Modified antagonist of receptor il-4 and purified polynucleotide intended for its production
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
The scope to which the invention relates.
This invention relates to a receptor antagonist mutein IL-4, attached to a non-protein polymer, such as polyethylene glycol. It also provides compositions, dosage forms and methods of their administration for therapeutic purposes. These modified receptor antagonists mutein IL-4 and compositions and methods of using them are suitable for treating individuals who suffer from severe asthma, chronic obstructive pulmonary disease and related conditions of the lungs.
Background of invention
Asthma is characterized by variable, reversible obstruction of the Airways and separatethem respiratory tract (AHR), associated with infiltration of the bronchial mucosa activated T-lymphocytes (T-cells and eosinophils. These cells, along with the resident fat cells of the respiratory tract secrete a variety of cytokines and mediators that play a major role in the pathogenesis of the disease. CD4+Th2 due to the release of specific cytokines (IL-4, IL-5, IL-9 and IL-13) manage the development of the disease. In particular, Th2 cytokines, IL-4 and IL-13 are considered to be important in the development and preservation of airway inflammation and hyperacute respiratory tract.
In some in vivo studies also supported the opinion about the main role of IL - and IL-13 in the pathogenesis of asthma. When using animals deficient cytokine or chemicals that neutralize the function or IL-4 or IL-13, there is an important role of these cytokines in the regulation of primary and secondary immune response, leading to inflammation of the Airways and hyperacute respiratory tract (3, 4). Taken together, these data suggest that IL-4 and IL-13 both can play overlapping and independent role in the allergic response of the respiratory tract and that targeting both cytokine can have a significant additional advantage in comparison with targeting any one cytokine.
Antagonists of IL-4 are described in the literature. Mutants of IL-4, which act as antagonists include mutein IL-4, IL-4/Y124D (N. Kruse, Tony, H.P., W. Sebald, Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement, Embo J. 11:3237-41, 1992) and 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)). Single mutein is a tyrosine substituted for aspartic acid at position 124 in the D-helix. Double mutein represents an arginine substituted for aspartic acid at position 121, and the tyrosine is replaced with an aspartic acid at position 124 in the D-helix. Changes in this part of the D-helix correlate well with changes in interactions on the second site svyazyvanie is.
Options mutants of IL-4 expressing agonism or antagonism of IL-4 wild type, can be useful in the treatment of conditions associated with one of the pleiotropic effects of IL-4. For example, antagonists of IL-4 would be suitable for treating conditions aggravated when producing IL-4, such as asthma, allergies or other inflammatory condition responses. Agonists of IL-4 may be useful for treating conditions in which the presence of IL-4 is associated with improvement or alleviation of 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 T cells helper (helper cells), types 1 and 2 (Th1, Th2). Uninfected CD4+T cells to differentiate subpopulations (Th1 or Th2 depending on the cytokine contained during stimulation. Agonist of IL-4 would be ideal to shift production towards T cells-helper cells, namely, in the direction of education Th2, thereby providing a therapeutic effect.
In the application PCT/US93/03613 described variant of IL-4 containing Phe-Leu or Tight-Leu sequence in the alpha region of the spiral and negatively charged amino acid between two amino acids in the 3'-5'-or 5'-3'-direction from the sequence Phe-Leu or Tyr-Leu, and this option has increased SRO is ETS to the IL-4 receptor a neutral amino acid, replacement of negatively charged amino acid. In this proposal also stated that the specific substitution of Trp-Leu or Phe-Leu in the a-helix of IL-4 in 2-residues negatively charged residue leads to increased affinity. Option is IL-4 fused protein (diphtheria toxin).
Recombinant mutein (IL-4RA), which is derived IL-4 human, mutated in two provisions of its amino acid sequence described in U.S. patent 6028176 and 6313272. IL-4RA binds with high affinity to the alpha chain of the IL-4 receptor human, important functional signal component of both receptor complexes of IL-4 and IL-13. This mutein has no agonist activity and acts as a potent competitive receptor antagonist of IL-4 and IL-13 in vitro (see U.S. patent 6028176 and 613272). A significant disadvantage of the use of IL-4RA is its relatively short half-life (approximately 3-6 hours). Pharmacokinetic/pharmacodynamic modeling of IL-4RA in a model of asthma in primates shows that the effective average stable concentration in achieving optimal therapeutic effect is about 60 ng/ml
One approach to overcome the short half-life is the frequent introduction mutein IL-4RA patient, however, the frequent introduction (usually by injection or intubation into the trachea) creates znachitelyonaja tolerability of the patient and therapy in a clinical setting.
The invention provides mutiny IL-4RA with a longer half-life than previously described Malinov. The invention also provides reagents and methods for inhibiting 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, as described below.
According to one variant, the invention provides a purified preparation of the modified receptor antagonist mutein IL-4, including receptor antagonist mutein IL-4, attached to a non-protein polymer selected from the group consisting of polyethylene glycol, polypropyleneglycol and polyoxyalkylene. According to one aspect of the invention the purified product contains a modified polypeptide receptor antagonist mutein IL-4, which is encoded in 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:7 or SEQ ID NO:8. According to another aspect, the polypeptide includes 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.
According to one variant of the modified polypeptide receptor antagonist mutein IL-4 could be paired with a non-protein polymer to amino acid residue in position 28, 36, 37, 38, 104, 105 the sludge is 106 IL-4. These positions are numbered according to the amino acid sequence of IL-4 wild-type (i.e. interleukin-4 people). According to one aspect of this variant amino acid residue in positions 28, 36, 37, 38, 104, 105 or 106 is a cysteine.
According to another variant of the modified receptor antagonist mutein according to the invention binds to the alpha chain of the IL-4 receptor with Kdabout 0.1 nm - 10 μm, about 0.5 nm - 1 μm, or about 1.0 nm to 100 nm.
According to another variant of the modified receptor antagonist mutein IL-4 inhibits the proliferative response of TF-1 cells to IL-4 with IC50approximately 0.1 nm - 10 μm, about 0.5 nm - 1 μm, or about 1.0 nm to 100 nm.
According to another variant of the modified receptor antagonist mutein IL-4 inhibits the proliferative response of TF-1 cells to IL-13 with IC50selected from intervals of approximately 0.1 nm - 10 μm, about 0.5 nm - 1 μm, or about 1.0 nm to 100 nm.
According to a further variant of the modified receptor antagonist mutein IL-4 inhibits the proliferative response of human cells to IL-4 with IC50selected from intervals of approximately 0.1 nm - 10 μm, about 0.5 nm - 1 μm, or about 1.0 nm to 100 nm.
According to another variant of the modified receptor antagonist mutein IL-4 inhibits the proliferative response of T cells to IL-4 with IC50 selected from intervals of approximately 0.1 nm - 10 μm, about 0.5 nm - 1 μm, or about 1.0 nm to 100 nm.
According to another variant of the modified receptor antagonist mutein IL-4 according to the invention has a half-life in plasma, which is at least 2-10 times longer half-life in plasma is not modified receptor antagonist mutein IL-4.
This invention also provides pharmaceutical compositions containing: (a) a modified receptor antagonist mutein IL-4, which binds to the receptor of IL-4, and (b) a pharmaceutically acceptable carrier. The invention also provides purified polynucleotide containing (a) the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or (b) a nucleotide sequence encoding the 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 polynucleotide according to the invention and cells of the host containing the expression vector according to the invention.
In addition, the invention provides methods of obtaining a modified receptor antagonist mutein IL-4, which includes stages: (a) culturing the host cell described is Anna above, the conditions under which the expression of the antagonist, and (b) purification of the antagonist from the culture of host cells. According to a particular aspect of the antagonist, obtained by the method according to the invention, may inhibit the activity mediated by IL-4 and IL-13, and combined with non-protein polymer selected from the group consisting of polyethylene glycol, polypropyleneglycol and polyoxyalkylene.
The invention also provides methods of treating disorders in humans associated with increased activity of IL-4 and IL-13, comprising the stage of: (a) selection of a person with a condition in which increased activity of IL-4 and IL-13, and (b) introducing this person an effective amount of the modified receptor antagonist mutein IL-4 according to the invention or pharmaceutical compositions according to the invention. According to one aspect of this infringement is asthma, chronic obstructive pulmonary disease (such as emphysema or chronic bronchitis) or related pulmonary disorders.
This invention also provides a method of obtaining modified receptor antagonist mutein IL-4 in the active form, antagonists, obtained in this way, compositions containing such antagonists, and a method of treatment of disorders in humans, including the introduction of such antagonists and pharmaceutical compositions containing such as tagonist. This method involves the following stages: (a) culturing a host cell as described above under conditions in which the expression of the antagonist; (b) the repeated folding of the antagonist in the presence of dithiothreitol and (C) purification of the antagonist from the culture of host cells. According to one variant, the method also comprises the stage of: (g) the combination of the antagonist with non-protein polymer, and (d) cleaning antagonist attached to a non-protein polymer.
Specific preferred variants of the present invention will become apparent from the following more detailed description of some aspects and in the claims.
The drawing shows a scheme of the reaction of paglierani.
Detailed description of the invention
This invention relates to a modified receptor antagonists mutein IL-4, containing the receptor mutein IL-4, attached to a non-protein polymer, preferably, to a molecule of polyethylene glycol.
Unless otherwise required by the context, words in the singular include the plural and the terms in the plural include the singular.
The terms "polynucleotide" or "nucleic acid sequence" or "nucleic acid molecule" refer to a sequence of DNA or RNA. These terms encompass molecules, education is developed from any of the known basic analogs of DNA or RNA, such as, without limitation, 4-acetylcysteine, 8-hydroxy-N6-methyladenosine, aziridination, pseudoisocyanine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl, dihydrouracil, inosine, N6-ISO-pentylamine, 1-methyladenine, 1-methylpseudouridine, 1-methylguanine, 1-methyl-inosine, 2,2-dimethylguanosine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine A 5-methylaminomethyl, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosidosis, 5'-methoxycarbonylmethyl, 5-methoxymethyl, 2-methylthio-N6-isopentenyladenine, uracil-5-exucuse acid methyl ester, uracil-5-oxiana acid, oxybutynin, pseudorutile, koozin, 2-tocitizen, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-exucuse acid methyl ester, uracil-5-oxiana acid, pseudorutile, koozin, 2-tocitizen and 2,6-diaminopurine.
The term "purified" or "isolated" polynucleotide refers to a nucleic acid according to the invention, which (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates or other materials with which it is contained when the total nucleic acid was isolated from the cells of the source, (2) is not associated with all or part of polynucleotide with which the "isolated nucleic acid is associated in nature, (3) associated with polynucleotide with which it is not linked in nature, or (4) does not occur in nature as part of a longer polynucleotide sequence.
Preferably, the selected nucleic acid according to the invention is substantially free of any other impurity nucleic acids or other contaminants that are found in its environment, which will participate with its use in obtaining polypeptide or therapeutic, diagnostic, prophylactic use or use for research purposes.
Under "numbering according to IL-4 wild-type" refers to the identification of the selected amino acids with reference to the position in which this amino acid is usually found in IL-4 wild-type.
The term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus)used to transfer coding information to a cell-master.
The term "expression vector" refers to a vector suitable for transformation of a host cell and contains nucleic acid sequence that direct and/or regulate expression of the inserted sequences heterologous nucleic acid. The expression includes, but is not limited to, processes such as transcription, translation and RNA splicing, if introns are found.
The term "transduction" denotes the transmission of genes from one bacterium to another, usually using a phage. "Transduction" refers to the acquisition and transfer of eukaryotic cellular sequences by retroviruses.
The term "transfection" refers to the absorption of foreign or exogenous DNA of the cell, and the cell transfected"when exogenous DNA has been introduced into the cell membrane. A number of methods of transfection are well known and described in the prior 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 fragments of exogenous DNA into the appropriate cell hosts.
The term "transformation" refers to the change in the genetic characteristics of the cell, the cell is transformed when it has been modified and contains a new DNA. For example, the cell is considered t is informirovannii, when it is genetically modified in its natural state. After transfection and transduction of transforming DNA can still recombine with the DNA of a cell by physically integrating into a chromosome of the cell, can be stored temporarily in the form of an episomal element without reproduction or can be played independently as a plasmid. It is believed that the cell is stably transformed, when DNA is reproduced by cell division.
The term "identity", as known from the prior art, relates to the relationship between sequences of two or more polypeptide molecules or between two or more molecules of nucleic acid is determined by comparing the sequences. In the prior art "identity" also means the degree of relatedness of the sequences between the molecules of nucleic acids or polypeptides, as may be determined by the match between the threads of two or more nucleotides or two or more amino acid sequences. "Identity" is measured by the percentage of identical matches between the lesser of two or more sequences with alignment gaps (if any) using a particular mathematical model or computer program (i.e., "algorithms").
The term "similarity" is a related concept, but in contrast to Eden what was mentioned" "likeness" refers to a measure of relatedness, which includes both identical matches and conservative pair substitution. If two polypeptide sequences are, for example, 10/20 identical amino acids, and the rest all are non-conservative substitutions, then the values of the degrees of identity and similarity both will be equal to 50%. If in the same example, there are five provisions that contain conservative substitutions, then the value of the degree of identity is equal to 50%, and the degree of similarity will be 75% (15/20). Therefore, in cases where there are conservative substitutions, the value of the degree of similarity between two polypeptides is greater than the magnitude of the degree of identity of these two polypeptides.
The degree of identity and similarity of related nucleic acids and polypeptides can be easily determined by the 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 is designed to obespechivayushchee match between test sequences. Methods of determining the degree of identity described in the 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 program BLASTX public in the National information center for biotechnology (NCBI) and other sources (BLAST Manual, Altschul et al., NCB/NLM/NIH Bethesda MD 20894; Altschul et al., 1990, supra). To determine the degree of identity is also possible to apply a well-known algorithm, Smith Waterman.
Some schemes alignment of two sequences of amino acids can lead to match only a short section of two sequences, and this small area can have a very high degree of sequence identity, even though there is no significant relationship between the two sequences with a total length of. Accordingly, in some embodiments, the selected alignment method (GAP) leads to alignment, which comprises at least 50 contiguous amino acids of the target polypeptide.
For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, WI), two polypeptide for which shall be determined by the degree of identity is barb sequences aligned for optimal matching of their respective amino acids (the"matching section", as determined by the algorithm). According to some variants, in combination with the algorithm uses a penalty for opening a gap (calculated as triple the amount of the average diagonal; where the "average diagonal" is the average value of the diagonal matrix used in the comparison; the "diagonal" means the score or number assigned to each full coincidence of amino acids using specific matrix comparison) and the penalty for extending a gap (which is usually 1/10 of the penalty for opening a gap), and matrix comparisons, such as RAM or BLOSUM62. According to some variants of the algorithm using standard matrix comparison (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, the parameters for comparison of polypeptide sequences include the following:
Algorithm: Needleman et al., 1970, J. Mol. Biol., 48:443-453;
Matrix comparison: BLOSUM 62Henikoff et al., 1992, supra;
The penalty for gap: 12;
The penalty for extending the gap: 4;
The similarity threshold: 0.
With the specified parameters may be used by the program GAP. In some embodiments, the aforementioned parameters are the default parameters for comparison of polypeptides (together with the lack of the receiving penalty for end gaps) when using the GAP algorithm.
In this application we use common twenty conventional amino acids and their abbreviations. Cm. IMMUNOLOGY - A SYNTHESIS. 2ndEdition (E.S.Golub and D.R.Gren, Eds.), Sinauer Associates: Sunderland, MA, 1991, this publication is incorporated in this application by reference. Suitable components for polypeptides of the invention can also be stereoisomers (for example, D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α, α-disubstituted amino acids, N-alkylaminocarbonyl, lactic acid and other unusual amino acids. Examples of unusual amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetylized, σ-phosphoserine, N-acetylserine, N-formylmethionine, 3-methyl-histidine, 5-hydroxylysine, σ-N-methylarginine and other similar aminokisloty (for example, 4-hydroxyproline). In the notation of the polypeptides used in this application, the left direction is the direction of the N-end amino group and the right direction is a direction From the end bearing the carboxyl group, it is standard notation. Remnants of natural origin can be divided into the following classes based on General 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) basic: His, Lys, Arg;
5) residues that influence the orientation of the CE is to her, Gly, Pro; and
6) aromatic: Trp, Tyr, Phe.
Conservative substitution of amino acids can involve the exchange of a member of one of these classes to another member of the same class. Conservative substitutions may cover the remains of the non-natural amino acids, which are usually introduced by chemical synthesis of peptides, and not by synthesis in biological systems. They include peptidomimetics and other reversible or inverted forms of amino acids.
Non-conservative substitutions can include the exchange of a member of one of these classes to a member of another class. Such substituted residues may be introduced into the parts of the human protein, which are homologues of nonhuman proteins, or non-homologous parts of the molecule.
When making such changes, according to some options you can consider hydropathicity index of amino acids. Each amino acid was assigned hydropathicity index on the basis of its hydrophobicity and characteristics of the charges. These amino acids: isoleucine (+4,5); valine (+4,2); leucine (is+3.8); phenylalanine (a+2.8); cysteine/cystine (+2,5); methionine (+1,9); alanine (+1,8); glycine (-0,4); threonine (a-0.7); serine (of-0.8); tryptophan (of-0.9); tyrosine (-1,3); Proline (of-1.6); histidine (-3,2); glutamate (for 3,5); glutamine (for 3,5); aspartate (for 3,5); asparagine (for 3,5); lysine (-3,9) and arginine (-4,5).
An important role hydropathical index of amino acids when comparing inter is the positive function of the protein observed in the prior art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can substitute for other amino acids having similar hydropathicity index or score and still retain a similar biological activity. When making changes based on hydropathical index according to some variants carry out the substitution of amino acids whose gidroopticheskie indexes equal to ±2. According to some variants use amino acids with the value of the specified index ±1 and according to other variants - with the value of the specified index ±0,5.
In the prior art it is known that the substitution of like amino acids can be performed efficiently on the basis of hydrophilicity, particularly where the biologically functional protein or peptide is intended for use in immunology, as described in this application.
According to some variants of the greatest local average hydrophilicity of a protein, adjustable hydrophilic adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.
Residues of these amino acids are assigned the following hydrophilicity values: 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 at (- 0.5±1); alanine (-0,5); a histidine at (- 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 on the basis of similar hydrophilicity values according to some variants of conduct amino acid substitution, the value of hydrophilicity which is ± 2, according to some versions, this value is equal to ± 1 and according to the other options, this value is equal to ± 0.5. You can also on the basis of hydrophilicity to identify epitopes from primary sequences of amino acids. These areas are also called "epitope cor-plots".
Examples of substitutions of amino acids are given in Table 1.
|Amino acid sequence.|
|Initial residues||Examples of the substituents||Preferred substituents|
|Ala||Val, Leu, Ile||Val|
|Arg||Lys, Gln, Asn||Lys|
|His||Asn, Gln, Lys, Arg||Arg|
|Ile||Leu, Val, Met, Ala, Phe, norleucine||Leu|
|Leu||Norleucine, Il, 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|
|Ser||Thr, Ala, Cys||Thr|
|Tyr||Trp, Phe, Thr, Ser||Phe|
|Val||Ile, Met, Leu, Phe, Ala, norleucine||Leu|
The person skilled in the art is able to determine suitable variants of the polypeptide, as described below with the use of well known methods. According to some variants, the specialist is able to identify suitable areas of the molecule that can be modified without destroying the activity of the targeted areas that are not considered to be important for activity. According to other variants, the technician can identify the remains and parts of molecules, which remain among such polypeptides. According to other variants, even those areas that may be important for biological activity or for structure may be subject to conservative substitutions of amino acids without destroying the biological activity or without adversely affecting the structure of the polypeptides.
In addition, a specialist can examine the work on the study of structure-function, which identified the remains of such polypeptides that are important for activity or structure. Given this comparison specialist who can predict the importance of amino acid residues in the protein, which correspond to amino acid residues important for activity or structure in similar proteins. The specialist may choose chemically similar to the substituents for the amino acids in the case of such predicted important amino acid residues.
The specialist may also analyze the three-dimensional structure and amino acid sequence in connection with the structure in similar polypeptides. With this information, the specialist may predict the alignment of amino acid residues of the polypeptide with respect to its three dimensional structure. According to some variants, the specialist may decide not to implement radical changes of amino acid residues, which, as predicted, are on the surface of the protein, as these residues can participate in important reactions interactions with other molecules. Moreover, a specialist can generate experimental variants containing a single substitution in the amino acids in each desired balance of amino acids. These variants can then be screened using the methods of determining the activity, known to specialists. Such variants can be used to gather information about suitable options. For example, if it finds that a change in a particular amino acid residue resulted in the disappearance of unwanted decrease of activity or the appearance of inappropriate the activity, such variants such changes should be avoided. In other words, based on the information obtained from such routine experiments, the expert can easily determine amino acids, further substitution which should be avoided by itself or in combination with other mutations.
The number of scientific publications has been devoted to the prediction of secondary structure. Cm. 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, currently available computer programs that help to predict the secondary structure. One method of secondary structure prediction based on homology modeling. For example, two of the polypeptide or protein, in which the degree of identity is equal to more than 30% or the degree of similarity more than 40%, often have similar structural topology. Extension base structural data of proteins (PDB) recently provided an improved predictability of secondary structure, including the potential number of folds in the structure of the polypeptide or protein. Cm. Holm et al., 1999, Nucl. Acid. Res. 27:244-247. It has been suggested that there is a limited number of folds in the polypeptide or protein and that once a critical number of structures is determined, dramatically increases the accuracy of structure prediction.
The stage is leitlinie methods of secondary structure prediction include "education threads" (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 link" (see Holm, 1999, supra and Brenner, 1997, supra).
In some cases, variants of proteins include glycosylation variants, in which the number and/or type of glycosylation sites have been altered compared to the amino acid sequences of the parental forms of the polypeptide. In other cases, protein include more or fewer N-linked glycosylation sites than the native protein. N-linked glycosylation site is characterized by the sequence Asn-X-Ser or Asn-X-Thr, where the remainder of the amino acids denoted by X may be any amino acid residue except Proline. The substitution of amino acid residues to obtain this sequence provides a potential new site for the addition of N-linked carbohydrate chain. Or substitution, which remove this sequence will remove the existing N-linked carbohydrate chain. There is also a rearrangement of N-linked carbohydrate chains, when one or more N-linked glycosylation sites (typically those that are of natural origin) are deleted and created one or more new N-linked sites. Additional preferred variants include cysteine variant is, for example, in which one or more cysteine residues deliciousy or substituted for another amino acid (e.g., serine) as compared with the sequence of the parental form amino acids. Cysteine variants can be useful when should be the refolding of proteins for the production of biologically active conformation such as occur after separation of insoluble inclusion bodies. Cysteine variants typically contain fewer cysteine residues than the native protein, and usually contain the same number to minimize interactions arising due to non-coaxial cysteines. According to additional aspects of the ways proteins can include mutations such as substitution, addition, deletion or any combination thereof, and is usually obtained by siteprovides mutagenesis using one or more mutagenic oligonucleotides in accordance with the methods described in this application, as well as methods known from the prior art (see, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed, 2001, Cold Spring Harbor. N.Y. and Berger and Kimmel, METHODS IN ENZYMOLOGY, Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press, Inc., San Diego, CA., included in this application as a reference).
According to some variants of the substitution of amino acids include those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, () change the affinity for binding in the formation of protein complexes, (4) change the affinity for binding and/or (5) add or modify other physicochemical or functional properties of such polypeptides.
According to other variants of single or multiple substitutions of amino acids (in some embodiments, conservative substitutions of amino acids) can be carried out in the sequence of natural origin (in some embodiments, the portion of the polypeptide outside the domain(s)forming (- ) intramolecular contacts). According to preferred variants, the conservative substitution of amino acids is usually slightly change the structural characteristics of the parent sequence (e.g., the amino acid substitution has no tendency to the destruction of a helix that occurs in the parent sequence, or break other types of secondary structure that characterizes 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 these publications is included in this application by reference.
Peptide analogs are commonly used in the pharmaceutical industry as ones medication with properties that analogion the mi properties of the peptide template. These types of ones connection called "peptide mimetics" or "peptidomimetics". Cm. 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 included in this application as a reference for any purpose. Such compounds are often created by computer molecular modeling. Peptide mimetics that are structurally similar to therapeutically applied peptides, can be used to produce similar therapeutic or prophylactic effect. Typically, peptidomimetics are structurally similar to the reference polypeptide (i.e polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide bonds, possibly substituted by a connection selected from-CH2-NH-, -CH2-S, -CH2CH2-, -CH=CH- (CIS and TRANS), -the PINES2-, -CH(OH)CH2- and-CH2SO-introduced well-known methods. Systematic substitution of one or more amino acids in the consensus sequence of D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used in some embodiments to obtain a more stable peptides. In addition, an inactive protein comprising the consensus sequence or substantially identical variant consensus placenta is successive, can be obtained by known methods (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61:387 included in this application as a reference for any purpose), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclist polypeptide.
(a) characteristics of the modified receptor antagonists mutein IL-4.
"Modified receptor antagonists mutein IL-4 in this application include mutein IL-4RA described in U.S. patent 6028176 and 6 313 272 (included in this application as a reference) with additional substitutions of amino acids, and these substitutions contribute sitespecifically combination (joining)at least one non-protein polymer, such as polypropyleneglycol, polyoxyalkylene or polyethylene glycol (PEG) with muteena. Sitespecific attaching the PEG, for example, allows to obtain a modified mutein, which has the advantages of polyethylene glycosylated (Pepiliyana) connection, namely, a longer half-life in plasma and reduced immunogenicity while maintaining higher activity compared to non-specific methods Pepiliyana, such as N-terminal Pepiliyana or Pepiliyana in the side chain of lysine. This invention is also inherent in the Bor specific site in the Deputy amino acids, which promotes the proper folding of the molecule followed by the expression. Modified receptor antagonists mutein IL-4 are associated with IL-4 and IL-13 with the loss of affinity is not more than 10 times compared with IL-4RA. Modified receptor antagonists mutein IL-4 inhibit mediated IL-4 and IL-13 activity with loss of activity is not more than ten-fold, compared with the activity of IL-4RA. In addition, the modified receptor antagonists mutein IL-4 are characterized by the magnitude of the half-life in plasma, which is at least 2-10 times greater than that of unmodified IL-4RA.
Mutiny IL-4 according to the invention can also be characterized by insertions, deletions, substitutions and modifications of amino acids in place of one or more websites or other residues of polypeptide chain of the native IL-4. According to this invention any such insertions, deletions, substitutions and modifications provide mutein IL-4, which retains the activity associated with IL-4.
An additional aspect of the present invention is a method under which happen expression and refolding as described in Example 2. Mutein IL-4 must be properly cleaned in order to allow efficient Pegylation. Cleaning method described below in Example 2. When refolding mutein in the presence sultry rolnego protective agent, such as beta-mercaptoethanol, glutathione or cysteine, peeled mutein cannot be Pageregion as active sulfhydryl in the cysteine introduced into IL-4, is inactivated by oxidized protective agent. Between the free cysteine mutein IL-4, and a protective agent forms a covalent disulfide bonds. In contrast, the use of sulfhydryl protective agent dithiothreitol (DTT), which is oxidized with the formation of stable disulfide bonds, will not form a covalent bond with the free cysteine mutein IL-4, which leaves its sulfhydryl group free to react with Permalien reagent. Mutiny IL-4, peeled after refolding in the presence of beta-mercaptoethanol, glutathione, or cysteine can react with the PEG, if they are treated with DTT, but a mixture is formed monopegylated and multipayline products that also involves Pepiliyana existing cysteine IL-4. Pepiliyana available cysteines will result in incorrect folding with the formation of products which are inactive.
Todmodified receptor antagonists mutein IL-4 to IL-4 receptor can be determined by any known method, including techniques such as Bimolecular interaction Analysis (BIA) (analysis of bimolecular interactions in real time, as described in the Example . BIA is a method for studying biospecific interactions in real time, without labeling any of the reagents (e.g., BIAcore™). Changes in the optical surface plasmon resonance (SPR) can be used to indicate reactions between biological molecules in real time.
The ability of modified receptor antagonists mutein IL-4 to inhibit the proliferative response of immune cells can be assessed using the method of proliferative analysis described in Example 5, this ability is expressed in the form of inhibiting 50% concentration (IC50).
In the method BIAcore™ modified receptor antagonists mutein IL-4 according to the invention bind specifically to the receptor of the human IL-4 with the preferred value of Kdin the range from about 1.0 nm to about 100 nm. According to preferred variants of the present invention, these antagonists bind to the receptor of the human IL-4 with Kdapproximately from 0.5 nm to 1.0 μm. According to a further preferred variants of these antagonists bind to the receptor of the human IL-4 with Kdequal to from about 0.1 nm to 10 μm.
In addition, the modified receptor antagonists mutein IL-4 according to the invention will be contacted with the receptor of the human IL-4 and neutralize his ability about aciravati proliferation of cells of the immune system with the preferred size of the IC 50in the range from about 1.0 nm to about 100 nm. Preferred antagonists man bind the IL-4 receptor and neutralize its ability to proliferation of cells of the immune system with the value of the IC50in the range of from about 0.5 nm to 1.0 μm, and most preferred antagonists according to the invention with the value of the IC50in the range from about 0.1 nm to about 10 microns.
Options modified receptor antagonists mutein IL-4 according to the invention are also characterized by the magnitude of the 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 value of half-life in plasma, which is 10-100 times greater than that of unmodified IL-4RA (see Example 7).
A number of modified receptor antagonists mutein IL-4 with the characteristics described above, were identified by screening using the above methods of analysis. Antagonists according to this invention include the polypeptide sequences given in Table 2 (SEQ ID NO: 10-16).
(b) Polynucleotide encoding the modified receptor antagonists mutein IL-4.
The invention also provides for polynucleotide encoding the modified antagonist is East of the receptor mutein IL-4. These polynucleotide can be applied, for example, to obtain quantitative antagonists according to the invention. Polynucleotide according to the invention can be easily obtained by different methods, including, without limitation, chemical synthesis, screening cDNA libraries or genomic screening libraries of expression and/or amplification of cDNA by polymerase chain reaction (PCR).
Recombination DNA described in this application is typically the 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). This invention provides nucleic acids and methods for their preparation, are described in this application.
One way of obtaining a suitable nucleic acid sequence is a polymerase chain reaction (PCR). According to this method, cDNA derived from poly(A)+RNA or RNA using reverse transcriptase. Two primers, typically complementary to two separate cDNA fragments of the modified receptor antagonist mutein IL-4, are added to the cDNA along with a polymerase such as Taq polymerase, and polymerase amplificare cDNA fragment between the two primers.
Another means of obtaining a nucleic acid according to the invention is chemical synthesis using methods that are well known Spa is ailisto, for example, described by Engels et al., 1989, Angew. Chem. Intl. Ed. 28:716-34. Along with other these methods include methods of synthesis of nucleic acids using phosphotriester, phosphoramidite and H-phosphonate. The preferred method of this chemical synthesis is a synthesis on polymeric carrier using standard techniques with phosphoramidites. Usually DNA contains several hundred nucleotide units. Nucleic acids containing more than about 100 nucleotide units, can be synthesized as multiple fragments with the use of these methods. Then these fragments can be legirovanyh together. You can use other methods, well known to the experts.
Polynucleotide according to the invention, which can be used to encode the modified receptor antagonists mutein IL-4, shown in Table 3 (SEQ ID NO:2-8).
The invention also provides expression vectors comprising polynucleotide according to the invention, and cells of the host containing the expression vector according to the invention.
Polynucleotide according to the invention can be inserted into a suitable expression vector using standard methods of subsidies. Usually choose a vector that is functional in the cell-the master (i.e. the vector of owls is Estim processes, taking place in the cells of the host, and therefore can cause gene amplification and/or expression of the gene). Polynucleotide according to the invention may be expressed in prokaryotic, yeast cells of the host, the host cell insects and/or eukaryotic cell hosts. Selection of host cells will depend on various factors such as the desired level of expression. Overview of expression vectors, see Meth. Enz. Vol.185 (D.V.Goeddel, ed., Academic Press, 1990).
Typically, expression vectors used in the cell-master, contain sequences for the conservation of plasmids for cloning and expression of exogenous nucleotide sequences. Such sequences, called "flanking sequences", in some cases typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences (amp), the source of replication, transcription, terminal (leaf) sequence, a complete intron sequence containing a donor - and acceptor-site splicing, sequence, encoding a basic sequence for secretion of the polypeptide, the binding site of the ribosome, polyadenylated sequence plot of polylinker to insert a nucleic acid that encodes a polypeptide that is to be expressed, and a selectable e is ement marker. Each of these sequences is discussed below.
Flanking sequences may be homologous (i.e. of the same fragments and/or strain as the host cell), heterologous (i.e fragments of different fragments of the host cell or strain), hybrid (i.e. a combination of flanking sequences from more than one source), or synthetic, or the flanking sequences may be native sequences which normally function by regulating the expression of the receptor antagonist mutein IL-4. As such, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any organism or spinal nepoznannogo, or any plant, provided that the flanking sequence is functional in the processes occurring in the cell host, and they can be activated.
Flanking sequences used in the vectors according to the invention, can be obtained by any of the methods well known in the prior art. Usually used according to the invention flanking sequences, other than the flanking sequences of the genes of the receptor antagonist mutein IL-4, had to be pre-identified by the compilation certaily by digestion with restriction endonuclease, and could be, therefore, isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequences may be known. In this application flanking sequence can be synthesized by the methods described for the synthesis or cloning 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 genomic libraries using appropriate oligonucleotide and/or fragment flanking sequences from the same or different species. If the flanking sequence of the unknown DNA fragment containing the flanking sequence may be selected from a larger segment of DNA, which may contain, for example, the coding sequence or even the other(s) of the gene or genes. The selection can be carried out by digestion with restriction endonuclease to obtain the desired DNA fragment followed by isolation by purification using gel agarose, Qiagen® chromatography on a column (Chatsworth, CA), or other methods known to the expert. The selection of appropriate enzymes to achieve this goal is obvious to a person skilled in this is blast.
The Replicator is usually a part of the prokaryotic expression vectors, are available commercially, and contributes to the amplification of the vector in the cell host. If the selected vector does not contain the source site replication, it can be synthesized based on the known sequence by chemical and ligitamate in the vector. For example, the Replicator from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and a variety of sources (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV) or papilloma viruses, such as HPV or BPV) are suitable for cloning vectors in mammalian cells. Usually Replicator does not require expression vectors mammals (e.g., SV40 is often used only because it contains the early promoter).
Transcriptional terminal sequence is usually located in the position 3' end of the coding segment of the polypeptide and is used for termination of transcription. Usually a transcription terminal sequence in prokaryotic cells is an enriched G-C fragment with subsequent 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 the methods of synthesis of nucleic acids, such as description of the districts in this application.
The selective marker gene encodes a protein necessary for the survival and growth of host cells grown in a selective culture medium. Typical selective marker genes encode proteins that (a) give resistance to antibiotics and other toxins; (b) complement auxotrophic defect cells; or (C) supply the necessary nutrients not available from complex environments. Preferred selective marker gene are resistant to kanamycin gene resistant to ampicillin, and gene resistant to tetracycline. Gene resistant to neomycin, can also be used for selection in prokaryotic and eukaryotic cells-hosts.
Other selective genes can be used for amplification of the gene that will be expressed. Amplification is the process in which genes, which are essential for the production of a protein critical for growth, repeated in tandem with the chromosomes of the next generations of recombinant cells. Examples of suitable selective marker for human cells include dihydrotetrazolo (DHFR) and timedancing. The transformed human cells is placed in conditions of pressure selection, where only the transformants are adapted to survival by selective gene contained in the vector. Giving is giving selection is created by culturing the transformed cells under conditions where the concentration of agent selection in the medium is successively changed, which leads to amplification, as selective gene and the DNA that encodes the modified receptor antagonist mutein IL-4. As a result of amplified DNA synthesized in increased amounts of the modified receptor antagonist mutein IL-4.
The binding site of the ribosome is usually required to initiate translation of an mRNA and is characterized by the sequence of the Shine-Dalgarno (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located at the end of the 3' relative to the promoter and 5' to the coding sequence of the modified receptor antagonist mutein IL-4. The sequence of the Shine-Dalgarno changed, but is typically polypurine (i.e. has a high content of A-G). Many sequences Shine-Dalgarno were identified, each of them can be easily synthesized using the methods described in this application, and is used in prokaryotic vector.
As a leader, or signal, sequence may be used to direct the modified receptor antagonist mutein IL-4 from the host cell. Typically, a nucleotide sequence encoding a signal sequence that is located on the encoding section of the nucleic acid molecule to the slots of the modified receptor antagonist mutein IL-4, or directly at the 5' end of the coding section of the modified receptor antagonist mutein IL-4. Many signal sequences have been identified, and any of them, which is valid in the selected cell as the host, can be used in combination with the nucleic acid molecule of the modified receptor antagonist mutein IL-4. Therefore, the signal sequence may be homologous (naturally occurring) or heterologous to the nucleic acid molecule of the modified receptor antagonist mutein IL-4. Additionally, the signal sequence may be synthesized by chemical methods described in this application. In most cases, the secretion of the modified receptor antagonist mutein IL-4 from the host cell due to the presence of a signal peptide will result in removal of the signal peptide of the selected modified receptor antagonist mutein IL-4. The signal sequence may be a component of the vector, or it may be part of a molecule of the modified receptor antagonist mutein IL-4, which insertion in the vector.
In many cases, transcription of the nucleic acid molecule increases due to the presence of one or more introns in the vector; this is especially true when the polypeptide is produced in eukaryotic cells-owners, especially in the cells of the host mammal. Used the introns can be of natural origin in the gene modified receptor antagonist mutein IL-4, especially when the gene used is a full sequence of the genome or its fragment. If the intron is intron of natural origin in a gene, it can be obtained from another source. The position of the intron with respect to flanking sequences and gene modified receptor antagonist mutein IL-4 is generally important, as the intron for efficient should be transcribed. Thus, when the nucleic acid molecule according to the invention is transcribed, the preferred position for the intron is 3' to the site of the start of transcription and a position 5' to the poly-A transcription terminal sequence. Preferably, the intron or introns were located on one side or on the other side (i.e., 5' or 3') cDNA, so that he (they) did 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, in which he incertitude. Used synthetic introns. The vector can be applied to more than one intron.
The expression vectors and cloning according to the invention usually contain a promoter that recognizes the I the host organism and is linked to a nucleic acid molecule according to the invention. Promoters are retranscribing sequences located in the upstream direction (i.e., 5') to the initial codon of a structural gene (generally 100-1000 base pairs)that control the transcription of the structural gene. Promoters usually refers to one of two classes: the inducible promoters and constitutive promoters. Inducible promoters increase the level of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient medium or the temperature change. On the other hand, constitutive promoters initiate continuous production of the gene product, then there is little control of gene expression or no. A large number of promoters recognized by a variety of potential cells of the host, is well known. Suitable promoter may be associated with the nucleic acid molecule according to the invention by removing the promoter from the source DNA digestion with restriction enzymes and interturbine desired promoter sequence into the vector. The sequence of the promoter receptor antagonist mutein native IL-4 can be used to control the amplification and/or expression of nucleic acids according to the invention. The heterologous promoter is preferred is sustained fashion, however, only if it provides greater transcription and higher yields downregulation of protein compared to the native promoter and if it is compatible with the system host cell that has been selected.
Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose system promoters; alkaline phosphatase system, tryptophan (trp) promoter, and hybrid promoters such as the tac-promoter. Suitable are also other known bacterial promoters. Their published sequence that allows the specialist to ligitamate their desired DNA sequence using linkers and adapters necessary to create a suitable restriction sites.
Suitable for use with yeast cells masters promoters well known in the prior art. Yeast enhancers preferably used with yeast promoters. Suitable promoters for host cells of the mammal are well known and include, without limitation, promoters obtained from the genomes of viruses such as virus polyoma, fowlpox virus, adenovirus (such as adenovirus 2), human papilloma virus of cattle, sarcoma virus of birds, cytomegalovirus, retroviruses, hepatitis b virus and most preferably simian vacuolating in the Rus 40 (SV40). Other suitable promoters include heterologous mammalian promoters, e.g., jitsukawa promoters (promoters of heat shock) and the actin promoter.
Other promoters of interest to control gene expression include, without limitation, the scope of the early SV40 promoter (Bernoist and Chambon, 1981, Nature 290:304-10), the CMV promoter, the promoter contained in the position 3' long terminalnode repeat of rous sarcoma virus (Yamamoto et al., Cell 22:787-97), the promoter timedancing herpes (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA, 78:1444-45), the regulatory sequences of the gene of metallothionine (Brinster et al., 1982, Nature, 296:39-42), the prokaryotic expression vectors such as the promoter of the beta-lactamase (Vella-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA, 75:3727-31) or tac-promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA, 80:21-25). Are also of interest the following areas of control transcription in animals that exhibit tissue specificity and have been utilized in transgenic animals: the scope of control of the elastase gene I, which 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); the scope of control of the insulin gene, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); area control immunoglobulin gene, which is active in lymphoid cells (Grosscheld et al., 1984, Cell 38:647-58, Adames et al., 1985, Nature 318533-38; Alexander et al, 1987, Mol. Cell. Biol, 7:1436-44); area control virus Bittner, which is active in testicular cells, the cells of the breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-95); area control albumen gene, which is active in liver (Pinkert et al, 1987, Genes and Devel. 1:268-76); area control gene alpha-fetoprotein, which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science, 235:53-58); area control gene alpha-1-antitrypsin, which is active in liver (Kelsey et al. Genes and Devel. 1:161-71); area control beta-globin gene, which is active in myeloid cells (Mogram et al., 1985, Nature, 315:338-40; Kollias et al., 1986, Cell 46:89-94); the scope of control of the gene of the basic protein of myelin, which is active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48:703-12); area control gene light chain 2 myosin, which is active in skeletal muscle (Sani, 1985, Nature, 314:283-86) and the control gene hormone secreting gonadotropin, which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-78).
Enhancer sequence may be insertion into the vector to increase the transcription of a nucleic acid according to the invention is higher eukaryotes. Enhancers are CIS-acting elements of DNA, usually a length of about 10-300 base pairs that act on a promoter to increase transcription. The enhancers are quite independent from the orientation and the position. It was the mustache is novlene, they are in the position 5' and 3' to the transcription unit. Several sequences such as enhancers, available from mammalian genes known (for example, globulin, elastase, albumin, alpha-fetoprotein, and insulin). However, usually use the enhancer of virus. The SV40 enhancer, the enhancer early promoter of cytomegalovirus enhancer, polyoma and enhancers adenoviruses are examples of amplifiers for activation of eukaryotic promoters. Although the enhancer may be playserver in the vector at position 5' or 3' to the nucleic acid molecule according to the invention, it is usually located on a site 5' from the promoter.
The expression vectors of the invention can be obtained from the original 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 legirovanyh in the vector. The methods used to obtain each of the flanking sequences, are well known to the specialist.
Preferred vectors in the practice of this invention must be compatible with bacterial cells-master cells of the host insects and mammals. These vectors, along with others, VK is ucaut 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, without limitation, Comedy, plasmids or modified viruses, and the specialist is obvious that the vector system must be compatible with the selected cell of the host. Such vectors include, without limitation, plasmids, such as a derivative of the plasmid Bluescript® (fahmida on the basis of ColEl with a large number of copies, Stratagene Cloning Systems, La Jolla, CA), PCR cloning plasmids for cloning Tag-amplified PCR products (e.g., derived plasmids TORO™ TA Cloning® Kit, PCR2.1®, Invitrogen, Carlsbad, CA) and the vectors, mammalian, yeast or viruses, such as baculovirus expression (derived plasmids Rusnak, Clontech, Palo Alto, CA).
After creating a vector and inserting a nucleic acid according to the invention in the proper site of the vector ready vector can be insertion into a suitable host cell for amplification and/or expression of the polypeptide. Transformation of the expression vector according to the invention in a selected host cell may be well-known methods including transfection, infection, use of calcium chloride, electroporation, microinjection, lipofection, the use of DEAE-dextran, or other met the water. The selected method is partly a function of the type of the host cell, which should be used. These and other methods are well known and described, for example, in Sambrook et al., supra.
Cell host may be prokaryotic, such as E. coli) or eukaryotic, such as yeast cells, insect or vertebrate). A host cell under cultivation in appropriate conditions, synthesizes a modified receptor antagonist mutein IL-4, which can subsequently be collected from the culture medium (if a host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of a suitable host cell depends on various factors such as the desired level of expression, modification of polypeptides, which are desirable or necessary for the emergence of activity (such as glycosylation or phosphorylation) and the ease of folding with the receipt.
A number of suitable host cells are known in the art and many are available at the American Type Culture Collection (ATCC), Manassas, VA. Examples include, without limitation, mammalian cells, such as cells of the Chinese hamster ovary (Cho)cells, Cho DHFR(-) (Urnaub et al., 1980, Proc. Natl. Acad. Sci. USA, 97:4216-20), 293 cells or T of human embryo kidney (SOME) or cells T. Selection of suitable cells of the host mammal and methods of transformation of cultivation, amplification, screening, obtain products and cleaning are known. Other suitable cell lines mammals are cell line COS-1 and COS-7 monkey and cell line CV-1. Other examples of host cells of mammals include cell lines primates and rodent cell lines, including transformed cell lines. Suitable are also normal diploid cells, cell strains from in vitro culture of primary tissue, primary explants. Cell-candidates may have genotypic defect selective gene or may contain dominant incumbent selective gene. Other suitable mammalian cell lines include, without limitation, cells of neuroblastoma N2A mouse, HeLa cells, L-929 mouse line T obtained in mice Swiss, Balb-c or NIH, cell lines KSS or NAC hamster. Each of these cell lines are known and available to the experts in the field of protein expression.
Suitable for this invention are cells of the host, which is the bacterial cells. For example, in the field of biotechnology is well known for various strains .li (for example, NW, DH5S, DH10, and MS). In this way you can apply different strains of B.subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp. etc.
Professionals are also known and available as host cells for rapid and polypeptides according to the invention many strains of yeast cells. Preferred yeast cells include, for example, Saccharomyces cervisae and Pichia pastoris.
Also, if this is desirable, in the methods according to the invention to use insect cells. 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).
Polynucleotide according to the invention contained in the cell host, can be isolated without the presence of other components of cells, such as membrane components, proteins, and lipids. Polynucleotide can be isolated from cells by standard methods of purification of nucleic acids or can be synthesized using the method of amplification, such as polymerase chain reaction (PCR), or automatic synthesizer. Methods selection of polynucleotides are routine and known in the prior art. Any such technique for obtaining polynucleotide can be used to obtain isolated polynucleotide coding antagonists according to the invention. For example, for isolation of polynucleotides that encode antagonists, can be used restriction enzymes and samples. Preferably, the isolated polynucleotide were in the form of drugs that do not contain or do not contain 70, 80 or 90% of other substances.
Experts obviously, Thu the nucleic acid and polypeptides, described in this application can be obtained recombinant and other methods. For example, cDNA modified receptor antagonist mutein IL-4 according to the invention can be obtained by standard methods of molecular biology, using mRNA as a template. Then cDNA can be replicated using molecular biology techniques known in the art and described in Example 1. Example 2 describes specific methods of recombinant expression and purification, used in obtaining the modified antagonists mutein according to the invention.
(C) Evaluation of therapeutic usefulness of human antagonists.
To assess the potential activity of a specific antagonist in the treatment of allergic asthma antagonist can be tested in vitro in the determination of cell proliferation, as described in detail in Examples 5 and 6. In addition, the half-life in plasma modified receptor antagonist mutein IL-4 can be measured in vivo in the study of pharmacokinetics in rats as described in Example 6.
(g) the Pharmaceutical composition.
Any of the modified receptor antagonists mutein IL-4, described above, may be in the form of pharmaceutical compositions containing a pharmaceutically acceptable carrier. Pharmaceutically acceptable carrier preferably is epeirogeny. The composition can be administered by itself or in combination with at least one other agent, such as a stabilizer, which may be entered in any sterile, biocompatible pharmaceutical carrier, including, without limitation, saline, buffered saline, dextrose, and water. You can apply different aqueous media, for example, 0.4% saline, 0.3% glycine and the like, These solutions are sterile and generally do not contain particles. These solutions can be sterilized by conventional, well known sterilization methods (for example, when filtering).
If required, the composition may contain pharmaceutically acceptable auxiliary substances. Acceptable excipients, preferably, are non-toxic to recipients at the dosages and concentrations. The pharmaceutical composition may contain auxiliary substances for modifying, maintaining or stabilizing, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Appropriate components of the compositions include, without limitation, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ASC is Runova acid, sodium sulfite or acidic sodium sulfite), buffering agents (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or organic acid), agents that adds volume (for example, mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin 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, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, soleobrazutaya counterions (such as sodium), preservatives (such as benzylaniline, benzoic acid, salicylic acid, thimerosal, finitely alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide), solvents (such as glycerin, polypropylenglycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspendresume agents, surfactants or wetting agents (such as pluronics, PEG, esters sorbitan, Polysorbate, such as Polysorbate 20 or Polysorbate 80, Triton, tromethamine, lecithin, cholesterol or tyloxapol), agents that increase their stability (such as sucrose or sorbitol), agents increasing toychest (such as halides of alkali metals, preferably sodium chloride or potassium or mannitol-sorbitol), media delivery, diluents, excipients and/or pharmaceutical adjuvants. Cm. Remington''s Pharmaceutical Sciences (18thEd., A.R.Gennaro, ed., Mack Publishing Company, 1990).
The concentration of the antagonist according to the invention in such pharmaceutical formulation can vary within wide limits, namely, from less than 0.5%, typically 1% or at least about 1%, up to 15 or 20% by weight and is selected primarily based on the volume of fluid, viscosity, etc. in accordance with the specific method of administration. If desired, the pharmaceutical composition may be included in more than one type of antagonist, for example, with different value of Kdthe IL-4 receptor.
The composition can 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 comprising excipients and adjuvants that facilitate the introduction of the active compounds into preparations which can be used pharmaceutically.
Acceptable drugs preferably are non-toxic to recipients at the dosages and concentrations.
Pharmaceutical the song can contain components for modification, preservation or stabilization, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Appropriate components of the compositions include, without limitation, amino acids (such as glycine, glutamine, asparagine, arginine or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite or acidic sodium sulfite), buffering agents (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or organic acid), agents that adds volume (for example, mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin 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, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, soleobrazutaya counterions (such as sodium), preservatives (such as benzylaniline, benzoic acid, salicylic acid, thimerosal, finitely alcohol, methylparaben, propylparaben, chlorine is exidy, sorbic acid or hydrogen peroxide), solvents (such as glycerin, polypropylenglycol or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspendresume agents, surfactants or wetting agents (such as pluronics, PEG, esters sorbitan, Polysorbate, such as Polysorbate 20 or Polysorbate 80, Triton, tromethamine, lecithin, cholesterol or tyloxapol), agents that increase the stability (such as sucrose or sorbitol), agents that increase toychest (such as halides of alkali metals, preferably sodium chloride or potassium or mannitol-sorbitol), media delivery, diluents, excipients and/or pharmaceutical adjuvants. Cm. Remington''s Pharmaceutical Sciences (18thEd., A.R.Gennaro, ed., Mack Publishing Company, 1990).
The optimal pharmaceutical composition may be obtained by the person skilled in the art depending, for example, the intended route of administration, forms of delivery and the desired dose. See, for example. Remington''s Pharmaceutical Sciences, supra. Such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of nucleic acid, or a modulator of bone density according to the invention.
Primary filler or carrier in the form of pharmaceutical compositions may be aqueous or nonaqueous. For example, a suitable carrier for injection can be water, saline solution or artificial cerebrospinal fluid, possibly supplemented with other substances, typical compositions for parenteral administration. Examples of carriers are also neutral buffered saline or saline mixed with serum albumin. Other examples of pharmaceutical compositions containing Tris buffer with pH around 7.0 to 8.5, or acetate buffer with a pH of around 4.0 to 5.5, which can also include sorbitol or a suitable substitute. According to one variant of the invention the pharmaceutical composition according to the invention can be prepared for storage by mixing the selected components with the desired degree of purity with possible additives (Remington''s Pharmaceutical Sciences, supra) in the form of dried sludge or water solution. In addition, the composition may be in the form of a lyophilisate containing the appropriate excipients such as sucrose.
The pharmaceutical compositions can be prepared for parenteral delivery. The composition can also be obtained for inhalation or for delivery through the digestive tract, for example, oral delivery. The preparation of such pharmaceutically acceptable compositions known in the art.
The components of the composition are contained in concentrations that are acceptable for injection. the example buffering agents are used to maintain the composition at physiological pH or slightly lower pH, typically in the range of from about 5 to about 8.
When used in parenteral administration, therapeutic compositions according to the invention can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired substance according to the invention in a pharmaceutically acceptable carrier. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the substance is obtained as a sterile, isotonic solution, properly sterilized.
Another medication may contain a substance according to the invention together with the same agent as injectable microspheres, biorstwami particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, which provide adjustable or prolonged release of the product, which can then be delivered via a depot injection. You can also use hyaluronic acid, which promotiom prolonged presence in the circulation system. Other suitable means for introducing the substances according to the invention include implantable system for drug delivery.
According to one variant of the invention is farmacevticheskaja composition can be obtained for administration by inhalation. For example, nucleic acid, or a modulator of bone density according to the invention can be composed of a dry powder for inhalation. Inhalation solutions may also be obtained with a propellant for aerosol delivery. According to another variant of the solutions may be introduced by means of a nebulizer. Introduction to light as described in the application WO 94/20069, where disclosed introduction in light of chemically modified proteins.
In other embodiments, the compositions can be administered orally. According to one variant of the invention, nucleic acids, or modulators of bone density according to the invention, which are introduced in this manner can be kept together with those carriers that are commonly used in the preparation of solid dosage forms such as tablets or capsules, or without those carriers. For example, can be obtained capsule to release the active portion of the composition in the place of the gastrointestinal tract, where the maximum bioavailability and minimized desistence decomposition. To ensure the absorption of the antagonist or modulator according to the invention may include additional agents. Can also be used diluents, flavorings, low melting waxes, vegetable oils, lubricating agents, suspendresume agents, disintegrators for tablets and binding.
Other pharmaceutical compositions can be in luceti effective amount of nucleic acids, or modulators of bone density according to the invention in a mixture with non-toxic excipients, which is suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable media can be obtained solutions in a single dosage form. Suitable excipients include, without limitation, inert diluents, such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; or binders, such as starch, gelatin or resin of acacia; or lubricating agents such as magnesium stearate, stearic acid or talc.
Other pharmaceutical compositions obvious to experts, these include nucleic acids, or modulators of bone density according to the invention in compositions for prolonged or controlled delivery. Methods of obtaining various other means for prolonged or controlled delivery, for example, liposomal carriers, biodegradable microparticles or porous granules and depot injections, are well known in the art. See, for example, PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
Additional examples of drugs with slow release include semi-permeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Matrix slow release can on the part polyesters, hydrogels, polylactide (U.S. patent No. 3773919 and patent EP No. 058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-56), poly-(2-hydroxyethylmethacrylate) (Langer et al., 1981, J.Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), a copolymer of ethylene with vinyl acetate (Langer et al., supra) or poly-D(-)-3-hydroxybutiric acid (patent EP No. 133988). The prolonged release composition may also include liposomes, which can be obtained in one of 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.
Pharmaceutical composition for administration in vivo typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using the specified method can be carried out either before or after drying and recovery. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile inlet, for example, a package for intravenous solution or container with a cork, through which is introduced hypodermal needle for injection.
The pharmaceutical compositions according to the invention can be introduced by any of the methods described in this application, including, without limiting the tion, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal.
After preparation of pharmaceutical compositions, they may be placed in an appropriate container with a label for the treatment of this condition. This label will include the quantity, frequency and method of administration.
(g) Therapeutic methods.
This invention provides a way to alleviate the symptoms of the disorder by binding the alpha chain of the IL-4 receptor and inhibiting the activity mediated by IL-4 and IL-13.
These disorders include, without limitation, the Hyper-reactivity of the Airways and inflammation of the respiratory tract, including recruitment and activation of mast cells, eosinophils and lymphocytes associated with asthma and other immunological or allergic disorders.
According to one variant of the invention a therapeutically effective dose of a modified receptor antagonist mutein IL-4 according to the invention and/or pharmaceutical compositions according to the invention is administered to a patient having a disorder characterized by increased activity of IL-4 and IL-13, as the disorders mentioned above.
(d) Determining terap whitesky effective dose.
Determination of therapeutically effective dose is known to specialists in this field. A therapeutically effective dose refers to the amount of the antagonist, which is used for the effective treatment of asthma, compared with efficiency, which is evident in the absence of therapeutically effective dose.
A therapeutically effective dose can be determined initially in animal models, usually rats, mice, rabbits, dogs, pigs or primates. The animal model can also be used to determine the appropriate concentration range and method of administration.
This information can then be used to determine appropriate doses and methods of introducing people.
Therapeutic efficacy and toxicity, for example, the ED50(the dose which is therapeutically effective in 50% of the population) and LD50(dose, which is lethal in 50% of population) human antagonist can be determined by standard pharmaceutical methods in cell cultures or experimental animals. The ratio of the toxic dose to the dose that is therapeutically effective, represents a therapeutic index and can be expressed as the ratio LD50/ED50.
Pharmaceutical compositions that possess high therapeutic indices are preferred. Data obtained from the study of animals used to determine the range of doses to humans. The dosage contained in such compositions are preferably in the range of circulating concentrations that include the ED50with little toxicity or no toxicity. Dose range in this interval, which depends on the applied dosage forms, the sensitivity of the patient and the method of administration.
The exact dose determined by the practitioner based on factors related to the patient who undergoes treatment. The dose and method of introducing regulated in such a way as to ensure sufficient levels of antagonist or maintain the desired effect. Factors that should be taken into account include the severity of the disease, General health status of the patient, age, weight and gender of the subject, diet, time and frequency of administration, combination(s) of drug sensitivity reactions and tolerance/response to therapy.
Pharmaceutical compositions of long-term action can be entered every 3-4 days, every week or once every two weeks depending on the size of half-life and rate of clearance of a particular composition.
Polynucleotide encoding the modified receptor antagonists mutein IL-4 according to the invention can be constructed and introduced into the cell ex vivo or in vivo using well-developed methods, in the including, without limitation, DNA transfer mediated by transferrin-polycation, transfection with naked or encapsulated nucleic acids, cell fusion mediated by liposomes, intracellular transportation of granules of latex coated with DNA, fusion of protoplasts, viral infection, electroporation, "gene gun" and transfection mediated by DEAE or calcium phosphate.
Effective in vivo dose of the antagonist is 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 patient's weight and from about 200 μg to about 250 μg/kg of patient's weight. With the introduction of polynucleotides coding antagonists are effective in vivo dose range from about 100 ng to about 200 ng, 500 ng to about 50 mg, from about 1 μg to about 2 mg, about 5 μg to about 500 μg and about 20 μg to about 100 μg of DNA.
Method of introduction of the pharmaceutical compositions according to the invention, containing modified receptor antagonists mutein IL-4, can be by any method of delivery of the antagonist to the owner. The pharmaceutical compositions according to the invention are particularly suitable for parenteral administration, namely, subcutaneous, intramuscular, intravenous, intratracheal or intranasal and other methods of introduction into the lungs.
Recombinant receipt of cysteine Malinov IL-4-RA and IL-4-RE.
For recombinant expression of IL-4 was chosen expression system pet Directional TOPO® (Invitrogen). This system uses a highly efficient one-step strategy "TOPO® Cloning" for direct cloning of PCR product with a blunt end and TAC promoter for IPTG-inducible expression with a high degree of the desired gene in E. coli. Additional features include lacI gene to reduce basal transcription source pBR322 for replication and preservation of plasmids and gene resistant to ampicillin for selection.
IL-4 was cloned in the vector pET101/D-TOPO to produce the recombinant protein of IL-4. Oligonucleotide primers are listed in Table 4. Was created forward PCR primer 5 SASS sticky end to facilitate direct clone, with a unique site of restriction enzyme NdeI to sublimirovanny and initial start codon ATG. Reverse PCR primer included two stop codon to stop the introduction of C-terminal tags and unique website Rees is reaktsionnogo enzyme BamHI to sublimirovanny. By using the previously cloned human IL-4 as a template was obtained PCR product of IL-4 with a blunt end. The product was purified on gel and incubated with a solution of salt and vector TORO® for 5 minutes at room temperature for direct cloning into the vector pET101/D-TOPO. The recombinant vector was transformed into chemically competent One Shot TOP10 E. coli. Recombinant plasmid DNA was used for DNA sequencing to confirm the correct sequence.
IL-4/pET101/D-TOPO served as a template to obtain cysteine of Malinov IL-4RE by means of a set QuikChange® Site-Directed Mutagenesis Kit, Strategene. Each cysteine mutein received with use of two oligonucleotide primers, each complementary to opposite strands of the vector, and contained the codon TGC or GCA for the introduction of desired mutations of cysteine. In Table 4 pericycle the s primers, used to obtain Malinov IL-4RE. Using primers cyclization and conditions specified in the Protocol of the manufacturer, was acquired mutation of the plasmid containing staggered nicks. This product was processed by DpnI endonuclease for 1 hour at 37°C to digest methylated neutropenia the parent template DNA. DNA treated with DpnI was transformed into supercompetent cells XL-1 Blue, which were restored nicks in the mutated plasmid. Mutagenic DNA plasmids were analyzed according to standard methods sequencing to confirm the correct sequence.
Recombinant expression and purification.
Cells BL21 Star (DE3) One Shot (Invitrogen), which were transformed using protein containing plasmids were characterized for optimal expression and were grown at 37°C until the value of OD600approximately 0.4 and induced with 1 mm IPTG (Invitrogen) for 3 h at 37°C. One liter of cells alloy preformed at a speed of 13000 rpm for 10 min, weighed and stored at -80°C. the Frozen tablets cells resuspendable in 8 ml of buffer, destructible cells (0.1 M phosphate buffer with a pH of 7.3, 0.1% of Triton X100, 1 mm EDTA) per gram of cells and treated with ultrasound for 4 for 1 min with intervals of 1 min, the Cell lysate was removed centrifuge holds is the key at 35000 g for 10 minutes Tablets cells were then washed 2-3x in suspension in 30 ml of buffer, destructible cells were treated with ultrasound for 1 min and centrifuged. The final tablets of cells, body turn, was stored at -20°C. the inclusion Body resuspendable in 5 ml solubilizing buffer (0.2m Tris, pH 9, 7M of guanidinate) per gram of cells. Added reagents sulfatase (0.16 g of sodium sulfite, 0.08 g of tetrathionate potassium per gram of cells) and mixed body of particles at room temperature for 2 hours. Undissolved components were then removed by centrifugation at 35000 g for 20 min, remained solubilization body inclusions. Body enable then passed on pressure column chromatography Superdex200 size (Akta) for isolation of the protein. The column was balanced with 2 column volumes (CV) 6M mixture of guanidine hydrochloride/PBS, pH 7, with a flow rate of 1 ml/min, the protein was suirable 1,5 CV. Collected peak fractions (each 1.5 ml) and subjected to gel electrophoresis on 12% or 4-20% Bis-Tris SDS. Collected fractions containing the protein was added to a final concentration of 7.5 mm DTT for reduction of protein molecules. After incubation for 2 h at room temperature the mixture was diluted 5x with water and dialyzed in 4.5 l of 3 mm NaH2PO4, 7 mm Na2HPO4, 2 mm KCl, 120 mm NaCl. Dialysis was continued for 3-4 days, feeding fresh buffer at least 3 times. Dial is used the product is then filtered through a 0.2 μm filter and brought the pH to 5 with acetic acid. The column was balanced by 10 CV of buffer 1 (25 mm ammonium acetate, pH 5), creating a gradient for 20 min to 100% buffer B (25 mm ammonium acetate, pH 5/ 1M NaCl). Collected peak fractions (1.5 ml each) and subjected to gel electrophoresis on 12% or 4-20% Bis-Tris SDS. The fractions containing the product were collected and diluted 2x with buffer A (0.1% of TFA/water). Then protein was chromatographically method GHUR with reversed phase C4 (Beckman system Gold), using a loop of 5 ml and a flow rate of 1 ml/min according to the following program: 10% buffer And during injection, 10 min gradient to 40% buffer B (0.1% of TFA/ACN), 30 min gradient to 50% buffer and 5 min gradient to 100% buffer C. Collected peak fractions (0.5 ml each) and subjected to gel electrophoresis on 12% or 4-20% Bis-Tris SDS. Fractions containing protein, dried and resuspendable in 0.1m MES, pH of 6.1 to analyze and definitions.
Site-specific Pegylation of cysteine and cleanup.
For Paglierani cysteine containing mutiny IL4 RA, a Protocol was developed using stable thioester communication between the sulfhydryl group of a protein and maleimides linear group derived methoxypolyethyleneglycol-maleimide 22 kD (Nektar Therapeutics). To 600M protein dissolved in the reaction buffer OLM MES, pH 6, was added 2-molar excess of MPEG-MAL 22 kD. After 0.5 h exposure at room temperature, the reaction was interrupted 2M excess of cysteine on the to compared to MPEG-MAL 22 kD (see the drawing). Targeted protein was purified from the unreacted MPEG-MAL 22 kD (treated with cysteine) and unreacted cysteine mutein IL4 RA using cation exchange and displacement chromatography. The crude reaction mixture was applied on a cation-exchange columns Vivapure Mini S (Vivascience), balanced 0.4 ml 0,1M MES, pH 6. The column washed twice with 0.4 ml of 0.1 m MES, pH 6, followed by centrifugation 2000 g after each washing. The samples were then washed from the column by centrifugation using 0.4 ml of 0.6m NaCl/0,1M MES, pH 6. Erwerbende fraction volume of 0.4 ml was loaded into the column GHUR TSK-GEL G2000SWYL (Tosoh Biosep), using a Beckman system Gold. The samples were again dissolved in a phosphate buffer saline solution (Dulbecco's PBS), a mobile phase with a flow rate of 1 ml/min for 30 minutes Peak fractions (0.5 ml) were collected and subjected to electrophoresis on the gel 4-12% Bis-Tris SDS to highlight Paglinawan protein. The fractions containing the product were collected and concentrated using the device Ultrafree Biomax-5 (Millipore) according to the manufacturer's Protocol, to obtain approximately 60 m (~1 mg/ml) for analysis and in vitro tests. The final concentration Pageregion protein was determined by the method of amino acid analysis. The final outputs are given in Table 5.
|The magnitude of the output Pageregion cysteine of Malinov IL4RA.|
|Mutein||Pageler. protein (mg, initial)||Pageler. protein (mg, end)||% output|
The reaction of the receptor binding of IL-4 using BiaCore.
The IL-4 receptor immobilizerpower on the sensor chip for research BiaCore CM by attaching amino groups. The surface of the sensor activated by EDC/NHS impulse. The receptor for IL-4 was dissolved in 10 mm acetate buffer (pH 5.0) was injected into the element 2 with a flowing electrolyte with subsequent pulsed supply 1.0 M ethanolamine-Hcl to deactivate the surface. The degree of immobilization of the receptor was 3000 RU. Element 1 with a flowing electrolyte also activated without ligand to function as a control. For the kinetic analysis used BiaCore Wizard. Candidates antagonists IL4RE diluted MBS-EP-dividing buffer) was injected at 30 μl/min for 30 min at the time of dissociation 15 minutes Regeneration of the chip was performed by two 30-second injection of 10 mm glycine, pH 2.5, the (flow rate of 100 µl/min) to baseline before the next injection in a series of concentrations. The values of dissociation constants (KD) was determined for each candidate, based on the kinetics of direct binding (table 5). The results show that all structures IL4-RE-A104C, IL4-RE-N105C and IL4-RE-Q106C dissociation constants less than 0.6 mm.
the Definition of a 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 assess functional antagonistic activity of IL-4RE. For this TF-1 cells were cultured for 2-4 days in the plate with 96 wells (1×104/well volume of 100 μl) in RPMI+10% serum IL-4, IL-13 and IL-4RE or without them. The processing of GM-CSF was used as a positive control. 24 hours before the last read to each well was added 10 μl of AlamarBlue (10 vol%). Measured fluorescence at 530/590 nm using a WALLAC Victor 2. Inhibitory concentration (IC50) was calculated by titration dose of the candidate IL-4RE. Data bioanalysis for TF-1 and inhibition of IL-4 and IL-13 are shown in Table 6. The results show that the design IL4-RE-CC, IL4-RE-N38C and IL4-RE-A104C have value IC50comparable to the IC50 for IL-4-RA in the presence of IL-4 or IL-13.
|Analysis of the binding of PEG-IL 4RE-BiaCore and assessment of bioactivity Pageregion of Malinov against IL4RA when determining the proliferation of TF-1 cells.|
|Mutein||Affinity by BiaCore, nm||TF-1/IL-4 IC50nm||TF-1/IL-13 IC50nm|
|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||of 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)|
Determination of proliferation of primary cells.
The proliferative response of primary human cells (T - and b-cells) IL-4 was assessed after pre-treatment IL-4RE. Peripheral blood mononuclear cells (PBMCs) were isolated from peri is aricescu blood and some RNA was treated for 4 days to induce the formation of blast T cells. PBMCSalso was treated with anti-CD40 activation activity In cells and immediately used. Cells were sown in tablets with 96 wells (105cells/well). Drugs blast T cells and b cells, treated RNA, stimulated for 3 days with IL-4 (10 ng/ml, 0,667 nm) in the presence of varying concentrations of IL-4RE. Introduction tritium-labeled 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 Pepiliyana construction had the largest IC50almost 5 times the IC50IL-4RA in two analyses of primary cells.
|Assessment of bioactivity of PEG-IL 4RE when determining the proliferation of b-cells and blast T cells.|
|Mutein||B-cell IC50nm||Blast T-cell IC50nm|
|BAY 16-9996 IL-4RA||0,86+0,42 (n=10)||3,22+3,26 (n=16)|
|IL-4RE-K37C||to 3.73+2,15 (n=2)||13,86+12,77 (n=2)|
|IL-4RE-A104C||3,29+1,46 (n=2)||4,67+4,65 (n=2)|
Pharmacokinetics study in rats.
Used adult male rats Spraque-Dawley. Rats were Coulibaly catheter in the jugular vein for collection of blood samples. In addition, rats in group with intravenous (IV) administration has introduced a catheter into the femoral vein for drug administration.
Rats were injected or IL-4RA, or modified receptor antagonist mutein IL-4 at doses of 1 and 0.5 mg/kg, respectively. Used and IV (intravenous) and SC (subcutaneous) administration methods. The IV dose was administered by injection directly into the catheter resides in the femoral vein. SC dose was administered by injection into the dorsal thoracic region. In each group there were 3 rats.
After injection of a bolus (IV or SC) took blood samples before the dose and at specified time points up to 168 hours after administration of the dose. Centrifugation of blood samples was started within 1 hour after sampling. Plasma was collected and placed on dry ice at about -70°C.
The concentration of IL-4RA and modified mutein in plasma were determined by quantitative enzyme-linked immunosorbent assay. Anti-1b-4-antibody was used as reagents for coating and detection. A lower limit to castenago determine 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 is the evaluation of the kinetics of absorption and elimination, the volume of distribution, and the absorbed amount.
1. Purified polynucleotide intended for obtaining modified receptor antagonist mutein IL-4, containing
(a) the nucleotide sequence listed as SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6, or
(b) a nucleotide sequence encoding the polypeptide containing the amino acid sequence listed as SEQ ID NO:12, or SEQ ID NO:13, or SEQ ID NO:14.
2. The expression vector including polynucleotide according to claim 1.
3. A host cell that is designated to receive the modified receptor antagonist mutein IL-4, containing the expression vector of claim 2.
4. The method of obtaining modified receptor antagonist mutein IL-4, which includes stages:
(a) culturing the host cell according to claim 3 under conditions in which the antagonist is expressed, and
(b) purification of the antagonist from the culture of the host cell.
5. The method of obtaining modified receptor antagonist mutein IL-4 in the active form, which includes stages:
a) culturing the host cell according to claim 3 under conditions when expressed antagonist;
b) refolding antagonist in prisutstvie is and dithiotreitol and
C) purification of the antagonist from the culture of the host cell.
6. The method according to claim 5, characterized in that it additionally includes the stage:
g) attaching antagonist to non-protein polymer and
d) cleaning antagonist attached to a non-protein polymer.
7. Modified receptor antagonist mutein IL-4, obtained by the method according to claims 4 and 5, in which amino acid residues 37, 38 or 104 are cysteine and which inhibits mediated IL-4 and IL-13 activity.
8. Modified receptor antagonist mutein IL-4, attached to a non-protein polymer obtained by the method according to claim 6, in which amino acid residues 37, 38 or 104 are cysteine and which inhibits mediated IL-4 and IL-13 activity.
9. Modified receptor antagonist mutein IL-4 according to claim 8, characterized in that the non-protein polymer selected from the group consisting of polyethylene glycol, polypropyleneglycol and polyoxyalkylene.
10. Modified receptor antagonist mutein IL-4 according to claim 9, characterized in that it is attached to a non-protein polymer through amino acid residue at position 37, 38 or 104 in IL-4.
11. Modified receptor antagonist mutein IL-4 according to 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 unmodified antagonist d is aptara IL-4.
12. Modified receptor antagonist mutein IL-4 according to claim 9, characterized in that the amino acid sequence represented by SEQ ID No. 12.
13. Modified receptor antagonist mutein IL-4 according to claim 9, characterized in that the amino acid sequence represented by SEQ ID No. 13.
14. Modified receptor antagonist mutein IL-4 according to claim 9, characterized in that the amino acid sequence represented by SEQ ID No. 14.
15. The modified receptor antagonist mutein IL-4 according to claims 7 to 14 for the manufacture of tools for the treatment of human disorders associated with increased activity of IL-4 and IL-13.
16. The application of clause 15, characterized in that the infringement is asthma, chronic obstructive pulmonary disease or related pulmonary condition.
17. The application of clause 16, wherein the chronic obstructive pulmonary disease is emphysema or chronic bronchitis.
SUBSTANCE: in dissolvent, which contains from 55% to 70% of water (wt/wt), precursor of insulin or precursor of insulin derivative is exposed to fermentative splitting at alkaline values of pH. In process of fermentative splitting, they use tripsin or lysil-specific protease, preferably Achromobacter lyticus protease I. Then without separation of intermediate product from reaction mixture, mentioned intermediate product is fermentatively complemented with nucleophilic compound, which represents aminoacid ether, aminoacid amide, peptide, peptide ether or peptide amide in reaction mixture, having water content in the range from 10% to 50% of water (wt/wt), at acidic values of pH, close to neutral pH value. If required, protective group (s) is/are removed.
EFFECT: preparation of insulin compound from its precursor by efficient improved method.
24 cl, 5 ex
SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.
EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.
16 cl, 3 tbl, 7 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.
EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.
3 cl, 1 tbl, 5 ex
SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.
EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.
29 cl, 5 dwg, 6 tbl, 7 ex
FIELD: medicine; microbiology.
SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.
EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.
3 cl, 3 dwg, 3 ex
FIELD: genetic engineering.
SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.
EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.
9 cl, 6 ex
SUBSTANCE: peptide is obtained from phage peptide library including set of static peptides with length of 12 aminoacid residues, by affine selection of phage clones containing peptide capable of specific linking to antibodies of benzo[α]pyrene and benzo[α]anthracene. Peptide displays specific interaction effect on antibodies of benzo[α]pyrene and benzo[α]anthracene and features molecular weight of 1.3 kDa and registered aminoacid sequence LHLPHHDGVGWG encoded by nucleotide sequence SEQ ID NO:1.
EFFECT: application in medicine as a base for peptide medicine development for immunologic prevention of malignant tumours of humans.
4 dwg, 3 ex
SUBSTANCE: present invention relates to genetic engineering, more specifically to obtaining anticoagulative protein extracted from nematodes (NAP) and can be used in medicine. To obtain a medicinal preparation based on NAP, methanotrophic yeast host cells are cultured, encoding rNAPc2 or rNAPc2/praline, until attaining the desired cell density. NAP is then extracted from the said yeast host cells through cation-exchange chromatography on an expanding layer. To purify the NAP medicinal preparation, hydrophobic-interaction chromatography is used. NAP is extracted and purified at pH levels below 4.
EFFECT: simple and more efficient method of obtaining anticoagulation proteins from nematodes.
25 cl, 8 dwg, 7 tbl, 6 ex
SUBSTANCE: claimed are expression vectors for obtaining IL-21 in E.coli cells. IL-21 coding nucleotide sequence, included in composition of novel vectors, contains modifications aimed at optimisation of codons and secondary mRNA structure for translation in E.coli. As a result of transformation with claimed vector structures E.coli strains suitable for industrial scale application have been obtained. Methods of wide scale IL-21 production which use said strains have been elaborated, allowing to obtain more than 1g/l of recombinant cytokine.
EFFECT: novel compounds possess useful biological properties.
14 cl, 1 dwg, 12 tbl, 19 ex
SUBSTANCE: hybrid protein - human insulin precursor consists of N-end fragment of human gamma-interferon connected through peptide linker with amino acid sequence of human proinsulin. Recombinant human insulin is obtained by cultivation of Escherichia coli JM109/pHINS11 strain-producer, carrying plasmid pHINS11, isolation of inclusion bodies and their dissolving in buffer which contains urea and dithiotreitole. Then hybrid protein re-naturation, sedimentation of admixture compounds, purification of re-naturated hybrid protein by ion-exchanging chromatography, combined fermentative hydrolysis of hybrid protein with tripsin and carbopeptidase B are carried out. At the last stage insulin purification with cation-exchanging chromatography and method of highly efficient reverse phase liquid chromatography are carried out.
EFFECT: simplification of obtaining highly purified recombinant human insulin and increase of its output.
6 cl, 1 dwg, 4 tbl, 5 ex
SUBSTANCE: integrative plasmid vector contains selective marker for selection of transformants in cells E.coli, site of replication start, site of integration and expression cassette, which consists of promotor, transcription terminator and selective marker, and integration site is represented by sequence of DNA that codes area 18S of messenger RNA.
EFFECT: improved productivity of strain.
3 cl, 4 dwg, 10 ex
SUBSTANCE: invention represents polypeptide, having α-L-arabinofuranosidase activity selected from the following polypeptides: polypeptide with SEQ ID No. 2, polypeptide, amino-acid sequence of which is located between positions 28 and 400 SEQ ID No. 2, fragment of polypeptide with SEQ ID No. 2, having activity of α-L-arabinofuranosidase, polypeptide having activity of α-L-arabinofuranosidase B and expressing 80% identity with polypeptide SEQ ID No. 2. Invention also relates to polynucleotide, which codes this polypeptide, expression cassette and vector, containing polynucleotide, and master organism that contains this polypeptide.
EFFECT: expanded arsenal of mediums for hydrolysis of α-L-arabinofuranosyl links in arabinofuranosyl-oligosaccharide compounds.
9 cl, 6 dwg, 2 tbl, 1 ex
SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.
EFFECT: applicable in medicine for preparing a vaccine.
52 cl, 32 dwg, 7 tbl, 29 ex
SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.
EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.
9 cl, 14 dwg, 2 tbl, 13 ex
SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.
EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.
57 cl, 45 dwg, 4 tbl, 8 ex
SUBSTANCE: genetic makers of siSTRIKE-neo vector producing interfering RNA (siRNA), are inhibitors of reproduction of human immunodeficiency virus type 1. Invention allows producing effective anti-HIV preparations of siRNA produced in cells by the administered genetic makers containing palindrome intended for formation of siRNA production and selected with using non-virus and virus models.
EFFECT: invention can be used in medicine and researches.
12 cl, 3 dwg, 3 tbl, 3 ex
SUBSTANCE: invention concerns biotechnology and represents a new alpha galactosidase, a DNA molecule coding it. Besides the invention concerns an expression vector containing such DNA molecule, and also a cell transformed by the vector. The invention also concerns the method for making alpha-galaktobiose disaccharides with using new alpha galactosidase.
EFFECT: invention allows for high-efficient alpha-galaktobiose disaccharides.
15 cl, 5 dwg, 1 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.
EFFECT: invention allows improving effectiveness of cancer prevention.
28 cl, 7 dwg, 2 tbl, 1 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex
SUBSTANCE: there is offered molecule of nucleic acid inducing CEA immune response, containing a nucleotide sequence that codes a fused protein on a basis of carcinoembryonal antigen (CEA) or its functional version fused with a subunit B of thermolabile enterotoxin E coli. There are described versions thereof, as well as the related purified protein. There is disclosed an expression vector containing said molecule of nucleic acid, and a host-cell containing specified vector. There are described adenoviral vaccinal vector for inducing the immune response and a vaccinal plasmid on the basis of the specified molecule.
EFFECT: application of the invention allows to inducing the immune response in a mammal which is stronger, than that induced with natural CEA that can find application in medicine for cancer treatment.
20 cl, 62 dwg, 20 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and is a method of growing transgenic carrot plants, which produce human intereleukin-10. Recombinant plasmid DNA pBi101-IL10 is constructed, which codes synthesis of human interleukin-10, and transfers it to the Agrobacterium strain. Tylosis is obtained, which is induced from mature embryos of carrot seeds using agarised culture medium MS, which contains 0.2 mg/l 2,4-D and 0.2 mg/l kinetin. Agrobacterial transformation of tylosis with pBi101-IL10 construction is carried out, obtaining transgenic explants. The obtained explants are cultured for inducing kanamycin-resistant tylosis using culture medium MS, containing 0.2 mg/l 2,4-D, 0.2 mg/l kinetin, 100 mg/l kanamycin and 500 mg/l cefotaxime. The formed embryoids are transferred for regeneration of plants on paper bridges in test-tubes using liquid medium MS, containing 100 mg/l kanamycin, 500 mg/l cefotaxime with subsequent growing of regenerated plants in nursery conditions.
EFFECT: method allows for simple and cheap growing transgenic carrot plants, which produce human interleukin-10.
4 dwg, 3 ex