LT2010012A - Linear oligomers of granulocyte stimulating protein having prolonged survival time - Google Patents

Abstract

The present invention provides structural variants – linear oligomers of recombinant ptotein, namely, granilocyte colony stimulating factor (G-CSF) having novel pharmacodynamic and pharmacokinetic characteristics such as prolonged survival (circulation) period in plasma and longer period of biological activity as compared to monomeric protein. Structural and technological pecularities enabling to obtain recombinant proteins with improved therapeutic charcteristics that may be used in biopharmacy and medicine are disclosed.@

Description

1

FIELD OF THE INVENTION This invention is within the field of biotechnology. It reveals structural variants of a recombinant granulocyte stimulating protein, such as granulocyte colony stimulating factor (G-CSF), namely linear dimers with new pharmacodynamic and pharmacokinetic properties, and most importantly, prolonged life (circulation) in plasma compared to natural monomeric protein. . The state of the art of the invention

The use of therapeutic proteins derived from recombinant DNA techniques has reached new heights over the last few decades. They are based on 15 biopharmaceutical products (erythropoietin - EPO, human growth hormone - hGH, interferon -IFN, insulin, granulocyte (macrophage) colony stimulating factors - G-CSF or GM-CSF, follicle stimulating factor - FSH, thrombopoietin - TPO, thrombolytics). ) The annual global production volume is already over tens of billions of dollars and continues to grow. The advancement of biotechnology encourages the improvement of the efficiency, safety, and delivery of innovative products (also known as first generation 20) on the market. The next (second and third) generation of biopharmaceutical products are being actively developed, with an extended (prolonged) therapeutic effect in vivo. Formulations of a proprietary drug pharmacy in which the active substance is chemically or genetically modified therapeutic proteins with extended circulation in vivo have obvious advantages. Such extended-release formulations are injected into the patient daily as a routine and once a week or a month. This provides the patient with more security guarantees and convenience. In general, the rate of depletion of the protein can be slowed down by increasing the molecular weight or negative charge of the protein. Thus, the in vivo protein lifetime can be varied in general by two ways of altering the protein structure: a) by increasing the molecule of therapeutic protein: this can be achieved by conjugating protein molecules with polymeric molecules of molecular nature or by conjugation of protein (chemically or genetically) with another inactive protein partner , which has a longer lifespan than the target protein, b) by altering the structure of the protein for therapeutic purposes: this can be achieved by addition of carbohydrate structures by glycosylation (in vivo or in vitro), which can be carried out both chemically, genetically and genetically. 2

Techniques for chemical modification are known to produce therapeutic protein prolongation effects. The latter can be conjugated with polyethylene glycols, dextran, other carbohydrates, or stitched with other proteins. The most advanced and actively patented (among others WO 2004/083242 A1, 2004) chemical modification method is 5 pegylation (polyethylene glycol, abbreviated - PEG single or multiple chain attachment). The major deficiency of pegylation tends to result in a frequent marked decline in the biological activity of the protein to be modified, mainly due to the sufficiently high hydrodynamic diameter of the PEG molecule, which can cause spatial interference with the protein interaction with the receptor. The size of the target protein molecule (spatial volume) and half-life in vivo can be raised not only by conjugation with polymers (PEG, dextran, etc.), but also with other higher molecular weight proteins, such as carriers, such as serum albumin. . [Poznansky M. J., In vitro and in vivo activity of soluble cross-linked uricase album polymers: a model for enzyme therapy. Life Sci. 1979 vol. 24 (2), p. 153-158]. 15 Chemical stitching techniques allow the development of long-acting therapeutic proteins using a variety of protein partners, including specific antibodies. There are a number of chemically linked immuno-molecular derivatives (in this case monoclonal antibodies - Mak), such as doxorubicin-Mak and calichemycin-Mak conjugates in Phase I clinical trials [Sievers E.L. et al., Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood, vol. 93 (11): p. 3678-84, 1999]

Recently, analogous structures have been created by fusing a target protein (fusion protein) with its partner, using recombinant DNA technology. Unlike 25 chemicals, the genetic method of producing fused protein enables products of repetitive quality to be obtained, which is particularly important for further clinical trials. However, any modification of the target protein may result in partial loss of its biological function / activity or cause undesirable immune response.

The target protein partner circle in fused constructs typically comprises: a) immunoglobulin domains: smallest parts of immunoglobulins are single-stranded antibody fragments (scFv) that can be expressed in many systems such as VL-VH pairs linked together with artificial linkers Gly / Ser. By combining cytokines or proteins with scFv, immunocytokines and immunotoxins for therapeutic use are obtained. Some immunotoxins, such as anti-Tac (Fv) -PE 38, have been used in 35 clinics to treat cancer patients who have failed other therapies [Kreitman R.J. et al., Phase I trial of recombinant immunotoxin anti-Tac (Fv) -PE38 (LMB-2) in patients with hematologic malignancies. J. Cin. Oncol. 2000; 18: 1614-36]. Toxicity and 3 immunogenicity limit the wider use of such fused products in therapy. To reduce the latter undesirable effects, the target protein in such a fused form is additionally pegylated through the amino group of the Lys residue or by introducing a Cys residue by point mutation. This methodological solution was used by Bolder BioTechnology to develop extended-action G-CSF, growth hormone, EPO and IFN immunostaining constructs with spot-specific pegylation. (b) for human and other animal albumin: \ t A group of French scientists have shown that chemical growth hormone and albumin conjugate have a slower in vivo release rate and soon announced the expression of recombinant CD4-albumin fusion protein (Lu H., et al., FEBS Lett., 1994) in Kluveromyces yeast. Thus, the half-life of soluble CD4 was increased by almost 140 times. The same group of scientists achieved the secretion of urokinase-type plasminogen activator and human albumin fusion protein in these yeasts. Human Genome Sciences and CoGenesys companies have published long-acting therapeutic protein EPO, hGH, G-CSF, IFN alpha forms, genetically fusing target proteins with human albumin, and their submission to clinical studies.

An alternative to the target protein chemical attachment or genetic fusion to albumin method is the generation of a prolongation effect by forcing the target protein to become an in vivo specific albumin ligand. Thus, the chemical conjugation pathway of polystyrene and maleic acid copolymer coupled to superoxide dismutase (SOD) gave the ability to specifically bind to albumin in vivo. As a result, the elimination of SOD from rats increased from 4 min. to 6 or. [Inoue M., et al., Synthesis of a superoxide dismutase derivative that circulates bound to albumin and accumulates in tissues, pH is revised, Biochemistry, 1989, 28 (16), p. 6619-6624],

Modification technologies have been developed to extend the in vivo duration of the proteins, and a series of therapeutic proteins have been tested, but few have been placed on the market. This is a prolonged-acting EPO - Aranesp (additional glycosylation) and G-CSF - Neulasta (pegylated G-CSF) developed by the Amgen Corporation, and pegylated interferon alpha 2a from Pseudo-alpha 2 - Pegasys and alpha-2b - PEG-Intron.

Existing technologies and methodological solutions to prolong the lifetime of therapeutic proteins have several inherent disadvantages. Increasing the size (volume) of the protein molecule by combining polymers or other proteins produces a sufficiently large final derivative in which the target protein can often lose (fully or partially) the ability to specifically interact with the cell receptor, thereby losing its biological efficacy. The lower the target protein, the more serious the problem is. Today, successful trials have been achieved with sufficiently large therapeutic proteins. However, even then, the biological activity of the modified protein is significantly lower than that of the intact unmodified protein 4, resulting in the amount of such modified derivative in drug doses exceeding the amount of unmodified protein.

In the case of additional glycosylation (i.e., introducing new, non-natural glycosylation targets in the amino acid sequence of the protein), technological solutions usually require changes in the genetic structure of the protein, such as the introduction of point mutations. Such a modified protein structure is already a new derivative, often leading to unexpected side reactions and toxic effects.

There is little material in the patent literature about structures of linear multimeric proteins. EP1334127 (A1) refers to the possibility of covalent attachment of multimeric proteins (mainly cytokines, including G-CSF) and addition of additional polymer chain (PEG or other) to multimeric protein. This patent does not provide information on the effect of the linker sequence on the biological activity of the multimeric protein. Patent Application WO9206116 (A1) discloses the ability of heterodimeric recombinant proteins to promote myeloid early and late differentiation of hemapoietic cells. The first molecule may be IL-3 or GM-CSF, the second one being EPO, G-CSF, IL-5 or M-CSF.

Patent Application WO0103737 (A1) discloses the ability and properties of fusion proteins consisting of a cytokine or growth factor fused to an immunoglobulin domain (e.g., IgG). 20 It is known [Paige A.G. et al. "Prolonged circulation of recombinant human granulocyte colony stimulating factor by covalent linkage to albumin through a heterobifunctional polyethylene glycol" Pharm. Res. 1995, 12 (12): 1883-8] for extended rhG-CSF circulation, when the protein is covalently bound to serum albumin via the heterobifunctional agent maleimido-carboxylpolyethylene glycol. A similar idea for the fusion of growth hormone and albumin 25 molecules was also disclosed in Poznansky [Poznansky, M.J. et al. “Grovvth hormone album conjugates. Reduced renal toxicity and altered plasma clearance. ”FEBS Lett. Oct. 24, 1988; 239 (1): 18-22].

Granulocyte colony-stimulating factor (G-CSF) has not been found in patent-pending structural variants (linear oligomers) characterized by prolonged plasma survival and in the non-patent literature.

As follows from the description of the prior art, the therapeutic effects of existing protein preparations are often short-lived and require additional portions of the protein, since circulating proteins in the body are rapidly degraded and lose their therapeutic effect. Previous methods of prolonging the life of a protein molecule by increasing their molecular weight suggest that non-protein sequences (e.g., polyethylene glycol) or other proteins (e.g., albumin) that have no therapeutic effect are attached to the therapeutic protein, but this usually significantly reduces the activity of the therapeutic molecule. Therefore, it would be useful to find a way to prolong the therapeutic effect by oligomerizing the molecules of the same therapeutic protein. This would not only increase the molecular weight of the protein (which, apparently, is one of the important factors in increasing its stability in vivo), but also retains or improves the biological activity of the novel protein, compared to the prior art, i.e. Protein therapeutic prolongation goals require constructing and purifying an active protein that, while remaining stable in vivo for a prolonged period of time, would have a structurally sustained therapeutic protein biological activity.

Thus, it is an object of the present invention to construct new polypeptide structures for second-generation therapeutic proteins such as G-CSF, with their targeted in vivo regulation of 10 (prolonged) lifespan and biological activity by genetically forming homo-multimeric protein forms of desired length, and to develop biopharmaceuticals based on their extended in vivo circulation duration.

In particular, one of the objects of the present invention was to provide an effective linear structure of the linear G-CSF dimer to produce a biologically active protein with a lifetime in vivo of 15 greater than that of the monomeric protein. The essence of the invention

The aforementioned objects are achieved by implementing the totality of the claims 1-5 of the present invention. The present invention includes linear oligomeric recombinant proteins composed of genetically linked monomers of the same protein, wherein the monomer is a granulocyte-stimulating protein, and the monomers are linked by a linker amino acid sequence.

In an optimal embodiment of the present invention, the granulocyte stimulating protein is a granulocyte colony stimulating factor G-CSF and the number of monomers is two. The optimum linker sequence of the present invention is selected from the group of amino acid sequences consisting of 25 (S-G4). where n = 2-7 and SGLEA- (EAAAK) m -ALEA- (EAAAK) m -ALEGS, where m = 2-8. The present invention includes linear oligomeric proteins having biological activity and extended circulation time in vivo.

Another object of the present invention is a pharmaceutical composition comprising an effective amount of the above-mentioned linear oligomeric protein, preferably a linear G-CSF dimer, and a pharmaceutically acceptable carrier and / or excipient.

Description of the drawing

FIG. Figure 1 shows Western blot analysis of dimeric G-CSF using monoclonal antibodies against G-CSF. Linker sequences are disclosed in Table 1 below. Tracks: 1.5-1 pg monomeric G-CSF under reduced conditions, reduced and non-reduced conditions, respectively; 6, 2 to 6 µg of dimeric G-CSF protein with linker La, under reduced and undigested conditions, respectively; 3, 7 - 1 µg of dimeric G-CSF protein with linker L5 under reduced and undigested conditions, respectively; 5, 8 - 1 µg of dimeric G-CSF protein with linker L7 under reduced and undigested conditions, respectively; K - Molecular Weight Standards (Ferment # SM671), from the bottom of the gel: 15, 25, 35, 40, 55, 70, 100 kDa.

FIG. Figure 2 shows high-pressure efficient chromatography of dimeric G-CSF-L5 reverse phases) (RP-HPLC) using C4 (Hi-Pore RP-304, 250x4.6 mm, 300 A, Bio-Rad, USA) reverse phases column. For elution, solutions A (0.1% trifluoroacetic acid (TFA) aqueous solution) and B (TFA-water-acetonitrile 0.1: 9.9: 90), component concentrations in solutions are expressed as volume percent. Protein peaks are detected at 215 nm. The protein peak was identified by retention time tR, and the amount of this protein form in the test sample 15 was determined by integration of chromatogram data.

FIG. Fig. 3 shows the change in the concentration of dimeric G-CSF protein in serum of rats after injection of protein (500 µg / kg) subcutaneously. A - dGCSF-L5 and control Neupogen ™ sample; B - dGCSF-L5, dGCSF-L7 dGCSF-La, control Neupogen ™ sample and buffer (placebo without protein) used for dimeric protein storage. Figure 4 depicts the effect of dimeric G-CSF proteins in vivo based on the change in blood neutrophil count after injection of the protein (500 µg / kg) in the rat subcutaneously. Control Neupogen ™ and Buffer (placebo, Protein-Free) used to store dimeric proteins.

DETAILED DESCRIPTION OF THE INVENTION The main object of the present invention are linear oligomeric recombinant proteins, examples of which are five new linear dimeric G-CSF structures which show longer in vivo (serum) time and similar or higher biological activity (neutrophil proliferation) compared to control monomeric. protein. Linear oligomers of the present invention can be obtained using a variety of cell-colony-stimulating cytokines such as: G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocytes and macrophage stimulating factor), M-CSF (macrophage stimulating factor). A genetically engineered linear oligomer may contain both an equal number of monomers and an odd number. The invention also describes how the structural variants of the dimeric protein dGCSF have been obtained with biological activity and prolonged serum life. The main stages of the preparation and testing of dimeric G-CSF (dGCSF) according to the present invention include the following steps: 7 - obtaining a genetic construct and constructing a bacterial strain producing the protein; - protein biosynthesis, purification and renaturing; - In vitro bioavailability granulocyte proliferation testing; 5 - In vivo kinetics testing of life expectancy; - In vivo testing for biological effects (proliferation of neutrophils). Linear dimers of the G-CSF gene were constructed by combining two copies of one gene through a linker sequence into a single DNA fragment cloned into expression plasmid pET21b and strain E. coli BL21 (DE3) by means of a polymerase chain reaction (PCR). The following recombinant protein constructs were obtained; dGCSF-L2, L3, L5, L7, La, where two G-CSF molecules are linked by linker sequences consisting of amino acids shown in Table 1: Table 1 15 Sequence of Dimeric G-CSF (dGCSF) linkers

Linker Amino Acid Sequence Amino Acids Number L2 (SG4) - (SG4) -S 11 L3 (SG4) - (SG4) -S 16 L5 (SG4) - (SG4) - (SG4) - (SG4) - (SG4) ) -S 26 L7 (SG4) - (SG4) - (SG4) - (SG4) - (SG4) - (SG4) - (SG4) -S 36 La SGLEA- (EEA) 4-ALEA- (EEA) alegs 54

The DNA sequences of all genetic constructs were tested by determining the primary nucleotide sequence corresponding to the intended dimeric coding for recombinant proteins consisting of 20 linearly spaced G-CSF protein monomeric elements linked together by the above linker linkages (monomer linking sequences). The latter were selected by increasing length from two to seven unstructured SGGGG elements (Ln), or structured linker La (with alpha-spiral domains). The La Linker amino acid sequence through which the linear oligomers are linked has the alpha-spiral domains of the EAAAC, the number of which may vary from two to eight. Such an amino acid sequence has been selected to maintain a specific linker structure with alpha-helical elements that recur periodically. Both shorter linker sequences (m = 2) and longer sequences (m = 8) form the same alpha-helical elements and provide an analogous structure to the monomer-linking sequence. 8

The structure of dimeric proteins, as mentioned above, was confirmed by several independent methods: the gene sequence encoding the proteins was determined (in all cases, the determined sequence corresponded to the intended dimeric protein sequence, Table 2). Table 2 5 The amino acid sequence and molecular weight of dimeric proteins

Dimeric protein Molecular weight kDa amino acid sequence Full Linkerinė-L3 sequence dGCSF 38.55 MTPLGPASSLPQSFLLKCLEQVRKIQ GDGAALQEKLCATYKLCHPEELVLLG HSLGIPWAPLSSCPSQALQLAGCLSQ LHSGLFLYQGLLQALEGISPELGPTLD TLQLDVADFATTIWQQMEELGMAPAL QPTQGAMPAFASAFQRRAGGVLVAS HLQSFLEVSYRVLRHLAQPSGGGGG SGGGGSGGGGSTPLGPASSLPQSFL LKCLEQVRKIQGDGAALQEKLCATYK LCHPEELVLLGHSLGIPWAPLSSCPS QALQLAGCLSQLHSGLFLYQGLLQAL EGISPELGPTLDTLQLDVADFATTIWQ QMEELGMAPALQPTQGAMPAFASAF QRRAGGVLVASHLQSFLEVSYRVLRH LAQP SGGGGGSGG GGSGGGGS dGCSF L5 39.12 MTPLGPASSLPQSFLLKCLEQVRKIQ GDGAALQEKLCATYKLCHPEELVLLG HSLGIPWAPLSSCPSQALQLAGCLSQ LHSGLFLYQGLLQALEGISPELGPTLD TLQLDVADFATTIWQQMEELGMAPAL QPTQGAMPAFASAFQRRAGGVLVAS HLQSFLEVSYRVLRHLAQPSGGGGS GGGGSGGGGSGGGGSGGGGSTPL GPASSLPQSFLLKCLEQVRKIQGDGA ALQEKLCATYKLCHPEELVLLGHSLGI PWAPLSSCPSQALQLAGCLSQLHSGL FLYQGLLQALEGISPELGPTLDTLQLD VADFATTIWQQMEELGMAPALQPTQ GAMPAFASAFQRRAGGyLVASHLQS FLEVSYRVLRHLAOP SGGGGSGGG GSGGGGSGG GGSGGGGS dGCSF-L7 39.75 MTPLGPASSLPQSFLLKCLEQVRKIQ GDGAALOEKLCATYKLCHPEE LVLLG HSLGIPWAPLSSCPSQALQLAGCLSQ LHSGLFLYQGLLQALEGISPELGPTLD TLQLDVADFATTIWQQMEELGMAPAL QPTQGAMPAFASAFQRRAGGVLVAS HLQSFLEVSYRVLRHLAQPSGGGGS GGGGSGGGGSGGGGSGGGGSGGG GSGGGGSTPLGPASSLPQSFLLKCLE QVRKIQGDGAALQEKLCATYKLCHPE ELVLLGHSLGIPWAPLSSCPSQALQLA GCLSQLHSGLFLYQGLLQALEGISPEL GPTLDTLQLDVADFATTIWQQMEELG MAPALQPTQGAMPAFASAFQRRAGG VLVASHLQSFLEVSYRVLRHLAQP SGGGGSGGG GSGGGGSGG GGSGGGGSG GGGSGGGGS 9-La dGCSF 42.52

MTPLGPASSLPQSFLLKCLEQVRKIQ GDGAALOEKLCATYKLCHPEELVLLG HSLGIPWAPLSSCPSQALQLAGCLSQ LHSGLFLYQGLLQALEGISPELGPTLD TLQLDVADFATTIWQQMEELGMAPAL QPTQGAMPAFASAFQRRAGGVLVAS HLQSFLEVSYRVLRHLAQPSGLEAEA AAKEAAAKEAAAKEAAAKALEAEAAA KEAAAKEAAAKEAAAKALEGSTPLGP ASSLPQSFLLKCLEQVRKIQGDGAAL OEKLCATYKLCHPEELVLLGHSLGIP WAPLSSCPSQALQLAGCLSQLHSGLF LYQGLLQALEGISPELGPTLDTLQLDV ADFATTIWQQMEELGMAPALQPTQG AMPAFASAFQRRAGGVLVASHLQSFL EVSYRVLRHLAQP

SGLEAEAAAKAAKAAKAAKAAA AAAAAKAA KEAAAKAAA EAAAKALEGA

The molecular weight of the dimeric protein was assessed by SDS-PAGE agility and compared to the calculated molecular mass (in all cases, the molecular weights of the dimers correspond to the theoretical motility in SDS-PAGE reduced conditions). Immunoblotting results confirm the structure of dimeric proteins after an interaction with G-CSF specific antibodies (Fig. 1). The active structure of dimeric proteins is confirmed by the determination of specific biological activity in an in vivo assay (Fig. 4) and in vitro proliferation assay using mouse myeloid leukemia cell line G-NFS-60 to determine the biological activity of newly obtained protein structures (Table 3, Example 3). The pharmaceutical composition based on linear oligomeric proteins according to the present invention may contain one or more of the following components: a carrier which may be polyhydric alcohols (e.g., mannitol, sorbitol), various monosaccharides (e.g. glucose), disaccharides (e.g., sucrose) ; - a buffering agent for maintaining the required pH value (e.g., acetate, phosphate, TRIS, 15 HEPES); - salt additives to maintain the isotonicity of the composition (e.g., sodium chloride); - detergent-protecting protein-degradation liquid-air within the boundary (eg polysorbate 80, polysorbate 20, various compounds of the Pluronic type); - stabilizers such as polyethylene glycols, polyvinylpyrrolidones, amino acids (e.g., L-20 methionine, L-arginine, L-histindine, L-glutathione acid, L-aspartic acid); - a chelating agent such as EDTA, EGTA, IDA; - an antimicrobial agent (e.g. benzene derivatives such as cresol, benzyl alcohol); - SH agent (e.g., glutathione, cysteine, acetylcysteine); - other suitable excipients. Examples of implementing the invention

The following are specific examples of the implementation of the invention. These examples do not limit, rather. only illustrates the scope of the invention. Example 5 1

Biosynthesis of dimeric G-CSF protein

Producer of dimeric G-CSF E. coli BL21 (DE3) p21 GCSF / L5 / GCSF [V. Chmeliauskaitė, R. Mikšytė, V. Lukša, E. Zareckaja, G. Zvirblis, Overexpression study of human colony stimulating factors in Escherichia coli. 2. Overexpression of human granulocyte colony 10 stimulating factor. Biology, 1996, No.2, p. 15-18, etc.] were grown in 2L flasks and / or fermenter (Sartorius AG, 5L working volume fermenter). The medium was used with 45.12g of M9 salt, 20g of yeast extract, 80ml of 20% glucose, 8mM MgSO4, 4ml of ampicillin 100mg / ml. Adjustable parameters in the fermenter: temperature 37 ° C; pH 6.8 (acid or alkali added), aeration (p02 25-30%; 1.0-1.31 / min, 02 concentration and 15 mixing intensity). The optical density of the culture, which shows the amount of extended biomass, was recorded separately. The target protein synthesis is induced by the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside). Induction is performed with ISTG (final concentration 100 mM) at 1.2 o. optical density. Biomass yield 30.31g wet biomass, i.e. 8.6 g dry weight of 1 L culture fluid. 20 Example 2

Purification of the dimeric protein by renaturing the solution

Isolation of dimeric G-CSF protein insert bodies was performed as follows: 10 g of E. coli biomass was homogenized in 100 ml of 0.1 M Tris-HCl, pH 7.0 at 5 mM EDTA. 0.1 g of lysozyme, 0.1 ml of Triton 100-100, 1.0 ml of 100 mM phenylmethylsulfonyl fluoride (PMSF) and 0.7 ml of 2-mercaptoethanol are added to the homogenate. The homogenate was stirred for 30 min. at room temperature under ultrasound in an ice bath and centrifuged at 24.500 g for 25 min. The liquid was drained, the precipitate was washed twice, homogenized in 100 ml of 1 M NaCl, 0.1% Tween 80 and once with 100 ml of distilled water. After each homogenization, the resulting suspension was centrifuged for 25 min. at 24500 g.

Method for oxidative renaturation of dimeric G-CSF in incubation solution in solution: 1.7 g of dimeric G-CSF insert bodies are dissolved in 80 ml of 7 M guanidine hydrochloride (GuHCI) solution in 10 mM Tris-HCl, pH 7.0, overnight at 4 ° C. The intramuscular solution is centrifuged at 40,000 g for 25 min. The supernatant is diluted with 10 mM 35 Tris-HCl buffer (pH 7.0) to 1 mg / ml protein (according to Bradford), 6 M GuHCI, added to a CuSO 4 solution to a concentration of 20 μ ir and stirred for 1 or. at room temperature. Then add the EDTA solution to a concentration of 10 mM and bring the pH to 5.4 with HCl.

The protein solution is centrifuged for 25 min. at 40,000 g. Approximately 145 ml of the protein solution is applied to a C 26/100 column (2.6 * 100 cm, Pharmacia, Uppsala, Sweden) filled with 500 ml sorbent Sephadex G-25 Medium and equilibrated with 25 mM Tris-maleic acid-NaOH, pH 6.5 , 0.25 M Na 2 SO 4. Protein application and elution with 25 mM Tris-maleic 5-acid -NaOH buffer, pH 6.5, 0.25 M Na2SO4 at 30 cm / h. flow rate. The target protein-containing eluate fractions were pooled and centrifuged at 40,000 g for 25 min. Dimeric G-CSF solution after chromatography on Sephadex G-25 Medium was purified by metal chelate chromatography using a sorbent Sepharose CL-6B with a covalently bonded yellow Yellow SA 2KT-Cu (II). Column C 16/20 (1.6 * 20 cm, 10 Pharmacia, Uppsala, Sweden) is filled with 20 ml of this sorbent and equilibrated with 25 mM Tris-maleic acid-NaOH, pH 6.5, 0.25 M Na2SO4. 120 ml of protein solution (concentration 0.30 mg / ml according to Bradford) are introduced into the column and the column is washed with 5 volumes of the initial buffer. The protein is desorbed by gradually increasing the concentration of imidazole in the column from 0 to 300 mM. During the chromatography, the eluent flow rate was 45 cm / h. I5 Fractions from the imidazole gradient containing the predominantly target protein are pooled and dialyzed against 25 mM Na-acetate buffer, pH 5.4 at 4 ° C. Dimeric G-CSF was further purified by ion exchange chromatography. 31 ml protein solution (Bradford concentration 0.13 mg / ml) is introduced into a C 10/10 column (1.0 * 10 cm, Pharmacia, Uppsala, Sweden) filled with 4 ml of sorbent SP-Sepharose and equilibrated with 25 mM Na-20 acetate. buffer, pH 5.4. The column was washed with 5 volumes of this buffer, and the protein was desorbed by gradually increasing the NaCl concentration from 0 to 500 mM. During the chromatography, the eluent flow rate was 45 cm / h. The target protein fractions were pooled and dialyzed at 4 ° C prior to 10 mM Na-acetate buffer, pH 4.0. The protein solution is centrifuged after dialysis and filtered through a 0.22 µm sterile filter. During the chromatography, the optical density of the eluate was observed at 280 nm using UV-1 detector (Pharmacia, Uppsala, Sweden). The protein concentration in the eluate fractions was determined by the Bradford method (Bradford, 1976). The Western blot using the monoclonal antibodies against G-CSF is used for the identification test (Fig.1). The quality of the purified protein is checked by reversed-phase high-pressure high performance chromatography (RP-HPLC) (Fig.2). Example 3 Determination of biological activity of dGCSF using a cell line proliferation assay

The biological activity of the recombinant dimeric G-CSF protein is determined using the 35-mouse myeloid leukemia line G-NFS-60 [Weinstein Y, Ihle JN, Lava S, Reddy EP, Truncation of the c-myb gene by retroviral integration in an interleukin 3-dependent. myeloid leukemia cell line. Proc. Natl. Acad. Sci. USA, 1986, vol. 83, p. 5010-5014]. The active G-CSF 12 protein promotes proliferation of this cell line after interaction with the G-CSF receptor on the surface of these cells. For the determination of the biological activity of dimeric G-CSF, cells G-NFS-60 are cultured in 96 well plates 48-72 or by adding different amounts (0.1-10 ng / ml) of the GCSF protein sample to the growth medium. Control G-CSF 5 Neupogen ™ (the active substance Filgrastim), whose biological activity is known, is used for monitoring. Cell proliferation is assessed by colorimetric method using MTS dye. The number of living cells is directly proportional to the biological activity of G-CSF. Data on biological activity of dimeric G-CSF protein are presented in Table 3.

The results obtained suggest that using the protein structures and purification techniques provided by the invention, a biologically active dimeric G-CSF protein is obtained. Table 3

Biological activity of dimeric GCSF (dGCSF) samples

No. Preparation Biological Activity TV / ml TV / mg 1. dGCSF L3 3.7-106 2.3-107 2. dGCSF-L5 21.7-106 1.57-107 3. dGCSF-L7 10.8-106 2, 16-10 '4. dGCSF-La 7.05-106 3.064-10' 15 Example 4

Pharmacokinetic study of serum levels of dGCSF

In the pharmacokinetic evaluation of recombinant proteins, experimental animals (Wistar rats) were divided into groups of 4-5 rats each. The test and control proteins were administered subcutaneously. The following amount of recombinant protein was used to calculate the body weight of the animal: 100 to 1100 pg / kg. Neupogen ™ (Filgrastim) was used for control at the same protein concentration as the animal's body weight. Blood serum was collected from animals at 3, 6, 12, and 18 hours after exposure to test or control proteins to test for recombinant protein levels. To this end, about 0.8-1.0 ml of blood was taken from the rat vein vein, which was 1 to 25 1.5 or more. incubated at room temperature followed by 12 or. - in a refrigerator (+ 4 ° C). For serum separation, the samples were centrifuged for 15 min. 1500 rpm at room temperature, serum from each tube was collected in 2-3 sterile tubes and frozen in a freezer (-80 ° C). Blood serum was tested according to the manufacturer's instructions using R & D company human G-CSF protein detection kits DuoSet for blood serum 30 analysis by ELISA. The results are shown in FIG. 3. 13 Example 5

Pharmacokinetic study of the biological effects of dGCSF

In the pharmacokinetic evaluation of recombinant proteins, experimental animals (Vistar rats) were divided into groups of 4-5 rats each. The test and control proteins were administered subcutaneously. The following test recombinant protein was used to calculate the body weight of the animal: 100 to 1100 µg / kg. Neupogen ™ (Filgrastim) was used to control the body weight of the animal at the same protein concentration. Blood serum was collected from animals at 24, 48, 72, 96 hours after exposure to test or control proteins to test for recombinant protein levels. Blood tests were performed using the Hemavet 950 blood analyzer. The pharmacodynamic effect of recombinant protein on the changes in the number of blood neutrophils (granulocytes) was measured before and after the recombinant protein injection. For this purpose, 20 pi of blood, mixed with 5 μΙ 7.5% EDTA, was taken from the rat vein vein and incubated for 10 min. and analyzed by said apparatus. The results are shown in FIG. 4, from which we conclude that the effect of dimeric protein (increase in neutrophil count) exceeds the control. From the examples given, it can be concluded that the tested dimeric GCSF protein structures have biological activity in vitro and in vivo similar to or greater than the control monomeric G-CSF protein (Neupogen ™), and the new G-CSF protein forms have valuable pharmacokinetic properties such as prolonged lifetime in experimental animals in comparison to control monomeric G-CSF protein (Neupogen ™). The pharmacokinetic profile of new therapeutic proteins differs significantly from control: the serum serum of dimeric protein increases by at least six or. (Fig. 3) compared to the control monomeric protein, and the biological effect of dimeric proteins in increasing the number of neutrophils is up to 72 hours when the control product is observed up to 48 or. after the injection (Fig.4).

Claims (5)

DEFINITION OF THE INVENTION 1. Linear oligomeric recombinant proteins composed of genetically linked monomers of the same protein, where the monomer is a granulocyte stimulating protein, and the monomers are linked by a linker amino acid residue. Linear oligomeric proteins according to claim 1, wherein the granulocyte stimulating protein is a granulocyte colony stimulating factor G-CSF and the number of monomers is two. Linear oligomeric proteins according to claim 1 or 2, wherein the linker sequence is selected from the group of amino acid sequences consisting of (S-G4) nS, where n = 2-7 and SGLEA- (EAAAK) m -ALEA- (EAAAK) m. -ALEGS, where m = 2-8. Linear oligomeric proteins according to any one of the preceding claims, characterized by prolonged lifetime in vivo. A pharmaceutical composition comprising an effective amount of a linear oligomeric protein according to any one of the preceding claims, preferably a linear G-CSF dimer, and a pharmaceutically acceptable carrier and / or excipient.