CN100549030C - The IL-4 mutein receptor antagonists of modifying - Google Patents

Abstract

The IL-4 mutein receptor antagonists that the present invention relates to modify comprises the IL-4 mutein receptor antagonists that is coupled to polyoxyethylene glycol.The related preparations and dosage and the medication that are used for the treatment of purpose also are provided.By suppressing the airway hyperreactivity and the eosinophilia of IL-4 and IL-13 mediation, IL-4 mutein receptor antagonists, composition and the method for these modifications provides treatment to select for those suffer the individuality of the hardship of respiratory disorder such as asthma.More particularly, owing to have longer plasma half-life, these antagonists have the effect that the time length increases than the IL-4RA of unmodified.

Description

The IL-4 mutein receptor antagonists of modifying

Cross reference

[0001] the application requires the U.S. Provisional Application 60/498,906 of submission on August 29th, 2003; The right of priority of the U.S. Provisional Application 60/530,182 that the U.S. Provisional Application 60/528,228 that on December 9th, 2003 submitted to and on December 17th, 2003 submit to, the disclosure of these applications is incorporated this paper by reference clearly into.

Technical field

[0002] the present invention relates to be coupled to the IL-4 mutein receptor antagonists of non--protein polymer such as polyoxyethylene glycol.In addition, also provide related preparations, dosage and the medication thereof that is used for the treatment of purpose.The IL-4 mutein receptor antagonists of these modifications, compositions related and method can be used for selecting for the individuality that suffers severe asthma, chronic obstructive pulmonary disease and the misery of relevant lung symptom (lung conditions) provides treatment.

Background technology

[0003] feature of asthma is changeable, reversibility airflow obstruction and airway hyperreactivity (AHR), and to soak into tunica mucosa bronchiorum relevant with activated T-lymphocyte (T-cell) and eosinophil.The air flue mastocyte of these cells and residence is secreted the various kinds of cell factor and medium (mediator) together, and they play basic effect in disease pathogenesis.By discharging specific cytokine (IL-4, IL-5, IL-9 and IL-13), the CD4+Th2 cell is being considered to the process (1,2) of disease.Especially, Th2 cytokine IL-4 and the IL-13 key that is considered to the development of airway inflammation and airway hyperreactivity and keeps.

[0004] research also supports IL-4 and IL-13 to have crucial effect in the asthma pathogeny in many bodies.By using the animal that lacks arbitrary cytokine, perhaps and the reagent of IL-4 or IL-13 function, observe the vital role of these cytokines, promptly, cause the primary immunne response of airway inflammation and airway hyperreactivity and the effect (3,4) in the secondary immune response in adjusting.Cumulatively, these data show that IL-4 and IL-13 may bring into play in the reaction of supersensitivity air flue not only overlapping but also independently act on, and show that these two kinds of cytokines of target may have more benefit with respect to the wherein independent a kind of cytokine of target.

[0005] antagonist of IL-4 is in the news in the literature.The IL-4 mutant that serves as the antagonist function comprises IL-4 antagonist muteins IL-4/Y124D (Kruse, N., Tony, H.P., Sebald, W., Conversion of humaninterleukin-4 into a high affinity antagonist by a single amino acid replacement, Embo J.11:3237-44,1992) and two mutain IL-4 (double mutein IL-4) [R121D/Y124D] (Tony, H., et al., Design of Human Interleukin-4 Antagonists in Inhibiting Interleukin-4-dependentand Interleukin-13-dependent responses in T-cells and B-cells with high efficiency, Eur.J.Biochem.225:659-664 (1994)).Tyrosine is substituted by aspartic acid on single mutation albumen 124 in the D-spiral.Arginine is substituted by aspartic acid on two mutains 121 in the D-spiral, and tyrosine is substituted by aspartic acid on 124.Variation in this D spiral fragment with in the interactional variation of second calmodulin binding domain CaM positive association is arranged.

[0006] demonstrates the IL-4 mutation variants that wild-type IL-4 is had excitability (agonism) or an antagonism (antagonism) and can be used for the treatment of wherein a kind of relevant symptom in the multiple-effect effect with IL-4.For example, the antagonist of IL-4 can be used for the treatment of the symptom that the generation owing to IL-4 worsens, such as asthma, allergy or other struvite related symptoms of replying.The agonist of IL-4 can be used for the treatment of such symptom, that is, the wherein improvement of the existence of IL-4 and disease or alleviate relevantly, described disease is autoimmune disease such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes etc. for example.One of these autoimmune diseases is characterized as the t helper cell group, has polarized action in the generation of 1 type and 2 types (Th1, Th2).Rely on the cytokine that exists between stimulation period, initial CD4+T cytodifferentiation becomes Th1 or Th2 hypotype.The IL-4 agonist will make this situation shift the T-helper of wanting for producing ideally, just produce Th2, thereby have result of treatment.

[0007] PCT/US93/03613 discloses a kind of IL-4 variant, it has Phe-Leu or Tyr-Leu sequence in the α-Luo Xuanjiegou territory, and have electronegative amino acid in two amino acid in upstream that is close to Phe-Leu or Tyr-Leu sequence or downstream, described variant has substituted electronegative amino acid has increase to the IL-4 acceptor avidity because of neutral amino acids.It also discloses, and in the alpha-helix of IL-4, in two residues of electronegative residue, the specificity of Trp-Leu or Phe-Leu substitutes and to cause producing the avidity that has improved.This variant is IL-4 fusion rotein (with diphtheria toxin).

[0008] before, United States Patent (USP) 6,028 was reported in 176 and 6,313,272 from recombination mutation albumen (IL-4RA) people IL-4, that sudden change is arranged on its two positions of aminoacid sequence.IL-4RA combines with people IL-4 receptor alpha chain with high-affinity, and the α chain is the important function signal conduction component of IL-4 and IL-13 acceptor complex body.This mutain does not have agonist activity, at external potent competitive IL-4 and the IL-13 receptor antagonist (referring to United States Patent (USP) 6,028,176 and 6,313,272) of serving as.The distinct disadvantage of using IL-4RA is the transformation period (about 3-6 hour) in its short relatively body.In the primate asthmatic model, pharmacokinetics/pharmacokinetic modeling of IL-4RA shows that effective average steady state concentration of optimum therapeuticing effect is approximately 60ng/ml.

[0009] overcoming a short method of transformation period is frequently to give the patient with the IL-4RA mutain, yet frequent drug administration (normally by injection and endotracheal intubation) produces very significantly obstacle to patient's acceptance of this treatment and treatment administration clinically.

Summary of the invention

[0010] the invention provides the IL-4RA mutain that has the longer transformation period than the mutain of previous report.The present invention also provides the reagent and the method for the immunne response that suppresses IL-4 and IL-13 mediation.This aspect of the present invention and other aspects are provided by one or more embodiments of listing below.

[0011] in one embodiment, the invention provides the purifying prepared product of the IL-4 mutein receptor antagonists of modification, it comprises the IL-4 mutein receptor antagonists that is connected to charged non-protein polymer, described charged non-protein polymer be selected from polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene (polyoxyalkylene, polyoxyalkylenes).Aspect of the present embodiment, the prepared product of purifying comprises the IL-4 mutein receptor antagonists polypeptide of modification, this polypeptide nucleotide sequence coded by shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7 or the SEQ ID NO:8.In one aspect, this polypeptide comprises the aminoacid sequence shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15 or the SEQ ID NO:16.

[0012] in one embodiment, the IL-4 mutein receptor antagonists polypeptide of modification can be coupled to charged non-protein polymer at the position 28,36,37,38,104,105 of IL-4 or 106 amino-acid residue place.Such position is to be numbered according to wild-type IL-4 (being human interleukin-4) aminoacid sequence.Aspect of this embodiment, 28,36,37,38,104,105 or 106 amino-acid residue is a halfcystine in the position.

[0013] in one embodiment, the mutein receptor antagonists modified of the present invention with about 0.1nM to about 10 μ M, about 0.5nM about 1 μ M or the about 1.0nM K of about 100nM extremely extremely _d, be incorporated into the IL-4 receptor alpha chain.

[0014] in another embodiment, the IL-4 mutein receptor antagonists of modification with about 0.1nM to about 10 μ M, about 0.5nM to about 1 μ M or about 1.0nM the IC of about 100nM extremely ₅₀, suppress the TF-1 cell proliferative of IL-4 replied.

[0015] in also having an embodiment, the IL-4 mutein receptor antagonists of modification suppresses the TF-1 cell and the proliferative of IL-13 is replied its IC ₅₀Be selected from about 0.1nM to about 10 μ M, about 0.5nM to about 1 μ M or about 1.0nM about 100nM extremely.

[0016] in further embodiment, the IL-4 mutein receptor antagonists of modification suppresses human B cell and the proliferative of IL-4 is replied its IC ₅₀Be selected from about 0.1nM to about 10 μ M, about 0.5nM to about 1 μ M or about 1.0nM about 100nM extremely.

[0017] in another embodiment, the IL-4 mutein receptor antagonists of modification inhibition human T-cell replys its IC to the proliferative of IL-4 ₅₀Be selected from about 0.1nM to about 10 μ M, about 0.5nM to about 1 μ M, about 1.0nM about 100nM extremely.

[0018] in also having an embodiment, the plasma half-life of the IL-4 mutein receptor antagonists that the present invention modifies be unmodified the IL-4 receptor antagonist plasma half-life at least about 2-10 doubly.

[0019] the invention provides pharmaceutical composition, comprise the IL-4 mutein receptor antagonists that (a) modifies, it is in conjunction with people IL-4 acceptor; (b) pharmaceutically acceptable carrier.

[0020] the invention provides purified polynucleotides, comprise (a) nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; Or (b) coding has the nucleotide sequence of polypeptide of the aminoacid sequence shown in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or the SEQ ID NO:16.

[0021] the present invention also provides the expression vector that comprises polynucleotide of the present invention, and the host cell that comprises expression vector of the present invention.

[0022] in addition, the invention provides the method for the IL-4 mutein receptor antagonists of preparation modification, comprise step: a) under the condition that described antagonist can be expressed, cultivate above-mentioned host cell; And b) is purified into described antagonist from described host cell culture.Aspect specific, the antagonist that produces by the inventive method can suppress the activity of IL-4 and IL-13 mediation, and is coupled to the charged non-protein polymer that is selected from polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

[0023] the present invention also provides the method for the treatment people disorder relevant with the IL-13 activity with the IL-4 that increases, and comprise step: the people with certain state (a) is provided, and the activity of IL-4 and IL-13 increases in described state; (b) give IL-4 mutein receptor antagonists or the pharmaceutical composition of the present invention that the present invention of the effective quantity of described people modifies.In one aspect, described disorder is asthma, chronic obstructive pulmonary disease (such as pulmonary emphysema or chronic bronchitis) or relevant lung state.

[0024] the present invention also provides the method for method, the antagonist by this method preparation, the composition that comprises this kind antagonist and the treatment people disorder of the IL-4 mutein receptor antagonists of the modification for preparing activity form, and this method comprises the pharmaceutical composition that gives this kind antagonist and comprise this kind antagonist.Described method comprises the steps: that (a) under the condition that antagonist is expressed, cultivates above-mentioned host cell; (b) make the antagonist refolding under the condition of dithiothreitol (DTT) existing; (c) be purified into antagonist from the host cell culture.In one embodiment, this method also comprises step: (d) antagonist is coupled to charged non-protein polymer; (e) purifying is coupled to the antagonist of charged non-protein polymer.

[0025] some preferred embodiment and claims of more describing in detail below stating by reading, concrete preferred embodiment of the present invention will become and be perfectly clear.

Description of drawings

[0026] Fig. 1 shows the schematically statement of chemistry of PEGization (PEGylation) reaction.

Detailed Description Of The Invention

[0027] the present invention relates to the IL-4 mutein receptor antagonists modified, comprises being coupled to charged non-protein polymer the IL-4 mutein receptor of---preferred peg molecule---.

[0028] unless context has requirement in addition, singular references will comprise plural number, and plural term also will comprise odd number.

[0029] chapter title used herein only is used for organizational goal, and is not interpreted as the described theme of restriction.

All lists of references that the application quotes are incorporated this paper by reference clearly into.

Definition

[0030] term " polynucleotides " or " nucleotide sequence " or " nucleic acid molecules " refer to DNA or RNA sequence. This term comprises the molecule that is formed by any known DNA and RNA base analogue, the base analogue of DNA and RNA is such as still being not limited to: the 4-acetylcytosine, 8-hydroxy-n 6-methyladenosine, aziridinyl-cytimidine, false iso-cytosine (pseudoisocytosine), 5-(carboxyl hydroxymethyl) uracil, 5 FU 5 fluorouracil, 5-bromouracil, 5-carboxyl methylamino methyl-2-thiouracil, 5-carboxyl-methylamino methyluracil, dihydrouracil, inosine, N6-is different-the pentenyl adenine, the 1-methyl adenine, 1-methyl pseudouracil, the 1-methyl guanine, M1I, 2,2-dimethyl-guanine, the 2-methyl adenine, the 2-methyl guanine, the 3-methylcystein, 5-methylcytosine, the N6-methyl adenine, the 7-methyl guanine, 5-methylamino methyluracil, 5-methoxyl group amino-methyl-2-thiouracil, β-D-MANNOSE Q nucleosides, 5 '-methoxycarbonyl-methyluracil, the 5-methoxyuracil, 2-methyl sulfo--N6-isopentenyl gland purine, uracil-5-oxy acetic acid methyl ester, uracil-5-fluoroacetic acid, oxybutoxosine, pseudouracil, Q nucleosides (queosine), 2-sulphur cytimidine, 5-methyl-2-thiouracil, the 2-thiouracil, the 4-thiouracil, methyl uracil, N-uracil-5-oxy acetic acid methyl ester, uracil-5-fluoroacetic acid, pseudouracil, the Q nucleosides, 2-sulphur cytimidine and 2,6-diaminopurine.

[0031] term " purifying " or " separation " polynucleotides refer to nucleic acid molecules of the present invention, open with protein, lipid, carbohydrate or other separating substances at least about 50% its (1), when TNA origin source cell is separated, these materials are natural discoveries, (2) be not attached to the whole part of polynucleotides or wherein a part of, described polynucleotides are and described " nucleic acid molecules of separation " the natural connection, (3) be operably connected to polynucleotides, described polynucleotides not with its natural connection, or (4) natural existence as the part of not larger polynucleotide sequence. Preferably, the nucleic acid molecules that the present invention separates basically (substantially) without nucleic acid molecules or other pollutants of any other pollution, and these pollutants are found to be present in its natural surroundings, and can disturb it in polypeptide production application or disturb its treatment, diagnosis, prevention or research purposes.

[0032] for " according to wild type IL-4 numbering ", we refer to differentiate selected amino acid according to the normal amino acid position that occurs in wild type IL-4.

[0033] term " carrier " is used to refer to any molecule (for example nucleic acid, plasmid or virus), and it is used for coded message is passed to host cell.

[0034] term " expression vector " refers to the carrier that is suitable for transformed host cell and contains nucleotide sequence, and described nucleotide sequence instructs and/or the expression of the heterologous nucleic acid sequence that regulation and control are inserted. Expression includes but not limited to such as the process of transcribing, translating, if there is introne, also comprises the RNA montage.

[0035] term " host cell " is used in reference to the cell that has been converted in this article, or can transform by enough nucleotide sequences, then expresses the cell of selected interested gene. This term comprises the offspring of parental cell, and no matter whether the offspring is identical with original parent in form or genetic constitution, and is just passable as long as selected gene exists.

[0036] term " transduction " is used in reference to gene and transfers to another bacterium from a bacterium, is usually finished by bacteriophage. " transduction " also refers to obtain and shift the eukaryotic sequence by retrovirus.

[0037] term " transfection " is used to refer to or foreign DNA external by Cell uptake, and when foreign DNA had been imported into cell membrane the inside, cell was just by " transfection ". Many rotaring dyeing technologies are known in the art, and open in this article. Referring to for example Graham et al., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods in Molecular Biology (Elsevier, 1986); With Chu et al., 1981, Gene 13:197. This type of technology can be used to one or more foreign DNAs are partly imported suitable host cell.

[0038] as used herein, the variation of term " conversion " phalangeal cell hereditary feature, when cell has been modified to when containing new DNA, cell just is converted. For example, when cell by its nature during by genetic modification, cell just is converted. After transfection or the transduction, be integrated into the chromosome of cell by entity, transforming DNA can be recombinated with cell DNA, or can be used as the episome element and kept by instantaneous, and does not copy, or can be used as plasmid and independently copy. When DNA is replicated along with cell division, can think that cell is by stable conversion.

[0039] road as known in the art, term " homogeneity (identity) " refer to the relation between the sequence of two or more peptide molecules or two or more nucleic acid molecules, and it is by comparative sequences and determined. In this area, " homogeneity " also refers to the Serial relation degree between nucleic acid molecules or the polypeptide, and this situation can be to measure by the coupling between two or more nucleotides or two or more amino acid sequences. " homogeneity " is measured is the percentage of the common identical match of sequence less in two or more sequences, and wherein breach comparison (if any) is processed by specific Mathematical Modeling or computer program (i.e. " algorithm ").

[0040] term " similitude " is a relevant concept, but different from " homogeneity ", " similitude " refers to that a kind of of correlation measures, and described correlation comprises that not only identical coupling also comprises the conservative coupling that substitutes. If two peptide sequences for example have 10/20 same amino acid, and remaining amino acid all is non-conservative substitute, and the percentage of homogeneity and similitude all is 50% so. In same example, if also have 5 positions that conservative substituting arranged, the percentage of homogeneity still is 50% so, but the percentage of similitude then will be 75% (15/20). Therefore, in having the conservative example that substitutes, the similitude percentage between two polypeptide will be higher than the homogeneity percentage between those two polypeptide.

[0041] utilizes currently known methods, can easily calculate the identity and the similarity of associated nucleic acid and polypeptide.Such method includes but not limited to the method for following document description: COMPUTATIONAL MOLECULARBIOLOGY, (Lesk, A.M., ed.), and 1988, Oxford University Press, New York; BIOCOMPUTING:INFORMATICS AND GENOME PROJECTS, (Smith, D.W., ed.), and 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A.M., and Griffin, H.G., eds.), and 1994, HuMana Press, New Jersey; VonHeinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, AcademicPress; SEQUENCE ANALYSIS PRIMER, (Gribskov, M.and Devereux, J., eds.), and 1991, M.Stockton Press, New York; Carillo et al., 1988, SIAM J.Applied Math., 48:1073; With Durbin et al., 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge UniversityPress.

[0042] preferred method of mensuration identity is designed to provide maximum coupling between tested sequence.The method of measuring identity is described in the available computer program of the public.The preferred computer program technic of measuring identity between two sequences includes but not limited to the GCG routine package, comprises GAP (Devereux et al., 1984, Nucl.Acid.Res., 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN and FASTA (Altschul et al., 1990, J.Mol.Biol., 215:403-410).The BLASTX program can (MD 20894 for BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda from National Center for Biotechnology Information (NCBI) and other sources; Altschul etal., 1990, supra) the open acquisition.Known Smith Waterman algorithm also can be used for measuring identity.

[0043] some the comparison scheme that is used to compare two aminoacid sequences can be created in the coupling in zone of the weak point of two sequences, and this little comparison zone can have very high sequence identity, although between these two full length sequences tangible relation not.Therefore, in certain embodiments, the comparison that selected comparison method (GAP program) will produce is the comparison of at least 50 continuous amino acids of coverage goal polypeptide.

[0044] for example, algorithm GAP (Genetics Computer Group uses a computer, University ofWisconsin, Madison, WI), treat that two polypeptide of determined sequence identity per-cent are joined by connection, to obtain their amino acid whose separately optimum matching (" (scope of coupling) matched span " is as determining by algorithm).In certain embodiments, (it multiply by 3 by average diagonal lines (averagediagonal) and calculates the open penalties in room (gap opening penalty); Wherein " average diagonal lines " is cornerwise mean value of the comparator matrix that just is being used; " diagonal lines " is score value or number of being given each accurate amino acid coupling by specific comparator matrix) and the room extend penalties (gap extension penalty) (its normally the open penalties in room 1/10th), and comparator matrix such as PAM250 or BLOSMM 62 and algorithm are united use.In certain embodiments, and the standard comparator matrix (referring to Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352 is about PAM 250 comparator matrixs; Henikoff et al., 1992, Proc.Natl.Acad.Sci USA, 89:10915-10919 is about BLOSMM 62 comparator matrixs) also adopted by algorithm.

[0045] in certain embodiments, the parameter of peptide sequence comparison comprises:

Algorithm: Needleman et al., 1970, J.Mol.Biol., 48:443-453;

Comparator matrix: from Henikoff et al., 1992, the BLOSMM 62 of supra;

Room penalties: 12

Room length penalties (Gap Length Penalty): 4

Similarity threshold value (Threshold of Similarity): 0

[0046] the GAP program can be used with reference to above-mentioned parameter.In certain embodiments, compare (terminal room is not had penalties) for the polypeptide that uses the GAP algorithm, above-mentioned parameter can be a default parameters.

[0047] as used herein, 20 conventional amino acid and their the abbreviation usage that observes a usual practice.Referring to IMMUNOLOGY--A SYNTHESIS, second edition, (E.S.Golub and D.R.Gren, Eds.), Sinauerland Associates:Sunderland, MA, 1991, it incorporates this paper into by reference to be used for any purpose.20 amino acid whose steric isomers (for example D-amino acid) commonly used; Alpha-non-natural amino acid, such as α-, α-two substituted amino acids, N-alkyl amino acid, lactic acid and other unconventional amino acid also can be the suitable compositions of polypeptide of the present invention.Unconventional amino acid whose example comprises: 4-oxyproline, Gla, ε-N, N, N-trimethyl lysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-Methyl histidine, 5-oxylysine, σ-N-methylarginine and other similar amino acid and imino-acid (for example 4-oxyproline).Polypeptide representation used herein, according to the usage and the convention of standard, left-hand is to being the amino-terminal end direction, right-hand lay is the C-terminal direction.

[0048] according to common side chain characteristic, naturally occurring residue can be divided into several types:

1) hydrophobic residue: nor-leucine (Nor), Met, Ala, Val, Leu, Ile;

2) residue of neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) tart residue: Asp, Glu;

4) Jian Xing residue: His, Lys, Arg;

5) influence the residue of chain orientation (chain orientation): Gly, Pro; With

6) aromatic residue: Trp, Tyr, Phe.

[0049] Bao Shou amino acid replacement can comprise that the member of the wherein class in these types is alternative with another member of same type.Conservative amino acid replacement can comprise the amino-acid residue that non-natural exists, and it is synthetic rather than by synthetic being impregnated in biosystem by chemical peptide usually.These comprise the amino acid moiety of peptide mimics and other upsets (reversed) or reversing (inverted) form.

[0050] nonconservative amino acid replacement can comprise that the member of the wherein class in these types substitutes a member of another type.Replaced like this residue can be imported in the human protein with non-human protein's matter homologous zone in, or be imported in the non-homogeneous zone of this molecule.

[0051] when producing this type of and change, according to some embodiment, can considered amino acid detest aqua index (hydropathic index).According to amino acid whose hydrophobicity and charge characteristic, every seed amino acid has been endowed one and has detested aqua index.They are: Isoleucine (+4.5); Xie Ansuan (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Halfcystine/Gelucystine (+2.5); Methionine(Met) (+1.9); L-Ala (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophane (0.9); Tyrosine (1.3); Proline(Pro) (1.6); Histidine (3.2); L-glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); L-asparagine (3.5); Methionin (3.9) and arginine (4.5).

[0052] amino acid detest the importance of aqua index in giving the biological function of protein interaction understood by this area (referring to for example Kyte et al., 1982, J.Mol.Biol.157:105-131).Know that some amino acid can be replaced by to have similarly detests aqua index or another amino acid of fractional, and still keeps similar biologic activity.When detesting aqua index and change, in certain embodiments, comprise detest aqua index ± 2 with interior amino acid whose replacement.In certain embodiments, comprise those ± 1 with interior, in certain embodiments, comprise those ± 0.5 with interior.

[0053] this area is understood, and similar amino acid whose substitute also can effectively be carried out according to wetting ability, particularly when consequent biological function albumen or peptide are intended to be used for the immunology embodiment, as disclosed herein.In certain embodiments, the local average wetting ability of protein maximum (local average hydrophilicity)---by wetting ability control of this proteic adjacent amino acid---is with its immunogenicity and antigenicity, promptly relevant with proteinic biological characteristics.

[0054] following hydrophilicity value has been endowed these amino-acid residues: arginine (+3.0); Methionin (+3.0); Aspartic acid (+3.0 ± 1); L-glutamic acid (+3.0 ± 1); Serine (+0.3); L-asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline(Pro) (0.5 ± 1); L-Ala (0.5); Histidine (0.5); Halfcystine (1.0); Methionine(Met) (1.3); Xie Ansuan (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5) and tryptophane (3.4).When changing based on similar hydrophilicity value, in certain embodiments, comprise hydrophilicity value ± 2 with interior amino acid whose replacement.In certain embodiments, comprise those ± 1 with interior, in certain embodiments, comprise that those replace with interior amino acid ± 0.5.The technician also can identify the epi-position of one-level aminoacid sequence according to wetting ability.These zones are also referred to as " epi-position nucleus (epitopic core regions) ".

[0055] exemplary amino acid replacement is shown in the table 1.

Table 1

Amino acid replacement

Original residue	Exemplary substituting	Preferred substituting
			Ala	Val、Leu、Ile	Val
Arg	Lys、Gln、Asn	Lys
			Asn	Gln	Gln
Asp	Glu	Glu
			Cys	Ser、Ala	Ser
Gln	Asn	Asn
			Glu	Asp	Asp
Gly	Pro、Ala	Ala
			His	Asn、Gln、Lys、Arg	Arg
Ile	Leu, Val, Met, Ala, Phe, nor-leucine	Leu
			Leu	Nor-leucine, Ile, Val, Met, Ala, Phe	Ile
Lys	Arg, 1,4 diamino-butyric acid, Gln, Asn	Arg
			Met	Leu、Phe、Ile	Leu
Phe	Leu、Val、Ile、Ala、Tyr	Leu
			Pro	Ala	Gly
Ser	Thr、Ala、Cys	Thr
			Thr	Ser	Ser
Trp	Tyr、Phe	Tyr
			Tyr	Trp、Phe、Thr、Ser	Phe
Val	Ile, Met, Leu, Phe, Ala, nor-leucine	Leu

[0056] use technique known, the technician can determine suitable polypeptide variants, as described herein.In certain embodiments, those skilled in the art can identify suitable molecular domains, can be considered to the unessential zone of activity is changed this zone by target, and saboteur's activity not.In other embodiments, the technician can identify residue and molecular moiety conservative in similar polypeptide.In further embodiment, even may replace, and can not destroy biologic activity or influence polypeptide structure unfriendly to biological activity or to the amino acid that the important zone of structure also can be guarded.

[0057] in addition, those skilled in the art can consult in similar polypeptide structure-functional study of identifying the important residue of activity or structure.According to this type of relatively, the technician can the importance of predicted amino acid residue in protein, described residue corresponding in analogous protein to activity or the important amino-acid residue of structure.Those skilled in the art can select chemically similarly amino acid replacement for predicted like this important amino acid residue.

[0058] those skilled in the art also can be according to the structure in the similar polypeptide, analyzing three-dimensional structure and aminoacid sequence.According to this type of information, those skilled in the art can be according to its three-dimensional structure, the comparison (alignment) of the amino-acid residue of prediction polypeptide.In certain embodiments, those skilled in the art can select predicted amino-acid residue on protein surface not to be carried out extreme change, because this type of residue may participate in the important interaction with other molecules.In addition, those skilled in the art are contained single amino acids alternate test variant at the amino-acid residue place that can be created in each expectation.Described variant screens with activity determination method well known by persons skilled in the art then.This type of variant can be used to collect the information about suitable variant.For example, if find change that particular amino acid residue is carried out is caused ruinate, reduced or inappropriate activity undesirably, can get rid of variant so with such change.In other words, based on the information of collecting from such normal experiment, those skilled in the art can easily determine such amino acid, that is, therein, further substitute and avoided, and perhaps suddenly change separately or with other.

[0059] many scientific publication things have been devoted to the prediction of secondary structure.Referring to Moult, 1996, Curr.Op.in Biotech.7:422-427; Chou et al., 1974, Biochemistry 13:222-245; Chou et al., 1974, Biochemistry 113:211-222; Chou et al., 1978, Adv.Enzymol.Relat.Areas Mol.Biol.47:45-148; Chou et al., 1979, Ann.Rev.Biocherm.47:251-276; With Chou et al., 1979, Biophys.J.26:367-384.And computer program also can be used for the aid forecasting secondary structure at present.A kind of method of prediction secondary structure is based on the homology model.For example, have and surpass 30% sequence identity, or two polypeptide or the albumen that have greater than 40% similarity usually have similar topological framework.The growth of protein structure proximity database (pdb) proximity recently provides enhanced secondary structure predictability, is included in possible folding number in polypeptide or the protein structure.Referring to Holm et al., 1999, Nucl.Acid.Res.27:244-247.Show (Brenner et al., 1997, Curr.Op.Struct.Biol.7:369-376), in given polypeptide or protein, have limited number of folds, in case very a large amount of structures is resolved, structure prediction will become obviously more accurate so.

[0060] additive method of prediction secondary structure comprises " method of trying to make a match (threading) " (Jones, 1997, Curr.Opin.Struct.Biol.7:377-87; Sippl et al., 1996, Structure 4:15-19), " preface type analysis method (profile analysis) " (Bowie et al., 1991, Science 253:164-170; Gribskov et al., 1990, Meth.Enzym.183:146-159; Gribskov et al., 1987, Proc.Nat.Acad.Sci.84:4355-4358) and " evolution connection method (evolutionary linkage) " (referring to Holm, 1999, ibid; And Brenner, 1997, ibid).

[0061] in certain embodiments, protein variant comprises the glycosylation variant, wherein compares with parent's amino acid sequence of polypeptide, and the quantity and/or the type of glycosylation site are changed.In certain embodiments, compared with natural protein, the glycosylation site that protein variant comprises or the N-of more or less quantity connects.The glycosylation site that N-connects is characterized by following sequence: Asn-X-Ser or Asn-X-Thr; Wherein, the amino-acid residue of called after X can be any amino-acid residue except proline(Pro).Connect carbohydrate chain and provide potential new site for adding N-for producing amino acid replacement that this sequence carries out.Selectively, eliminate substituting of this sequence and will remove the carbohydrate chain that already present N-connects.Rearranging of carbohydrate chain that N-connects also is provided, and the glycosylation site (normally those are naturally occurring) that wherein one or more N-connect is eliminated, and the site that one or more new N-connect is generated.Other preferred variants comprise the halfcystine variant, deleted or alternative another amino acid of wherein one or more cysteine residues (for example Serine), and this is compared with parent's aminoacid sequence.When protein must be folded into the biologic activity conformation again, such as after the separatin non-soluble inclusion body, the halfcystine variant may be useful.The halfcystine variant has still less cysteine residues than natural protein usually, and has the even number halfcystine usually, so that the interaction that produces because of azygous halfcystine is minimized.

[0062] in other embodiments, protein variant can comprise sudden change, such as substituting, add, rejecting or their any combination, usually by using one or more mutagenic oligonucleotides, produce by site-directed mutagenesis, this can carry out (reference example such as Sambrook et al., MOLECULAR CLONING:A LABORATORY MANUAL, 3rd Ed. according to method and the methods known in the art described herein, 2001, Cold SpringHarbor, N.Y. and Berger and Kimmel, METHODS IN ENZYMOLOGY, Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press, Inc., San Diego, CA., they incorporate this paper by reference into).

[0063] according to some embodiment, amino acid replacement is that with following effect those substitute: (1) reduces the susceptibility to proteolysis; (2) reduction is to the susceptibility of oxygenizement; (3) change the binding affinity that is used to form protein complex; (4) change binding affinity, and/or; (5) give or change other physical chemistry or the functional performance of this type of polypeptide.According to some embodiment, single or multiple amino acid replacements (in certain embodiments, conservative amino acid replacement) can (in some embodiment, in the polypeptide portion that forms the overseas portion of intermolecular contacting structure) carry out in the sequence of natural generation.In preferred embodiments, the constitutional features that conservative amino acid replacement generally can substantially not change the parent sequence (for example, replace amino acid and should not trend towards destroying the spiral that is present in the parent sequence, or destroy the secondary structure of the other types that characterize the parent sequence).The example of approved polypeptide secondary and tertiary structure is described in the following document: PROTEINS, and STRUCTURESAND MOLECULAR PRINCIPLES, (Creighton, Ed.), 1984, W.H.Freeman andCompany, New York; INTRODUCTION TO PROTEIN STRUCTURE (C.Branden andJ.Tooze, eds.), 1991, Garland Publishing, New York, N.Y.; With Thornton et al., 1991, Nature 354:105, each piece of writing in these documents is incorporated herein by reference.

[0064] peptide analogs is used for pharmaceutical industries at large, and it is used as non-peptide medicine, has and the similar characteristic of the characteristic of template peptide.The non-peptide compound of these types is called " peptide mimics (peptide mimetics) " or " peptide mimics (peptidomimetics) " referring to Fauchere, and 1986, Adv.Drug Res.15:29; Veber ﹠amp; Freidinger, 1985, TINS is p.392; With Evans et al., 1987, J.Med.Client.30:1229, these documents are incorporated this paper into by reference with any purpose.Such compound usually is to develop under the help of computerized molecule modeling.Be similar to the peptide mimics for the treatment of upward useful peptide on the structure and can be used to produce similar treatment or preventive effect.In general, peptide mimics structurally is similar to example polypeptide (that is, having the polypeptide of biochemical characteristic or pharmacological activity), such as people's antibody, is replaced but one or more peptide bond can randomly be selected from following key :-CH ₂-NH-,-CH ₂-S-,-CH ₂-CH ₂-,-CH=CH-(cis and trans) ,-COCH ₂-,-CH (OH) CH ₂-and-CH ₂SO-, this can utilize method well known in the art to carry out.In some embodiment, can substitute the one or more amino acid (for example D-Methionin is replaced L-Methionin) in the consensus sequence with the D-amino acid of same type, to produce more stable peptide systemicly.In addition, the configuration peptide (constrained peptides) that comprises the variation of consensus sequence or substantially the same consensus sequence can produce (Rizo ﹠amp by methods known in the art; Gierasch, 1992, Ann.Rev.Biochem.61:387, its with any purpose incorporated herein by reference), for example undertaken by adding the inside cysteine residues can form the intramolecular disulfide bridged bond of peptide cyclisation.

(a) The feature of the IL-4 mutein receptor antagonists of modifying

[0065] " the IL-4 mutein receptor antagonists of modification " that is used for this paper comprise have extra amino acid replacement, be described in United States Patent (USP) 6,028,176 and 6,313, IL-4RA mutain in 272 (incorporating this paper into) by reference in its entirety, wherein, described substituting makes and at least one charged non-protein polymer such as polypropylene glycol, polyoxyalkylene or polyoxyethylene glycol (PEG) molecular locus can be coupled to mutain specifically.Site-specific coupling PEG, for example, make it possible to produce the mutain of modification, it has the advantage of Pegylation (PEGization) molecule, promptly, the plasma half-life that increases and the immunogenicity of minimizing are kept bigger effectiveness (potency) simultaneously, compared with not implementing specificity PEGization strategy turns the time spent into such as N-end and lysine side-chain PEG situation.Being also included among the present invention is the selection in the specificity site of amino acid replacement, and this makes it possible to carry out correct molecular folding after expressing.Compared with IL-4RA, the IL-4 mutein receptor antagonists of modification is to be not more than 10 times avidity loss in conjunction with IL-4 and IL-13.Compared with IL-4RA, the IL-4 mutein receptor antagonists of modification is to be not more than 10 times the loss of effectiveness inhibition IL-4 and the activity of IL-13 mediation.In addition, compared with the IL-4RA of unmodified, the IL-4 mutein receptor antagonists of modification has at least 2 to 10 times plasma half-life.

[0066] IL-4 mutain of the present invention also can be characterized by in other residues of natural IL-4 polypeptide chain or the aminoacid insertion in one or more sites at other residue places, rejecting, substitute and modify.According to the present invention, any this type of inserts, rejects, substitutes and modify the IL-4 mutain that all should produce the IL-4 related activity that keeps it.

[0067] another aspect of the present invention has provided method, expresses the folded protein of laying equal stress on this method, as described in embodiment 2.The IL-4 mutain must carry out suitable purifying, turns usefulness into to carry out effective PEG.Purifying for example is described in the following examples 2.When mutain at mercapto-protective agent; when carrying out refolding under existing such as beta-mercaptoethanol, Triptide or halfcystine; the mutain of purifying can not be by PEGization, and this is the cause because of the oxidized protective material inactivation of the sulfhydryl-group activity in the halfcystine that is introduced on the IL-4.Covalent disulfide bonds is formed between the free cysteine and protective material of IL-4 mutain.On the contrary; use mercapto-protective agent dithiothreitol (DTT) (DTT)---its oxidation forms stable disulfide linkage---can not form covalent linkage with the free cysteine of IL-4 mutain; thereby make that its mercapto groups is free, can with PEG maleimide reagent react.If handle with DTT, in the presence of beta-mercaptoethanol, Triptide or halfcystine after the refolding IL-4 mutain of purifying can with the PEG reagent react, but produced single PEGization and mixture of products many PEGization, this shows that already present IL-4 halfcystine also changed by PEG.The PEG of already present halfcystine turns the product with the false folding that will cause producing non-activity into.

[0068] the IL-4 mutein receptor antagonists of Xiu Shiing is to the K of IL-4 acceptor _dCan measure with any method in this area, comprise technology (BIA), as described in embodiment 4 such as real-time bimolecular transactional analysis (Bimolecular InteractionAnalysis).BIA studies the interactional technology of biologic specificity in real time, without any interactant of mark (BIAcore for example ^TM).The variation of optical phenomena surface plasma resonance (SPR) also can be as the real time reaction between the indicator organism molecule.

[0069] ability that the IL-4 mutein receptor antagonists inhibition immune cell propagation of modifying is replied can be evaluated and tested with the proliferation assay method of describing among the embodiment 5, and this ability is expressed as 50% inhibition concentration (InhibitoryConcentration 50%) (IC ₅₀).

[0070] at BIAcore ^TMIn the mensuration, the IL-4 mutein receptor antagonists that the present invention modifies is specifically in conjunction with people IL-4 acceptor, preferred K _dAt about 1.0nM to about 100nM scope.More preferably embodiment of the present invention is with the K of about 0.5nM to about 1.0 μ M _dIn conjunction with people IL-4 acceptor.Still more preferably embodiment of the present invention with about 0.1nM to the K of about 10 μ M _dIn conjunction with people IL-4 acceptor.In addition, according to expectation arrives, preferably will be with the IC of about 1.0nM to about 100nM scope ₅₀, the IL-4 mutein receptor antagonists that the present invention modifies is in conjunction with people IL-4 acceptor, and inhibition people IL-4 acceptor promotes the ability of immune cell propagation.More preferably, people's antagonist is with the IC in about 0.5nM to 1 μ M scope ₅₀, in conjunction with the IL-4 acceptor, and suppress its immune cell propagation ability, most preferably, antagonist of the present invention is with the IC of about 0.1nM to about 10 μ M ₅₀, in conjunction with and suppress the IL-4 acceptor.

[0071] the present embodiment of the IL-4 mutein receptor antagonists of the present invention's modification has also shown at least 2 to 10 times the plasma half-life of the IL4RA that is preferably unmodified, and the most preferred embodiment of the present invention has shown the plasma half-life (referring to embodiment 7) doubly of 10-100 at least for the IL-4RA of unmodified.

[0072] many IL-4 mutein receptor antagonists with modification of above-mentioned feature have utilized above-mentioned analytical procedure to be obtained by evaluation by the screening material standed for.Embodiment of the present invention have the peptide sequence described in the table 2 (SEQ ID NOS:10-16).

Table 2

Peptide sequence

SEQ ID No.	Title	Sequence
			9	IL4RA	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAA SKNTTEKETFCRAATVLRQFYSHHEKDTRCLGAT AQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE

		ANQSTLENFLERLKTIMDEKDSKCSS
			10	IL4-RE-T28C	MHKCDITLQEIIKTLNSLTEQKTLCTELCVTDIFA ASKNTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANQSTLENFLERLKTIMDEKDSKCSS
11	IL4-RE-S36C	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFA ACKNTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANQSTLENFLERLKTIMDEKDSKCSS
			12	IL4-RE-K37C	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFA ASCNTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANQSTLENFLERLKTIMDEKDSKCS8
13	IL4-RE-N38C	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTT)IFA ASKCTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANQSTLENFLERLKTIMDEKDSKCSS
			14	IL4-RE-A104C	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFA ASKNTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKECNQSTLENFLERLKTIMDEKDSKCSS
15	IL4-RE-N105C	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFA ASKNTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEACQSTLENFLERLKTIMDEKDSKCSS
			16	IL4-RE-Q106C	MHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFA ASKNTTEKETFCRAATVLRQFYSHHEKDTRCLG ATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCP VKEANCSTLENFLERLKTIMDEKDSKCSS

(b) The polynucleotide of the IL-4 mutein receptor antagonists that coding is modified

[0073] the present invention also provides the polynucleotide of the IL-4 mutein receptor antagonists of coding modification.These polynucleotide can be used to for example provide a large amount of antagonists, to be used for the treatment of purposes.

[0074] polynucleotide of the present invention can easily obtain with several different methods, and method includes but not limited to the pcr amplification of chemosynthesis, cDNA or genomic library screening, expression library screening and/or cDNA.

[0075] the recombinant DNA method general description of describing herein is in following document: Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989) and/or Current Protocols in Molecular Biology (Ausubel et al., eds., Green PublishersInc.and Wiley and Sons 1994).The invention provides nucleic acid molecule of describing and the method that obtains this quasi-molecule herein.

[0076] a kind of method that obtains suitable nucleotide sequence is polymerase chain reaction (PCR).In the method, use the enzyme reversed transcriptive enzyme, be prepared into cDNA from poly-(A)+RNA or total RNA.---usually with the cDNA of the IL-4 mutein receptor antagonists of modifying two distinct area complementations---and polysaccharase join cDNA such as the Taq polysaccharase then with two primers, then the cDNA zone between two primers of this polymeric enzymatic amplification.

[0077] another method for preparing nucleic acid molecule of the present invention is a chemical synthesis process, and this chemical synthesis process uses method well known to those skilled in the art, such as Engels et al., and 1989, the method that Angew Chem.Intl.Ed.28:716-34 describes.These methods comprise and are used for nucleic acid synthetic phosphotriester, phosphoramidite and H-phosphonate method.The preferred method of this type of chemosynthesis is to use synthetic (the polymer-supported synthesis) by the polymkeric substance support of the phosphoramidite chemical process of standard.Usually, DNA length will be a hundreds of Nucleotide.Use these methods, it is synthetic to be divided into some fragments greater than the nucleic acid of about 100 Nucleotide.Fragment can link together then.The additive method of also can the use technology personnel knowing.

[0078] can be used to the to encode polynucleotide of the present invention of the IL-4 mutein receptor antagonists modified are illustrated in (SEQ ID NOS:2-8) in the table 3.

Table 3

Polynucleotide sequence

SEQ ID No.	Title	Sequence
			1	IL4RA	ATGCACAAGTGCGATATCACCTTACAGGAGATC ATCAAAACTTTGAACAGCCTCACAGAGCAGAA GACTCTGTGCACCGAGTTGACCGTAACAGACAT CTTTGCTGCCTCCAAGAACACAACTGAGAAGGA AACCTT CTGCAGGGCTGCGACTGTGCTCCGGCA GTTCTACAGCCACCATGAGAAGGACACTCGCTG CCTGGGTGCGACTGCACAGCAGTTCCACAGGCA CAAGCAGCTGATCCGATTCCTGAAACGGCTCGA CAGGAACCTCTGGGGCCTGGCGGGCTTGAATTC CTGTCCTGTGAAGGAAGCCAACCAGAGTACGTT GGAAAACTTCTTGGAAAGGCTAAAGACGATCAT GGACGAGAAAGACTCAAAGTGTTCGAGCTAAT AA
2	IL4-RE-T28C	ATGCACAAGTGCGATATCACCTTACAGGAGAT

		CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGTGCGTAACAGAC ATCTTTGCTGCCTCCAAGAACACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAAGCCAACCA GAGTACGTTGGAAAACTTCTTGGAAAGGCTAA AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA
			3	IL4-RE-S36C	ATGCACAAGTGCGATATCACCTTACAGGAGAT CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGACCGTAACAGAC ATCTTTGCTGCCTGCAAGAACACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAAGCCAACCA GAGTACGTTGGAAAACTTCTTGGAAAGGCTAA AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA
4	IL4-RE-K37C	ATGCACAAGTGCGATATCACCTTACAGGAGAT CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGACCGTAACAGAC ATCTTTGCTGCCTCCTGCAACACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAAGCCAACCA GAGTACGTTGGAAAACTTCTTGGAAAGGCTAA AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA
			5	IL4-RE-N38C	ATGCACAAGTGCGATATCACCTTACAGGAGAT CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGACCGTAACAGAC ATCTTTGCTGCCTCCAAGTGCACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAAGCCAACCA GAGTACGTTGGAAAACTTCTTGGAAAGGCTAA

		AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA
			6	IL4-RE- A104C	ATGCACAAGTGCGATATCACCTTACAGGAGAT CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGACCGTAACAGAC ATCTTTGCTGCCTCCAAGAACACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAATGCAACCA GAGTACGTTGGAAAACTTCTTGGAAAGGCTAA AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA
7	IL4-RE- N105C	ATGCACAAGTGCGATATCACCTTACAGGAGAT CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGACCGTAACAGAC ATCTTTGCTGCCTCCAAGAACACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAAGCCTGCCA GAGTACGTTGGAAAACTTCTTGGAAAGGCTAA AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA
			8	IL4-RE- Q106C	ATGCACAAGTGCGATATCACCTTACAGGAGAT CATCAAAACTTTGAACAGCCTCACAGAGCAGA AGACTCTGTGCACCGAGTTGACCGTAACAGAC ATCTTTGCTGCCTCCAAGAACACAACTGAGAA GGAAACCTTCTGCAGGGCTGCGACTGTGCTCC GGCAGTTCTACAGCCACCATGAGAAGGACACT CGCTGCCTGGGTGCGACTGCACAGCAGTTCCA CAGGCACAAGCAGCTGATCCGATTCCTGAAAC GGCTCGACAGGAACCTCTGGGGCCTGGCGGGC TTGAATTCCTGTCCTGTGAAGGAAGCCAACTG CAGTACGTTGGAAAACTTCTTGGAAAGGCTAA AGACGATCATGGACGAGAAAGACTCAAAGTGT TCGAGCTAATAA

[0079] the present invention also provides expression vector that comprises polynucleotide of the present invention and the host cell that comprises expression vector of the present invention.

[0080] use the standard interconnection technique, polynucleotide of the present invention can be inserted in the suitable expression vector.Be typically chosen in the carrier that has function in the host cell that is utilized (that is, carrier and host cell system are compatible, and gene can be amplified and/or gene can be expressed like this).Polynucleotide of the present invention can be expressed in prokaryotic cell prokaryocyte, yeast, insect (rhabdovirus system) and/or eukaryotic host cell.The selection of host cell will be depended on various factors, such as the expression level of expectation.About the summary of expression vector, referring to Meth.Enz., vol.185 (D.V.Goeddel, ed., Academic Press 1990).

[0081] usually, the expression vector that is used for host cell contains and is useful on the sequence of keeping plasmid, and clone and express the sequence of extraneous nucleotide sequence.Such sequence---is referred to as " flanking sequence (flankingsequences) "---typically will comprise one or more following nucleotide sequences: promotor in certain embodiments, one or more enhancer sequence, replication orgin, transcription termination sequence, the complete intron that contains donor and acceptor splice site, coding is used for the sequence of the leading strand of secrete polypeptide, ribosome bind site, the polyadenylic acid sequence, being used to insert coding treats by the polylinker zone of the nucleic acid of polypeptide expressed, with the selected marker element.In these sequences each is discussed below.

[0082] flanking sequence can be homologous (promptly, from species and/or the bacterial strain same) with host cell, can be allogenic (promptly, from the species outside host cell species or the bacterial strain), can be heterozygote (that is, from the combination of the flanking sequence in source more than), maybe can be the synthetic sequence, or flanking sequence can be natural sequence, and it is under normal circumstances brought into play function and regulates the IL-4 mutein receptor antagonists and express.Like this, the source of flanking sequence can be any protokaryon or most eukaryotes, and any vertebrates or invertebrates, or any plant are as long as flanking sequence is brought into play function and can be activated just passable in the host cell system.

[0083] flanking sequence that is used for carrier of the present invention can obtain with any method of Several Methods known in the art.Usually, available flanking sequence---except IL-4 mutein receptor antagonists gene flanking sequence---was before identified by mapping (mapping) and/or digestion with restriction enzyme herein, and therefore can use suitable restriction enzyme to separate from suitable tissue source to obtain.In some cases, the full nucleotide sequence of flanking sequence can be known.Here, by using the synthetic or cloning process of the nucleic acid of describing herein, can synthesize flanking sequence.

[0084] when whole flanking sequence or when only some flanking sequence is known, it can use PCR and/or obtain by using from the suitable oligonucleotide of same species or another species and/or the method in flanking sequence fragment screening-gene group library.When flanking sequence was unknown, the dna fragmentation that contains flanking sequence can separate from bigger dna fragmentation and obtains, and this bigger dna fragmentation can contain for example encoding sequence or even another gene or a plurality of gene.Separation can be finished by following step: produces suitable dna fragmentation by digestion with restriction enzyme, uses the sepharose purifying then, (Chatsworth, CA), or additive method known to the skilled separates column chromatography.The selection that is used to realize the suitable enzyme of this purpose will be apparent to those skilled in the art.

[0085] replication orgin generally is the part of the prokaryotic expression carrier buied of those commerce, and replication orgin helps amplification vector in host cell.If the carrier of selecting does not contain replication origin, can be according to replication origin of known sequences chemosynthesis, and connect into carrier.For example, from plasmid pBR322 (NewEngland Biolabs, Beverly, MA) replication orgin is suitable for most gram-negative bacteria, and various replication orgin (for example SV40, polyomavirus, adenovirus, herpes stomatitis virus (VSV) or papillomavirus such as HPV or BPV) are used in cloning vector in the mammalian cell.Usually, the starting point of duplicating component is not mammalian expression vector necessary (for example, usually using the SV40 starting point only to be because it contains early promoter).

[0086] transcription termination sequence generally is positioned at the 3 ' end in peptide coding zone, and is used for stopping transcribing.Usually, the transcription termination sequence in the prokaryotic cell prokaryocyte is the fragment that is rich in G-C, and heel has poly-T sequence.This sequence is cloned from the library easily and is obtained, even buys as the part of carrier and by the commercial channel and to obtain, and simultaneously, its is also easily by using all methods as described in this article of nucleic acid synthetic method synthetic.

[0087] the selected marker's component numbering host cell grown survival and necessary protein of growing in selective medium.The such protein of typical selectable marker gene coding, its (a) gives the resistance of prokaryotic host cell to microbiotic or other toxin such as penbritin, tsiklomitsin or kantlex; (b) remedy the auxotroph defective of cell; Or (c) provide from complex medium the crucial nutrition that can not obtain.Preferred selected marker is kalamycin resistance gene, ampicillin resistance gene and tetracycline resistance gene.Neomycin resistance gene also can be used for selecting at protokaryon and eukaryotic host cell.

[0088] other selection gene can be used to the gene that increases and will be expressed.Amplification is process so, therein, in order to produce the gene that greater amount ground needs to growing crucial protein, is repeated by series connection in the karyomit(e) of the reconstitution cell of continuous passage.For Mammals, the example of suitable selected marker comprises Tetrahydrofolate dehydrogenase (DHFR) and thymidine kinase.The mammalian cell transformant is placed under the selective pressure, wherein owing to the selection gene that is present in the carrier, has only transformant could adapt to survival uniquely.By with cultivating under the selective reagents concentration continually varying condition of cell transformed in substratum, apply selective pressure, cause selecting the amplification of the group and the DNA of the IL-4 mutein receptor antagonists of coding modification thus.The IL-4 mutein receptor antagonists of the modification of accelerating as a result, is synthetic by the DNA of amplification.

[0089] normally the mRNA translation initiation is necessary for ribosome bind site, is characterized by Shine-Dalgarno sequence (prokaryotic organism) or Kozak sequence (eukaryote).This element generally is positioned at 3 of promotor ' end, 5 of the sequence of the IL-4 mutein receptor antagonists that coding is modified ' end.The Shine-Dalgarno sequence is changeable, but generally is (polypurine) (that is, having high A-G content) of polypurine.Many Shine-Dalgarno sequences are identified, and each in them can synthesize with the method for describing herein and be applied in the prokaryotic vector easily.

[0090] leader sequence or the signal sequence IL-4 mutein receptor antagonists that can be used for modifying is guided out host cell.Usually, the nucleotide sequence of coded signal sequence is positioned at the coding region of the IL-4 mutein receptor antagonists nucleic acid molecule of modification, or is located immediately at 5 ' end of the IL-4 mutein receptor antagonists coding region of modification.Identified many signal sequences, those have any signal sequence in the signal sequence of function to unite use with the IL-4 mutein receptor antagonists nucleic acid molecule of modifying in selected host cell.Therefore, signal sequence can be a homologous (naturally occurring) or allogenic with the IL-4 mutein receptor antagonists nucleic acid molecule of modifying.In addition, signal sequence can be used the method chemosynthesis of describing herein.Under most of situation, by means of the existence of signal peptide, the IL-4 mutein receptor antagonists of modification is secreted from host cell, and the IL-4 mutein receptor antagonists that also will cause signal peptide to be modified from excretory is got rid of.Signal sequence can be the composition of carrier, or it can be a part that is inserted into the IL-4 mutein receptor antagonists nucleic acid molecule of the modification in the carrier.

[0091] in many cases, because of there are one or more introns in carrier, transcribing of nucleic acid molecule is increased, and this occurs in polypeptide especially in eukaryotic host cell, especially under the situation that polypeptide produces in mammalian host cell.The intron that uses can be the intragenic intron of IL-4 mutein receptor antagonists that is present in modification natively, especially when the gene that uses is full-length gene group sequence or its fragment.If intron is not natural being present in the gene, intron can obtain from its another source so.Intron is normally important with respect to the position of the IL-4 mutein receptor antagonists gene of flanking sequence and modification, because intron must be transcribed so that have effectiveness.Therefore, when nucleic acid molecule of the present invention was transcribed, the intron preferred positions was 3 ' direction with respect to transcription initiation site, with respect to 5 ' direction of poly-A transcription termination sequence.Preferably, intron or a plurality of intron will be positioned on one side or the other side of cDNA (promptly 5 ' or 3 '), and it can the interrupt encoder sequence like this.The intron in any source comprises the intron in virus, protokaryon and eucaryon (plant or animal) organism source may be used to implement the present invention, as long as it and its host cell of being inserted into is compatible just passable.Also comprise the synthetic intron herein.At random, in carrier, can use more than one intron.

[0092] expression of the present invention and cloning vector generally contain discerned by host organisms and operability be connected to promotor on the nucleic acid molecule of the present invention.Promotor is the sequence of not transcribed, and is positioned at the upstream (i.e. 5 ' direction) (usually about 100 to 1000bp) of the initiator codon of structure gene, its control texture gene transcription.Promotor is divided into two classes traditionally: inducible promoter and constitutive promoter.When some in the reply culture condition changed, under the control of inducible promoter, the DNA of the initial increase level of inducible promoter transcribed, and some of culture condition changes such as having or do not exist nutrition or variation of temperature.On the other hand, the initial lasting gene product of constitutive promoter produces; That is to say that genetic expression is not almost had or not control.A large amount of promotor by various potential host cell identifications is known.By Restriction Enzyme digestion promotor is removed from source DNA, and the promoter sequence of expectation is inserted in the carrier, can with suitable promotor operability be connected to nucleic acid molecule of the present invention.Natural IL-4 mutein receptor antagonists promoter sequence can be used to instruct the amplification and/or the expression of nucleic acid molecule of the present invention.Yet, compared with natural promoter, if allogeneic promoter can produce bigger transcriptional level and produce the expressing protein of higher output yield, and if it is compatible with the host cell systems of selecting to use, preferred allogeneic promoter.

[0093] promotor that is suitable for prokaryotic hosts comprises β-Nei Xiananmei and lactose promoter systems; Alkaline phosphatase; Tryptophane (trp) promoter systems; And hybrid promoter, such as the tac promotor.Other known bacterium promotors also are suitable for.Their sequence is disclosed, therefore makes those skilled in the art they can be connected on the dna sequence dna of expectation, and this provides any useful restriction site to carry out by using joint (linkers) or fit (adapters) as required.

[0094] the suitable promotor that is used for yeast host also is known in the art.It is favourable that the yeast enhanser uses with Yeast promoter.The suitable promotor that is used for mammalian host cell is known, include but not limited to the promotor that those obtain from viral genome, such as polyomavirus, bird pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and the promotor of simian virus 40 (SV40) most preferably.Other suitable mammalian promoters comprise the allos mammalian promoter, for example thermal shock promotor and actin promoter.

[0095] in expressing, controlling gene may interested extra promotor include but not limited to: SV40 early promoter district (Bernoist and Chambon, 1981, Nature 290:304-10); The CMV promotor; Be included in the promotor (Yamamoto, et al., 1980, Cell 22:787-97) during 3 of Rous sarcoma virus ' length is terminal repetition; The herpesvirus thymine deoxyriboside kinase promotor (Wagner et al., 1981, Proc.Natl.Acad.Sci.U.S.A.78:1444-45); The regulating and controlling sequence of metallothionein gene (Brinster et al., 1982, Nature 296:39-42): prokaryotic expression carrier such as β-Nei Xiananmei promotor (Villa-Kamaroff et al., 1978, Proc.Natl.Acad.Sci.U.S.A., 75:3727-31): or tac promotor (DeBoer et al., 1983, Proc.Natl.Acad.Sci.U.S.A., 80:21-25).Also interested is following animal transcripting controling area territory, and it demonstrates tissue specificity and has been used to transgenic animal: activated elastoser I Gene Handling zone (Swift et al., 1984, Cell 38:639-46 in pancreatic cell; Ornitz et al., 1986, Cold Spring Harbor Symp.Quant.Biol.50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); In pancreatic beta cell, has active insulin gene control area (Hanahan, 1985, Nature 315:115-22); In lymphocyte, have active immunoglobulin gene control area (Grosschedl et al., 1984, Cell 38:647-58; Adames etal., 1985, Nature 318:533-38; Alexander et al., 1987, Mol.Cell.Biol., 7:1436-44); Activated mouse mammary tumor virus control area in testis, breast, lymphocyte and mastocyte (Leder etal., 1986, Cell 45:485-95); Activated albumin gene control area in liver (Pinkert et al., 1987, Genes and Devel.1:268-76); Activated a-fetoprotein gene control area (Krumlauf et al., 1985, Mol.Cell.Biol., 5:1639-48 in liver; Hammer et al., 1987, Science 235:53-58); In liver activated alpha1-antitrypsin Gene Handling zone (Kelsey et al., 1987, Genesand Devel.1:161-71); Have active betaglobulin Gene Handling zone (Mogram et al., 1985, Nature 315:338-40 at medullary cell; Kollias et al., 1986, Cell 46:89-94); In the brain oligodendrocyte activated myelin basic protein Gene Handling zone (Readhead et al., 1987, Cell 48:703-12); In skeletal muscle activated myosin light chain-2 Gene Handling zone (Sani, 1985, Nature 314:283-86); With in hypothalamus, have active gonadotropin releasing hormone Gene Handling zone (Mason et al., 1986, Science 234:1372-78).

[0096] enhancer sequence can be inserted in the carrier, to increase higher eucaryote transcribing nucleic acid molecule of the present invention.Enhanser is cis-functional element of DNA, and normal length is about 10-300bp, and it acts on the promotor, transcribes with increase.Enhanser is relatively direction and position independent form.They have been found in 5 of transcription unit ' and 3 ' direction.Some enhancer sequence that obtain from mammalian genes are known (for example enhansers of sphaeroprotein, elastoser, albumin, alpha-fetoprotein and Regular Insulin).Yet, usually, can use enhanser from virus.SV40 enhanser, the sub-enhanser of cytomegalovirus early promoter, polyomavirus enhanser and adenovirus enhanser are the exemplary enhancing elements that activates promoter in eukaryote.Although enhanser can 5 of nucleic acid molecule of the present invention ' or the montage of 3 ' position in carrier, it generally is positioned on the position of 5 ' side of promotor.

[0097] expression vector of the present invention can be formed by the vector construction that initial vector such as commerce is buied.Examples of such carriers can maybe needn't contain the flanking sequence of all expectations.When one or more flanking sequences of describing herein also were not present in the carrier, they can obtain separately and connect in the carrier.The method that is used to obtain each flanking sequence is known for those skilled in the art.

[0098] being used to implement preferred vector of the present invention is those carriers compatible with bacterium, insect and mammalian host cell.Examples of such carriers comprises pCRII, pCR3 and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (PharmaciaBiotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub.No.WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY).

[0099] other suitable carriers includes but not limited to the virus of coemid, plasmid or modification, but those skilled in the art will recognize that carrier system must be compatible with the host cell of selecting.Examples of such carriers include but not limited to plasmid such as

Plasmid derivative thing (phasmid based on ColEl of high copy number, Stratagene cloning system, La Jolla CA), be designed for the PCR cloned plasmids (TOPO for example of clone's Taq amplification PCR products ^TMTA

Test kit, The plasmid derivative thing, Invitrogen, Carlsbad, CA) and Mammals, yeast or virus vector such as baculovirus expression system (pBacPAK plasmid derivative thing, Clontech, Palo Alto, CA).

[00100] after the site that plasmid is fabricated and nucleic acid molecule of the present invention insertion carrier is suitable, the carrier of finishing can be inserted in the proper host cell, is used for amplification and/or expression of polypeptides.The host cell that expression vector of the present invention is transformed into selection can be finished with known method, and method comprises these methods such as transfection, infection, calcium chloride, electroporation, microinjection, fat transfection, DEAE-dextran method or other known methods.Method selected partly depends on the type of host cell to be used.These methods and other suitable methods are that the technician knows, and are set forth in for example Sambrook et al., among the supra.

[00101] host cell can be prokaryotic host cell (such as intestinal bacteria (E.coli)) or eukaryotic host cell (such as yeast, insect or vertebrate cells).When being incubated at appropriate condition following time, the IL-4 mutein receptor antagonists of host cell synthetic modification, it can be collected from substratum subsequently and obtain (if host cell is secreted into it the substratum) or directly obtain (if it is not talked about by excretory) from the host cell that produces it.The selection of proper host cell will be depended on various factors, such as the expression level of expectation, be supposed in order to have activity or essential peptide modified (such as glycosylation or phosphorylation) and be folded into the easiness of bioactive molecules.

[00102] many proper host cell are known in the art, and many can be from American TypeCulture Collection (ATCC), Manassas, VA obtains.Example includes but not limited to mammalian cell such as Chinese hamster ovary cell (CHO), CHO DHFR (-) cell (Urlaub et al., 1980, Proc.Natl.Acad.Sci, U.S.A.97:4216-20), human embryonic kidney cell (HEK) 293 or 293T cell or 3T3 cell.The selection of suitable mammalian host cell and conversion, cultivation, amplification, screening, product produce and the method for purifying is known in the art.Other suitable mammal cell lines are monkey COS-1 and COS-7 clone and CV-1 clone.Other exemplary mammalian host cell comprises primates zooblast system and rodent zooblast system, comprises cell transformed system.Normal diploid cell, from the cell strain of the vitro culture thing of original structure, and original explant (primary explants) also is suitable for.Candidate cell can be that selected gene is the genotype defective, maybe can contain the dominance action selected gene.Other suitable mammal cell lines include but not limited to mouse neurocytoma N2A cell, HeLa, mouse L-929 cell, from Swiss, the 3T3cells of Balb-c or NIH mouse, BHK or HaK hamster cell system.In these clones each is known for the technician of field of protein expression, and is available.

[00103] bacterial cell equally can be as being suitable for host cell of the present invention.For example various coli strains (for example HB101, DH5, DH10 and MC1061) are known host cells at biological technical field.Subtilis (B.subtilis), Rhodopseudomonas certain (Pseudomonas spp.), certain (Bacillus spp.) of other bacillus and the various bacterial strains of streptomyces certain (Streptomyces spp.) and similar kind also can be used for present method.

[00104] many yeast cell strains well known by persons skilled in the art also can be used as host cell, are used to express polypeptide of the present invention.Preferred yeast cell comprises for example yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and pasteur pichia yeast (Pichia pastoris).

[00105] in addition, if desired, insect cell system can be used for method of the present invention.This type of system description is in following document for example: Kitts et al., 1993, Biotechniques, 14:810-17; Lucklow, 1993, Curr.Opin.Biotechnol.4:564-72; With Lucklow et al., 1993, J.Virol., 67:4566-79.Preferred insect cell is Sf-9 and Hi5 (Invitrogen).

[00106] polynucleotide of the present invention in host cell can be separated with other cell compositions, such as membrane component, protein and lipid.Polynucleotide can use the nucleic acid purification technology of standard to obtain from cellular segregation, or use amplification technique such as polymerase chain reaction (PCR), or are synthesized into by automatic DNA synthesizer DNA.The method that is used to separate polynucleotide is conventional and is known in the art.Any this type of technology that is used to obtain polynucleotide may be used to obtain the isolating polynucleotide of code book invention antagonist.For example, restriction enzyme and probe can be used to separate the polynucleotide of antagonist of encoding.Preferably, isolating polynucleotide are in such prepared product,, do not have other molecules or at least 70%, 80% or 90% ground does not have other molecules that is.

Those skilled in the art will recognize that [00107] nucleic acid of Miao Shuing and peptide molecule can produce by reorganization and other modes herein.For example, the IL-4 mutein receptor antagonists cDNA molecule of modification of the present invention can use mRNA to get as the template preparation with the Protocols in Molecular Biology of standard.Afterwards, the cDNA molecule can duplicate with the Protocols in Molecular Biology of describing among known in the art and the embodiment 1.Embodiment 2 has described specific recombinant expressed and purification technique, and they are used to produce the mutain antagonist that the present invention modifies.

(c) The evaluation that the treatment of people's antagonist is used

[00108] in order to estimate the potential utility of specific antagonist in allergic asthma treatment, antagonist can be in the cell proliferating determining method external the test, describe in detail as embodiment 5 and 6.In addition, measure in the mouse pharmacokinetic study body with embodiment 6 plasma half-life of the IL-4 mutein receptor antagonists of modification.

(d) Pharmaceutical composition

[00109] the IL-4 mutein receptor antagonists of above-mentioned any modification can provide with the form of the pharmaceutical composition that comprises pharmaceutically acceptable carrier.Pharmaceutically acceptable carrier preferably is non-pyrogen.Said composition can be individually dosed or with at least a other reagent such as the stable compound Combined Preparation, it can place any aseptic, biocompatible pharmaceutical carrier by administration, carrier includes but not limited to salt solution, buffer saline, dextrose and water.Can use various aqueous carriers, for example 0.4% salt solution, 0.3% glycine and analogue.These solution are aseptic, and do not have particulate matter usually.These solution can be sterilized with sterilising technology routine, that know (for example filtering).

[00110] if desired, composition can contain pharmaceutically acceptable auxiliary substance.Acceptable auxiliary substance is preferably nontoxic to the recipient at employed dosage and concentration.Pharmaceutical composition can contain auxiliary substance, changes, keeps or preserve being used for, for example the pH of composition, osmotic pressure, viscosity, transparency, color, isotonicity, smell, sterility, stability, dissolving or release rate, absorptivity or perviousness.The appropriate formulation material includes but not limited to that amino acid is (such as glycine, glutamine, l-asparagine, arginine or Methionin), antimicrobial reagent, antioxidant is (such as xitix, S-WAT or sodium bisulfite), damping fluid is (such as borate, supercarbonate, Tris-HCl, Citrate trianion, phosphoric acid salt or other organic acids), swelling agent (such as N.F,USP MANNITOL or glycine), sequestrant (such as ethylenediamine tetraacetic acid (EDTA) (EDTA)), complexing agent is (such as caffeine, Polyvinylpyrolidone (PVP), beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), weighting agent, monose, disaccharides and other carbohydrate are (such as glucose, seminose or dextrin), protein is (such as serum albumin, gelatin or immunoglobulin (Ig)), tinting material, correctives and thinner, emulsifying agent, hydrophilic polymer (such as Polyvinylpyrolidone (PVP)), low molecular weight polypeptide, salt formation counterion (salt-forming counterion) (such as sodium), sanitas is (such as benzalkonium chloride, phenylformic acid, Whitfield's ointment, Thiomersalate, phenylethyl alcohol, methyl hydroxybenzoate, Propyl Hydroxybenzoate, chlorhexidine (chlorhexidine), Sorbic Acid or hydrogen peroxide), solvent is (such as glycerol, propylene glycol or polyoxyethylene glycol), sugar alcohol (such as N.F,USP MANNITOL or sorbyl alcohol), suspension agent, tensio-active agent or wetting agent are (such as pluronics (pluronics); PEG; Isosorbide Dinitrate (sorbitan esters); Polysorbate such as polysorbate20 or polysorbate80; Triton; Trometamol; Yelkin TTS; Cholesterol or tyloxapol), stabilizing reinforcer (such as sucrose or sorbyl alcohol), osmotic pressure toughener (---preferred sodium-chlor or Repone K---or N.F,USP MANNITOL, sorbyl alcohol), transmit medium, thinner, vehicle and/or medicine adjuvant such as alkali metal halide.Referring to Remington ' sPharmaceutical Sciences (18th Ed., A.R.Gennaro, ed., Mack Publishing Company1990.

[00111] concentration of the antagonist of the present invention in such pharmaceutical preparation can extensively change, promptly calculate by weight, about 0.5% from being less than, usually about 1% or at least about 1% to nearly 15 or 20%, and will be mainly based on fluid volume, viscosity etc., select according to selected specific administration mode.If wish the antagonist of more than one types for example with to the IL-4 receptors bind can be had different K _dAntagonist be included in the pharmaceutical composition together.

[00112] composition can give the patient separately, or makes up with other reagent, medicine or hormone and to give the patient.Except active ingredient, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carrier, comprise vehicle and auxiliary agent, and it helps active compound to be processed as pharmaceutically operable preparation.

[00113] acceptable preparation material preferably in employed dosage and concentration to the avirulent material of recipient.

[00114] pharmaceutical composition can contain preparation material, to change, to keep or preserve, and for example pH of composition, osmotic pressure, viscosity, transparency, color, isotonicity, smell, sterility, stability, dissolving or release rate, absorptivity or perviousness.The appropriate formulation material includes but not limited to that amino acid is (such as glycine, glutamine, l-asparagine, arginine or Methionin), antimicrobial reagent, antioxidant is (such as xitix, S-WAT or sodium bisulfite), damping fluid is (such as borate, supercarbonate, Tris-HCl, Citrate trianion, phosphoric acid salt or other organic acids), swelling agent (such as N.F,USP MANNITOL or glycine), sequestrant (such as ethylenediamine tetraacetic acid (EDTA) (EDTA)), complexing agent is (such as caffeine, Polyvinylpyrolidone (PVP), beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), weighting agent, monose, disaccharides and other carbohydrate are (such as glucose, seminose or dextrin), protein is (such as the blood albumin that disappears, gelatin or immunoglobulin (Ig)), tinting material, correctives and thinner, emulsifying agent, hydrophilic polymer (such as Polyvinylpyrolidone (PVP)), low molecular weight polypeptide, salt formation counterion (salt-formingcounterion) (such as sodium), sanitas is (such as benzalkonium chloride, phenylformic acid, Whitfield's ointment, Thiomersalate, phenylethyl alcohol, methyl hydroxybenzoate, Propyl Hydroxybenzoate, chlorhexidine (chlorhexidine), Sorbic Acid or hydrogen peroxide), solvent is (such as glycerol, propylene glycol or polyoxyethylene glycol), sugar alcohol (such as N.F,USP MANNITOL or sorbyl alcohol), suspension agent, tensio-active agent or wetting agent are (such as pluronics (pluronics); PEG; Isosorbide Dinitrate (sorbitan esters); Polysorbate such as polysorbate20 or polysorbate80; Triton; Trometamol; Yelkin TTS; Cholesterol or tyloxapol), stabilizing reinforcer (such as sucrose or sorbyl alcohol), osmotic pressure toughener (---preferred sodium-chlor or Repone K---or N.F,USP MANNITOL, sorbyl alcohol), transmit medium, thinner, vehicle and/or medicine adjuvant such as alkali metal halide.Referring to Remington ' s Pharmaceutical Sciences (18th Ed., A.R.Gennaro, ed., Mack Publishing Company 1990.

[00115] Zui Jia pharmaceutical composition can be determined by the technician, the dosage of route of administration, transfer mode and expectation that this for example depends on to be planned.Referring to for example Remington ' s Pharmaceutical Sciences, supra.This based composition can influence the interior rate of release of physical condition, stability, body and the interior clearance rate of body of nucleic acid molecule of the present invention or bone density instrumentality (bone density modulator).

[00116] main medium in the pharmaceutical composition or carrier can be aqueous or water-free under natural situation.For example, suitable medium that is used to inject or carrier can be water, normal saline solution or synthetic cerebrospinal fluid, can replenish with other common materials in the administered parenterally composition.With serum albumin blended neutral buffered saline or salt solution be other exemplary medium.Other exemplary pharmaceutical compositions comprise the Tris damping fluid of about pH 7.0-8.5 or the acetate buffer of about pH 4.0-5.5, and it can also comprise sorbyl alcohol or suitable surrogate.In one embodiment of the invention, pharmaceutical composition of the present invention can will have the composition and the optional reagent preparation (Remington ' s Pharmaceutical Sciences of the selection of expectation purity, supra) mixing is prepared, so that preserve with the form of lyophilized cake piece or aqueous solution.And, can use suitable vehicle such as sucrose, composition is mixed with freeze-dried preparation (lyophilizate).

[00117] pharmaceutical composition can be selected for parenteral delivery.Perhaps, composition can be selected for suck or by digestive tube such as oral delivery.Pharmaceutically acceptable preparation of compositions like this is within the art technology scope.

[00118] concentration that exists of formulation component is acceptable for the administration position.For example, use damping fluid composition being maintained physiological pH or lower pH, general pH about 5 to about 8 scopes.

[00119] when considering administered parenterally, be used for therapeutic composition of the present invention and can be pyrogen-free, parenteral is acceptable, the aqueous solution form, it is included in the molecule of the present invention's expectation in the pharmaceutically acceptable medium.The specially suitable medium that is used for parenteral injection is a sterile distilled water, and therein, described molecule is mixed with that be suitable for preserving, aseptic, isoosmotic solution.Another kind of prepared product can comprise the molecule of described expectation and the preparation that following substances forms: such as injectable microsphere, biological erodable particle, polymerizable compound (such as poly(lactic acid) or polyglycolic acid), pearl or liposome, by the product that these materials provide controllable release or continued to discharge, it can be transferred by depot injection (depot injection) then.Also can use hyaluronic acid, it can have the promotion effect of the time length in circulation.Other suitable manner of introducing the molecule of expectation comprise implantable drug delivery device.

[00120] in one embodiment, pharmaceutical composition can be used for sucking by preparation.For example, nucleic acid molecule of the present invention or bone density instrumentality can be formulated as the dry powder that can suck.Suck solution and also can be prepared, it has and is used for the propeller that aerosol transmits.In another embodiment, solution can be atomized.Pulmonary administration also is described among the PCT international publication WO 94/20069, and its proteic lung of having described chemically modified transmits.

[00121] in other embodiments, some preparation can oral administration.In one embodiment of the invention, can be with some carriers or do not prepare with the nucleic acid molecule of the present invention of this mode administration or bone density instrumentality with these carriers, wherein said carrier is to be used to prepare solid dosage such as tablet and capsular carrier traditionally.For example, capsule can be designed to the active part at a certain position of intestines and stomach delivery formulations, and this moment, bioavailability was maximized, and the degraded (pre-systemic degradation) before the systematicness degraded is minimized.Can comprise other reagent, to help to absorb molecule of the present invention or instrumentality.Also can use thinner, correctives, low melt wax, vegetables oil, lubricant, suspension agent, tablet decomposition agent and binding agent.

[00122] another kind of pharmaceutical composition can comprise the nucleic acid molecule of the present invention or the bone density instrumentality of effective quantity, itself and the non-toxic excipients formation mixture that is suitable for preparing tablet.By with tablet dissolved in sterilized water, or in another suitable medium, solution can be prepared as unit dosage form.Suitable vehicle includes but not limited to inert diluent, such as lime carbonate, yellow soda ash or sodium bicarbonate, lactic acid or calcium phosphate; Or binding agent, such as starch, gelatin or gum arabic; Or lubricant such as Magnesium Stearate, stearic acid or mica.

[00123] the other drug composition will be tangible for those skilled in the art, comprise such preparation, nucleic acid molecule wherein of the present invention or bone density instrumentality are included in slowly-releasing or the controlled release preparation (sustained-orcontrolled-delivery formulations).Be used to prepare the technology of other various slowly-releasings or controlled release thing,, also those skilled in the art will know that such as liposome vectors, biological erodable microparticle or porous pearl and depot injection.Referring to as PCT/US93/00829, it has described the porous polymer microparticle of the controllable release that is used to transmit pharmaceutical composition.

[00124] other example of slowly-releasing prepared product includes the semipermeable polymers matrix of shaped object (shaped articles) form, such as film or microcapsule.Sustained-release matrix can comprise polyester, hydrogel, polylactide (United States Patent (USP) 3,773,919 and European patent 058481), multipolymer (the Sidman etal. of L-L-glutamic acid and γ ethyl-L-glutamate, 1983, Biopolymers 22:547-56), poly-(2-hydroxyethyl-methacrylic ester) (Langer et al., 1981, J.Biomed.Mater.Res.15:167-277 and Langer, 1982, Chem.Tech.12:98-105), ethylene-vinyl acetate (Langer et al., ibid) or poly--D (-)-3-hydroxybutyric acid (European patent 133988).Slow releasing composition also can comprise liposome, and it can get by any method preparation in the Several Methods known in the art.Referring to for example Eppstein et al., 1985, Proc.Natl.Acad.Sci.USA 82:3688-92; With European patent 036676,088046 and 143949.

[00125] pharmaceutical composition that will be used to vivo medicine-feeding generally must be aseptic.This can utilize filter method to finish by aseptic filter membrane.When composition during, use this method to sterilize and before or after freeze-drying and reconstruct, to carry out by freeze-drying.The composition that is used for administered parenterally can be preserved with lyophilized form or with the solution form.In addition, parenteral composition is placed in the container with sterile port usually, for example the intravenous solution bag or the bottle, it has the stopper that hypodermic needle can puncture.

[00126] pharmaceutical composition of the present invention can be with any administration of describing herein, include but not limited in oral, intravenously, intramuscular, intra-arterial, the marrow, in the sheath, in the ventricle, in skin, subcutaneous, intraperitoneal, nose, parenteral, part, hypogloeeis or rectal administration.

[00127] after pharmaceutical composition had been produced, they can place suitable containers and mark in addition, so that treat specified situation.This kind mark will comprise administration quantity, frequency and method.

(d) Methods of treatment

[00128] the invention provides by in conjunction with the IL-4 receptor alpha chain and suppress IL-4 and the activity of IL-13-mediation, improve the method for the symptom of illness.These illnesss include but not limited to airway hyperreactivity and airway inflammation, comprise and asthma and other immunity or relevant mastocyte, eosinophil and LR and the activation of anaphylactic disease.

[00129] in one embodiment of the invention, treatment being gone up IL-4 mutein receptor antagonists that the present invention of effective dose modifies and/or pharmaceutical composition of the present invention suffers from and shows as the IL-4 that increases and the patient of the active illness of IL-13 such as above-mentioned those illnesss.

(e) Effective dose determines in the treatment

[00130] the determining fully within those skilled in the art's ability of effective dose in the treatment.Effective dose is meant the antagonist quantity that is used for effectively treating asthma in the treatment, and this is the effect that manifests during with effective dose on lacking this treatment.

[00131] effective dose can be estimated in animal model earlier in the treatment, and animal model is rat, mouse, dog, pig or non-human primate normally.Animal model also can be used for determining proper concentration and route of administration.This type of information can be used for determining people's useful dosage and route of administration then.

[00132] the treatment effectiveness and the toxicity of people's antagonist, for example ED ₅₀(dosage that 50% of colony is had result of treatment) and LD ₅₀(is lethal dosage to 50% of colony) can be measured in cell culture or laboratory animal by the pharmacy program of standard.Toxicity is therapeutic index to the dosage rate of result of treatment, and it can be expressed as such ratio: LD ₅₀/ ED ₅₀

[00133] preferably demonstrates the pharmaceutical composition of big therapeutic index.The dosage that the data that obtain from zooscopy are used to prepare certain limit uses to the people.Be included in dosage in such composition preferably in certain circulation composition scope, this concentration range comprises ED ₅₀, have a small amount of toxicity or nontoxicity this moment.Depend on the formulation of use, patient's susceptibility and route of administration, dosage changes in this scope.

[00134] implementer will consider and the relevant factor of patient that needs treatment, determine accurate dose.Dosage and administration are adjusted so that the antagonist of enough levels to be provided, or to keep desired effects.The factor that can be considered comprises age, body weight and sex, diet, administration time and frequency, drug regimen, reaction sensibility and the tolerance/reaction to treating of the common health condition of seriousness, the object of morbid state, object.The transformation period and the clearance rate that depend on particular formulations, depot drug product composition (long-acting pharmaceutical composition) can every 3-4 days, weekly or per 2 week administrations.

[00135] uses the technology of having set up, the polynucleotide of the IL-4 mutein receptor antagonists that the code book invention is modified can be fabricated and introduce cell by mode in stripped or the body, and described technology includes but not limited to that the DNA of Transferrins,iron complexes-polycation mediation shifts, with nucleic acid transfection exposed or parcel, liposome-mediated cytogamy, the DNA bag is by the intracellular transport of latex bead (intracellular transportation of DNA-coated latexbeads), protoplastis merges, virus infection, electroporation, " particle gun ", transfection with DEAE or calcium phosphate mediation.

[00136] in effective body of antagonist the scope of dosage be the about 5 μ g of every kg of patient body weight to about 50 μ g, about 50 μ g to about 5mg, about 100 μ g extremely about 500 μ g and about 200 to about 250 μ g.For the administration of polynucleotide of coding antagonist, effectively in the body dosage about 100ng to about 200ng, 500ng to about 50mg, about 1 μ g extremely about 500 μ g and about 20 μ g extremely in the scope of the DNA of about 100 μ g of about 2mg, about 5 μ g extremely.

[00137] administering mode of the pharmaceutical composition of the IL-4 mutein receptor antagonists that contains modification of the present invention can be any approach that is suitable for antagonist is passed to the host.Pharmaceutical composition of the present invention can be used for administered parenterally especially, in promptly subcutaneous, intramuscular, intravenously, the tracheae or in the nose and other pulmonary administration modes.

[00138] this paper is clearly incorporated in used patent of being quoted in the disclosure and patent application by reference into.Above-mentioned disclosure has been described the present invention prevailingly.By with reference to following specific embodiment, can obtain a more complete understanding, the purpose that embodiment is provided is not in order to limit the scope of the invention just for exemplary role.

Embodiment

Embodiment 1

The proteic reorganization of IL-4-RA and IL-4-RE cysteine mutation produces

[00139] selects pET Directional

Expression system (Invitrogen) comes recombinant expressed IL-4.This system use efficiently single stage method "

Cloning " strategy comes directed cloning to equal terminal PCR product and T7lac promotor, so that this interested gene obtains high-caliber in intestinal bacteria and the expression IPTG induction type.Other features comprise the lacI gene that reduces basal transcription, are used to duplicate and keep the pBR322 starting point of plasmid and the ampicillin resistance gene that is used to select.

[00140] in order to produce reorganization IL-4 albumen, IL-4 is cloned into the pET101/D-TOPO carrier.Oligonucleolide primers is shown in Table 4.Forward PCR design of primers becomes to have 5 ' CACC overhang, and to help directed cloning, the NdeI restriction enzyme sites of the uniqueness that is used for subclone is followed in the back, and ATG initiator codon originally.The inverse PCR primer comprises two terminator codons, to guarantee unconformability the c-end mark is arranged, and this primer also comprises the BamHI restriction enzyme sites of the uniqueness that is used for subclone.The people IL-4 that uses previous clone produces flat terminal IL-4PCR product as template.Product is carried out gel-purified, and with salts solution and

Carrier is gone in the pET101/D-TOPO carrier to allow directed cloning room temperature incubation 5 minutes.Recombinant vectors is transformed into chemoreception attitude One Shot TOP10 intestinal bacteria.Recombinant plasmid dna is sent to carries out dna sequencing, to prove conclusively correct sequence.

Table 4

Be used to produce the proteic Oligonucleolide primers of IL-4RE cysteine mutation

SEQ ID No.	The oligonucleotide that is used for IL4RE	Sequence
			17	The T28C forward	GAAGACTCTGTGCACCGAGTTGTGCGTAACA GACATCTTTGC
18	T28C is reverse	GCAAAGATGTCTGTTACGCACAACTCGGTGC ACAGAGTCTTC
			19	The S36C forward	GTAACAGACATCTTTGCTGCCTGCAAGAACA CAACTGAG
20	S36C is reverse	CTCAGTTGTGTTCTTGCAGGCAGCAAAGATGT CTGTTAC
			21	The K37C forward	CCGTAACAGACATCTTTGCTGCCTCCTGCAAC ACAACTGAGAAGG
22	K37C is reverse	CCTTCTCAGTTGTGTTGCAGGAGGCAGCAAA GATGTCTGTTACGG
			23	The N38C forward	GACATCTTTGCTGCCTCCAAGTGCACAACTGA GAAGGAAACC
24	N38C is reverse	GGTTTCCTTCTCAGTTGTGCACTTGGAGGCAG CAAAGATGTC
			25	The A104C forward	GAATTCCTGTCCTGTGAAGGAATGCAACCAG AGTACGTTGG
26	A104C is reverse	CCAACGTACTCTGGTTGCATTCCTTCACAGGA CAGGAATTC

27	The N105C forward	CCTGTGAAGGAAGCCTGCCAGAGTACGTTGG AAAACTTC
			28	N105C is reverse	GAAGTTTTCCAACGTACTCTGGCAGGCTTCCT TCACAGG
29	The Q106C forward	CCTGTCCTGTGAAGGAAGCCAACTGCAGTAC GTTGGAAAACTTC
			30	Q106C is reverse	GAAGTTTTCCAACGTACTGCAGTTGGCTTCCT TCACAGGACAGG

[00141] in order to utilize Strategene's

Site-directed mutagenesis test kit (Site-DirectedMutagenesis Kit) produces IL-4RE cysteine mutation albumen, and IL-4/pET101/D-TOPO is used as template.Each cysteine mutation albumen produces by using two Oligonucleolide primers, the wherein opposite strand complementation of each bar primer and plasmid, and contain codon TGC or GCA, the cysteine mutation of wanting with introducing.Table 4 has been listed the primer that is used to produce the IL-4RE mutain.Use the loop parameter and the condition that provide in the experimental program of manufacturers, produce the mutant plasmid that contains staggered breach.Product was handled methylated to digest, nonmutationed mother body D NA template with the DpnI endonuclease 1 hour at 37 ℃.The DNA that DpnI is handled is transformed into the super competent cell of XL-1Blue, and wherein the breach in the mutant plasmid is repaired.According to the sequencing technologies of standard, analyze the plasmid DNA of mutagenesis, to confirm correct sequence.

Embodiment 2

Recombinant expressed and purifying

[00142] there is BL21 Star (DE3) the One Shot cell (Invitrogen) of protein-contg plasmid to characterize to conversion,, makes it be grown in 37 ℃, up to OD to realize optimum expression ₆₀₀Arrive approximately 0.4, induced 3 hours at 37 ℃ with 1mM IPTG (Invitrogen).With 13,000rpm centrifugation (pellet) 10 minutes is weighed and is kept at-80 ℃ with 1 liter cell.The refrigerated cell precipitation is suspended in the cell rupture damping fluid (0.1M phosphate buffered saline buffer pH7.3,0.1%Triton X100,1mM EDTA) again every gram cell 8ml damping fluid, ultrasonication 4 times, each 1 minute, each minor tick 1 minute.By centrifugal 10 minutes, remove cell lysate at 35000g.Then by in the cell rupture damping fluid that is suspended in 30ml again, ultrasonication 1 minute is carried out centrifugally again, and cell precipitation is washed 2-3 time.With final cell precipitation, inclusion body is kept at-20 ℃.Inclusion body is suspended in the solubilising damping fluid (0.2M Tris pH9,7M Guanidinium hydrochloride) again every gram cell 5ml damping fluid.Add Sulphotolysis reagent (every gram cell 0.16 gram S-WAT, 0.08 gram potassium tetrathionate), inclusion body was stirred 2 hours at room temperature.By centrifugal 20 minutes, remove undissolved component then, obtain dissolved inclusion body with 35000g.Then inclusion body is gone up operation at Superdex200 volume-exclusion post (Akta), with isolated protein.Pillar is with the 6M Guanidinium hydrochloride/PBS pH7 of 2 column volumes (CV), carries out balance with the flow velocity of 1ml/min, and uses the 1.5CV elute protein.Collect peak fraction (Peak fractions) (each 1.5ml), with 12% or the 4-20%Bis-Tris-SDS gel electrophoresis screen.Merge and contain proteinic fraction, add the DTT of final concentration 7.5mM, with the reduction protein molecule.At the room temperature incubation after 2 hours, with 5 times of mixture dilute with waters, and with 4.5L 3mM NaH ₂PO ₄, 7mM Na ₂HPO ₄, 2mM KCl, 120mM NaCl dialyse.Dialysis continues 3-4 days, at least 3 replacing fresh buffer.Then with the dialysis material filter by 0.2 μ m strainer, and with acetate with pH regulator to 5.With damping fluid 1 (25mM ammonium acetate pH5) the balance pillar of 10CV, after injection, be increased to 100% buffer B (25mM ammonium acetate pH5/lM NaCl) then at 20 minutes inside gradients.Collect peak fraction (each 0.5ml), by 12% or the 4-20%Bis-Tris-SDS gel electrophoresis screen.Merge the part that contains product, carry out 2 times of dilutions with buffer A (0.1%TFA/ water).Then, use the flow velocity of 5ml injection annulus (Loop) and 1ml/min, go up the chromatography isolated protein at C4 reversed-phase HPLC (Beckman systemGold), use following program: 10% buffer A is used for injection period (durationof injection), be increased to 40% buffer B (0.1%TFA/ACN) through 10 minutes gradients, be increased to 50% buffer B through 30 minutes gradients, and be increased to 100% buffer B through 5 minutes gradients.Collect peak fraction (each 0.5ml), by 12% or the 4-20%Bis-Tris-SDS gel electrophoresis screen.To contain proteinic fraction drying, and be suspended in again among the 0.1M MES pH6.1, and be used for analyzing and measuring.

Embodiment 3

Locus specificity halfcystine PEGization and purifying

[00143] set up and be used for the scheme that PEGization (PEGylate) contains the IL4 RA mutain of halfcystine, this finishes by stable thioether bond between the maleimide base group of proteinic sulfydryl and line style 22kD methoxyl group-polyoxyethylene glycol-maleimide derivatives (NektarTherapeutics).Be dissolved in reaction buffer 0.1M MES with what the excessive mPEG-MAL 22kD reagent of 2 times of molar weights joined 60 M, in the protein of pH6.After 0.5 hour, using with respect to mPEG-MAL 22kD is the excessive halfcystine termination reaction (Fig. 1) of 2 times of molar weights at room temperature.By cation-exchange chromatography and volume-exclusion chromatography, protein purifying from unreacted mPEG-MAL 22kD (using the halfcystine cancellation) and unreacted IL4RA cysteine mutation albumen of PEGization is come out.To Vivapure Mini S cationic exchange coloum (Vivascience), this exchange column is with the 0.1M MES of 0.4mL, pH6 balance with sample on the crude product mixture.Pillar is with the 0.1MMES of 0.4mL, the pH6 washed twice, then after each washing with 2,000xg is centrifugal.By centrifugal, with the 0.6M NaCl/0.1M MES of 0.4mL, pH6 elutes sample from pillar.Use Beckman HPLCsystem Gold, with 0.4mL eluate application of sample to TSK-GEL G2000SWXL HPLC fractionation column (sizingcolumn) (Tosoh Biosep).With the flow velocity of 1ml/min, use phosphate buffered saline buffer (Dulbecco ' s PBS) to make moving phase, sample separation 30 minutes.Collect peak fraction (0.5ml), use the 4-12%Bis-Tris-SDS gel electrophoresis, estimate the protein of PEGization.Merge the fraction contain product, use Ultrafree Biomax-5 device (Millipore), according to the experimental program of manufacturers, be concentrated to about 6 μ M (or～1mg/ml), be used for analyzing and external test.By amino acid analysis, measure the proteinic ultimate density of PEGization.Ultimate capacity is described in the table 5.

Table 5

The proteic purifying output of the IL4RA cysteine mutation of PEGization

Mutain	(mg, initial) of PEGization	(mg, final) of PEGization	% reclaims
				IL4RA	ND	ND	ND
T28C	0.267	0.064	24.0
				S36C	0.392	0.057	14.5
K37C	0.264	0.011	4.2
				N38C	0.387	0.083	21.4
A104C	0.213	0.010	4.7
				N105C	0.289	0.044	15.2
Q106C	0.125	0.023	18.4
				On average	0.176	0.042	14.6

Embodiment 4

BiaCore IL-4 receptors bind is measured

[00144] by the amine coupling, the IL-4 acceptor is fixed on BIAcore CM5 research grade sensor chip.The surface of transmitter EDC/NHS pulse activation.The IL-4 acceptor is dissolved in the acetate buffer (pH 5.0) of 10mM, and is injected into flow passage 2, use the pulse of 1.0M thanomin-HCL then, make surperficial inactivation.The immobilization level of acceptor is～300RU.Flow passage 1 also is activated, and does not wherein have part, to serve as blank.BiacoreWizard is used to carry out dynamic analysis.Candidate IL4RE antagonist is diluted with HBS-EP (operation damping fluid), and injected 3 minutes with 30ul/ minute flow velocity, the time of dissociating is 15 minutes.Before carrying out ensuing injection with a series of concentration, chip to be regenerated, this 10mM glycine pH2.5 (flow velocity 100 μ l/ minutes) injection by twice 30 seconds makes it be back to baseline and just finishes.Based on direct binding kinetics,, calculate dissociation constant (K to each material standed for _D) value (table 6).The result shows that construction IL4-RE-A104C, IL4-RE-N105C and IL4-RE-Q106C produce the dissociation constant that is lower than 0.6nM.

Embodiment 5

The TF-1 cell proliferating determining

[00145] the TF-1 cell to IL-4 (0.5ng/ml, 0.033nM) or IL-13 (5ng/ml, proliferative 0.416nM) is replied, and is used to assess the functional antagonistic activity of IL-4RE molecule.In this was measured, the TF-1 cell was at 96 orifice plates (1 * 10 ⁴/ hole, 100 μ l volumes) the middle cultivation 2-4 days, cultivate in RPMI+10% serum, there are or do not have IL-4 or IL-13 and IL-4RE molecule.GM-CSF handles and is used as positive control.In the end read 24 hours before, and added 10 μ l AlamarBlue (10% volume) to each hole.At 530/590nm, use WALLAC Victor 2 to measure fluorescence.Based on the dosage titre of candidate IL-4RE molecule, calculate 50% inhibition concentration (IC ₅₀).The TF-1 bioassay results that IL-4 and IL-13 suppress is summarized in the table 6.The result shows that under the situation of IL-4 or IL-13 existence, construction IL4-RE-K37C, IL4-RE-N38C and IL4-RE-A104C demonstrate the IC suitable with IL-4-RA ₅₀Value.

Table 6

PEG-IL4RE BIAcore in conjunction with mensuration and in the TF-1 cell proliferating determining mutain of PEGization and the evaluated biological activity of IL4RA.

Mutain	The BIAcore affinity, nM	TF-1/IL-4 IC 50，nM	TF-1/IL-13 IC 50，nM
				BAY16-9996IL-4RA	0.11	0.56+0.86(n＝17)	1.17+1.77(n＝6)
IL4-RE-T28C	0.89	2.35+0.75(n＝2)	2.87+0(n＝1)
				IL4-RE-S36C	1.15	1.20+0.02(n＝2)	1.21+0(n＝1)
IL4-RE-K37C	0.74	0.82+0.01(n＝2)	1.22+0.58(n＝2)
				IL4-RE-N38C	0.77	0.70+0.18(n＝2)	1.24+0.58(n＝2)
IL4-RE-A104C	0.56	0.55+0.10(n＝2)	1.34+1.21(n＝2)
				IL4-RE-N105C	0.59	2.26+0.20(n＝2)	2.11+0(n＝1)
IL4-RE-Q106C	0.52	2.44+0.68(n＝2)	1.95+0(n＝1)

Embodiment 6

The primary cell proliferation assay

[00146] people's primary cell (T cell and B cell) is replied also the proliferative of IL-4 and is estimated after the pre-treatment of IL-4RE molecule.(PBMCs) separates from peripheral blood with peripheral blood lymphocytes, and some peripheral blood lymphocytes were handled 4 days with PHA, forms with the inducing T cell parent cell.PBMCs also handles with anti-CD40, exists side by side with activation B cytoactive and promptly uses.With cell inoculation in the 96-orifice plate (10 ⁵Individual cells/well).Under the situation that the IL-4RE of different concns molecule exists, (10ng/ml 0.667nM) stimulated PHAT cell parent cell and B cellular preparations 3 days with IL-4.At last 20 hours of incubation, mix and contain the tritium thymidine, this is as the indicator of propagation.The results are shown in the table 7 of these mensuration.The result shows, measures for two kinds of primary cells, and the construction of all PEGization shows its IC ₅₀IC than IL-4RA ₅₀Greatly, but the former is all less than 5 times of the latter.

Table 7

The bioactive assessment of PEG-IL4RE in B cell and T cell parent cell proliferation assay

Mutain	B cell IC50, nM	T cell parent cell IC50, nM
			BAY 16-9996 IL-4RA	0.86+0.42(n＝10)	3.22+3.26(n＝16)
IL4-RE-K37C	3.73+2.15(n＝2)	13.86+12.77(n＝2)
			IL4-RE-N38C	3.33+2.68(n＝2)	9.94+8.99(n＝2)
IL4-RE-A104C	3.29+1.46(n＝2)	4.67+4.65(n＝2)

Embodiment 7

The mouse pharmacokinetic study

[00147] uses the bull Sprague-Dawley rat that weighs 250 to 300 grams.With the jugular vein conduit mouse is carried out conduit and insert, to obtain the blood sample gleanings.In addition, with femoral venous catheter the mouse of intravenously (IV) dosage group is carried out conduit and insert, so that carry out drug administration.

[00148] respectively with 1 and the dosage of 0.5mg/kg give the IL-4 mutein receptor antagonists of mouse IL-4RA or modification.Use IV and SC (subcutaneous) route of administration.By being injected directly into the femoral venous catheter of indwelling, give IV dosage.By being injected into territory, regio pectoris, back, award SC dosage.Each dosage group is used three mouse.

[00149] single bolus injection (single bolus injection) (IV or SC) afterwards, the predetermined time behind (predose) and the dosage before dosage---until 168 hours, collect blood sample.In 1 hour after collecting, begin sample centrifugal.Results blood plasma, and place on the dry ice, be kept at-70 ℃ approximately then.

[00150] quantizes with the plasma concentration of enzyme-linked immunoassay method the mutain of IL-4RA and modification.Anti--IL-4 antibody is used as bag quilt and detection reagent.The lower limit of quantitation of this mensuration is 0.2ng/ml.(Pharsight, Mountain view CA), analyze by non-chamber (non-compartmental), obtain pharmacokinetic parameter to use WinNonlin.Interested especially is to absorb and eliminate the quantity of dynamic (dynamical) evaluation, distribution volume and absorption.

Reference

1.Ying，S.，M.Humbert，J.Barkans，C.J.Corrigan，R.Pfister，G.Menz，M.Larche，D.S.Robinson，S.R.Durham and A.B.Kay.1997.Expression of IL-4and IL-5mRNAand protein product by CD4+and CD8+T cells，eosinophils，and mast cells in bronchialbiopsies obtained from atopic and nonatopic(intrinsic)asthmatics.J.Immunol.158：3539-3544.

2.Huang，S.K.，H.Q.Xiao，J.Kleine-Tebbe，G.Paciotti，D.G.Marsh，L.M.Lichtenstein and M.C.Liu.1995.IL-13 expression at the sites of allergen challenge inpatients with asthma.J.Immunol.155：2688-2694.

3.Zhu，Z.，R.J.Homer，Z.Wang，Q.Chen，G.P.Geba，J.Wang，Y.Zhang and J.A.Elias.1999.Pulmonary expression of interleukin-13 causes inflammation，mucushypersecretion，subepithelial fibrosis，physiologic abnormalities，and eotaxin production.J.Clin.Invest.103：779-788.

4.Henderson，W.R.J.，E.Y.Chi and C.R.Maliszewski.2000.Soluble IL-4receptorinhibits airway inflammation following allergen challenge in a mouse model of asthma.J.Immunol.164：1086-1095

5.Sambrook(1989)C _URRENT P _{ROTOCOLS IN} M _OLECULAR B _IOLOGY，John Wiley &Sons，New York，N.Y.，1995.

Claims (20)

1. purified polynucleotides, it is selected from

A) at the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:5 or the SEQ ID NO:6; Or

B) nucleotide sequence of the polypeptide of the aminoacid sequence of coding shown in SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

2. expression vector, it comprises the described polynucleotide of claim 1.

3. host cell, it comprises the described expression vector of claim 2.

4. prepare the method for the IL-4 mutein receptor antagonists of modifying, comprise step:

A) under the condition that described antagonist is expressed, cultivate the described host cell of claim 3; With

B) from described host cell culture, be purified into described antagonist.

5. the method for IL-4 mutein receptor antagonists of the modification of preparation activity form comprises step:

A) under the condition that described antagonist is expressed, cultivate the described host cell of claim 3;

B) in the presence of dithiothreitol (DTT), make described antagonist refolding; With

C) from described host cell culture, be purified into described antagonist.

6. method as claimed in claim 5 further comprises step:

D) described antagonist is coupled to charged non-protein polymer; With

E) purifying is coupled to the described antagonist of described charged non-protein polymer.

7. by the IL-4 mutein receptor antagonists of the modification of each described method generation in the claim 4 to 6, wherein 37,38 or 104 amino-acid residue is a halfcystine in the position, and wherein said antagonist suppresses the activity of IL-4 and IL-13 mediation.

8. the IL-4 mutein receptor antagonists of modification as claimed in claim 7, it is coupled to charged non-protein polymer, and described charged non-protein polymer is selected from polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

9. the IL-4 mutein receptor antagonists of Xiu Shiing, it is coupled to charged non-protein polymer in the position 37,38 of people's wild-type IL-4 or 104 amino-acid residue place, and wherein said charged non-protein polymer is polyoxyethylene glycol, polypropylene glycol or polyoxyalkylene.

10. the IL-4 mutein receptor antagonists of modification as claimed in claim 8, the IL-4 mutein receptor antagonists of wherein said modification is coupled to charged non-protein polymer at position 37,38 or 104 places of IL-4.

11. the IL-4 mutein receptor antagonists of Xiu Shiing as claimed in claim 8 or 9, the aminoacid sequence of wherein said antagonist is the aminoacid sequence shown in the SEQ ID NO:12.

12. the IL-4 mutein receptor antagonists of Xiu Shiing as claimed in claim 8 or 9, the aminoacid sequence of wherein said antagonist is the aminoacid sequence shown in the SEQ ID NO:13.

13. the IL-4 mutein receptor antagonists of Xiu Shiing as claimed in claim 8 or 9, the aminoacid sequence of wherein said antagonist is the aminoacid sequence shown in the SEQ ID NO:14.

14. pharmaceutical composition, it comprises:

A) the IL-4 mutein receptor antagonists of each described modification in the claim 7 to 13; With

B) pharmaceutically acceptable carrier.

15. the IL-4 mutein receptor antagonists of each described modification in the claim 7 to 13 preparation be used for the treatment of with the active pharmaceutical composition that increases relevant human disorders of IL-4 and IL-13 in application.

16. the described application of claim 15, wherein said illness are asthma or chronic obstructive pulmonary disease.

17. the described application of claim 16, wherein said chronic obstructive pulmonary disease are pulmonary emphysema or chronic bronchitis.

18. the described pharmaceutical composition of claim 14 preparation be used for the treatment of with the active medicine that increases relevant human disorders of IL-4 and IL-13 in application.

19. application as claimed in claim 18, wherein said illness are asthma or chronic obstructive pulmonary disease.

20. application as claimed in claim 19, wherein said chronic obstructive pulmonary disease are pulmonary emphysema or chronic bronchitis.

Images