RU2389796C2 - Modified antagonist of receptor il-4 and purified polynucleotide intended for its production - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.

EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.

17 cl, 1 dwg, 7 tbl, 7 ex

Description

FIELD OF THE INVENTION

This invention relates to an IL-4 mutein receptor antagonist attached to a non-protein polymer, such as polyethylene glycol. In addition, it provides compositions, dosage forms and methods for their administration for therapeutic purposes. These modified mutein IL-4 receptor antagonists and compositions and methods using them are useful in treating individuals suffering from severe asthma, chronic obstructive pulmonary disease, and related lung conditions.

BACKGROUND OF THE INVENTION

Asthma is characterized by variable, reversible airway obstruction and airway hyperresponsiveness (AHR) associated with bronchial mucosal infiltration by activated T-lymphocytes (T-cells) and eosinophils. These cells, together with resident mast cells of the respiratory tract, secrete various cytokines and mediators, which play a major role in the pathogenesis of the disease. CD4 + Th2 cells, through the release of specific cytokines (IL-4, IL-5, IL-9 and IL-13), control the development of the disease. In particular, Th2 cytokines, IL-4, and IL-13 are believed to be central to the development and maintenance of airway inflammation and airway hyperresponsiveness.

A number of in vivo studies also support the notion of the main role of IL-4 and IL-13 in the pathogenesis of asthma. When using animals with a deficiency of cytokine or reagents that neutralize the function of either IL-4 or IL-13, an important role of these cytokines in the regulation of the primary and secondary immune response, leading to airway inflammation and airway hyperresponsiveness, is observed (3, 4). Taken together, these data suggest that IL-4 and IL-13 can both play overlapping and independent roles in the allergic response of the respiratory tract and that targeting both cytokines can have a significant added advantage over targeting any single cytokine.

IL-4 antagonists are described in the literature. IL-4 mutants that act as antagonists include the mutein IL-4, IL-4 / Y124D (Kruse N., Tony HP, Sebald W., Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement , Embo J. 11: 3237-41, 1992) and the double mutein IL-4 [R121D / Y124D] Tony H. et al., Design of Human Inerieukin-4 Antagonist in Inhibiting hierleukin-4-dependent and Inerleukin-13-dependent responses in T-cells and B-cells with high efficiency, Eur. J. Biochem. 225: 659-664 (1994)). A single mutein is tyrosine substituted with aspartic acid at position 124 in the D helix. The double mutein is arginine substituted by aspartic acid at position 121, and tyrosine substituted by aspartic acid at position 124 in the D helix. Changes in this part of the D helix correlate well with changes in interactions in the second binding site.

Variants of IL-4 mutants exhibiting agonism or antagonism of wild-type IL-4 may be useful in treating conditions associated with one of the pleiotropic effects of IL-4. For example, IL-4 antagonists will be useful in treating conditions exacerbated by the production of IL-4, such as asthma, allergies, or other inflammatory response states. IL-4 agonists may be useful in treating conditions in which the presence of IL-4 is associated with an improvement or amelioration of the disease, for example, autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes mellitus, etc. These autoimmune diseases are characterized by polarization when producing populations of helper T cells (helpers), types 1 and 2 (Th1, Th2). Uninfected CD4 + T cells differentiate into subpopulations of Th1 or Th2 depending on the cytokine contained during stimulation. An IL-4 agonist will ideally shift production toward T helper cells, namely towards Th2 formation, thereby exerting a therapeutic effect.

PCT / US93 / 03613 describes an IL-4 variant comprising a Phe-Leu or Tug-Leu sequence in the alpha region of the helix and a negatively charged amino acid among two amino acids in the 3'-5'-direction or 5'-3'-direction from the Phe-Leu or Tyr-Leu sequence, this variant having increased affinity for the IL-4 receptor due to the neutral amino acid replacing the negatively charged amino acid. This application also indicates that the specific substitution of Trp-Leu or Phe-Leu in the a-helix of IL-4 in the 2 residues of the negatively charged residue leads to increased affinity. The variant is an IL-4 fusion protein (with diphtheria toxin).

Recombinant mutein (IL-4RA), a derivative of human IL-4, mutated in two positions of its amino acid sequence, is described in US patents 6028176 and 6313272. IL-4RA binds with high affinity to the alpha chain of the human IL-4 receptor, an important functional a signal component of both complexes of IL-4 and IL-13 receptors. This mutein does not have agonist activity and acts as a potent competitive antagonist of the IL-4 and IL-13 receptors in vitro (see US Patents 6028176 and 613272). A significant disadvantage of using IL-4RA is its relatively short half-life (approximately 3-6 hours). The pharmacokinetic / pharmacodynamic modeling of IL-4RA in the primates asthma model shows that the effective average stable concentration when the optimal therapeutic effect is achieved is about 60 ng / ml.

One of the approaches to overcoming the short half-life is the frequent administration of IL-4RA mutein to the patient, however, the frequent administration (usually by injection or intubation into the trachea) creates significant barriers to patient tolerance and clinical treatment.

SUMMARY OF THE INVENTION

The invention provides IL-4RA muteins with a longer half-life than previously described muteins. The invention also provides reagents and methods for inhibiting the immune responses mediated by IL-4 and IL-13. This and other aspects of the present invention are provided in one or more embodiments of the invention indicated below.

In one embodiment, the invention provides a purified preparation of a modified IL-4 mutein receptor antagonist, comprising an IL-4 mutein receptor antagonist, attached to a non-protein polymer selected from the group consisting of polyethylene glycol, polypropylene glycol and polyoxyalkylene. According to one aspect of the invention, the purified preparation comprises a modified IL-4 mutein receptor polypeptide antagonist, which is encoded by the nucleotide sequence indicated below 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. According to another aspect, the polypeptide comprises the amino acid sequence shown below 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 SEQ ID NO :16.

In one embodiment, the modified IL-4 mutein receptor polypeptide antagonist may be coupled to a non-protein polymer at the amino acid residue at position 28, 36, 37, 38, 104, 105, or 106 of IL-4. These positions are numbered according to the amino acid sequence of wild-type IL-4 (i.e., human interleukin-4). In one aspect of this embodiment, the amino acid residue at positions 28, 36, 37, 38, 104, 105, or 106 is cysteine.

In yet another embodiment, the modified mutein receptor antagonist of the invention binds to the alpha chain of the IL-4 receptor with a K _{d of} about 0.1 nM - 10 μM, about 0.5 nM - 1 μM, or about 1.0 nM - 100 nM.

In another embodiment, the modified mutein IL-4 receptor antagonist inhibits the proliferative response of TF-1 cells to IL-4 with an IC ₅₀ of about 0.1 nM - 10 μM, about 0.5 nM - 1 μM, or about 1.0 nM - 100 nM.

In yet another embodiment, the modified mutein IL-4 receptor antagonist inhibits the proliferative response of TF-1 cells to IL-13 with an IC ₅₀ selected from ranges of about 0.1 nM - 10 μM, about 0.5 nM - 1 μM, or about 1 , 0 nM - 100 nM.

According to a further embodiment, the modified mutein IL-4 receptor antagonist inhibits the proliferative response of human B cells to IL-4 with an IC ₅₀ selected from ranges of about 0.1 nM - 10 μM, about 0.5 nM - 1 μM, or about 1.0 nM - 100 nM.

In yet another embodiment, the modified mutein IL-4 receptor antagonist inhibits the proliferative response of human T cells to IL-4 with an IC ₅₀ selected from ranges of about 0.1 nM - 10 μM, about 0.5 nM - 1 μM, or about 1, 0 nM - 100 nM.

In yet another embodiment, the modified IL-4 mutein receptor antagonist of the invention has a plasma half-life that is at least 2-10 times the plasma half-life of the unmodified IL-4 mutein receptor antagonist.

The invention also provides pharmaceutical compositions comprising: (a) a modified IL-4 mutein receptor antagonist that binds to the human IL-4 receptor, and (b) a pharmaceutically acceptable carrier. The invention also provides a purified polynucleotide containing (a) the nucleotide sequence shown below in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, or (b) a nucleotide sequence encoding a polypeptide containing the amino acid sequence shown below 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 SEQ ID NO: 16.

The present invention also provides expression vectors containing the polynucleotide of the invention and host cells containing the expression vector of the invention.

In addition, the present invention provides methods for producing a modified IL-4 mutein receptor antagonist, comprising the steps of: (a) culturing a host cell as described above under conditions under which expression of the antagonist occurs, and (b) purifying the antagonist from the host cell culture . According to a specific aspect, an antagonist obtained by the method of the invention can inhibit IL-4 and IL-13 mediated activity and is combined with a non-protein polymer selected from the group consisting of polyethylene glycol, polypropylene glycol and polyoxyalkylene.

The invention also provides methods for treating a disorder in humans associated with increased activity of IL-4 and IL-13, comprising the steps of: (a) selecting a person with a condition in which the activity of IL-4 and IL-13 is increased, and (b) administering this to humans, an effective amount of a modified mutein IL-4 receptor antagonist of the invention or a pharmaceutical composition of the invention. In one aspect, the disorder is asthma, a chronic obstructive pulmonary disease (such as emphysema or chronic bronchitis), or related pulmonary disorders.

The invention also provides a method for producing a modified IL-4 mutein receptor antagonist in active form, antagonists obtained by this method, compositions containing such antagonists, and a method for treating disorders in humans, comprising administering such antagonists and pharmaceutical compositions containing such antagonists. This method includes the steps of: (a) culturing a host cell, as described above, under conditions under which antagonist expression occurs; (b) re-folding the antagonist in the presence of dithiothreitol; and (c) purifying the antagonist from the culture of the host cell. In one embodiment, the method also includes the steps of: (d) combining the antagonist with a non-protein polymer and (e) purifying the antagonist attached to the non-protein polymer.

Specific preferred embodiments of the present invention will become apparent from the following more detailed description of certain aspects and from the claims.

The drawing shows a diagram of the progress of the pegylation reaction.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to modified IL-4 mutein receptor antagonists comprising an IL-4 mutein receptor attached to a non-protein polymer, preferably a polyethylene glycol molecule.

Unless otherwise required by context, singular terms include the plural and plural terms include the singular.

Definitions

The terms “polynucleotide” or “nucleic acid sequence” or “nucleic acid molecule” refer to a DNA or RNA sequence. These terms encompass molecules derived from any of the known basic analogues of DNA or RNA, such as, but not limited to, 4-acetylcysteine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5 -carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpsevdouracil, 1-methylguanine, 1-methyl-inosine, 2,2-2-dimethyl -methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- ethylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid methyl ether, 5-hydroxyacetic acid , pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-hydroxy acetic acid methyl ester, uracil-5-hydroxy acetic acid, pseudouracil, , 2-thiocytosine and 2,6-diaminopurin.

The term “purified” or “isolated” polynucleotide refers to a nucleic acid of the invention that (1) has been separated from at least about 50% of the proteins, lipids, carbohydrates or other materials with which it is contained when the total nucleic acid is isolated from source cells, (2) is not associated with all or part of the polynucleotide with which the “isolated nucleic acid” is associated in nature, (3) is associated with a polynucleotide with which it is not associated in nature, or (4) is not found in nature as part of a longer polynucleotide in sequence.

It is preferred that the isolated nucleic acid of the invention is substantially free of any other impurity nucleic acids or other contaminants that are found in its environment that will be involved in its use in the preparation of the polypeptide or in its therapeutic, diagnostic, prophylactic or research use.

By "numbering according to wild-type IL-4" is meant the identification of the selected amino acid with reference to the position in which the amino acid is usually located in wild-type IL-4.

The term “vector” refers to any molecule (eg, nucleic acid, plasmid or virus) used to transmit coding information to a host cell.

The term “expression vector” refers to a vector that is suitable for transforming a host cell and contains nucleic acid sequences that direct and / or regulate the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if there are introns.

The term “host cell” is used to refer to a cell that has been transformed or that is capable of being transformed using a nucleic acid sequence and then to express the selected gene. The term includes the progeny of the parent cell, regardless of whether the progeny are identical in morphology or organization of genetic material to the original parent, as long as there is a selected gene.

The term "transduction" refers to the transfer of genes from one bacterium to another, usually using phage. "Transduction" also refers to the acquisition and transmission of eukaryotic cell sequences by retroviruses.

The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell, and the cell is "transfected" when exogenous DNA has been introduced into the cell membrane. A number of transfection methods are well known and described in the art. See, 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); and Chu et al., 1981, Gene 13: 197. Such methods can be used to introduce one or more exogenous DNA fragments into suitable host cells.

The term "transformation" refers to a change in the genetic characteristics of a cell; a cell is transformed when it has been modified and contains new DNA. For example, a cell is considered transformed when it is genetically modified in its natural state. Following transfection and transduction, the transforming DNA can recombine with the DNA of the cell by physical integration into the chromosome of the cell, can be stored temporarily as an episomal element without reproduction, or can be reproduced independently as a plasmid. It is believed that a cell is transformed stably when DNA is reproduced with cell division.

The term "identity", as is known from the prior art, refers to the relationship between sequences of two or more molecules of a polypeptide or two or more nucleic acid molecules, as determined by comparison of sequences. In the prior art, “identity” also means the degree of sequence affinity between nucleic acid molecules or polypeptides, as this can be determined by the coincidence between the strands of two or more nucleotides or two or more amino acid sequences. “Identity” is measured in the percentage of identical matches between the smaller of two or more sequences with gap alignment (if any) using a specific mathematical model or computer program (that is, “algorithms”).

The term “similarity” is a related term, but in contrast to “identity”, “similarity” refers to a measure of kinship that includes both identical matches and conservative substitution pairs. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the rest are all non-conservative substitutions, then both degrees of identity and similarity will be 50%. If in the same example there are five more provisions that contain conservative substitutions, then the value of the degree of identity remains equal to 50%, and the value of the degree of similarity will be 75% (15/20). Therefore, in cases where there are conservative substitutions, the magnitude of the degree of similarity of the two polypeptides will be greater than the magnitude of the degree of identity of these two polypeptides.

The degrees of identity and similarity of related nucleic acids and polypeptides can be easily determined by known methods. Such methods include, but are not limited to, described in COMPUTATIONAL MOLECULAR BIOLOGY (Lesk, A. M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS (Smith, D. W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQENCE DATA, Part 1 (Griffin, A. M., and Griffin, H. G., eds.), 1994; Humana Press, New Jersey; von Heinje, G., SEQENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQENCE ANALYSIS PRIMER (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. Applied Math., 48: 1073; and Durbin et al., 1998, BIOLOGICAL SEQENCE ANALYSIS, Cambridge University Press.

Preferred methods for determining the degree of identity are designed to provide the greatest match between the test sequences. Methods for determining the degree of identity are described in available computer programs. Preferred methods for determining the degree of identity of two sequences using computer programs include, but are not limited to, the GCG software package, including 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 is publicly available at the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda MD 20894; Altschul et al., 1990, supra) The well-known Smith Waterman algorithm can also be used to determine the degree of identity.

Some alignment schemes for two amino acid sequences can result in only a short region of two sequences coinciding, and this small region can have a very high degree of sequence identity, even though there is no significant relationship between the two full-length sequences. Accordingly, in some embodiments, the selected alignment method (GAP program) results in an alignment that includes at least 50 contiguous amino acids of the target polypeptide.

For example, using the GAP computer algorithm (Genetics Computer Group, University of Wisconsin, Madison, WI), two polypeptides, for which the degree of sequence identity is to be determined, are aligned to optimally match their respective amino acids (“matching region” as determined by the algorithm). According to some options, in conjunction with the algorithm, a penalty is used for opening a gap (which is calculated as a triple value of the average diagonal; where “average diagonal” is the average diagonal of the used comparison matrix; “diagonal” means the score or number assigned to each complete amino acid match using a specific comparison matrix) and a penalty for widening the gap (which is usually 1/10 of the penalty for opening a gap), as well as a comparison matrix such as RAM250 or BLOSUM62. According to some variants, a standard comparison matrix is also used in the algorithm (cM.Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5: 345-352 for the PAM 250 comparison matrix; Henikoffet al., 1992, Proc. Natl. Acad . Sci. USA, 89: 10915-10919 for the BLOSUM 62 comparison matrix).

In some embodiments, parameters for comparing polypeptide sequences include the following:

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

Comparison Matrix: BLOSUM 62 Henikoff et al., 1992, supra;

Penalty for a gap: 12;

Gap lengthening penalty: 4;

Similarity Threshold: 0.

With the specified parameters, the GAP program can be used. In some embodiments, the above parameters are the default parameters for comparing polypeptides (together with no penalty for end gaps) using the GAP algorithm.

In this application, the generally accepted twenty common amino acids and their abbreviations are used. See IMMUNOLOGY - A SYNTHESIS. 2 ^nd Edition (ESGolub and DRGren, Eds.), Sinauer Associates: Sunderland, MA, 1991, this publication is incorporated herein by reference. Suitable components for the polypeptides of the invention may also be stereoisomers (e.g., D-amino acids) of twenty common amino acids, unnatural amino acids, such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid and other unusual amino acids. Examples of unusual amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimethyl lysine, ε-N-acetyl lysine, σ-phosphoserine, N-acetylserin, N-formylmethionine, 3-methyl-histidine, 5-hydroxylisine, σ-N-methylarginine and other similar amino acids (e.g. 4-hydroxyproline). In the polypeptide naming system used in this application, the left direction is the direction to the N-terminus with an amino group and the right direction is the direction to the C-terminus bearing a carboxyl group, these are standard notation. Residues of natural origin can be divided into the following classes based on the common properties of the side chains:

1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, Ile;

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

3) acidic: Asp, Glu;

4) main: His, Lys, Arg;

5) residues that affect the orientation of the chains: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchanging a member of one of these classes for another member of the same class. Conservative substitutions may encompass residues of amino acids of a non-natural origin, which are usually introduced by chemical synthesis of peptides rather than synthesis in biological systems. These include peptidomimetics and other reversible or inverted forms of amino acids.

Non-conservative substitutions may include exchanging a member of one of these classes for a member of another class. Such substituted residues can be introduced into regions of the human protein that are homologues of non-human proteins, or into non-homologous regions of the molecule.

When making such changes, according to some options, you can consider the hydropathic index of amino acids. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. These amino acids are isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

The important role of the hydropathic amino acid index in comparing the interactive function of a protein is noted in the prior art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that some amino acids can replace other amino acids having a similar hydropathic index or score, and still maintain a similar biological activity. When making changes based on the hydropathic index according to some options, amino acids are substituted whose hydropatic indices are ± 2. In some embodiments, amino acids with a specified index of ± 1 are used, and in other embodiments, with an index of ± 0.5.

It is known from the prior art that the substitution of such amino acids can be carried out efficiently on the basis of hydrophilicity, especially when the resulting biologically functional protein or peptide is intended for use in immunology, as described in this application.

In some embodiments, the largest local average hydrophilicity of a protein, regulated by the hydrophilicity of neighboring amino acids, correlates with its immunogenicity and antigenicity, that is, with the biological property of the protein.

The following hydrophilicity values are assigned to the residues of these amino acids: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0 ± 1); glutamate (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4).

When making changes based on similar hydrophilicity values, according to some options, amino acid substitution is carried out, the hydrophilicity of which is ± 2, according to some options this value is ± 1 and according to other options this value is ± 0.5. Based on hydrophilicity, it is also possible to identify epitopes from sequences of primary amino acids. These sites are also called “epitope core sites”.

Examples of amino acid substitutions are shown in Table 1.

Table 1 Amino Acid Sequences. Initial balances Examples of substituents Preferred Substituents Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gin 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, norleucine Leu Leu Norleucine, Ilé, Val, Met, Ala, Phe He 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, Norleucine Leu

A person skilled in the art is able to determine suitable polypeptide variants as described below using well-known methods. In some embodiments, one of skill in the art can determine suitable regions of the molecule that can be changed without destroying the activity by targeted regions that are not considered important to the activity. In other embodiments, one of skill in the art can identify residues and parts of molecules that are retained among similar polypeptides. In other embodiments, even those regions that may be important for biological activity or for structure can undergo conservative amino acid substitutions without destroying biological activity or without adversely affecting the structure of the polypeptides.

In addition, one skilled in the art may study work on the structure-function, in which residues in similar polypeptides that are important for activity or structure are identified. Given this comparison, one skilled in the art can predict the value of amino acid residues in a protein that correspond to amino acid residues that are important for activity or structure in such proteins. One skilled in the art can select chemically similar substituents for amino acids in the case of such predicted important amino acid residues.

One of skill in the art can also analyze the three-dimensional structure and amino acid sequence in connection with the structure in similar polypeptides. Having such information, a specialist can predict the alignment of amino acid residues of a polypeptide with respect to its three-dimensional structure. In some embodiments, a person skilled in the art may decide not to make radical changes to amino acid residues that are predicted to be on the surface of the protein, since such residues may participate in important reactions of interactions with other molecules. Moreover, one skilled in the art can generate test variants containing one amino acid substitution at each desired amino acid residue. These options can then be screened using activity determination methods known to those skilled in the art. Such options can be used to collect information about suitable options. For example, if it is found that a change in a particular amino acid residue leads to disappearance, an undesirable decrease in activity or the appearance of inappropriate activity, such variants with such changes should be avoided. In other words, based on the information obtained from such routine experiments, a specialist can easily determine amino acids in which further substitution should be avoided by itself or in combination with other mutations.

A number of scientific publications have been devoted to the prediction of secondary structure. See Moult, 1996, Curr. Op. in Biotech. 7: 422-427; Chou et al., 1974, Biochemistry 13: 222-245; Chou et al., 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-148; Chou et al., 1979, Ann. Rev. Biochem. 47: 251-276 and Chou et al., 1979, Biophys. J. 26: 367-384. Moreover, computer programs are currently available to help predict the secondary structure. One of the methods for predicting the secondary structure is based on homology modeling. For example, two polypeptides or proteins in which the degree of identity is greater than 30% or the degree of similarity is greater than 40% often have similar structural topologies. The expansion of the protein structural data base (PDB) has recently provided improved predictability of the secondary structure, including the potential number of folds in the structure of the polypeptide or protein. See Holm et al., 1999, Nucl. Acid Res. 27: 244-247. It has been suggested that there is a limited number of folds in a given polypeptide or protein and that as soon as a critical number of structures is determined, the accuracy of the structure prediction sharply increases.

Additional methods for predicting the secondary structure include “filament formation” (Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-87; Sippi et al., 1996, Structure 4: 15-19), “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 “evolutionary relationships” (see Holm, 1999, supra and Brenner, 1997, supra).

In some cases, protein variants include glycosylation variants in which the number and / or type of glycosylation sites have been changed compared to the amino acid sequences of the parent forms of the polypeptide. In other cases, protein variants include more or less N-linked glycosylation sites than the native protein. The N-linked glycosylation site is characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the amino acid residue denoted by X may be any amino acid residue other than proline. Substitution of amino acid residues to obtain this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Or, substitutions that remove this sequence will remove the existing N-linked carbohydrate chain. A rearrangement of N-linked carbohydrate chains is also contemplated when one or more N-linked glycosylation sites (usually those of natural origin) are deleted and one or more new N-linked sites are created. Additional preferred variants include cysteine variants in which one or more cysteine residues are deleted or substituted with another amino acid (eg, serine) compared to the sequence of the parent form of the amino acid. Cysteine variants may be useful when protein refolding must be carried out to obtain a biologically active conformation, such as arising after isolation of insoluble inclusion bodies. Cysteine variants usually contain less cysteine residues than the native protein, and usually contain the same number to minimize interactions arising from unpaired cysteines. According to additional aspects, protein variants may include mutations, such as substitutions, additions, deletions, or any combination thereof, and are usually obtained by site-directed mutagenesis using one or more mutagenic oligonucleotides in accordance with the methods described in this application, as well as methods known from prior art (see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed, 2001, Cold Spring Harbor. NY and Berger and Kimmel, METHODS IN ENZYMOLOGY, Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press, Inc., San Diego, CA., which are incorporated herein by HONORS links).

According to some substitutional options, amino acids include those that (1) decrease the susceptibility to proteolysis, (2) decrease the susceptibility to oxidation, (3) change the binding affinity during the formation of protein complexes, (4) change the binding affinity and / or (5) confer or modify other physicochemical or functional properties of such polypeptides.

In other embodiments, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) can be carried out in a sequence of natural origin (in some embodiments, in the part of the polypeptide that is outside the domain (s) forming the intramolecular contacts). In preferred embodiments, conservative amino acid substitution usually slightly changes the structural characteristics of the parent sequence (for example, amino acid substitution does not tend to disrupt the helix that occurs in the parent sequence, or to break other types of secondary structure that characterize the parent sequence). Examples of known secondary and tertiary structures of polypeptides are described in PROTEINS, STRUCTURES AND MOLECULAR PRINCIPLES (Creighton, Ed.), 1984, W. H. Freeman and Company, New York; INTRODUCTION TO PROTEIN STRUCTURE (C. Branden and J. Tooze, eds.), 1991, Garland Publishing, New York, N.Y. and Thornton et al., 1991, Nature 354: 105, each of which is incorporated herein by reference.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of a peptide template. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics." See Fauchere, 1986, Adv. Drug Res. 15:29; Veber & Freidinger, 1985, TINS p. 392 and Evans et al., 1987, J. Med. Chem. 30: 1299, which are incorporated herein by reference for any purpose. Such compounds are often created using computer molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to provide a similar therapeutic or prophylactic effect. Typically, peptidomimetics are structurally similar to a reference polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as a human antibody, but contains one or more peptide bonds, possibly substituted by a bond selected from -CH ₂ —NH—, —CH ₂ - S, -CH ₂ CH ₂ -, -CH = CH- (cis- and trans-), -CHCH ₂ -, -CH (OH) CH ₂ - and -CH ₂ SO-, introduced by well-known methods. The systematic substitution of one or more amino acids in a consensus sequence by a D-amino acid of the same type (for example, D-lysine instead of L-lysine) can be used in some embodiments to produce more stable peptides. In addition, sedentary proteins comprising a consensus sequence or a substantially identical variant of the consensus sequence can be obtained by known methods (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61: 387, incorporated herein by reference for any purpose) for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the polypeptide.

(a) Characteristics of modified IL-4 mutein receptor antagonists.

“Modified IL-4 mutein receptor antagonists” in this application include the IL-4RA mutein described in US Pat. at least one non-protein polymer, such as polypropylene glycol, polyoxyalkylene or polyethylene glycol (PEG) with mutein. Site-specific addition of PEG, for example, allows to obtain a modified mutein that has the advantages of a polyethylene glycosylated (PEGylated) compound, namely, a longer plasma half-life and reduced immunogenicity while maintaining higher activity compared to non-specific PEGylation methods such as N-terminal PEGylation or PEGylation in the lysine side chain. it is also inalienable to select a specific site in an amino acid substituent that promotes proper folding of the molecule following expression. Modified mutein IL-4 receptor antagonists bind to IL-4 and IL-13 with an affinity loss of no more than 10 times compared with IL-4RA. Modified IL-4 mutein receptor antagonists inhibit IL-4 and IL-13 mediated activity with a loss of activity of not more than ten-fold compared with IL-4RA activity. In addition, modified mutein IL-4 receptor antagonists are characterized by plasma half-lives that are at least 2-10 times greater than for unmodified IL-4RA.

The IL-4 muteins of the invention may also be characterized by insertions, deletions, substitutions, and amino acid modifications at the site of one or more sites or at other residues of the native IL-4 polypeptide chain. According to the present invention, any such insertions, deletions, substitutions and modifications provide for the mutein IL-4, which retains the activity associated with IL-4.

An additional aspect of the present invention is a method according to which expression and refolding occur as described in Example 2. The IL-4 mutein must be properly purified so that effective PEGylation can be accomplished. The purification method is described, for example, in Example 2 below. When mutein is refolded in the presence of a sulfhydryl protective agent such as beta-mercaptoethanol, glutathione or cysteine, the purified mutein cannot be pegylated, since the active sulfhydryl in cysteine introduced in IL-4 , inactivated by an oxidized protective agent. A covalent disulfide bond is formed between the free cysteine of mutein IL-4 and the protective agent. In contrast, the use of the dithiothreitol sulfhydryl protective agent (DTT), which oxidizes to form a stable disulfide bond, will not form a covalent bond with the cysteine free mutein IL-4, which leaves its sulfhydryl group free to react with the PEG malimide reagent. IL-4 muteins purified after refolding in the presence of beta-mercaptoethanol, glutathione or cysteine can react with PEG if they are treated with DTT, but a mixture of mono-PEGylated and muti-PEGylated products is formed, which also involves the PEGylation of existing IL-4 cysteines. PEGylation of existing cysteines will lead to improper folding with the formation of products that are inactive.

K _d of the modified mutein receptor antagonists to the IL-4 receptor IL-4 can be determined by any known method, including techniques such as Bimolecular interaction Analysis (BIA) (bimolecular interaction analysis) in real time as described in Example 4. BIA is a technique for study of biospecific interactions in real time without labeling any of the reagents (for example, BIAcore ™). Changes in the optical resonance of a surface plasmon (SPR) can be used to indicate real-time reactions between biological molecules.

The ability of modified IL-4 mutein receptor antagonists to inhibit the proliferative response of immune cells can be assessed using the proliferative analysis method described in Example 5, this ability is expressed as a 50% inhibitory concentration (IC ₅₀ ).

In the BIAcore ™ method, the modified mutein IL-4 receptor antagonists of the invention bind specifically to the human IL-4 receptor with a preferred K _d value ranging from about 1.0 nM to about 100 nM. In more preferred embodiments of the invention, these antagonists bind to the human IL-4 receptor with a K _d of between about 0.5 nM and 1.0 μM. In even more preferred embodiments, these antagonists bind to the human IL-4 receptor with a K _d of between about 0.1 nM and 10 μM.

In addition, the modified mutein IL-4 receptor antagonists of the invention will bind to the human IL-4 receptor and neutralize its ability to promote cell proliferation of the immune system with a preferred IC ₅₀ value in the range of about 1.0 nM to about 100 nM. More preferred human antagonists bind the IL-4 receptor and neutralize its ability to proliferate cells of the immune system with an IC ₅₀ value ranging from about 0.5 nM to 1.0 μM, and the most preferred antagonists of the invention with an IC ₅₀ value within from about 0.1 nM to about 10 μM.

Variants of modified mutein IL-4 receptor antagonists of the invention are also characterized by a half-life in plasma, which is preferably at least 2-10 times greater than that of unmodified IL-4RA, and the most preferred variants of the invention have a half-life in plasma, which is 10-100 times greater than that of unmodified IL-4RA (see Example 7).

A number of modified IL-4 mutein receptor antagonists with the characteristics described above were identified by screening using the above analysis methods. The antagonists of this invention contain the polypeptide sequences shown in Table 2 (SEQ ID NO: 10-16).

(b) Polynucleotides encoding modified mutein IL-4 receptor antagonists.

The invention also provides polynucleotides encoding modified mutein IL-4 receptor antagonists. These polynucleotides can be used, for example, to quantify the antagonists of the invention. The polynucleotide of the invention can be easily prepared in a variety of ways, including, without limitation, chemical synthesis, cDNA or genome library screening, expression library screening and / or cDNA amplification by polymerase chain reaction (PCR).

The DNA recombination described in this application is usually carried out by methods described in 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 Publishers Inc. and Wiley and Sons 1994). The present invention provides nucleic acids and methods for their preparation described in this application.

One method for obtaining a suitable nucleic acid sequence is polymerase chain reaction (PCR). According to this method, cDNA is obtained from poly (A) + RNA or RNA using reverse transcriptase. Two primers, usually complementary to two separate cDNA fragments of a modified IL-4 mutein receptor antagonist, are added to the cDNA together with a polymerase, such as Taq polymerase, and the polymerase amplifies the cDNA fragment between the two primers.

Another means of obtaining the nucleic acid of the invention is chemical synthesis using methods that are well known to those skilled in the art, for example, those described by Engels et al., 1989, Angew. Chem. Intl. Ed. 28: 716-34. Among other methods, these methods include nucleic acid synthesis using phosphotriether, phosphoramidite and H-phosphonate. A preferred method for such chemical synthesis is polymer supported synthesis using a standard phosphoramidite procedure. Typically, DNA contains several hundred nucleotide units. Nucleic acids containing more than about 100 nucleotide units can be synthesized in several fragments using these methods. Then these fragments can be ligated together. Other methods known to those skilled in the art can be used.

Polynucleotides of the invention that can be used to code for modified IL-4 mutein receptor antagonists are shown in Table 3 (SEQ ID NO: 2-8).

The invention also provides expression vectors comprising the polynucleotide of the invention and host cells containing the expression vector of the invention.

The polynucleotide of the invention can be inserted into a suitable expression vector using standard donation techniques. Typically, a vector is selected that is functional in the particular host cell used (i.e., the vector is compatible with the processes occurring in the host cell, and therefore gene amplification and / or gene expression may occur). The polynucleotide of the invention can be expressed in prokaryotic, yeast host cells, in host insect cells and / or eukaryotic host cells. The choice of host cells will depend on various factors, such as the desired degree of expression. For a review of expression vectors, see Meth. Enz. Vol. 185 (D.V. Goeddel, ed., Academic Press, 1990).

Typically, the expression vectors used in the host cell contain sequences for the preservation of plasmids and for cloning and expression of exogenous nucleotide sequences. Such sequences, called "flanking sequences", in some cases usually include one or more of the following nucleotide sequences: a promoter, one or more enhancer (enhancer) sequences, a replication source, a transcriptional, terminal (terminal) sequence, a complete intron sequence containing a donor and a splice acceptor site, a sequence encoding a basic sequence for secretion of a polypeptide, a ribosome binding site, polyadenylation sequence, a polylinker site for insertion of nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Flanking sequences can be homologous (i.e., from the same fragments and / or the same strain as the host cells), heterologous (i.e., from fragments different from the fragments of the host cells or strain), hybrid (i.e., a combination of flanking sequences from more than one source) or synthetic or flanking sequences can be native sequences that function normally by regulating the expression of an IL-4 mutein receptor antagonist. As such, the source of the flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in the processes occurring in the host cell and can be activated by them.

The flanking sequences used in the vectors of the invention can be obtained by any of the methods well known in the art. The flanking sequences commonly used according to the invention, other than the flanking sequences of the IL-4 mutein receptor antagonist genes, must have been previously identified by mapping or by digestion with restriction endonuclease, and could thus be isolated from a suitable tissue source using appropriate restriction enzymes. endonuclease. In some cases, the complete nucleotide sequence of the flanking sequence may be known. In this application, the flanking sequence can be synthesized by the methods described for the synthesis or cloning of a nucleic acid described in this application.

If all or only part of the flanking sequence is known, it can be obtained by PCR and / or by screening the genomic library using a suitable oligonucleotide and / or fragment of the flanking sequence from the same or another species. If the flanking sequence is not known, a DNA fragment containing the flanking sequence can be isolated from a larger region of DNA, which may contain, for example, a coding sequence or even other gene (s) or genes. Isolation can be accomplished by restriction endonuclease digestion to produce the desired DNA fragment, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to the skilled person. The selection of suitable enzymes to achieve this goal is obvious to a person skilled in this field.

The replicator is usually a part of prokaryotic expression vectors, commercially available, and contributes to the amplification of the vector in the host cell. If the selected vector does not contain a source of the replication site, it can be synthesized based on a known sequence chemically and ligated into the vector. For example, the replicator from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and various sources (for example, SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV) or papilloma viruses such as HPV or BPV ) are suitable for cloning vectors in mammalian cells. Typically, the replicator does not need mammalian expression vectors (for example, SV40 is often used only because it contains an early promoter).

The transcriptional terminal sequence is usually located at position 3 'of the end of the coding region of the polypeptide and serves to terminate transcription. Typically, the transcriptional terminal sequence in prokaryotic cells is a G-C enriched fragment followed by a poly-T sequence. Although the sequence is easily cloned from the library and can even be purchased as part of a vector, it can be easily synthesized using nucleic acid synthesis methods such as those described in this application.

A selective marker gene encodes a protein that is necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selective marker genes encode proteins that (a) confer resistance to antibiotics and other toxins; (b) complement auxotrophic cell defects; or (c) supply necessary nutrients that are not available from a complex environment. Preferred selective markers are the kanamycin resistant gene, the ampicillin resistant gene, and the tetracycline resistant gene. The neomycin-resistant gene can also be used for selection in prokaryotic and eukaryotic host cells.

Other selective genes can be used to amplify the gene to be expressed. Amplification is a process in which genes, which are very necessary for the production of a protein critical for growth, are repeated in tandem with the chromosomes of subsequent generations of recombinant cells. Examples of suitable selective markers for human cells include dihydrofolate reductase (DHFR) and thymidine kinase. Transformants of human cells are placed under the conditions of selection pressure, and only the transformants adapt to survival due to the selective gene contained in the vector. The selection pressure is created by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium changes sequentially, which leads to amplification of both the selective gene and the DNA that encodes a modified mutein IL-4 receptor antagonist. As a result, increased amounts of a modified IL-4 mutein receptor antagonist are synthesized from amplified DNA.

The ribosome binding site is usually necessary to initiate mRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located at the 3 ′ end with respect to the promoter and 5 ′ with the coding sequence of the modified IL-4 mutein receptor antagonist. The Shine-Dalgarno sequence is altered, but is usually polypurine (i.e., has a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using the methods described in this application, and is used in a prokaryotic vector.

The leading, or signal, sequence can be used to direct the modified IL-4 mutein receptor antagonist from the host cell. Typically, a nucleotide sequence encoding a signal sequence is located on the coding region of a modified IL-4 mutein receptor antagonist nucleic acid molecule or directly on the 5 ′ end of the coding region of a modified IL-4 mutein receptor antagonist. Many signal sequences have been identified, and any one that acts in a selected host cell can be used in combination with a modified IL-4 mutein receptor antagonist nucleic acid molecule. Therefore, the signal sequence can be homologous (of natural origin) or heterologous with respect to the nucleic acid molecule of the modified mutein IL-4 receptor antagonist. Additionally, the signal sequence can be synthesized by the chemical methods described in this application. In most cases, the secretion of the modified IL-4 mutein receptor antagonist from the host cell due to the presence of the signal peptide will remove the signal peptide from the isolated modified IL-4 mutein receptor antagonist. The signal sequence may be a component of the vector, or it may be part of a molecule of a modified IL-4 mutein receptor antagonist that is inserted into the vector.

In many cases, the transcription of a nucleic acid molecule is increased due to the presence of one or more introns in the vector; this is especially true when the polypeptide is produced in eukaryotic host cells, especially in mammalian host cells. The introns used may be of natural origin in the modified IL-4 mutein receptor antagonist gene, especially when the gene used is the entire genome sequence or fragment thereof. If the intron is not a naturally-occurring intron in a gene, it can be obtained from another source. The position of the intron with respect to the flanking sequences and the gene of the modified mutein IL-4 receptor antagonist is usually important, since the intron must be transcribed to be effective. Thus, when the nucleic acid molecule of the invention is transcribed, the preferred position of the intron is position 3 ′ to the transcription start site and position 5 ′ to the poly-A transcriptional terminal sequence. Preferably, the intron or introns are located on one side or on the other side (i.e. 5 ′ or 3 ′) of the cDNA so that it (they) does not interrupt the coding sequence. Any intron from any source, including viral, prokaryotic and eukaryotic (plant or animal) organisms, can be used in the practice of this invention, provided that it is compatible with the host cell into which it is inserted. Synthetic introns are also used. More than one intron can be used in a vector.

The expression and cloning vectors of the invention typically comprise a promoter that is recognized by the host organism and which is linked to the nucleic acid molecule of the invention. Promoters are non-transcribed sequences located in the upstream direction (namely, towards 5 ') to the original codon of the structural gene (usually 100-1000 base pairs) that control the transcription of the structural gene. Promoters usually belong to one of two classes: inducible promoters and constitutive promoters. Inducible promoters increase the degree of transcription from DNA under their control in response to some changes in cultivation conditions, such as the presence or absence of a nutrient medium or a change in temperature. On the other hand, constitutive promoters initiate the continuous production of a gene product, i.e. there is little control over gene expression or none at all. A large number of promoters recognized by various potential host cells are well known. A suitable promoter may be coupled to the nucleic acid molecule of the invention by removing the promoter from the DNA source by digestion with a restriction enzyme and inserting the desired promoter sequence into the vector. The sequence of the native IL-4 mutein receptor antagonist promoter can be used to control amplification and / or expression of a nucleic acid of the invention. A heterologous promoter is preferred, however, only if it provides a greater degree of transcription and higher yields of the expressed protein compared to the native promoter and if it is compatible with the host cell system that was selected.

Promoters suitable for use with prokaryotic hosts include beta-lactamase and lactose promoter systems; alkaline phosphatase, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences are published, which enables the specialist to ligate them with the desired DNA sequence using linkers and adapters necessary to create suitable restriction sites.

Suitable promoters for use with yeast host cells are also well known in the art. Yeast enhancers are preferably used with yeast promoters. Suitable promoters for mammalian host cells are well known and include, without limitation, promoters derived from virus genomes such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), cattle papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus and, most preferably, vacuolating monkey virus 40 (SV40). Other suitable promoters include heterologous mammalian promoters, for example, hitshock promoters (heat shock promoters) and the actin promoter.

Other promoters of interest for controlling gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature, 290: 304-10), the CMV promoter, the promoter, located at position 3 'of the long terminal repeat of the sarcoma virus Rousa (Yamamoto et al., Cell 22: 787-97), herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA, 78: 1444-45), regulatory sequences of the metallothionine gene (Brinster et al., 1982, Nature, 296: 39-42), prokaryotic expression vectors such as the beta-lactamase promoter (Vella-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA, 75: 3727-31) or tac-pro motor (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA, 80: 21-25). Also of interest are the following transcriptional control regions in animals that exhibit tissue specificity and have been used in transgenic animals: an elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); MacDonald, 1987, Hepatology 7: 425-515); an insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-22); an immunoglobulin gene control region that is active in lymphoid cells (Grosscheld et al., 1984, Cell 38: 647-58, Adames et al., 1985, Nature 318: 533-38; Alexander et al, 1987, Mol. Cell. Biol 7: 1436-44); the Bittner virus control region that is active in testicular cells, breast cells, lymphoid and mast cells (Leder et al, 1986, Cell, 45: 485-95); the control region of the albumin gene that is active in the liver (Pinkert et al, 1987, Genes and Devel. 1: 268-76); an alpha fetoprotein gene control region that is active in the liver (Krumlauf et al, 1985, Mol. Cell. Biol., 5: 1639-48; Hammer et al., 1987, Science, 235: 53-58); an alpha-1-antitrypsin gene control region that is active in the liver (Kelsey et al. Genes and Devel. 1: 161-71); a beta globin gene control region that is active in myeloid cells (Mogram et al., 1985, Nature, 315: 338-40; Kollias et al., 1986, Cell, 46: 89-94); the control region of the gene for the myelin basic protein, which is active in oligodendrocyte brain cells (Readhead et al., 1987, Cell 48: 703-12); the control region of the myosin light chain-2 gene, which is active in skeletal muscle (Sani, 1985, Nature, 314: 283-86) and the control region of the gonadotropin releasing hormone gene, which is active in the hypothalamus (Mason et al., 1986, Science, 234: 1372-78).

An enhancer sequence can be inserted into a vector to increase transcription of the nucleic acid of the invention by higher eukaryotes. Enhancers are cis-acting DNA elements, typically about 10-300 base pairs long, that act on the promoter to increase transcription. Enhancers are quite independent of orientation and position. It was found that they are in position 5 'and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globulin, elastase, albumin, alpha-fetoprotein and insulin). However, a virus enhancer is usually used. The SV40 enhancer, cytomegalovirus early promoter enhancer, enhancer, polyomas and adenovirus enhancers are examples of amplifiers for activating eukaryotic promoters. Although the enhancer can be spliced into a vector at the 5 ′ or 3 ′ position with respect to the nucleic acid molecule of the invention, it is usually located at the 5 ′ site of the promoter.

The expression vectors of the invention can be obtained from a starting vector, for example, commercially available. Such vectors may or may not contain all of the desired flanking sequences. If one or more flanking sequences described in this application is not yet present in the vector, they can be individually obtained and ligated in the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

Preferred vectors in the practice of this invention should be compatible with bacterial host cells, insect and mammalian host cells. Such vectors, among others, include pCRII, pCR3, and pcDNA3.l (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (Phannacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NJ).

Other suitable vectors include, but are not limited to, cosmids, plasmids, or modified viruses, it being apparent to one skilled in the art that the vector system should be compatible with the selected host cell. Such vectors include, but are not limited to, plasmids such as Bluescript® plasmid derivatives (ColEl large copy phagemid, Stratagene Cloning Systems, La Jolla CA), PCR cloning plasmids for cloning Tag-amplified PCR products (e.g., TOPO plasmid derivatives ™ TA Cloning® Kit, PCR2.1®, Invitrogen, Carlsbad, CA) and mammalian, yeast or virus vectors, such as the baculovirus expression system (plasmid derivatives pBacPA, Clontech, Palo Alto, CA).

After the vector has been created and the nucleic acid of the invention is inserted into an appropriate vector site, the finished vector can be inserted into a suitable host cell for amplification and / or expression of the polypeptide. Transformation of the expression vector of the invention in a selected host cell can be accomplished by well-known methods, including such as transfection, infection, use of calcium chloride, electroporation, microinjection, lipofection, use of DEAE-dextran or other methods. The method chosen is partly a function of the type of host cell that should be used. These and other methods are well known and described, for example, in Sambrook et al., Supra.

Host cells can be prokaryotic (such as E. coli) or eukaryotic (such as yeast, insect or vertebrate cells). When cultured under appropriate conditions, the host cell synthesizes a modified IL-4 mutein receptor antagonist, which can subsequently be collected from the culture medium (if the host cell secrets it into this medium) or directly from the host cell that produces it (if it is not secreted ) The selection of a suitable host cell depends on various factors, such as the desired degree of expression, modifications of the polypeptides that are desirable or necessary for the appearance of activity (such as glycosylation or phosphorylation) and the ease of folding to obtain.

A number of suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), Manassas, VA. Examples include, without limitation, mammalian cells such as Chinese hamster ovary (CHO) cells, CHF DHFR (-) cells (Urnaub et al., 1980, Proc. Natl. Acad. Sci. USA, 97: 4216-20), 293 or 293T cells of human embryo kidneys (HEK) or 3T3 cells. Selection of suitable mammalian host cells and methods of transformation, cultivation, amplification, screening, preparation of products and purification are known. Other suitable mammalian cell lines are monkey cell lines COS-1 and COS-7 and cell line CV-1. Other examples of mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains from in vitro primary tissue culture, and primary explants are also suitable. Candidate cells may have a genotypic defect of a selective gene or may contain a dominant active selective gene. Other suitable mammalian cell lines include, without limitation, murine N2A neuroblastoma cells, HeLa, mouse L-929 cells, 3T3 lines obtained from Swiss, Balb-c or NIH mice, hamster BHK or HAC cell lines. Each of these cell lines is known and accessible to those skilled in the art of protein expression.

Also suitable according to the invention are host cells that are bacterial cells. For example, various strains of E. coli are well known in the field of biotechnology (for example, HB101, DH5S, DH10 and MC1061). Various strains of B. subtilis, Pseudomonas spp., Other Bacillus spp., Streptomyces spp. Can also be used in this method. etc.

Many strains of yeast cells are also known and are available to those skilled in the art as host cells for expression of the polypeptides of the invention. Preferred yeast cells include, for example, Saccharomyces cervisae and Pichia pastoris.

Furthermore, if desired, insect cell systems may be used in the methods of the invention. Such systems are described, for example, in Kitts et al., 1993, Biotechniques, 14: 810-17; Lucklow, 1993, Curr. Opin. Biotechnol. 4: 564-72; and Lucklow et al., 1993, J. Virol., 67: 4566-79. Preferred insect cells are Sf-9 and Hi5 (Invitrogen).

The polynucleotides of the invention contained in a host cell can be isolated without other cell components, such as membrane components, proteins, and lipids. Polynucleotides can be isolated from cells using standard nucleic acid purification methods or can be synthesized using an amplification method such as polymerase chain reaction (PCR) or an automatic synthesizer. Polynucleotide isolation methods are routine and known in the art. Any such method for producing polynucleotide can be used to obtain isolated polynucleotides encoding the antagonists of the invention. For example, restriction enzymes and probes can be used to isolate polynucleotides encoded by antagonists. Preferably, the isolated polynucleotides are in the form of preparations that do not contain at all or do not contain 70, 80 or 90% of other substances.

It will be apparent to those skilled in the art that the nucleic acid and polypeptides described herein can be prepared by recombinant and other methods. For example, cDNA of a modified mutein IL-4 receptor antagonist of the invention can be obtained by standard molecular biology methods using mRNA as a template. Then, the cDNA can be replicated using molecular biology methods known in the art and described in Example 1. Example 2 describes specific recombinant expression and purification methods used to produce the modified mutein antagonists of the invention.

(c) Evaluation of the therapeutic utility of human antagonists.

To assess the potential activity of a particular antagonist in the treatment of allergic asthma, the antagonist can be tested in vitro to determine cell proliferation, as described in detail in Examples 5 and 6. In addition, the half-life in plasma of the modified mutein IL-4 receptor antagonist can be measured in vivo with the study of pharmacokinetics in rats, as described in Example 6.

(g) Pharmaceutical compositions.

Any of the modified mutein IL-4 receptor antagonists described above may be formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is preferably non-pyrogenic. The composition may be administered alone or in combination with at least one other agent, such as a stabilizer, which may be administered in any sterile biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Various aqueous carriers can be used, for example, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and usually do not contain particles. These solutions can be sterilized by conventional, well-known sterilization methods (for example, by filtration).

If desired, the compositions may contain pharmaceutically acceptable excipients. Suitable excipients are preferably non-toxic to recipients at the applicable doses and concentrations. The pharmaceutical composition may contain excipients for modifying, maintaining or stabilizing, for example, pH, osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable components of the formulations include, without limitation, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium acid sulfite), buffering agents (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or organic acids), bulking agents (e.g. mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextr n or hydroxypropyl beta-cyclodextrin), fillers, mono-saccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrins), proteins (such as serum albumin, gelatin or immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorine xidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, polypropylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20 or polysorbate 80, triton, tromethamine, lecithin, cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, preferred tionary, sodium or potassium chloride-or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences (18 ^th Ed., ARGennaro, ed., Mack Publishing Company, 1990).

The concentration of the antagonist according to the invention in such a pharmaceutical composition can vary within wide limits, namely, from less than 0.5%, usually 1% or at least about 1%, to 15 or 20% by weight and is mainly selected based on the volume of fluid, viscosity, etc. in accordance with the particular chosen method of administration. If desired, more than one type of antagonist may be included in the pharmaceutical composition, for example, with different K _d values for IL-4 receptor.

The compositions may be administered to a patient alone or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and adjuvants, which facilitate the incorporation of the active compounds into preparations that can be used pharmaceutically.

Acceptable formulations are preferably non-toxic to recipients at the applicable doses and concentrations.

The pharmaceutical composition may contain components for modifying, maintaining or stabilizing, for example, pH, osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable components of the formulations include, without limitation, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or sodium acid sulfite), buffering agents (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or organic acids), bulking agents (e.g. mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextr n or hydroxypropyl beta-cyclodextrin), fillers, monosaccharides, disaccharides and other carbohydrates (such as glucose, mannose or dextrins), proteins (such as serum albumin, gelatin or immunoglobulins), colorants, flavors and diluents, emulsifiers, hydrophilic polymers ( such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorine xidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, polypropylene glycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 or polysorbate 80, triton, tromethamine, lecithin, cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, preferred specifically sodium or potassium chloride or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences (18 ^th Ed., ARGennaro, ed., Mack Publishing Company, 1990).

The optimal pharmaceutical composition can be obtained by a specialist in this field depending, for example, on the intended route of administration, form of delivery and the desired dose. See for example. Remington's Pharmaceutical Sciences, supra. Such compositions may affect the physical condition, stability, in vivo release rate and in vivo nucleic acid clearance rate or bone density modulator of the invention.

The primary excipient or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, a suitable vehicle for injection may be water, saline or artificial cerebrospinal fluid, optionally supplemented with other substances typical of parenteral compositions. Examples of carriers are also neutral buffered saline or saline mixed with serum albumin. Other examples of pharmaceutical compositions contain Tris buffer with a pH of about 7.0-8.5 or acetate buffer with a pH of about 4.0-5.5, which may also include sorbitol or a suitable substitute for it. According to one embodiment of the invention, the pharmaceutical compositions of the invention can be prepared for storage by mixing selected components with the desired degree of purity with possible additives (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized precipitate or aqueous solution. In addition, the composition may be in the form of a lyophilisate containing the appropriate excipients, such as sucrose.

Pharmaceutical compositions can be prepared for parenteral delivery. Compositions can also be prepared for inhalation or for delivery through the digestive tract, for example, oral delivery. The preparation of such pharmaceutically acceptable compositions is known to those skilled in the art.

The components of the composition are contained in concentrations that are acceptable for the injection site. For example, buffering agents are used to maintain the composition at a physiological pH or slightly lower pH, typically in the range of about 5 to about 8.

When parenteral administration is used, the therapeutic compositions of the invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired substance of the invention in a pharmaceutically acceptable carrier. A particularly suitable carrier for parenteral injection is sterile distilled water, in which the substance is obtained in the form of a sterile isotonic solution, properly sterilized.

Another preparation may contain a substance according to the invention together with an agent such as injectable microspheres, biodegradable particles, polymer compounds (such as polylactic acid or polyglycolic acid), granules or liposomes, which provide controlled or prolonged release of the product, which can then be delivered using injection depot. Hyaluronic acid, which promotes a prolonged presence in the circulation system, can also be used. Other suitable means for administering a substance of the invention include implantable drug delivery systems.

According to one embodiment of the invention, the pharmaceutical composition can be prepared for administration by inhalation. For example, a nucleic acid or bone density modulator of the invention may be formulated as a dry powder for inhalation. Inhalation solutions can also be prepared with a propellant for aerosol delivery. In another embodiment, the solutions may be administered using a nebulizer. An introduction to the lungs is described in WO 94/20069, which discloses an introduction to the lungs of chemically modified proteins.

In other embodiments, the compositions may be administered orally. In one embodiment of the invention, the nucleic acids or bone density modulators of the invention that are administered in this way can be contained with or without carriers which are commonly used in the preparation of solid dosage forms, such as tablets or capsules. For example, a capsule can be prepared to release the active part of the composition in that part of the gastrointestinal tract where bioavailability is maximized and pre-systemic decomposition is minimized. Additional agents may be included to provide absorption of the antagonist or modulator of the invention. Diluents, flavorings, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and binders may also be used.

Other pharmaceutical compositions may include an effective amount of the nucleic acids or bone density modulators of the invention in admixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, solutions can be prepared in unit dosage form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or acacia resin; or lubricants, such as magnesium stearate, stearic acid or talc.

Other pharmaceutical compositions are readily apparent to those skilled in the art; they include nucleic acids or bone density modulators of the invention as part of sustained or controlled delivery compositions. Methods of obtaining various other means for prolonged or controlled delivery, for example, liposome carriers, biodegradable microparticles or porous granules and depots for injection, are well known in the art. See, for example, PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions.

Further examples of sustained release formulations include semipermeable polymer matrices in the form of molded articles, for example, films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (US Pat. No. 3,773,919 and EP Patent No. 058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547- 56), poly- (2-hydroxyethyl methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene copolymer with vinyl acetate (Langer et al., supra) or poly-D (-) - 3-hydroxybutyric acid (EP patent No. 133988). Sustained release compositions may also include liposomes, which can be prepared by one of the known methods. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3688-92; and European Patent Nos. 036676, 088046 and 143949.

The pharmaceutical composition for in vivo administration should usually be sterile. This can be achieved by filtration through sterile filter membranes. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and recovery. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions are usually placed in a container having a sterile inlet, for example, an intravenous solution bag or a container with a stopper through which a hypodermal injection needle is inserted.

The pharmaceutical compositions of the invention can be administered by any of the methods described herein, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual.

After preparing the pharmaceutical compositions, they can be placed in an appropriate container with a label for the treatment of this condition. Such a label will include an indication of quantity, frequency and method of administration.

(d) Therapeutic methods.

The present invention provides methods for alleviating the symptoms of a disorder by binding the alpha chain of IL-4 receptor and inhibiting IL-4 and IL-13 mediated activity.

These disorders include, without limitation, airway hyperreactivity and airway inflammation, including recruitment and activation of mast cells, eosinophils and lymphocytes associated with asthma and other immunological or allergic disorders.

According to one embodiment of the invention, a therapeutically effective dose of a modified IL-4 mutein receptor antagonist of the invention and / or a pharmaceutical composition of the invention is administered to a patient having a disorder characterized by increased activity of IL-4 and IL-13, as the disorders indicated above.

(e) Determination of a therapeutically effective dose.

The determination of a therapeutically effective dose is known to those skilled in the art. A therapeutically effective dose refers to the amount of antagonist that is used to effectively treat asthma, compared with the effectiveness that is apparent in the absence of a therapeutically effective dose.

A therapeutically effective dose may be determined initially in animal models, usually rats, mice, rabbits, dogs, pigs or primates. An animal model can also be used to determine the appropriate concentration range and method of administration.

Such information can then be used to determine appropriate doses and methods of administration to humans.

Therapeutic efficacy and toxicity, e.g., ED ₅₀ (the dose therapeutically effective in 50% of the population) and LD ₅₀ (the dose is lethal in 50% of the population) of a human antagonist, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The ratio of toxic dose to therapeutically effective dose is the therapeutic index and can be expressed as the ratio of LD ₅₀ / ED ₅₀ .

Pharmaceutical compositions that have high therapeutic indices are preferred. The data obtained in the study of animals are used to determine the dose range for humans. Doses contained in such compositions are preferably within the range of circulating concentrations that include ED ₅₀ with little or no toxicity. Doses vary in this range, which depends on the dosage form used, patient sensitivity and method of administration.

The exact dose is determined by the practitioner based on factors related to the patient being treated. Dosages and route of administration are adjusted to provide sufficient antagonist levels or to maintain the desired effect. Factors to consider include the severity of the disease, the general health of the patient, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination (s), sensitivity of the response, and tolerability / response of therapy.

Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and the clearance rate of a particular formulation.

Polynucleotides encoding the modified mutein IL-4 receptor antagonists of the invention can be constructed and introduced into an ex vivo or in vivo cell using well-designed methods, including, without limitation, transferrin-mediated polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids acids, liposome-mediated cell fusion, intracellular transport of DNA coated latex granules, protoplast fusion, viral infection, electroporation, gene gun and transfec iju mediated DEAE or calcium phosphate.

Effective in vivo doses of the antagonist range from about 5 μg to about 50 μg / kg, from about 50 μg to about 5 μg / kg, from about 100 μg to about 500 μg / kg of the patient’s weight, and from about 200 μg to about 250 mcg / kg of patient weight. When polynucleotides encoding antagonists are administered, effective in vivo doses range from about 100 ng to about 200 ng, from 500 ng to about 50 mg, from about 1 μg to about 2 mg, from about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA.

The method of administering the pharmaceutical compositions of the invention containing modified mutein IL-4 receptor antagonists may be any method for delivering the antagonist to the host. The pharmaceutical compositions of the invention are particularly suitable for parenteral administration, namely, subcutaneous, intramuscular, intravenous, intratracheal or intranasal and other methods of administration to the lungs.

All patents and patent applications cited in this application are incorporated herein by reference. The above description discloses the invention in general. It is described in more detail in the following specific examples, which are given only to illustrate the invention and do not limit its scope.

Examples.

Example 1

Recombinant production of cysteine muteins IL-4-RA and IL-4-RE.

For recombinant expression of IL-4, the Directional TOPO® pET expression system (Invitrogen) was selected. This system uses the highly efficient, one-step TOPO® Cloning strategy to directly clone a blunt-ended PCR product and the T71ac promoter for IPTG-induced expression with a high degree of desired gene in E. coli. Additional features include the lacI gene to reduce basal transcription, the source of pBR322 for replication and preservation of the plasmid, and the ampicillin resistant gene for selection.

IL-4 was cloned into pET101 / D-TOPO vector to produce recombinant IL-4 protein. Oligonucleotide primers are shown in Table 4. A forward PCR primer with a 5'ACCC sticky end was created to facilitate direct cloning, with a unique NdeI restriction enzyme site for subcloning and an ATG start codon. The reverse PCR primer included two stop codons to prevent the introduction of c-terminal labels and a unique BamHI restriction enzyme site for subcloning. Using the previously cloned human IL-4 as a template, a blunt-terminated PCR product of IL-4 was obtained. The product was gel purified and incubated with salt solution and TOPO® vector for 5 minutes at room temperature to effect direct cloning into pET101 / D-TOPO vector. The recombinant vector was transformed into the chemically competent One Shot TOP10 of E. coli. Recombinant plasmid DNA was used for DNA sequencing to confirm the correct sequence.

IL-4 / pET101 / D-TOPO served as a template for the production of IL-4RE cysteine muteins using the QuikChange® Site-Directed Mutagenesis Kit, Strategene. Each cysteine mutein was obtained using two oligonucleotide primers, each of which is complementary to the opposite strands of the vector, and contained a TGC or GCA codon to introduce the desired cysteine mutation. Table 4 lists the primers used to produce IL-4RE muteins. Using cyclization primers and the conditions specified in the manufacturer's protocol, a mutation plasmid containing step nicks was obtained. This product was treated with DpnI endonuclease for 1 hour at 37 ° C to digest the methylated, unmutated template of parental DNA. DpnI-treated DNA was transformed in super-competent XL-1 Blue cells, where nicks in the mutated plasmid were reduced. The mutagenic DNA of the plasmid was analyzed in accordance with standard sequencing methods to confirm the correct sequence.

Example 2

Recombinant expression and purification.

BL21 Star (DE3) One Shot (Invitrogen) cells, which were transformed with a protein containing plasmids, were characterized for optimal expression and grown at 37 ° C until an OD ₆₀₀ of about 0.4 was obtained and induced with 1 mM IPTG (Invitrogen) for 3 hours at 37 ° C. One liter of cells was pelletized at a speed of 13000 rpm for 10 min, weighed and stored at -80 ° C. Frozen cell tablets were resuspended in 8 ml of cell disruption buffer (0.1 M phosphate buffer, pH 7.3, 0.1% Triton X100, 1 mM EDTA) per gram of cells and treated with 4x ultrasound for 1 min at 1 min intervals . The cell lysate was removed by centrifugation at 35,000 g for 10 minutes. The cells were then washed 2-3x in suspension in 30 ml of cell disruption buffer, sonicated for 1 min and centrifuged. The final cell tablets, inclusion bodies, were stored at -20 ° C. Inclusion bodies were resuspended in 5 ml of solubilizing buffer (0.2 M Tris, pH 9, 7 M guanidine chloride) per gram of cells. Sulfotolysis reagents (0.16 g of sodium sulfite, 0.08 g of potassium tetrathionate per gram of cells) were added and inclusion bodies were stirred at room temperature for 2 hours. The undissolved components were then removed by centrifugation at 35,000 g for 20 minutes, solubilized inclusion bodies remained. Inclusion bodies were then passed on a Superdex200 size (Akta) size exclusion chromatography column to isolate the protein. The column was balanced with 2 column volumes (CV) of a 6M guanidine hydrochloride / PBS mixture, pH 7, at a flow rate of 1 ml / min, the protein was eluted with 1.5 CV. Peak fractions (each 1.5 ml) were collected and gel electrophoresed with 12% or 4-20% Bis-Tris-SDS. Protein containing fractions were collected and a final concentration of 7.5 mM DTT was added to reduce protein molecules. After incubation for 2 h at room temperature, the mixture was diluted with 5x water and dialyzed in 4.5 L of 3 mM NaH ₂ PO ₄ , 7 mM Na ₂ HPO ₄ , 2 mM KCl, 120 mM NaCl. Dialysis was continued for 3-4 days, feeding fresh buffer at least 3 times. The dialyzed product was then filtered through a 0.2 μm filter and the pH was adjusted to 5 with acetic acid. The column was equilibrated with 10 CV of buffer 1 (25 mM ammonium acetate, pH 5), a gradient was created over 20 minutes to 100% buffer B (25 mM ammonium acetate, pH 5 / 1M NaCl). Peak fractions (1.5 ml each) were collected and gel electrophoresed with 12% or 4-20% Bis-Tris-SDS. Fractions containing the obtained product were collected and diluted with 2x buffer A (0.1% TFA / water). Then the protein was chromatographed using C4 reverse phase HPLC (Beckman system Gold) using a 5 ml loop and a flow rate of 1 ml / min according to the following program: 10% buffer A during injection, 10 min gradient to 40% buffer B (0.1 % TFA / ACN), 30 min gradient to 50% buffer B and 5 min gradient to 100% buffer B. Peak fractions (0.5 ml each) were collected and gel electrophoresed with 12% or 4-20% Bis-Tris- SDS Protein containing fractions were dried and resuspended in 0.1 M MES, pH 6.1 for analysis and determinations.

Example 3

Site-specific PEGylation of cysteine and purification.

To pegylate cysteine containing IL4 RA muteins, a protocol was developed using a stable thioether linkage between the sulfhydryl group of the protein and the malimide group of the 22 kD linear methoxypolyethylene glycol maleimide derivative (Nektar Therapeutics). To a 600M protein dissolved in the OLM MES reaction buffer, pH 6, a 2 molar excess of mPEG-MAL 22 kD was added. After 0.5 h at room temperature, the reaction was terminated with a 2M excess of cysteine with respect to mPEG-MAL 22 kD (see drawing). The pegylated protein was purified from unreacted mPEG-MAL 22 kD (treated with cysteine) and unreacted cysteine mutein IL4 RA by cation exchange and size exclusion chromatography. The crude reaction mixtures were fed to Vivapure Mini S cation exchange columns (Vivascience), equilibrated with 0.4 ml of 0.1 M MES, pH 6. The columns were washed twice with 0.4 ml of 0.1 M MES, pH 6, followed by centrifugation of 2000 x g after each washing. Samples were then washed from the column by centrifugation with 0.4 ml of 0.6 M NaCl / 0.1 M MES, pH 6. Eluted fractions of 0.4 ml were loaded onto a TSK-GEL G2000SWYL GLC column (Tosoh Biosep) using a Gold Beckman system. Samples were redissolved in phosphate buffered saline (Dulbecco's PBS), which is a mobile phase with a flow rate of 1 ml / min for 30 minutes. Peak fractions (0.5 ml) were collected and gel electrophoresed with 4-12% Bis-Tris-SDS to isolate the PEGylated protein. Fractions containing the resulting product were collected and concentrated using an Ultrafree Biomax-5 device (Millipore) according to the manufacturer's protocol, to obtain approximately 60 μM (or ~ 1 mg / ml) for analysis and in vitro tests. The final concentration of PEGylated proteins was determined by amino acid analysis. The final outputs are shown in Table 5.

Table 5 The yield of pegylated cysteine muteins IL4RA. Mutein Pegylir. protein (mg, initial) Pegylir. protein (mg, final) % yield IL4RA Nd Nd Nd T28S 0.267 0,064 24.0 S36c 0.392 0,057 14.5 K37S 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 Average 0.176 0,042 14.6

Example 4

Binding reaction to the IL-4 receptor using BiaCore.

The IL-4 receptor was immobilized on a sensor chip for BiaCore CM5 studies by attachment of an amino group. The sensor surface was activated by an EDC / NHS pulse. The IL-4 receptor was dissolved in 10 mM acetate buffer (pH 5.0) and injected into cell 2 with flowing electrolyte, followed by a pulsed feed of 1.0 M ethanolamine-Hcl to deactivate the surface. The degree of immobilization of the receptor was 3000 RU. Element 1 with flowing electrolyte was also activated without a ligand so that it functions as a control. For the kinetic analysis used BiaCore Wizard. IL4RE antagonists were diluted with MBS-EP (separation buffer) and injected at a rate of 30 μl / min for 30 minutes with a dissociation time of 15 minutes. The chip was regenerated by two 30-second injections of 10 mM glycine, pH 2.5, (flow rate 100 μl / min) to the baseline before the next injection in a series of concentrations. The values of the dissociation constant (K _D ) were determined for each candidate, based on the kinetics of direct binding (table 5). The results show that for all constructs IL4-RE-A104C, IL4-RE-N105C and IL4-RE-Q106C, dissociation constants are less than 0.6 mM.

Example 5

Determination of proliferation of TF-1 cells.

The proliferative response of TF-1 cells to IL-4 (0.5 ng / ml, 0.033 nM) or IL-13 (0.5 ng / ml, 0.416 nM) was used to evaluate the functional antagonistic activity of IL-4RE. For this, TF-1 cells were cultured for 2-4 days in a 96-well plate (1 × 10 ⁴ / well, 100 μl) in RPMI + 10% serum with IL-4, IL-13 and IL-4RE or without them. GM-CSF treatment was used as a positive control. 24 hours before the last reading, 10 μl of AlamarBlue (10 vol%) was added to each well. Fluorescence was measured at 530/590 nm using WALLAC Victor 2. The inhibitory concentration (IC ₅₀ ) was calculated by the titration dose of candidate IL-4RE. Bioassay data for TF-1 and inhibition of IL-4 and IL-13 are shown in Table 6. The results show that constructs IL4-RE-K37C, IL4-RE-N38C and IL4-RE-A104C have IC ₅₀ values comparable to IC50 for IL-4-RA in the presence of IL-4 or IL-13.

Table 6 PEG-IL 4RE-BiaCore Binding Assay and Evaluation of the Bioactivity of PEGylated Muteins Against IL4RA in Determining TF-1 Cell Proliferation. Mutein Affinity for BiaCore, nM TF-1 / IL-4 IC ₅₀ , nM TF-1 / IL-13 IC ₅₀ , nM BAY 16-9996 IL-4RA 0.11 0.56 + 0.86 (n-17) 1.17 + 1.77 (n = 6) IL-4RE-T28C 0.89 2.35 + 0.75 (n = 2) 2.87 + 0 (n = 1) IL-4RE-S36C 1.15 1.20 + 0.02 (n = 2) 1.21 + 0 (n-1) IL-4RE-K37C 0.74 0.82 + 0.01 (n = 2) 1.22 + 0.58 (n = 2) IL-4RE-N38C 0.77 0.70 + 0.18 (n = 2) 1.24 + 0.58 (n = 2) IL-4RE-A104C 0.56 0.55 + 0.10 (n = 2) 1.34 + 1.21 (n = 2) IL-4RE-N105C 0.59 2.26 + 0.20 (n = 2) 2.11 + 0 (n = 1) IL-4RE-Q106C 0.52 2.44 + 0.68 (n = 2) 1.95 + 0 (n = 1)

Example 6

Determination of proliferation of primary cells.

The proliferative response of human primary cells (T and B cells) to IL-4 was evaluated after pretreatment with IL-4RE. Peripheral mononuclear blood cells (PBMCs) were isolated from peripheral blood and some were treated with PHA for 4 days to induce the formation of blast T cells. PBMC _{S was} also treated with anti-CD40 to activate B cell activity and used immediately. Cells were seeded in 96 well plates (10 ⁵ cells / well). Preparations of blast T cells and B cells treated with PHA were stimulated for 3 days with IL-4 (10 ng / ml, 0.667 nM) in the presence of varying concentrations of IL-4RE. Administration of tritiated thymidine in the last 20 hours of incubation was used as an indicator of proliferation. The results of these determinations are shown in Table 7. These results show that all PEGiligirovannye structure had an _IC50 value of almost 5 times greater than the _IC50 IL-4RA in the two primary cell assays.

Table 7 Evaluation of the bioactivity of PEG-IL 4RE in determining proliferation of B cells and blast T cells. Mutein B cell IC ₅₀ , nM Blast T-cell IC ₅₀ , nM BAY 16-9996 IL-4RA 0.86 + 0.42 (n = 10) 3.22 + 3.26 (n = 16) IL-4RE-K37C 3.73 + 2.15 (n = 2) 13.86 + 12.77 (n = 2) IL-4RE-N38C 3.33 + 2.68 (n-2) 9.94 + 8.99 (n = 2) IL-4RE-A104C 3.29 + 1.46 (n = 2) 4.67 + 4.65 (n = 2)

Example 7

The study of pharmacokinetics in rats.

Used adult male Spraque-Dawley rats. Rats were cannulated with a catheter into the jugular vein to collect blood samples. In addition, rats in the intravenous (IV) group were injected with femoral catheters for drug administration.

Rats were injected with either IL-4RA or a modified IL-4 mutein receptor antagonist at doses of 1 and 0.5 mg / kg, respectively. Both IV (intravenous) and SC (subcutaneous) administration methods were used. An IV dose was administered by injection directly into a catheter resident in the femoral vein. The SC dose was administered by injection into the dorsal thoracic region. There were 3 rats in each group.

After injection in the form of a bolus (IV or SC), blood samples were taken before the dose and at specified points in time up to 168 hours after the dose. Centrifugation of blood samples began within 1 hour after sampling. Plasma was collected and placed on dry ice at about -70 ° C.

Plasma concentrations of IL-4RA and modified mutein were quantified by enzyme-linked immunosorbent assay. Anti-1b-4 antibody was used as a reagent for coating and detection. The lowest limit for quantification by this method was 0.2 ng / ml. Pharmacokinetic parameters were determined by the method of compartmental analysis using WinNonlin (Pharsight, Mountain view, CA). Of particular interest are the estimation of the kinetics of absorption and elimination, distribution volumes, as well as absorbed amounts.

Claims (17)

1. Purified polynucleotide designed to obtain a modified mutein receptor antagonist IL-4, containing
(a) the nucleotide sequence indicated as SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or
(b) a nucleotide sequence encoding a polypeptide containing the amino acid sequence indicated as SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14. 2. An expression vector comprising a polynucleotide according to claim 1. 3. A host cell for producing a modified IL-4 mutein receptor antagonist, comprising the expression vector of claim 2. 4. A method for producing a modified mutein IL-4 receptor antagonist, comprising the steps of:
(a) culturing the host cell according to claim 3 under conditions under which the antagonist is expressed, and
(b) purifying the antagonist from the culture of the host cell. 5. A method for producing a modified IL-4 mutein receptor antagonist in active form, comprising the steps of:
a) culturing the host cell according to claim 3 under conditions when an antagonist is expressed;
b) refolding of the antagonist in the presence of dithiothreitol and
c) purification of the antagonist from the culture of the host cell. 6. The method according to claim 5, characterized in that it further comprises the steps of:
g) attaching the antagonist to a non-protein polymer and
d) purification of the antagonist attached to a non-protein polymer. 7. A modified IL-4 mutein receptor antagonist obtained by the method of claims 4 and 5, wherein amino acid residues 37, 38 or 104 are cysteine and which inhibits IL-4 and IL-13 mediated activity. 8. A modified IL-4 mutein receptor antagonist attached to a non-protein polymer obtained by the method of claim 6, wherein amino acid residues 37, 38 or 104 are cysteine and which inhibits IL-4 and IL-13 mediated activity. 9. The modified mutein IL-4 receptor antagonist of claim 8, wherein the non-protein polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol and polyoxyalkylene. 10. The modified mutein IL-4 receptor antagonist according to claim 9, characterized in that it is attached to a non-protein polymer via an amino acid residue at position 37, 38 or 104 in IL-4. 11. The modified mutein IL-4 receptor antagonist of claim 8 or 9, characterized in that it has a half-life in plasma, which is at least 2-10 times greater than that of the unmodified IL-4 receptor antagonist. 12. The modified mutein IL-4 receptor antagonist according to claim 9, characterized in that the amino acid sequence is represented by SEQ ID No. 12. 13. The modified IL-4 mutein receptor antagonist according to claim 9, characterized in that the amino acid sequence is represented by SEQ ID No. 13. 14. The modified mutein IL-4 receptor antagonist according to claim 9, characterized in that the amino acid sequence is represented by SEQ ID No. 14. 15. The use of a modified mutein IL-4 receptor antagonist according to claims 7-14 for the manufacture of a medicament for treating a human disorder associated with increased activity of IL-4 and IL-13. 16. The application of clause 15, wherein the violation is asthma, chronic obstructive pulmonary disease or related pulmonary conditions. 17. The application of clause 16, wherein the chronic obstructive pulmonary disease is emphysema or chronic bronchitis.