilic granulocyte (the 0th day the-the 84th day)

[k/mm3]	Daclizumab N=88 (77%)	Placebo N=27 (23%)
[k/mm3]	Daclizumab N=88 (77%)	Placebo N=27 (23%)	Baseline
Meansigma methods (s.e.)	0.2(0.02)	0.22(0.02)	Baseline
Meansigma methods (s.e.)	0.2(0.02)	0.22(0.02)	N	87	26
The 84th day			N	87	26
The 84th day			Meansigma methods (s.e.)	0.14(0.01)	0.23(0.03)
N	74	25	Meansigma methods (s.e.)	0.14(0.01)	0.23(0.03)
N	74	25	% apart from the baseline variation
Meansigma methods (s.e.)	-0.05(0.02)	0.02(0.03)	% apart from the baseline variation
Meansigma methods (s.e.)	-0.05(0.02)	0.02(0.03)	N	73	24
P value in the group	0.003	0.45	N	73	24
P value in the group	0.003	0.45	P value between group		0.04

4. causal analysis

Also according to logarithmic odd number result than the two distortion amounts of acquisition: % increases the weight of, Sx reduces＞=and 25%, FEV1 raising＞=10%.The logarithm odd number ratio of all three variablees is about 1, and this helps daclizumab treatment patient.Though each data does not have significance,statistical, this general trend that helps daclizumab is further supported the curative effect of its treatment asthma.

5. pharmacokinetics feature (PK)

Listed the main PK parameter that the II phase measures in studying in the table 19.

Table 19: main PK parameter brief summary

	V1 (mL/kg)	CL (mL/hr/kg)	C _max (μg/mL)	β_HL (hr)	C _ssavg (μg/mL)	AUC _0-inf (hr*μ g/mL)	V _ss (mL/kg)
	V1 (mL/kg)	CL (mL/hr/kg)	C _max (μg/mL)	β_HL (hr)	C _ssavg (μg/mL)	AUC _0-inf (hr*μ g/mL)	V _ss (mL/kg)	Median	48.6	0.1297	39.9	432	22.9	82892	77.9
Meansigma methods	47.0	0.1339	42.2	473	23.9	86117	79.5	Median	48.6	0.1297	39.9	432	22.9	82892	77.9
Meansigma methods	47.0	0.1339	42.2	473	23.9	86117	79.5	SD	12.7	0.0448	12.6	157	7.4	29493	25.5
CV％	27.0％	33.5％	29.9％	33.3％	31.2％	34.2％	32.1％	SD	12.7	0.0448	12.6	157	7.4	29493	25.5
CV％	27.0％	33.5％	29.9％	33.3％	31.2％	34.2％	32.1％	N
^*	16	32	16	32	32	32	16	N

Estimated 32 patients' PK pattern.The result shows that clearance rate is low, volume of distribution is low and the elimination half-life reaches about 20 days.Originally the low volume of distribution near the blood plasma volume shows originally do not have drug distribution beyond blood circulation.Do not observe drug accumulation after loading dosage.

(comprise 6 patients) in a clinical place from the same position blood sampling of administration.This causes 5 minutes generation deviation high concentrations behind the dosage sample value, appreciable impact C _Max, V1 and V _SsThe calculating of value.All 6 patients in this place are got rid of at V1, V _SsWith the first dosage C _MaxOutside the statistical result of value.

The model curve (i.e. the semilog level curve of simulation group mean P K pattern) that shows cell mean is closely related with viewed mean P K value.Behind the 126th day last dosed administration, be reduced to the serum-concentration of about 1.5 μ g/mL daclizumabs to the 210th day observed PK value.This reduction and this model curve were closely related, and this model curve was reduced to 1 μ g/mL serum-concentration greatly about the 214th day.

6. immunogenicity

With the anti-daclizumab antibody in the bridging ELISA screening blood serum sample.Then, in confirmatory experiment, further estimate the positive that screens.In 113 patients of the anti-daclizumab antibody of test, screening test filters out 10 positive patients.Among these patients, in daclizumab specificity confirmatory experiment, confirmed 6 positive.Accept among the patient of daclizumab 4.7% (4/86) and confirm to have detectable antibody.

7. conclusion

Above-mentioned II phase result of study proof daclizumab can effectively be treated and be sucked the not good chronic persistence asthma adult patients of corticosteroid control.Specifically, the patient demonstrates pulmonary function (vital capacity) and improves, as estimating by the baseline percent change of FEV1 during the treatment phase 1 (the 0th day the-the 84th day).Other vital capacity determination is consistent with these results.The rescue of diary symptom score, β2Ji Dongji use and night the peak value exhalation flow rate disclosed in the group and the variation of group distance baseline has significance,statistical.In placebo group, do not observe in tangible group and change.

In addition, the patient who accepts daclizumab demonstrates and is developed to the time lengthening that needs oral corticosteroid rescue asthma to increase the weight of and has significance,statistical.And the periphery eosinophil count of daclizumab treatment group obviously reduces, and has clear with a consistent signal in all clinical pharmacodynamics terminal points.

In addition, the toleration with the daclizumab treatment is good usually.Between daclizumab and placebo group, sum frequency and seriousness that side reaction takes place have no difference.

Embodiment 2: comprise the II phase data result that inhaled steroid reduces gradually during the treatment phase 2.

Present embodiment is according to the 1 described II phase of embodiment that obtained by the 140th day efficacy result of study (albumen Design Laboratory scheme is numbered DAC-1003) data description.In brief, begin the 85th day (promptly treating the beginning day of phase 2), the patient takes the dosage of imbedibility triamcinolone (TAA), finish 25% of treatment phase 1 o'clock dosage every reducing the patient two weeks (the 84th, 98 and 112 day), fully phased out the steroid (the 126th day) that sucks up to them, accept daclizumab 1mg/kg or infusion with placebo (the 84th, 98,112 and 126 day) simultaneously, input in 15 minutes.As (also referring to Fig. 3 summary) as described in the embodiment 1, dosage, assessment and measurement drug effects that per two weeks are observed a patient.

The result

Proof is compared with placebo patients, the time lengthening (p=0.024) that the asthma that the patient of daclizumab treatment is developed to needs whole body to give the steroid rescue increases the weight of.And, compare placebo group, the daclizumab group at 20 all steroid stable and steroid reduce the phase gradually (11.6% and 28.6%, p=0.09) in the speed reduction that increases the weight of of the state of an illness.Daclizumab treatment patient shows that the periphery eosinophil count is reduced to the 20th week (30 ± 20/mm from baseline ³And placebo is+60 ± 30/mm ³, p=0.004).During the pro-56 days, compare with placebo, the daclizumab treatment patient's that serum eosinophile cationic protein (sECP) baseline raises sECP obviously reduces (p＜0.01) from baseline.Improve with the baseline that has the daclizumab treatment patient who once increases the weight of at least and to compare, do not have the daclizumab patient's that asthma increases the weight of periphery eosinophilic granulocyte obviously to reduce (p=0.032).

Embodiment 3: the analysis of serious asthmatic patient subgroup

Analyze the treatment of daclizumab described in the embodiment 1 especially and study curative effect in serious asthmatic patient subgroup the asthma curative effect II phase.The patient that its data are used for this analysis is at " the ATS symposium collection of thesis of obstinate asthma: present intelligible recommendation and do not answer a question " (Proceedings of the ATS workshop on refractoryasthma:current understanding, recommendations, and unanswered questions), American Thoracic Society, defined " obstinate asthma " patient among Am J Respir Crit Care Med.162 (6): the 2341-51 (2000).The parameter of determining is: TAA dosage＞200mcg/ days, need every day or almost use the symptoms of asthma and FEV1 predicted value＜80% of fugitive beta-agonists every day.33 patients studying of II phase meet the definition of this obstinate asthma.The efficacy result of this obstinate asthma subgroup when listing for the 12nd week in the following table 20.Compare with listed total asthmatic patient colony among the embodiment 1, these results support the conclusion of daclizumab better efficacy (comparing with placebo) in serious asthmatic patient.

Table 20: the efficacy analysis in the serious asthmatic patient colony

	Daclizumab (N=27)		Placebo (N=6)		The p value
	Daclizumab (N=27)		Placebo (N=6)			The curative effect assay method		N		N
FEV1	+8.5％	21	-9.3％	6		The curative effect assay method		N		N	0.01
FEV1	+8.5％	21	-9.3％	6	No asthma natural law	15％	26	0％	6	0.015	0.01
PM_PEFR (L/ branch)	+15.7％	18	-13.5％	6	No asthma natural law	15％	26	0％	6	0.015	0.028
PM_PEFR (L/ branch)	+15.7％	18	-13.5％	6	Symptom (Sx)	-0.5％	19	+1.2％	6	0.1	0.028
β2Ji Dongji MDI uses (pressure-vaccum/sky)	+0.3％	17	+1.3％	6	Symptom (Sx)	-0.5％	19	+1.2％	6	0.1	0.404
β2Ji Dongji MDI uses (pressure-vaccum/sky)	+0.3％	17	+1.3％	6	AM_PEFR (L/ branch)	+9％	19	-10％	6	0.138	0.404

The asthma of also having analyzed serious asthmatic patient colony increases the weight of data.Table 21 provides the result of treatment phase 1 (the 0th day the-the 84th day) to show the incidence rate lower (comparing with placebo) that increases the weight of in the serious asthmatic patient of daclizumab treatment.

Table 21: the asthma in the serious asthma colony increases the weight of

	Daclizumab (N=27)	Placebo (N=6)	The p value ^*
	Daclizumab (N=27)	Placebo (N=6)	The p value ^*	The patient that report increases the weight of	3/21(14.3％)	2/6(33.3％)	0.303
Report needs the patient that increases the weight of of CCS rescue	2/20(10.0％)	2/6(33.3％)	0.218	The patient that report increases the weight of	3/21(14.3％)	2/6(33.3％)	0.303
	2/20(10.0％)	2/6(33.3％)	0.218	The p value of Fisher degree of accuracy check ^*

Though the preferred implementation with reference to this paper has been described the present invention, it should be understood that, do not deviate from spirit of the present invention can make various modifications.Include all publications, patent, patent application and website in this paper as a reference with integral body, identical when its degree and each patent, patent application or website are quoted especially and individually and included this paper as reference in full in.

Claims

1. method that the patient who needs treatment of respiratory diseases is treated, described method comprise and give the pharmaceutical preparation of described patient treatment effective dose, described pharmaceutical preparation contain can with the bonded antibody of IL-2 receptor-specific.

2. the method for claim 1 is characterized in that, described respiratory tract disease is selected from: asthma, allergic rhinitis, atopic dermatitis, nasal polyp, Churg-Strauss syndrome, sinusitis and COPD.

3. the method for claim 1 is characterized in that, described antibody is humanized antibody.

4. method as claimed in claim 3 is characterized in that described humanized antibody is a daclizumab.

5. the method for claim 1 is characterized in that, described antibody and daclizumab are in conjunction with identical epi-position.

6. method as claimed in claim 5 is characterized in that, the aminoacid sequence of described antibody is identical with the aminoacid sequence of daclizumab at least 80%.

7. the method for claim 1 is characterized in that, outside gastrointestinal tract, intravenous, intramuscular or subcutaneously give described pharmaceutical preparation.

8. method as claimed in claim 7, it is characterized in that, described pharmaceutical preparation is liquid, the tension force buffer agent that it contains the 100mg/ml daclizumab of having an appointment, the succinate buffer of the about 5.5-6.5 of about 20-60mM pH, about 0.01%-0.1% polysorbate and produces isotonicity.

9. the method for claim 1 is characterized in that, described treatment effective dose is about 0.001mg/kg-10mg/kg.

10. the method for claim 1 is characterized in that, described treatment effective dose is about 0.5mg/kg-4.0mg/kg.

11. the method for claim 1 is characterized in that, described treatment effective dose is the fixed dosage between about 100mg and the 200mg.

12. a method for the treatment of patient's asthma, described method comprises: give the pharmaceutical preparation of described patient treatment effective dose, described pharmaceutical preparation contain can with the bonded antibody of IL-2 receptor-specific.

13. method as claimed in claim 12 is characterized in that, described asthma is chronic persistence asthma.

14. method as claimed in claim 12 is characterized in that, described asthma is that moderate is to severe asthma.

15. method as claimed in claim 12, also comprise give the patient one or more be selected from down the group medicine: beclometasone, budesonide, flunisolide, fluticasone, triamcinolone, mometasone and Nai De.

16. method as claimed in claim 12 is characterized in that, the binding affinity of described antibody and described human IL-2's receptor is at least 10 ⁸M ^-1

17. want 12 described methods, it is characterized in that the binding affinity of described antibody and described human IL-2's receptor is at least 10 as right ⁹M ^-1

18. method as claimed in claim 12 is characterized in that, described antibody is monoclonal antibody.

19. method as claimed in claim 12 is characterized in that, described antibody is chimeric antibody.

20. method as claimed in claim 12 is characterized in that, described antibody is people's antibody.

21. method as claimed in claim 12 is characterized in that, described antibody is humanized antibody.

22. method as claimed in claim 21 is characterized in that, described humanized antibody is a daclizumab.

23. method as claimed in claim 12 is characterized in that, described antibody and daclizumab are in conjunction with identical epi-position.

24. method as claimed in claim 23 is characterized in that, the aminoacid sequence of described antibody is identical with the aminoacid sequence of daclizumab at least 80%.

25. method as claimed in claim 24 is characterized in that, the aminoacid sequence in described antibody CDR district is identical with the aminoacid sequence at least 95% in daclizumab CDR district.

26. method as claimed in claim 12 is characterized in that, outside gastrointestinal tract, intravenous, intramuscular or subcutaneously give described pharmaceutical preparation.

27. method as claimed in claim 22, it is characterized in that, described pharmaceutical preparation is liquid, the tension force buffer agent that it contains succinate buffer, the about 0.01%-0.1% polysorbate of the 100mg/ml daclizumab of having an appointment, the about 5.5-6.5 of about 20-60mM pH and keeps isotonicity.

28. method as claimed in claim 12 is characterized in that, described treatment effective dose is about 0.001mg/kg-10mg/kg.

29. method as claimed in claim 12 is characterized in that, described treatment effective dose is about 0.5mg/kg-4.0mg/kg.

30. method as claimed in claim 12 is characterized in that, described treatment effective dose is about the fixed dosage between 100mg and the 200mg.

31. the method that the patient who needs the cell-mediated atopic disease therapeutics of Th2 is treated, described method comprises the pharmaceutical preparation that gives described patient treatment effective dose, described pharmaceutical preparation contain can with the bonded antibody of IL-2 receptor-specific.

32. method as claimed in claim 31 is characterized in that, described disease is selected from: asthma, allergic rhinitis, atopic dermatitis, nasal polyp, Churg-Strauss syndrome and sinusitis.

33. method as claimed in claim 31 is characterized in that, described antibody is humanized antibody.

34. method as claimed in claim 33 is characterized in that, described humanized antibody is a daclizumab.

35. method as claimed in claim 33 is characterized in that, described antibody and daclizumab are in conjunction with identical epi-position.

36. method as claimed in claim 33 is characterized in that, the aminoacid sequence of described antibody is identical with the aminoacid sequence of daclizumab at least 80%.