Recombinant fused human protein epo-fc with prolonged time of half-life and increased erythropoetic activity in vivo (versions), dimeric protein construction, dimeric protein, pharmaceutical composition, sequence of nucleic acid (versions), expression vector, cell, method of obtaining protein and method of erythropoesis stimulation in mammal

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

 

Related applications

This application is based on patent application U.S. No. 11/340 661, filed January 27, 2006

The technical field to which the invention relates.

This invention relates to fused protein human erythropoietin.

The level of technology

Human erythropoietin (EPO), the representative of a family of growth factors system of erythropoiesis, is synthesized mainly in the kidneys of adult and fetal liver in response to tissue hypoxia due to reduced availability of oxygen in the blood [1]. The principal function of EPO is to act directly on specific progenitors and precursors of erythrocytes (red blood cells, RBC) in the bone marrow to stimulate the synthesis of hemoglobin and Mature RBC. It also regulates the proliferation, differentiation and maturation of RBC. Was obtained recombinant EPO having the amino acid sequence of natural EPO, which was approved for use in treatment is associated with impaired renal anemia, cancer and other pathological conditions [2]. The results of recent studies [3] showed that EPO, in addition to associated with erythropoiesis properties, there are also cells from the bone marrow, such as neurons, suggesting the presence of EPO other possible physiological/pathological functions in the Central nervous system (CNS) the other organs/systems. Because EPO receptors have been found in many different organs, EPO may have many biological functions, such as acting as anti-apoptotic agent.

Human EPO is a glycoprotein with a molecular weight of 30.4 kDa. Carbohydrates account for approximately 39% of its total mass. Gene EPO is located on chromosome 7q11-22 and occupies an area with a length of 5.4 thousand pairs of nucleotides (tysp) 5 exons and 4 introns [4]. Predecessor EPO consists of 193 amino acids. Cleavage of the leader sequence and the last amino acids Arg during post-translational modification gives a Mature EPO, with 165 amino acids. Glycosylation, with three N-sites at positions Asn 24, Asn38, Asn83 and one O-site at position Ser-126, plays a crucial role in the biosynthesis, the formation of tertiary structure and biological activity in vivo EPO [5]. EPO works by binding to the erythropoietin receptor is glycosylated and phosphorylated transmembrane a polypeptide with molecular weight 72-78 kDa. This binding triggers homodimerization receptors, which leads to activation of several signal transduction pathways: system JAK2/STAT5, G protein, calcium channel and kinase. In order to achieve optimal activation of the receptor requires the simultaneous binding of two molecules of the protein EPO with one molecule of the receptor [6].

As per the initial growth factor system hematopoesis, allowed to treat people, recombinant human EPO (rHuEPO) has been used to treat anemia resulting from chronic renal failure, cancer (primary anemia associated with chemotherapy, autoimmune diseases, AIDS, surgical complications, bone marrow transplantation, myelodysplastic syndromes, etc. Interestingly, recent studies have also shown that rHuEPO has functions that are not related to the blood system, and has potential for use as neuroprotective drugs for the treatment of cerebral ischemia, brain injury, inflammatory diseases and neuro-degenerative disorders [7].

Currently on sale are three types of rHuEPO or analogues rHuEPO, namely rHuEPO-α, gnaro-β and darbepoietin-α([8]. These three recombinant protein bound to the same receptor, erythropoietin, but differ in structure, degree of glycosylation, the affinity in binding to the receptor and metabolism in vivo. Co time start using rHuEPO-α in the 1980s, clinicians quickly found that a significant drawback of medicines is the need for frequent injections dose. Average times the half-life in vivo rHuEPO-α and rHuEPO-β by intravenous or subcutaneous injection, respectively only 8.5 and 17 h [9, 10]. Therefore, patients need to inject the hedgehog the day, twice a week or three times a week, which creates difficulties for both patients and health workers. Therefore, there is a long-standing need in the development of recombinant EPO analogues with longer half-life in vivo and/or increased activity in erythropoiesis.

In previous practice, attempts have been made genetically altered or chemically modify the structure of a native protein EPO to either slow down its metabolism in vivo, or to improve its therapeutic properties. For example, it turns out that there is a direct correlation between the number in the molecule EPO carbohydrates containing sialic acid and its metabolism and functional activity in vivo. Therefore, the increase in the content of carbohydrates in the molecule of EPO leads to prolonged half-life and increased activity in vivo [11, 12]. The firm Amgen constructed similar rHuEPO darbepoietin-α to include in rHuEPO in position 5N two additional carbohydrate chain.

Darbepoietin-α is also known as the "New protein that stimulates erythropoiesis" (Erythropoiesis Stimulating Protein, NESP), and sold under the trade name AranespTM. Darbepoietin-α differs from native human EPO in five positions of amino acids (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, Pro90Thr)that allows you to attach two additional N-attached oligosaccharide to an asparagine residues in put the s 30 and 88. Darbepoietin-α binds to the EPO receptor is identical to native EPO manner inducyruya intracellular signal circuit involving phosphorylation of the tyrosine kinase JAK-2 and the same intracellular molecules Ras/MAP-k, P13-K and STAT-5. Because of high content of carbohydrates, the half-life of darbepoetin-α in human and animals is almost three times longer than that of rHuEPO-α (25,3 h compared with 8.5 h) [9]. It turns out that darbepoietin-α (AranespaTM) has increased biological activity compared to natural or recombinant human EPO in vivo [13] and was approved for use by the American pharmacological Committee (FDA) as a drug rHuEPO the second generation. This medication must be entered only once a week in order to get the same therapeutic effect as with injections 2-3 times a week rHuEPO[10, 14, 15].

Made other attempts to extend the half-life of EPO focused on increasing the molecular weight of the protein of EPO by chemical conjugation with polyethylene glycol ("Pegylation") and similar methods. Targeted EPO has a much higher molecular weight, are protected from removal from the bloodstream and therefore has a longer half-life in plasma [16]. However, Pegylation can alter the structure of the protein, which can lead to undesirable changes in the function and specificity of the part of Romolecules. There is also information about the increase in molecular weight of EPO by other methods such as a binding molecule of EPO protein carrier (human albumin), or by homodimerization two EPO molecules with peptide bridges (length 3-17 amino acids) or chemical cross-linking reagents [17,18, 19, 20]. Although all of these methods have led to some success in the extension of time half-life and increase the activity of EPO, the combination of a molecule of EPO with the Fc fragment of human immunoglobulin (IgG) in fused protein, as described in this application provides unique advantages.

Human immunoglobulin IgG consists of four polypeptides (two identical copies of the light chain and heavy chains linked covalently by disulfide bonds. Proteolysis of IgG molecules with papain yields two fragments Fab and one Fc fragment. The Fc fragment is composed of two polypeptides that are connected by disulfide bonds. Each polypeptide from the N-Terminus to the C-end is composed of the hinge region, CH2 domain and domain snz. The structure of the Fc fragment were almost the same among all subtypes of human immunoglobulin. IgG is the most prevalent protein in human blood and ranges from 70 to 75% of all immunoglobulins in human serum. The half-life of IgG in the blood stream the highest among all five types of immunoglobulin and can gain the AMB 21 days.

Modern bioengineering technology has been applied with success to create a fused protein consisting of fragments of therapeutic proteins, such as cytokines and soluble receptors, and Fc fragment of human IgG[21, 22, 23, 24]. These fused proteins have significantly longer half-life in vivo and retain their biological and therapeutic properties. Therefore containing the Fc fragment fused proteins were successfully developed as a biological medicinal substances and FDA-approved for treatment of rheumatoid arthritis and chronic plaque psoriasis [25, 26].

Previous studies have shown that the dimer of two molecules of EPO, United or chemical curing, or polypeptide, have increased activity in vivo and prolonged half-life [17, 19]. Increased activity may be due to more efficient binding of EPO dimer with a single receptor, and prolonged half-life in vivo may be due to the large size of the protein dimer. However, the process of chemical crosslinking is not very effective and hard to control. In addition, the peptide bridge in the dimer EPO can modify the three-dimensional structure of a molecule of EPO, and the peptide can stimulate immunogenic response in vivo. These shortcomings reduce therapeutic potential of EPO dimers, especially for patients with a disorder of the kidneys, to the which substitution therapy EPO lasts a lifetime.

So there is a need in the EPO analogues with longer half-life and increased eritropoeticescoe activity in vivo, but not with enhanced immunogenic properties.

The invention

In accordance with the invention describes a recombinant protein containing eritropoetinovmi peptide part of the peptide of human erythropoietin attached to the immunoglobulin peptide part. Protein has a prolonged half-life in vivo in comparison with natural or recombinant native human eritropoetina. In one variant of the invention, the protein has a half-life in vivo, at least 3 times the half-life of native human erythropoietin. Protein may also have increased eritropoeticescoe biological activity compared to native human erythropoietin.

In one variant of the invention, the immunoglobulin peptide portion is an Fc fragment, such as a fragment of lgG1. The Fc fragment contains domains CH2 and CH3, and hinge region. EPO peptide portion can be directly attached to the hinge region. Preferably, the hinge region has a length of at least 9 amino acids. In one of the embodiments of the invention EPO peptide portion has OST the current cysteine, closest to it is the end, and the hinge region includes a cysteine residue located closest to the EPO peptide. Preferably these two cysteine residue separated by at least 12 amino acids. In one of the embodiments of the invention EPO peptide portion can represent a complete molecule of EPO, directly attached to the immunoglobulin of part (that is, between EPO and immunoglobulin parts no alien peptide bridges).

The invention relates also to a multimeric protein structures containing multiple units fused protein according to the present invention.

For example, two fused protein can be mounted in the form of a dimer, with the hinge region of proteins connected by disulfide bonds. The dimer has the General shape of the IgG molecule and more stable than free molecules of EPO.

The invention relates also to a nucleic acid that encodes a protein, corresponding to amino acid sequence and to the lines transfected cells and methods of producing fused protein. Further, the invention includes pharmaceutical compositions containing the protein, and methods of using fused protein and/or pharmaceutical compositions, for example, to stimulate erythropoiesis in need of treatment of individuals.

Briefly the description of figures

It is understood that the figures illustrating various embodiments of the invention should not be construed in a limiting sense.

Figa is a schematic diagram showing the overall structure of recombinant human fused protein EPO-Fc (rHuEPO-Fc) according to the present invention.

Figb - the list of sequences, showing the nucleotide sequence and deduced from it the amino acid sequence of the protein rHuEPO-Fc. Full length DNA equal 1281 digested derived protein sequence 426 amino acids (AA) include 27 AK for the signal peptide and 399 AK for complete protein rHuEPO-Fc. Complete protein rHuEPO-Fc consists of the domain of the human EPO (166 AA), hinge region (16 AK, underlined) and domains CH2 and CH3 (217 AA), Fc fragment of human lgG1. The calculated molecular weight of the Mature polypeptide fused protein rHuEPO-Fc equal to 44.6 kDa, which part of 18.5 kDa (41,4%) refers to the fragment of EPO and 26.1 kDa (58,6%) is a piece of lgG1 Fc. Glycosilated formed by two disulfide bonds between two cysteine residues (boxed) in the hinge region. In position 172 Mature fused protein (i.e. in the 6th amino acid hinge region) native cysteine residue was replaced by glycine (in bold).

Figure 2 - schematic diagram showing the structure and features Express the dominant plasmids mammalian pCD1, used for introducing a DNA sequence that encodes a polypeptide fused protein rHuEPO-Fc, and for transfection of cells SNO expressing the protein rHuEPO-Fc.

Figure 3 - image areas after electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE)showing the installed analysis in SDS-PAGE sizes dimeric form of pure protein rHuEPO-Fc in the absence of recovery and Monomeric forms of pure protein rHuEPO-Fc during recovery. Purified protein rHuEPO-Fc from the supernatant fluids of cultivated cell line Cho expressing rHuEPO-FC, exists mainly in the form of a dimeric form and has a molecular weight of approximately 180 kDa in 8% gel buffer bis-Tris without recovery. When restoring (100 mm dithiothreitol, DTT)disrupting disulfide bond, a dimer splits into 2 identical Monomeric units with a molecular weight of 75 kDa.

Figures 4A and 4B - graphs showing dose-dependent increase of hemoglobin (Hb) in normal mice treated 3 times per week subcutaneous (s/C) injection of rHuEPO-Fc or rHuEPO. Each point represents the average Hb level in the group (6 mice). Levels at day 0 represent the levels of Hb before impact. A: mice received rHuEPO-Fc. B: mice received native rHuEPO.

Figures 5A and 5B - graphs showing dose-dependent increase in levels of hemoglobi is on (Hb) in normal mice received 1 week p/C rHuEPO-Fc or rHuEPO. Each point represents the average Hb level in the group (6 mice). Levels at day 0 represent the levels of Hb before impact. A: mice received rHuEPO-Fc. B: mice received native rHuEPO.

Figures 6A and 6B - graphs showing increased levels of hemoglobin (Hb) in normal mice treated with intravenous (IV) injection of 12.5 mcg/kg of rHuEPO-Fc or rHuEPO. Each point represents the average Hb level in the group (6 mice). Levels at day 0 represent the levels of Hb before impact. A: mice received an injection of 1 time per week. B: mice received injections 3 times a week.

Fig.7 is a graph showing the dose-dependent increase of hemoglobin (Hb) in rats based 5/6 receiving p/1 weekly rHuEPO-Fc, rHuEPO or darbepoetin-α (designation Darbe). Each point represents the average Hb level in the group. Normal control presents normal rats with injection mortar carrier. Model control is rat with based 5/6 treated with injections of a solution of the carrier. Levels at week 0 represent the Hb levels before stimulation. With * marked weeks after exposure.

Fig is a graph showing the dose-dependent increase of hemoglobin (Hb) in rats based 5/6 receiving p/1 time per 2 weeks of rHuEPO-Fc, rHuEPO or darbepoetin-α (designation Darbe)Each point represents the average Hb level in the group. Normal control presents normal rats with injection mortar carrier. Model control is rat with based 5/6 treated with injections of a solution of the carrier. Levels at week 0 represent the Hb levels before stimulation. With * marked weeks after exposure.

Fig.9 is a graph showing the dose-dependent increase of hemoglobin (Hb) in rats based 5/6 receiving/1 time per 2 weeks at 62.5 μg/kg of rHuEPO-Fc, rHuEPO or darbepoetin-α (designation Darbe). Each point represents the average Hb level in the group. Normal control presents normal rats with injection mortar carrier. Model control is rat with based 5/6 treated with injections of a solution of the carrier. Levels at week 0 represent the Hb levels before stimulation. With * marked weeks after exposure.

Figures 10A through 10B show a comparison of the activity of rHuEPO-Fc, rHuEPO and darbepoetin-α to stimulate the formation of colonies of CFU-E and BFU-E in rats based 5/6, which was administered different doses in different modes. The introduction of rHuEPO-Fc and darbepoetin-α (designation Darbe) showed the same dose-dependent activity in the formation of colonies of CFU-E and BFU-E and rHuEPO was less active. A: p/introduction 1 once a week. B: p/C introduction 1 every 2 weeks. In: in/introduction 1 every 2 weeks.

11 is a graph showing the th levels in serum rHuEPO-Fc and rHuEPO after intravenous injection of rhesus monkeys 5 μg/kg of rHuEPO-Fc or rHuEPO (average in 5 monkeys).

Fig - the list of sequences, showing the nucleotide sequence and deduced amino acid sequence of the protein rHuEPO-FcC wild type. Details of the sequences are the same as shown in figure 1, except that at position 172 Mature fused protein (i.e. in position 6 amino acids of the hinge region) contains the cysteine residue corresponding native protein wild-type.

Fig is a graph showing the dose-dependent increase of hemoglobin (Hb) in normal mice treated 3 times per week p/C injection of rHuEPO-Fc mutant protein according to the present invention), rHuEPO - FcC (protein wild-type) and rHuEPO. Each point represents the average Hb level in the group (8 mice). Normal control presents normal mice, which were injected solution media. Day 0 represents the Hb levels before exposure.

Fig is a graph showing the dose-dependent increase of hemoglobin (Hb) in normal mice exposed to 1 time per week s/C injection of rHuEPO-Fc, rHuEPO-FcC and rHuEPO. Each point represents the average Hb level in the group (8 mice). Normal control presents normal mice, which were injected solution media. Day 0 represents the Hb levels before exposure.

A detailed description of the invention

Throughout the following description for best pone the mania of the invention provides specific details. However, the invention may be practiced without these details. In other instances, well known elements have not been shown or described in detail to avoid accumulation of parts, which will be useful to understand the present invention. Therefore, the description and the figures should be considered illustrative, and not in a limiting sense.

This invention relates to a new fused protein having eritropoeticescoe properties. Protein, designated here as rHuEPO-Fc, contains a molecule of the human erythropoietin (EPO) using recombinant technology connected to the Fc fragment of immunoglobulin. As discussed later, the protein may be in the form of a dimer consisting of two identical polypeptide subunits. In the embodiment of the invention, schematically presented in figa, each polypeptide subunit from N-Terminus to the C-end consists of a polypeptide sequence of human EPO molecule and polypeptide sequence of the hinge region, CH2 domain and CH3 domain of the Fc fragment of human immunoglobulin lgG1. Two polypeptide subunit for the formation of dimeric structures are connected to each other by disulfide bonds between the respective hinge regions. Therefore, the dimer has the same General shape as the IgG molecule and has better stability than the free m is likely EPO, as discussed in the examples below.

As will be obvious to the person skilled in the art, the hinge area of intact immunoglobulin squirrel gives sufficient flexibility for efficient binding of the antigen-antibody. Similarly, in the present invention the hinge area is included in the design fused protein rHuEPO-Fc, in order to preserve its flexibility, especially when the protein is dimeric form. As described below, this provides the normal binding of the EPO fused protein rHuEPO-Fc with EPO receptors to activate the biological functions of EPO. It is safe to assume that the dimeric form of the fused protein rHuEPO-FC, providing two molecules of EPO, is able to induce optimal activation of the EPO receptor (for example, promoting the cross-linking of receptors).

As shown in the following examples, protein rHuEPO-Fc was successfully synthesized using techniques of recombinant DNA. In studies in mice, rats and primates, it was found that protein has a prolonged half-life in vivo and increased eritropoeticescoe activity compared to natural or recombinant native human EPO. The terms "native human erythropoietin" and "native human EPO"as used in this patent application, refer to the EPO with unmodified page is the established levels of wild-type. As is clear to a person skilled in the art, native human EPO may be natural or obtained by recombinant techniques (for example rHuEPOα). The term "native human EPO does not include analogs of rHuEPO, such as darbepoietin-α, where the structure of EPO was significantly modified, as for example by hyperglobulinaemia.

The sequence of the nucleic acid fused protein rHuEPO-Fc according to the present invention is presented as SEQ ID No. 1. The corresponding deduced amino acid sequence presented in SEQ ID No. 2. Full length slit protein rHuEPO-Fc is a 399 amino acids. As shown in figb full of protein rHuEPO-Fc consists of domain EPO (166 amino acids), hinge region (16 amino acids underlined) and domains CH2 and CH3 (217 amino acids). Signal and leader peptide sequence consisting of 27 amino acids, also shown in figb. The signal peptide is cleaved during synthesis of rHuEPO-Fc. The sequence of the nucleic acid and amino acid sequence of rHuEPO-Fc, including the signal or leader peptide, shown respectively in SEQ ID No. 3 and SEQ ID No. 4.

As can be seen best on fehb and in the sequence SEQ ID No. 2, the domain of the EPO contains a cysteine residue near its C-end at position 161 amino acid sequence. The hinge area includes 2 residue C is steina at positions 178 and 181 amino acid sequence, which figb boxed. The remains of the cysteine of the hinge region to form a disulfide bond between the polypeptide subunits of glycosilated, as discussed above. Natural hinge region fragment of a human lgG1 also has a cysteine residue in position 6 of the part of the hinge region (counted from the N-Terminus). In the present invention, the remainder of the 6 cysteine in the hinge region was replaced nicotinoyl balance. In particular, in the embodiment of the invention, presented at figb and in the sequence SEQ ID No. 2, the amino acid cysteine was replaced by glycine (position 172 amino acid sequence of rHuEPO-Fc, which corresponds to residue 6 of the hinge region). As will be obvious to the person skilled in the art, a cysteine at this position can also be replaced by other nicotianamine residues, in order to prevent formation of disulfide bonds.

In the result of amino acid substitutions at position 172 of the first cysteine residue of the hinge region (at position 178) separated by 17 amino acids from the above-described residue cysteine domain of EPO (at position 161). Applicants believe that the minimum distance between the cysteine residue 161 domain EPO and the first cysteine residue of the hinge region should be at least 12 amino acids, in order to ensure the successful Assembly and/or linking Rotz the torus EPO of glycosilated rHuEPO-Fc. To is, if the residue at position 172 is a cysteine residue, it is possible the formation of unwanted disulfide bonds, such as between cysteine residues 161 and 172. This may alter the three-dimensional structure of a molecule of EPO, which will lead to the disappearance or reduction of biological activity.

In one of the embodiments of the present invention domain EPO attached directly to the part corresponding to the Fc fragment fused protein. The exception alien peptide-bridge maintains the preferred three-dimensional structure of the fused peptide rHuEPO-Fc and minimizes unwanted immunogenic reactions. The hinge region of Fc fragment preferably has a length of at least 9 amino acids, and preferably its length is approximately 10-20 amino acids.

The following examples will further illustrate the invention in more detail, although it should be recognized that the invention is not limited to the specific examples.

Examples

Example 1. Designing recombinants plasmids pCdEpo-Fc. encodes a protein HuEPO-Fc

The DNA molecule full length, which encodes the amino acid sequence of the polypeptide rHuEPO-Fc, was created by overlapping polymerase chain reaction (PCR) using the following oligonucleotide primers (QIAGEN Inc., US):

Sequences of the above primers are presented, respectively, in the sequences with SEQ ID No. 5 through SEQ ID No. 8.

The restriction sites EcoR I and Not I introduced respectively in EF5 and EF3.

For optimal expression of the protein HuEPO-Fc in mammalian cells in EF5 also introduced a Kozak sequence (GCCACCATGG). EFL5 and EFL3 is complementary to the sequence comprising the 3'end DNA sequence of EPO (22 BP) and 5'-terminal sequence of the DNA for lgG1 hinge (22 BP).

First, the DNA fragment of EPO 0.6 tysonite amplified in PCR (Platinum Taq DNA Polymerase High Fidelity) with primers EF5 and EFL3 of plasmids re containing the full length cDNA for human EPO, and the Fc fragment size of 0.7 tysonite amplified with primers EF3 and EFL5 with plasmid pD containing the cDNA sequence of full length human lgG1 (re and pD were obtained in the laboratory applicants). Then both fragments were purified and mixed in equal quantities. Using the mixture as the template, DNA rHuEPO-Fc full length size 1.3 tysonite amplified with primers EF5 and EF3. The purified fragment length 1,3 tysonite were digested with restrictase EcoR I and Not I (New England Biolab Inc. US) and then cloned in split EcoR I/Not I expressing vector mammals pCDI (figure 2). The obtained recombinant vector was named pCdEpo-Fc entered into it, later etelnost nucleic acid, encoding the amino acid sequence of HuEPO-Fc was confirmed by DNA sequencing.

Example 2. Obtaining a cell line expressing the rHuEPO-Fc

As host cells for the expression of rHuEPO-Fc used the ovarian cells of the Chinese hamster defective in dihydrofolate-reductase (dhfr) (CHO/dhfr-, ATCC No. CRL-9096), which is FDA-approved for the production of biological substances.

Cells CHO/dhfr-was transfusional recombinant vector pCdEpo-Fc using lipofectamine (Gibco, Cat. No: 18292-037, USA). The supernatant fluid from cultures of selected clones were analyzed by ELISA method (Roche, Cat. No: 1-693417, Canada) for determining the activity of EPO. Next, the positive clones were subjected to screening with increasing load methotrexate (MTX). One cell line with the highest expression of protein rHuEPO-Fc was selected as expressing the rHuEPO-Fc cell line Cho and gradually adapted to the environment without serum (CD Cho Medium, Gibco, Cat. No: 10743-029, USA). This expressing rHuEPO-Fc cell line Cho was used for the production of protein rHuEPO-Fc.

Example 3. The protein purification rHuEPO-Fc

Protein molecules rHuEPO-Fc contained in the supernatant fluids collected after cultivation expressing rHuEPO-Fc cells SNO in medium without serum, were selected initially using affinity chromatography on protein A (Amersham, Cat. No: 17-0402 - 01, Canada). Selected proteins were further is entrusted purified by gel-chromatography on a column (HiLoad 16/60 Superdex 200pg (Amersham, Cat. No: 17-1069-01, Canada). The purity of the protein rHuEPO-Fc was more than 98% based on electrophoresis.

Example 4. Determination of the amount of pure protein rHuEPO-Fc

First, to determine the amount of pure protein rHuEPO-Fc was performed SDS-PAGE. As shown in figure 3, during electrophoresis in an 8% gel with bis-Tris buffer without recovery, which allows us to measure the total amount of protein with disulfide bonds received one zone with a molecular weight of approximately 180 kDa. This indicates that the main part of the protein molecules rHuEPO-Fc produced in dimeric form, as expected on the basis of the design fused protein. If the analysis SDS-PAGE was performed in reducing conditions (100 mm DTT) for the destruction of disulfide bonds, was identified only one line with a molecular weight of 75 kDa, which is consistent with the evaluation of the molecular weight for a single polypeptide chain composition of HuEPO-hinge region-CH2-CH3.

The exact molecular weight of pure fused protein rHuEPO-Fc with glycosylation defined mass spectrometry (MALDI-TOF-MS), was 111099 Yes (111,1 kDa). In this analysis, we observed only one protein peak, which indicates that close to 100% purity of the protein rHuEPO-Fc. Sequence analysis of the protein allowed to determine the 15 amino acid N-Terminus of pure protein rHuEPO-Fc as APPRLICDSRVLERY. This is consistent with the sequence of the first 15 amino acids of the polypeptide native to the human ER and confirms, what a purified protein rHuEPO-Fc really is correct and complete sequence of the EPO molecule, as predicted by the DNA sequence that encodes the amino acid sequence of the fused protein rHuEPO-Fc.

Example 5. Increased eritropoeticescoe activity rHuEPO-Fc in normal mice

To confirm the saving eritropoeticescoe activity of the protein rHuEPO-Fc and determination of its activity compared with rHuEPO and darbepoietin-α were conducted in vivo studies in mice. In order to compare all three doses used in the described experiments on animals EPO: rHuEPO-Fc in accordance with the present invention, rHuEPO (native human EPO) and darbepoetin-α were expressed by the quantities of the molecules of EPO on the basis of the size of the molecule. For protein rHuEPO-Fc part of the EPO is 41,4% of the total molecular weight of rHuEPO-Fc, which is calculated based on the weight of amino acids of EPO and weight of all amino acids in a molecule of rHuEPO-Fc (160 AK from a total of 399 AK). Therefore, the number of EPO in rHuEPO-Fc was considered equal to 41.4% of the total number of protein rHuEPO-Fc.

rHuEPO-Fc (the concentration of the working solution of 0.5 mg/ml, purity 98.6 per cent) and native human rHuEPO (i.e. with the structure of natural human EPO) (6000 units/0.5 ml, the production company Kirin Brewery Co., Japan) was diluted in media solution (2.5 mg/ml human serum albumin, and 5.8 mg/ml sodium citrate, 0.06 mg/ml lemon is Oh acid and 5.8 mg/ml sodium chloride, pH 5.5 and 5.6). Quantitative dose of rHuEPO was calculated according to the ratio of the activity/number. Mouse BALB/c mice (age 6-8 weeks, weight 18-22 g, equal number of males and females obtained from the Experiment Animal Center, AMMS, China) were randomly divided into groups of 6 mice in each group. Each group of mice received a single combination of one dose(0,1, 0,5, 2,5, 12,5, 62,5 µg/kg), one route of administration (in/in the tail vein or p/C) and one mode of injection (3 times a week or 1 time per week). The control group of mice were administered an equal volume of a solution of the carrier. The effect lasted for 3 weeks, and the total observation time was 5 weeks. Samples of peripheral blood from the tail vein for measurement were taken before exposure, on the 4th and 7th day of each week for 5 weeks. The number of Hb was measured indicator of absorbtiometry. The average standard deviation was calculated for each data group, for comparison of different groups used the r-test.

The introduction of EPO mice 3 times per week, provided that these EPO have normal eritropoeticescoe activity, will be with saturation to induce stimulation of erythropoiesis. As shown in figure 4, in both groups exposed to 3 times a week, we saw significant increase in levels even at the dose of 2.5 µg/kg This experience has shown that erythropoetic the Kai activity rHuEPO-Fc in vivo as high as with rHuEPO. The increase of Hb levels in exposed groups was dose dependent. However, the saturation in the increase of Hb levels was achieved in mice at the dose of rHuEPO-Fc 12.5 µg/kg, whereas the same intense increase of Hb levels with the introduction of rHuEPO was achieved only at a dose of 62.5 mg/kg Induced by rHuEPO-Fc in a dose of 2.5 mg/kg increased levels of Hb was also higher than the increase in the induction of rHuEPO at a dose of 2.5 mg/kg, These results suggest a stronger stimulation of erythropoiesis under the action of rHuEPO-Fc, than the effect of rHuEPO.

Eritropoeticescoe potential rHuEPO-Fc was then used to reduce the number of s/C injection up to 1 week. As shown in figure 5, in receiving rHuEPO-Fc groups were observed in a dose-dependent increase of Hb levels at doses of 12.5 or 62.5 μg/kg of Both doses of 12.5 and 62.5 µg/kg) rHuEPO also induced increased levels of Hb to close values, which was much lower than the levels induced at 62.5 μg/kg of rHuEPO-Fc. It undoubtedly indicates that rHuEPO-Fc has increased eritropoeticescoe activity in vivo. Perhaps this is a consequence of any extended time half-life of rHuEPO-Fc in vivo or enhance binding/activation of the EPO receptor by dimers of molecules of EPO protein rHuEPO-Fc, or the combined effects of both factors.

If the same dose (12.5 µg/kg) of rHuEPO-Fc or rHuEPO was administered intravenously or 3 times a week or 1 time a week,in all exposed groups was observed increased levels of Hb (6). However in/in the introduction of rHuEPO-Fc 1 week induced a greater and more persistent increase of Hb levels, which lasted longer after exposure. These data also support the view of higher eritropoeticescoe activity of the protein rHuEPO-Fc compared with rHuEPO, having the structure of natural EPO protein.

Example 6. Increased eritropoeticescoe activity rHuEPO-Fc in rats with 5/6-nephrectomy

Experiments in normal mice confirmed the increased eritropoeticescoe activity rHuEPO-Fc in vivo. To further verify the effectiveness of rHuEPO-Fc stimulation of erythropoiesis, studies have been conducted pharmacodynamics in rats with experimental renal anemia, obtained by 5/6 nephrectomy. The effectiveness of rHuEPO-Fc was compared with the efficacy of rHuEPO and darbepoetin-α (60 μg/ml, batch No. N079, produced by Kirin Brewery Co., Japan).

In this invention to obtain a model of anemia due to disorders of the kidney by a two-stage nephrectomy [27] used Wistar rats (equal number of males and females, weight 160-180 g, obtained from Vitalriver Experiment Animal Inc., Beijing, China. Licence No. SCXKI 1-00-0008). In rats under General anesthesia was performed 5/6 nephrectomy in two separate operations under sterile conditions. After resection of 2/3 of the left kidney of the rats were left to recover for 20 days. Then gently removed the right kidney. To prevent the Deposit of infection after each operation administered antibiotics. Were eventually removed 5/6 renal tissue. In rats after nephrectomy gradually evolved failure of kidney function and anemia. Stable state of anemia in rats was achieved after 50 days after nephrectomy, after which they were randomly split by group (9 rats per group) for injection of EPO. Each group of rats received a single combination of one dose (of 2.5 to 12.5, 62,5 µg/kg), one route of administration (in/in through the tail vein or p/C) and one mode of injections (1 per week or 1 every 2 weeks). The control group and model group rats were administered an equal volume of a solution of the carrier. The exposure was continued for 4 weeks, and the total observation time was 6 weeks.

All doses of 2.5 to 12.5, 62,5 µg/kg) of rHuEPO-Fc, injected subcutaneously 1 week, induced a dose-dependent increase of Hb levels compared with model control group, where there was no introduction of EPO. Doses of 12.5 and 62.5 μg/kg of rHuEPO or darbepoetin, injected subcutaneously 1 week, also induced increased levels of Hb. Elevated levels of Hb in both groups treated with 12.5 or 62.5 μg/kg of rHuEPO-Fc, were significantly higher than those in the groups treated with 12.5 or 62.5 μg/kg of rHuEPO. The Hb levels in the group treated at 62.5 μg/kg of rHuEPO-Fc, were also slightly higher than the levels in the group treated at 62.5 μg/kg darbepoetin. After termination of exposure reduction level is th Hb group, receiving at 62.5 μg/kg of rHuEPO-Fc, was much slower, and Hb levels remained higher until the end of follow-up (2 weeks after termination of exposure)than the levels in the groups of normal control and model control. This indicated more severe and/or prolonged stimulation of erythropoiesis (data summarized on Fig.7).

For the effects of subcutaneous injections of 1 times in 2 weeks have introduced 3 types of EPO only in doses of 12.5 or 62.5 μg/kg (Fig). The dose of 12.5 mcg/kg of rHuEPO weakly increased Hb levels compared with model control group and in the group treated at 62.5 μg/kg of rHuEPO, weak eritropoeticescoe reaction is not allowed to increase Hb levels to normal values in comparison with normal control group. Effects of rHuEPO-Fc or darbepoietin at doses of 12.5 or 62.5 μg/kg induced a significant increase of Hb levels that were higher than levels in normal control group. This served as proof of the effective correction anemic status as rHuEPO-Fc and darbepoietin. Between identical doses of rHuEPO-Fc and darbepoetin there were no significant differences in terms of effectiveness. High dose of 62.5 mg/kg led to a steady increase erythropoiesis until the end of follow-up (2 weeks after exposure). This supported the assumption that rHuEPO-Fc and darbepoietin possess the property a long time with emulerat erythropoiesis in vivo, which in turn can be used to reduce the frequency of injections of medications to patients in the clinic.

While darbepoietin was approved for clinical use with less frequent injections for better adherence of patients to treatment and reduce the workload for medical staff, the experimental data presented here strongly indicate that disclosed in the present invention rHuEPO-Fc has at least the same potential benefits. As discussed above, darbepoietin, as mutant similar to the human EPO molecule, containing more sugar components (increased glycosylation)may bring an increased risk of induction of immunogenesis in vivo due to the modified three-dimensional structure. Only long-term observation of patients treated with darbepoietin, can give a decisive opinion on immunogenic risks darbepoetin. In contrast, rHuEPO-Fc, without being modified with a molecule of EPO, is a carbohydrate that is identical or very close to the content in native human EPO. The number of sialic acids in the proposed applicants pure protein rHuEPO-Fc was about 10.0 mmol to 1 mmol of EPO, which is consistent with the published parameters for rHuEPO. The Fc part of the molecule rHuEPO-Fc does not have alien amino acids and/or Chu is arodnogo peptide bridge and is the main structure of the human lgG1. Theoretically, this should not lead to immunological reactions. rHuEPO-Fc, if it is approved for clinical use, may be more beneficial to patients than the commonly used rHuEPO and EPO analogues, especially for patients requiring long-term administration of drugs.

For intravenous injection of 1 times in 2 weeks and rHuEPO-Fc and darbepoietin (62,5 µg/kg) were able to induce the same increase of Hb levels in rats with renal anemia is much higher than normal levels of Hb in rats in the normal control (figure 9). This is further demonstrated persistent stimulation of erythropoiesis rHuEPO-Fc, since the efficiency darbepoetin has been proven in clinical trials.

Data obtained in experiments on the cultivation of bone marrow cells taken from rats with 5/6 nephrectomy after exposure (p/or/1 time a week or 1 every 2 weeks), showed that rHuEPO-Fc, rHuEPO and darbepoietin all stimulated the formation of CFU-E and BFU-E. Activity rHuEPO-Fc and darbepoetin were similar and superior to the activity of rHuEPO (figure 10).

The levels of urea nitrogen (BUN) and creatinine in the blood were similar in the exposed group and in the group model control. In all exposed groups, the level of iron (Fe) in serum was higher than in the model control. Pathological examination showed the sun is x receiving EPO rats increased content related to red blood cells (RBC) cells in bone marrow and in the spleen.

Example 7. Pharmacokinetic studies of rHuEPO-Fc in rhesus monkeys

As discussed above, applicants have designed rHuEPO-Fc in such a way that the part of the EPO fused protein retains the functional properties of natural EPO, such as the ability to stimulate erythropoiesis, and the Fc fragment of human lgG1 provides a stable existence of a fused protein in the bloodstream, which prolongs its half-life in vivo. The above animal studies have shown that eritropoeticescoe activity rHuEPO-Fc higher than that of rHuEPO. Applicants also conducted pharmacokinetic studies for comparison of time half-life of rHuEPO-Fc and rHuEPO in vivo. To obtain the data used primates, because they are very similar to people.

The research design was based on literature data and the experiments carried out according to the General rules pharmacokinetic studies. Two groups of rhesus monkeys with 5 monkeys in each group (weight 3-5 kg, obtained from the Experiment Animal Center, AMMS, China) was inserted in the/with the injection of 5 μg/kg of rHuEPO-Fc or rHuEPO. Blood samples were taken before injection and after 0,017, 0,167, 0,5, 1, 2, 4, 8, 12, 24, 48, 96, 168, 240 h after injection. Serum was collected by centrifugation and determined the levels of serum rHuEPO-Fc or rHuEPO use kits for enzyme-linked immunoassay (ELISA) for human erythropoietin (obtained from R&D Systems Minneapolis, MN). The average half-life (t1/2) rHuEPO-Fc and after rHuEPO in/injection respectively 35,24±5,15 h and 8,72±1,69 h (data summarized on 11).

To determine the bioavailability of rHuEPO-Fc, 5 μg/kg of rHuEPO-Fc was administered by injection p/5 rhesus monkeys. Blood samples were taken before injection and after 1, 2, 5, 8, 10, 12, 15, 24, 48, 72, 96, 168, 240 h after injection. Levels of rHuEPO-Fc in serum were determined using kits R&D. the Calculated value of the index of biological availability was equal 35,71±5.37 percent when s/C injection. This value is identical to the published data on the biological availability darbepoetin (AranespTMin patients with chronic renal failure [9, 15].

These data indicate that rHuEPO-Fc has significantly prolonged the half-life of primates, and the half-life of rHuEPO-Fc in vivo at least 4 times the half-life of rHuEPO, manufactured by Kirin Beer Brewing Co., Japan. Extended half-life in vivo likely contributes to increased eritropoeticescoe activity rHuEPO-Fc.

Example 8. The immunogenicity of rHuEPO-Fc in Masasa fascicularis

As indicated above, attention to the design of fused protein rHuEPO-Fc was intentionally caused by the need to eliminate or minimize changes immunogenic properties of the fused protein rHuEPO-Fc. Applicants have avoided the inclusion/addition of any alien amino acids(any alien amino acids) or compounds of a bridge peptide sequences fused protein. The claimed protein HuEPO-Fc in the embodiment of the invention on figb contains only the polypeptide sequence of the natural protein EPO and Fc fragment (hinge region, CH2, CH3) of human lgG1 and theoretically should not induce an immunogenic response and production of antibodies to the protein of rHuEPO-Fc. As will be recognized by an experienced specialist in this field, other embodiments of having alternative structures are also covered by the present invention.

To determine the immunogenicity of the protein rHuEPO-Fc following studies were conducted on primates. Ten-eating crabs makaka (Masasa fascicularis, 5 males and 5 females, aged about 5 years, the average weight of males of 4.0±0.3 kg, females of 2.9±0.4 kg, obtained from the Laboratory Animal Center, AMMS, China), was injected s/C injection of 5 mg/kg of purified rHuEPO-Fc 3 times a week for 4 weeks, two monkeys were administered an equal volume of solution media (control). Serum was collected 1 day a week for 5 weeks (starting 1 week after exposure) and examined for the presence of specific antibodies against rHuEPO-Fc by ELISA method, using as the coating antigen purified rHuEPO-Fc (5 μg/ml). In addition, during the period of experience in peripheral blood was determined by the number of RBC and Hb levels. The obtained data show that despite the stimulated amplification of erythropoiesis in patients received rHuEPO-Fc macaques (the Central number of RBC increased from 4.74×10 9/ml up to 6.67×109/ml, and mean levels of Hb with 12.2 g/DL to 13.7 g/DL), rHuEPO-Fc did not induce appreciable quantities of specific antibodies to the fused protein. These results indicate that protein rHuEPO-Fc does not cause immunogenicity in primates.

Example 9. The study of the acute toxicity of rHuEPO-Fc in normal mice

To evaluate the safety fused protein rHuEPO-Fc, studies have been conducted acute toxicity in animals.

Two groups of BALB/c mice (n=20, an equal number of males and females, aged 5-6 weeks, the average weight of females to 15.8±0.4 g, males of 15.9±0.6 g, obtained from the Chinese Academy of Medicine, China) was administered in a single/in the injection into the tail vein excessive amount of rHuEPO-Fc (males and 13.3 mg/kg, females 13.2 mg/kg) or equal volume of solution media. In addition, within 14 days of daily controlled and registered General behavior and status, activity, eating and defecation, in order to establish a strong reaction after the injection. All mice were also weighed on the 7th day and 14th day. On 15-th day after injection was performed anatomical examination of the main organs of mice. If I had observed any unusual or suspicious changes of bodies, it was necessary to conduct pathological examination.

No mice in the 2 groups was not observed any noticeable resistant reaction after injection. During the period of 14 days not nabludatelnij behavior changes, activity, modes of food and faeces. Moreover, during the period of observation, the weight of the mice in both groups was continuously increased, and on the 7th and 14th days after injection, there was no obvious difference between the two groups. In the brain, lung, heart, liver and kidneys were not detected any abnormal or pathological changes. These results show that the introduction of excessive amounts of rHuEPO-Fc, much larger than is required for the manifestation of normal eritropoeticescoe functions safely and gives no obvious toxic effects.

Example 10. Comparison of fused proteins EPO wild-type and mutant

We also conducted studies to compare proteins EPO wild-type and mutant versions. As described above, one of the embodiments of the present invention include a single amino acid mutation at position 172 (C172G). For comparison we have also prepared protein wild-type, having in position 172 cysteine (Fig). Protein wild type was prepared as described above in examples 1-3. To construct the recombinant plasmid used the following oligonucleotide primers (QIAGEN Inc., US) (changes of amino acids in EFL5w and EFL3w compared with primers in example 1 are marked in bold):

Sequence pry the development EFL5w and EFL3w specified respectively in SEQ ID SEQ ID No.9 and No.10.

For comparison eritropoeticescoe activity of the fused protein of the wild type (indicated here rHuEPO-FcC) with mutant fused protein (i.e., the above-described protein rHuEPO-Fc according to the present invention) and with recombinant human EPO (rHuEPO) were conducted in vivo studies in mice. To compare all doses used in this example, three proteins, namely rHuEPO-Fc, rHuEPO-FcC and rHuEPO were expressed by a number of only part of the EPO molecule in molar terms. In terms of protein rHuEPO-Fc and rHuEPO-FcC part EPO is 41,4% of the total molecular weight is calculated based on the weight of amino acids in EPO-to-weight ratio of all amino acids in the intact molecules rHuEPO-Fc and rHuEPO-FcC (i.e. 166 AK from 399 AK).

rHuEPO-Fc concentration in the working solution of 300 μg/ml), rHuEPO-FcC (concentration in the working solution of 90 mg/ml) and rHuEPO with the structure of natural human EPO (6000 units/0.5 ml, produced by Kirin Brewery Co., Japan) were diluted in a solution medium (2.5 mg/ml human serum albumin, and 5.8 mg/ml sodium citrate, 0.06 mg/ml citric acid and 5.8 mg/ml sodium chloride, pH 5.5 and 5.6). The dose of rHuEPO in quantitative terms was calculated based on the ratio of the activity/number. Mouse BALB/c mice (age 9-10 weeks, weight 18-22 g, equal number of males and females obtained from the Experiment Animal Center, AMMS, China) were divided randomly into groups of 8 mice in each group. Each group is ISA got one combination of one dose (2,5, 12,5, 62,5 µg/kg), one route of administration (p/C) and one mode of injection (3 times a week or 1 time per week). The control group of mice were administered an equal volume of a solution of the carrier. The effect lasted for 26 days. Peripheral blood samples for measurement were taken from the tail vein prior to exposure, and on the 2nd, 6th, 9th, 13th, 16th, 19th, 22nd and 26th days of exposure. The Hb level was measured as an index of absorbtiometry. Mean values ± standard deviations were calculated according to the data for each group for comparison of different groups was applied t-test.

As shown in Fig, the introduction of all three proteins EPO in mode 3 times a week stimulated erythropoiesis. And in the dose of 2.5 μg/kg, and the dose of 12.5 mcg/kg of rHuEPO-Fc induced a more significant increase of Hb levels than rHuEPO. The highest rise in Hb levels was achieved at a dose of 12.5 mcg/kg of rHuEPO-Fc. Both doses of rHuEPO-FcC (2.5 mg/kg and 12.5 mg/kg) induced much weaker erythropoiesis than equivalent doses of rHuEPO and rHuEPO-Fc, which manifested itself in a much weaker increase of Hb levels in the groups treated with rHuEPO-FcC. Indeed, the dose of 12.5 mcg/kg of rHuEPO-FcC induced a weaker rise in Hb levels than 2.5 μg/kg of rHuEPO. These results suggest that HuEPO-FcC has weaker eritropoeticescoe activity in vivo than rHuEPO with the natural sequence of the EPO molecule. In contrast, protein rHuEPO-Fc according infusion is his invention exhibits much stronger eritropoeticescoe activity. The introduction of three proteins EPO in mode 3 times a week allows you to eliminate the contribution of differences in the half-life of proteins.

Eritropoeticescoe activity rHuEPO-Fc and rHuEPO-FcC additionally evaluated by reducing the frequency of injections to 1 per week n/K. As shown in Fig, receiving rHuEPO-Fc groups at doses of 12.5 mg/kg or 62.5 mg/kg detected more significant increase in Hb levels than in the groups treated with rHuEPO. In contrast, rHuEPO-FcC induced much weaker rise in Hb levels than rHuEPO. For example, a dose of 12.5 mcg/kg of rHuEPO induced greater rise in Hb levels than the dose of 62.5 μg/kg of rHuEPO-FcC in most times. This further indicates that at lower frequency the introduction, when they begin to affect differences in time-life, rHuEPO-FcC shows much weaker eritropoeticescoe activity than rHuEPO having a molecular sequence of natural EPO, and the protein rHuEPO-Fc in accordance with the present invention.

Together, these results indicate that rHuEPO-FcC received by the merger of the natural molecular sequence of human EPO and human Fc fragment (hinge, CH2 and CH3), shows a much weaker eritropoeticescoe activity in vivo than rHuEPO having a molecular sequence of natural EPO. In particular, eritropoeticescoe activity of the fused protein rHuEPOFcC is less than 1/5 of the activity of molecules of natural EPO. This indicates that the fusion molecule of EPO with the natural sequence human Fc fragment affects the functional properties of the molecule EPO. A single substitution of the first cysteine in the hinge region of Fc fragment of another amino acid gives protein rHuEPO-Fc in accordance with the present invention, containing a sequence of natural molecules EPO and mutant Fc fragment and with a much higher eritropoeticescoe activity in vivo than the molecule of natural EPO. These data suggest that the first cysteine residue in the hinge region of Fc fragment of wild-type interferes with the EPO molecule, causing, apparently, structural changes in the molecule of EPO, and this in turn affects the functional properties of the molecule EPO stimulation of erythropoiesis.

As will be obvious to those skilled in this field from the preceding description, in practice the implementation of the present invention may be many changes and modifications without departing from the scope of the idea and scope of the present invention.

1. A protein having the activity is using erythropoietin, having a prolonged half-life in vivo in comparison with natural or recombinant native human erythropoietin containing:
a) the natural human erythropoietin molecule containing a cysteine residue near its C-end; and
b) the Fc fragment of human IgG containing the hinge region, and N is the end of the specified Fc fragment attached to a specified With-the end of the specified molecule of erythropoietin, and the specified Fc-fragment is natural, except for mutations, consisting in the substitution of a cysteine residue in the specified hinge region that is closest to the specified eritropoetinovmi molecule, nicotinoyl balance, resulting in the first cysteine residue of the specified hinge region located closest to the specified N-end, separated by at least 17 amino acids from the specified residue cysteine specified erythropoietin molecule.

2. Protein as defined in claim 1, where the half-life of the specified protein at least 3 times more time specified half-life of native human erythropoietin.

3. Protein as defined in claim 2, where the half-life of the specified protein at least 4 times more time specified half-life of native human erythropoietin.

4. Fused protein according to claim 2, where the specified protein has a high erythropoetin the second biological activity compared with the specified native human erythropoietin.

5. Protein as defined in claim 1, where the specified Fc fragment is a fragment of IgG1.

6. Protein as defined in claim 5, where the specified Fc fragment contains the specified hinge region and domains CH2 and CH3.

7. Protein as defined in claim 1, where this protein contains the amino acid sequence represented in SEQ ID NO:2.

8. Protein has a prolonged half-life in vivo in comparison with natural or recombinant native human erythropoietin containing natural human erythropoietin molecule containing a cysteine residue near the C-end, and the Fc fragment of human IgG containing the hinge region, and N is the end of the specified Fc fragment attached to a specified With-the end of the specified molecule of erythropoietin, and the specified Fc-fragment is natural, except for mutations, consisting in the substitution of a cysteine residue in the specified hinge region that is closest to the specified eritropoetinovmi molecule, nicotinoyl balance, resulting in the first cysteine residue of the specified hinge region located closest to the specified N-end, separated by at least 17 amino acids from the specified residue cysteine specified erythropoietin molecule, where this protein contains the amino acid sequence represented in SE ID NO:2, or sequence whose degree of identity with it is at least 98%, and this protein has a balance nezateilivii amino acid at position 172.

9. Dimeric protein design with the activity of erythropoietin containing two fused protein as defined in claim 1, connected by a disulfide bond between the two hinge regions.

10. Dimeric protein containing two polypeptide, each of which contains natural human erythropoietin molecule containing a cysteine residue near the C-end, and the Fc fragment of human IgG containing the hinge region, and N is the end of the specified Fc fragment attached to a specified With-the end of the specified molecule of erythropoietin, and the specified Fc-fragment is natural, except for mutations, consisting in the substitution of a cysteine residue in the specified hinge region that is closest to the specified eritropoetinovmi molecule, nicotinoyl balance, resulting in the first cysteine residue of the specified hinge region located closer only to the specified N-end, separated by at least 17 amino acids from the specified residue cysteine specified erythropoietin molecule and has the amino acid sequence represented in SEQ ID NO:2, or a sequence whose degree of identity with it is p is at least 98%, moreover, this protein has the rest nezateilivii amino acid at position 172.

11. Protein as defined in claim 10, where the dimer contains a disulfide bond between the respective hinge domains of these polypeptides.

12. Protein as defined in claim 10, where each of these polypeptides has a molecular mass of about 75 kDa.

13. Protein as defined in claim 10, where the dimer has a molecular mass of about 180 kDa.

14. Pharmaceutical composition having activity of erythropoietin protein according to claim 1 in an effective amount together with a pharmaceutically acceptable carrier, adjuvant or diluent.

15. The sequence of a nucleic acid encoding a polypeptide having a degree of identity of at least 98% with SEQ ID NO:2, and the specified polypeptide has a prolonged half-life in vivo as compared to native human erythropoietin and contains a fragment of a molecule of human immunoglobulin, N-end is directly attached to the C-end of the erythropoietin molecule, and thus the hinge area of the specified molecule of the immunoglobulin has a mutation, by which is closest to the specified N-the end of a cysteine residue substituted nicotinoyl balance.

16. The sequence is a nucleic acid having a sequence which made the Lenna in SEQ ID NO:1, or a sequence having in relation to it a degree of identity of at least 98%encoding a polypeptide containing a fragment of a molecule of human immunoglobulin, N-end is directly attached to the C-end of the erythropoietin molecule, and thus the hinge area of the specified molecule of the immunoglobulin has a mutation, by which is closest to the specified N-the end of a cysteine residue substituted nicotinoyl balance.

17. The expression vector containing the sequence of nucleic acid according to item 15 or 16.

18. Cell for producing fused protein according to claim 1, transfusiona expression vector by 17.

19. Cell as defined in p where the specified cell is a CHO cell.

20. The way to obtain protein as defined in claim 1, comprising culturing cells on p and cleaning encoded by the polypeptide.

21. Method of stimulating erythropoiesis in a mammal, comprising the introduction of a given mammal protein as defined in claim 1, or a pharmaceutical composition as defined in 14.

22. The method, as defined in item 21, where the specified mammal is a Primate.

23. The method as defined in article 22, where specified, the Primate is a human.

24. The method, as defined in item 21, stimulating erythropoiesis in a mammal, comprising the introduction of a specified m capitalsim pharmaceutical composition, as defined in 14.

25. The method, as defined in paragraph 24, where the specified mammal is a Primate.

26. The method as defined in A.25, where specified, the Primate is a human.

27. The method, as defined in item 21, where the half-life of the specified protein in the specified mammal at least 3 times the half-life of native human EPO with intravenous or subcutaneous injection.

28. The method, as defined in item 27, where the half-life of the specified protein in the specified mammal at least 4 times the half-life of native human EPO with intravenous or subcutaneous injection.

29. Protein as defined in claim 8, where the specified nechitailova amino acid is a neutral amino acid.

30. Protein as defined in clause 29 where the specified nechitailova amino acid is a glycine.

31. Protein as defined in claim 1, where the half-life of the specified protein when administered to a mammal at least 3 times the half-life of native human erythropoietin entered the specified mammal in the same way.

32. Protein as defined in p, where the half-life of the specified protein when administered to a mammal at least 4 times the half-life of native human e is Atropatena, put the specified mammal in the same way.

33. Protein as defined in p, where specified, the mammal is a human.

34. The expression vector as defined in 17 containing the nucleic acid sequence presented in SEQ ID NO:1.

35. Dimeric protein as defined in claim 10, containing two polypeptide, each of which has the amino acid sequence represented in SEQ ID NO:2.

36. Protein has a prolonged half-life in vivo no comparison with natural or recombinant native human erythropoietin containing:
a) a portion of the peptide erythropoietin containing adjacent to it-the end of a cysteine residue; and
b) the Fc fragment, containing the hinge region, and N is the end of the specified hinge region directly connected to the specified S-the end of the specified portion of the peptide erythropoietin, and specified the hinge region has a mutation in adjacent to the specified N-end position amino acids, consisting in the substitution of the cysteine residue nicotinoyl the residue, and the first cysteine residue of the specified hinge region located closest to the specified N-end, separated by at least 12 amino acids of the cysteine residue of the specified portion of the peptide erythropoietin.

37. Protein as defined in p, glaucosoma hinge region has a length of at least 9 amino acids.

38. Protein as defined in clause 37, where this hinge region is a variant of a human Fc fragment having necesairly residue in position 6 amino acids, measured from N-hinged end of the specified field.

39. Protein as defined in § 38, where this hinge region has the amino acid sequence VEPKSGDKTSTCPPCP or a sequence with relation to the degree of identity of at least 90%.

40. Protein as defined in p, where this protein has the amino acid sequence specified in SEQ ID NO:2, or a sequence with relation to the degree of identity of at least 98%.

41. Protein as defined in p where a specified part of the peptide erythropoietin is a complete human erythropoietin molecule.

42. Protein as defined in p, where specified, the Fc fragment is an Fc fragment of human IgG containing the specified hinge region and domains CH2 and CH3.

43. Protein as defined in § 42, where the specified Fc fragment of IgG is an IgG1 fragment.



 

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FIELD: medicine.

SUBSTANCE: plasmid vector pE-Trx-Aur is constructed for expression of aurelin in cells of Escherichia coli in composition of hybrid protein Trx-Aur, consisting of two DNA fragments, whose nucleotide sequence is given in description. By means of said vector parent strain of Escherichia coli is transformed, obtaining strain-producent of hybrid protein Trx-Aur. In order to obtain peptide aurilin cultivation of cells of obtained strain-producent is carried out, after that, performed are: cell lysis, affine purification of hybrid protein Trx-Aur on metal-chelate carrier, decomposition of hybrid protein Trx-Aur with bromine cyan by residue of methionine, introduced between sequences of aurelin and thioredoxin, and purification of target peptide by method of reversed-phase HPLC.

EFFECT: invention makes it possible to obtain biologically active aurelin by simplified technology and without application of hard-to-obtain natural raw material.

3 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: biologically active peptide is proposed with the common formula: , where OBu - residue of 2-oxobutyric acid, Abu - residue of 2-aminobutyric acid, Dha - residue of 2,3-didehydroalanine, Dhb - residue of 2,3-didehydroaminobutyric acid, besides residues are in sequence linked with amide links, residues Abu3-Ala7, Abu22-Ala27 and Abu24-Ala31 are also linked pairwise with thioether links with formation of three residues of 3-methyllanthionine, residues Ala11-Ala21 are linked with thioether link to form residue of lanthionine. This peptide has antimicrobial effect at gram-positive bacteria Bacillus subtilis, strains L1 and 1621; Bacillus pumilus, strain 2001; Bacillus globigii, strain I; Bacillus amyloliquefaciens, strain I; Bacillus megaterium, strain VKM41; Mycobacterium smegmatis, strain 1171; Mycobacterium phlei, strain 1291; Micrococcus luteus, strain B 1314; Staphylococcus aureus, strain 209p; Rhodococcus sp., strain SSI.

EFFECT: increased efficiency of peptide antimicrobial action.

4 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: as feedstock for fractioning used is anticytomegalovirus plasma with activity index not less than 70%. Process of production includes: ultrafiltration of immunoglobulin at ion force not higher than 0.02 and pH value 4.0-5.7 with further diafiltration against distilled water with pH value 3.5-6.0. Immunoglobulin is incubated with maltose of 2-11% concentration at temperature 32-40°C and pH value 4.0-4.8 for 10-36 hours, or with pepsin in dose 1:10-6-1:10-5 in presence of 2-11% maltose. After that inactivation/removal of viruses is carried out by immunoglobulin incubation at pH value 4.0-4.4 and/or with application of depth filtration, and/or nanofiltration. Afterpurification of immunoglobulin is performed by methods of: ultrafiltration and/or depth filtration, and/or nanofiltration, and/or anion-exchange chromatography, and/or dialysis. Immunoglobulin stabilisation is carried out with application of sodium chloride and/or maltose, and/or proline, and/or glucose and/or glycerol, whose content provides preparation osmolarity within 270-400 mOcm/kg. In obtained immunoglobulin preparation pH value 5.0-7.5 and protein content 4.5-10.5% are installed.

EFFECT: invention allows to obtain highly avid immunoglobulin preparation against cytomegalovirus for intravenous introduction with improved and stable qualitative quaracteristics.

7 cl, 5 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a pharmaceutical composition which contains TAT-HOXB4 protein as an effective component. Said composition has stimulating effect on production of hematopoietic stem cells. More specifically, the recombinant protein TAT-HOXB4 enhances acceptance of intramedullary transplants, hematopoietic reconstruction, repopulation and number of circulating stem cells, specifically after chemotherapy or exposure.

EFFECT: higher protein output and stability.

24 cl, 11 dwg, 2 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to optimised fused protein for blocking BLyS or APRIL, which contains extracellular region of N-end of truncated TACI (transmembrane activator and CAML-partner) and Fc sequence IgG. TACI segment of fused protein contains sequence of amino-end region of extracellular region, starting with 13-th amino acid residue, complete sequence of stem area from TACI and is obtained from native sequence of TACI between 12-th and 120-th amino acids. Segment Fc of immunoglobulin IgG of fused protein contains hinge region, CH2 region and CH3 region, TACI segment and Fc segment are fused either directly or through linker sequence. In addition, claimed is DNA sequence which codes fused protein, expression vector, host-cell, pharmaceutical composition, containing fused protein, and application of fused protein for blocking BLyS or APRIL. Obtained fused protein does not degrade in process of expression, possesses high biological activity and high level of expression.

EFFECT: fused protein in accordance with claimed invention can be used in treatment of diseases, associated with abnormal immunologic functions and in treatment of diseases caused by abnormal proliferation of B-lymphocytes.

10 cl, 6 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology and biotechnology. Claimed are: versions of antibody and antigen-binding fragments of antibody to receptor Il-6 of humans. Considered are: isolated molecule of nucleic acid and vector which contains it. Described are: system "host-vector" and method of obtaining antibody or its antigen-binding fragment, as well as application of antibody or its antigen-binding fragment for obtaining medication.

EFFECT: invention application provides novel antibodies to receptor IL-6 of humans, which can be applied in therapy of IL-6- mediated diseases.

11 cl, 5 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and particularly to obtaining guanylate cyclase receptor-activating peptides and can be used in medicine. A recombinant technique or chemical synthesis is used to obtain a polypeptide consisting of an amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys.

EFFECT: invention enables to obtain a guanylate cyclase-activating polypeptide, and can be used for efficient treatment of gastrointestinal disorders or visceral pain in a patient.

17 cl, 55 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: genetically engineered technique is applied to produce in Escherichia coli cells the recombinant polypeptides with human plasminogen properties, containing a catalytic plasminogen domain, 5th plasminogen kringle domains, and a modified fragment of an amino acid sequence preceding to the 5th plasminogen kringle domain.

EFFECT: invention allows producing the polypeptides of high fibrinolytic human plaminogen activity and high yield in expression in Escherichia coli cells.

7 cl, 1 dwg, 11 ex

FIELD: medicine.

SUBSTANCE: genetically engineered technique is applied to produce in Escherichia coli cells the recombinant polypeptides with human plasminogen properties containing a catalytic plasminogen domain, 5th and 4th plasminogen kringle domains, and a modified fragment of an amino acid sequence preceding to 4 plasminogen kringle domains.

EFFECT: invention allows producing the polypeptides of high fibrinolytic human plaminogen activity and yield 40-60 mg per 1 l of the culture in expression in Escherichia coli cells.

7 cl, 1 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: in order to obtain protein PS-CFP2/TurboYFP_MBP7, constructed is recombinant plasmid DNA PS-CFP2/TurboYFP_MBP7 with size 4916 bp, coding hybrid protein, containing sequence of proteins PS-CFP2 and Turbo YFP, bound with fragment of myelin basic protein 80-104. Composition of plasmid DNA also includes promoter of T5 PHK-polymerase transcription, site of ribosome binding; fragment of DNA plasmid gen of β-lactamase, determining stability of Escherichia coli cells to ampicillin, as genetic marker. Obtained plasmid DNA is used to transform cells of Escherichia coli strain BL21(DE3) to obtain strain-producent of hybrid protein PS-CFP2/TurboYFP_MBP7. In order to obtain protein PS-CFP2/TurboYFP_MBP7 cultivation of strain-producent of Escherichia coli BL21 (DE3)/pQe30_PS-CFP2/TurboYFP_MBP7 is performed, cells are destroyed and target protein is purified by method of affine and gel-filtration chromatography.

EFFECT: invention makes it possible to increase biosensor sensitivity and stability and extend its specificity in respect to pool of catalytic antibodies.

3 cl, 7 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: there are offered: IL-6 receptor antibody, a coding gene, a vector and a host cell for producing the antibody, a method for producing the antibodies, and a pharmaceutical composition for treating IL-6-related diseases containing the antibody.

EFFECT: use of the invention provides new humanised IL-6 receptor antibodies that can find further application in therapy of the IL-6-mediated diseases.

8 cl, 22 dwg, 8 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing fused proteins. The fused construct consists of an amino acid sequence of glycosyl phosphatidylinositol anchored tissue inhibitor of metalloproteinase.

EFFECT: cictrisation prevention during skin injures treatment when using the fused construct.

10 cl, 33 dwg, 18 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to optimised fused protein for blocking BLyS or APRIL, which contains extracellular region of N-end of truncated TACI (transmembrane activator and CAML-partner) and Fc sequence IgG. TACI segment of fused protein contains sequence of amino-end region of extracellular region, starting with 13-th amino acid residue, complete sequence of stem area from TACI and is obtained from native sequence of TACI between 12-th and 120-th amino acids. Segment Fc of immunoglobulin IgG of fused protein contains hinge region, CH2 region and CH3 region, TACI segment and Fc segment are fused either directly or through linker sequence. In addition, claimed is DNA sequence which codes fused protein, expression vector, host-cell, pharmaceutical composition, containing fused protein, and application of fused protein for blocking BLyS or APRIL. Obtained fused protein does not degrade in process of expression, possesses high biological activity and high level of expression.

EFFECT: fused protein in accordance with claimed invention can be used in treatment of diseases, associated with abnormal immunologic functions and in treatment of diseases caused by abnormal proliferation of B-lymphocytes.

10 cl, 6 dwg, 8 ex

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