Methods and drugs to stimulate growth and differentiation of megakaryocytes

 

(57) Abstract:

The invention relates to the field of medicine and biotechnology, namely to new proteins, which factors in the growth and development of megakaryocytes (MGDFs; mostly labeled Mp1-ligands), the biological activity of which is to stimulate the growth of megakaryocytes and their differentiation or maturation, which ultimately leads to the formation of platelets. The invention relates also to derived MGDF containing molecules MGDF, United with water-soluble polymers such as polyethylene glycol, and to methods of producing such derivatives. In addition, the invention relates to methods for MGDFs in the form of homogeneous preparations from natural sources, as well as to methods for MGDFs of mammals (including humans) using recombinant genetic engineering technologies. The advantage of the invention lies in the fact that these proteins have therapeutic applications due to their increased stability and time of their circulation, and chemical modification of these proteins prevents their degradation. 16 s and 23 C.p. f-crystals, 25 ill., 12 table.

The invention relates to a new protein, designated as the Mpl-ligand or MGDF, which leads to an increase in the number of platelets. In addition, the invention relates to methods of producing these proteins in homogeneous form from natural sources and methods of genetic engineering.

On the other hand, the present invention is closely related to the new class MGDF-derivatives, in which the molecule MGDF is connected with a water-soluble polymer. Also described methods for obtaining such derivatives. In addition, in the present invention are described MGDF-derivatives, in which the molecule MGDF is connected to one or more polietilenglikolya ("PEG") groups, as well as methods for their preparation.

The invention concerns at least two broad areas of research. The first of them is the development of megakaryocytes with subsequent formation of platelets, and the second properties of the polypeptide included in the family of receptors for growth factors, designated as Mpl-receptor and its ligands. Each of these research areas will be discussed below in more detail.

A. the Formation of platelets from megakaryocytes

The platelets of the blood is circulating cells, which play an important role in preventing blood loss and blood clotting. The megakaryocytes are the cellular source of platelets and originate from a common bone-Cerebro is known as pluripotent stem cell or PPSC.

Hierarchy megakaryocytic precursor cells was determined on the basis of the time of occurrence and size megakaryocytic (MK) colonies formed in culture systems in vitro in response to exposure of the respective growth factors. Burst-forming megakaryocytes cell (BFU-MK) is the most primitive megakaryocyte cell-precursor. It is believed that BFU-MK gives rise to numerous colony-forming the megakaryocytes (CPU - MK), which is a more differentiated MK precursor cells.

As MK cells undergo successive differentiation, they lose the ability to mitosis, but also acquire the ability to endoreduplication. Endoreduplication (or indomitus) is the phenomenon of nuclear fission in the absence of cell division. Endoreduplication inevitably leads to the formation of polyploid MK cells. Further maturation of MK leads to the acquisition of cytoplasmic organelles and components of the membrane, characteristic of platelets.

Platelets are formed from a Mature MK through poorly studied process, which, as it is a consequence of the physical fragmentation MK. There are other possible mechanisms. Detection is, according to which the separating membrane system separates the formed platelet inside the cell. Another model of clot formation is based on the fact that the megakaryocytes form long cytoplasmic processes, roughly corresponding to the diameter of the size of the platelet. Presumably, these processes and detached under the action of platelets in the blood flow in the bone marrow and/or in the lungs. These cytoplasmic processes were named Becker and Debruno prothrombintime to emphasize their intended role of precursors in the formation of platelets. CM.Becker and DeBruyn, Amer.J.Anat. 145: 183 (1976).

In Fig. 1 presents various precursor cells involved in the development of megakaryocytes and platelets. The leftmost cell of Fig. 1 - PPSC, to the right of it - BFU-MK, followed by CFU-MK. Cell undergoing the endoreduplication and located immediately to the right of PPSC is a Mature megakaryocyte cell. As a result of endomitosis this cell becomes polyploid. Further to the right shows the structure with long cytoplasmic processes, formed from a polyploid nucleus Mature megakaryocyte cells. In the rightmost part of Fig. 1 izobrazheniia are some important publications relevant to the stated description of the maturation of megakaryocytes and education platelet count:

1. Williams, N and Levine, R. F., British Journal of Haematology 52: 173-180 (1982).

2. Levin, J., Molecular Biology and Differentiation of Megakaryocytes, pub. Wiley-Liss, Inc.: 1-10 (1990).

3. Gewirtz, A. M. The Biology of Hematopoiesis, pub.Wiley - Liss, Inc.: 123-132 (1990).

4. Han, Z. C., et al., Int.J.Hematol. 54: 3-14 (1991).

5. Nieuwenhuis, H, K. and Sixma, J., New Eng.J. of Med. 327: 1812-1813 (1992).

6. Long.M., Stem Cells 11: 33-40 (1993).

C. Regulation of clot formation

A large amount of data obtained in different laboratories indicates that the formation of platelets is regulated by humoral factors. The complexity of this biological process was initially underestimated, but at the present time, it became clear that a number of human growth factors possess such ability.

Regulation of growth of megakaryocytes occurs at multiple cellular levels. Some cytokines enhance the formation of platelets by increasing the pool of progenitor cells. Another group of humoral growth factors is the maturation factors, acting on more differentiated cells and accelerating the endoreduplication. In addition, in the regulation of these processes, there are two independent who anywayt an important impact on the maturation of MK.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, IL-11, the suppression factor of leukemia (LIF) and erythropoietin (EPO) is independently and individually to accelerate the maturation of MK in vitro, what is judged by the size of the MC, their number and ploidy. The effects of LIF, IL-6 and IL-11 on the maturation of MK partially (LIF and IL-6) or fully (IL - 11) supplements such action of IL-3. The data in the following publications suggest that to enhance the maturation of MK in vivo may be a combination of cytokines.

Here are some of the major publications devoted to the regulation of the formation of megakaryocytes and platelets.

7. Hoffman, R. et al. Blood Cells 13: 75-86 (1987).

8. Murphy, M. J. , Hematology/Oncology Clinics of North America 3 (3): 465-478 (1988).

9. Hoffman, R., Blood 74 (4): 1196-1212 (1989).

10. Mazur, E. M. and Cohen, J. L., Clin.Pharmacol.Ther., 46(3): 250-256 (1989).

11. Gewirtz, A. M. and Calabretta. B., Int.J.Cell Cloning 8: 267-276 (1990).

12. Williams, N., Progress in Growth Factor Research 2: 81-95 (1990).

13. Gordon, M. S. and Hoffman, R., Blood 80 (2): 302- 307 (1992).

14. Hunt, P. et al., Exp.Hematol.21: 372-381 (1993).

15. Hunt, P. et al., Exp.Hematol.21: 1295-1304 (1993).

It was also reported about (see reference 16), the human aplastic serum contains megakaryocyte colony-stimulating aktivirovannoi environment lymphocytes. However, determining the activity of the molecule is not selected and is not described up to the present time.

16. Mazur.E.M., etal. Blood 76:290-297 (1990).

C. Receptor Mpl

Myeloproliferative leukemia virus (MPLV) is replication defective retrovirus mice, which infected them mammals acute leukemia. It is shown that expressed MPLV gene consists of a fragment of the gene encoding the retroviral surface protein associated with the sequence of related receptors of cytokines, including the receptors for GM-CSF, G-CSF and EPO.

Expression of the indicated gene MPLV in mouse cells - the precursors of different types leads to rapid acquisition of independence from growth factors, which are normally required for proliferation and final maturation. In addition, the fact that in some cultures, bone marrow cells, transformed MPLV, some megakaryocytes, specifies the relationship between the genome MPLV and growth and differentiation of megakaryocytes.

Currently, it is shown that viral gene MPLV (designated as v-Mpl) is homolog in mammalian cells, called cell genome Mpl (or c-Mpl). Using samples on the basis of the v-Mpl managed preklonyau sequences showed the protein encoded by the gene of c-Mpl, belongs to the highly conserved superfamily of receptors of cytokines, as well as homologous to the gene product of the v-Mpl.

Conclusion about the functional role of cellular gene c-Mpl in hematopoesis is based on the fact that its expression is detected in the bone marrow, spleen and fetal liver of normal mice, but not in other tissues. In particular, c-Mpl is expressed in megakaryocytes. It is also shown that the human c-Mpl is expressed in CD34-positive cells, including treated megakaryocytes and platelets. CD34 is an antigen characteristic of early hematopoietic precursor cells. In addition, treatment of CD34-positive cells by synthetic oligonucleotides, antimuslim in relation to c-Mpl mRNA, significantly inhibited colony-forming ability of CFU-MK megakaryocytic predecessors, but has no effect on erythroid and granulocyte-macrophage precursors.

The results indicate that c-Mpl encodes a cell surface molecule, designated as Mpl-receptor. The binding of the ligand activates the receptor and probably leads to the formation and/or development of megakaryocytes.

mouse. The product of this gene, which, as mentioned, is the receptor consists of at least three areas or domains: the extracellular domain, transmembrane domain and intracellular (or cytoplasmic) domain. Together, these domains are intact receptor Mpl. In the above-mentioned PCT publication also refers to a soluble form of the receptor, which largely corresponds to the extracellular domain of the Mature protein c-Mpl. The intracellular domain contains a hydrophobic region, and when connecting it via the transmembrane domain from the extracellular domain of the entire protein becomes insoluble. On the other hand, when the extracellular domain of the gene product of c-Mpl is separated from the transmembrane and intracellular domains, the protein becomes soluble, therefore, the extracellular form of the protein designated as "soluble" form of the receptor.

The following are the main publications receptor v-Mpl and c-Mpl and their genes.

17. Wendling. F., et al., Leukemia 3 (7): 475-480 (1989).

18. Wendling. F., et al., Blood 73 (5): 1161-1167 (1989).

19. Souyri. M.,et al., Cell 63: 1137-1147 (1990).

20. Vigon. l., Proc.Natl.Acad.Sci USA 89: 5640-5644 (1992).

21. Skoda. R. C., et al. The EMBO Journal 12 (7): 2645-2653 (1993).

22. Ogawa. M., Blood 81 (11): 2844-2853 (1993).

obnam to encourage the formation of platelet

There are reports that the transfusion of platelets are produced in ever-greater number of cases in medical centres in North America, Western Europe and Japan. see Gordon, M. S. and Hoffman, R., Blood 80 (2): 302-307 (1992). This is largely due to improvements in medical techniques and the spread of new technologies in cardiac surgery and bone marrow transplants, heart and liver. Increasing doses of chemotherapeutic drugs in the treatment of cancer patients and the spread of HIV - 1 infection has also contributed to the increasing demand for platelets.

Transfusion of platelets carries with it the spread of many diseases, blood-borne, as well as the possibility of alloimmunization. In addition, getting treated platelets procedure is expensive and increasingly frequent use leads to an increase in total medical costs. Therefore there is an urgent need for new and improved methods to obtain platelet transfusion people.

Here are some examples of experimental approaches to increase the formation of platelets.

In U.S. patent N 5,032,396 describes the ability of interleukin-7 (IL-7) to encourage the formation of trsi growth predecessors of T - and B-cells in the bone marrow. In published PCT application 88/03747 (filed October 19, 1988) and in the application for the European patent 88309977.2 (filed October 24, 1988) described DNA vectors and the necessary ways to obtain IL-7 mammal by methods of genetic engineering. The data presented in U.S. patent suggests that IL-7 can increase the number of circulating platelets in normal and sublethal irradiated mice.

In U.S. patent N 5,087,448 shown that the megakaryocytes and platelets mammals can be stimulated to proliferation by interleukin-6 (IL-6). Recombinant human IL-6 is a glycoprotein with a maul. weight of 26,000, with multiple biological activities. Presented in this patent data indicate that IL-6 increases the education megakaryocytic colonies in in vitro tests.

None of the cited patents are not mentioned Mpl-ligand, described in this invention.

Despite these achievements, the need for new stimulants of megakaryocytes and/or platelets in mammals remains acute.

That is the prerequisite of obtaining a chemically modified MGDF

Proteins for therapeutic use in the present BP is abinanti DNA. Chemical modification of these proteins can effectively prevent physical contact of proteolytic enzymes to the protein frame, thereby preventing degradation. Under certain conditions, may occur and other benefits, such as increasing the stability and circulation time of therapeutic protein and decrease its immunogenicity. However, it must be emphasized that the effect of modification in respect of each specific protein is difficult to predict. Francis published review article is devoted to the modification of proteins, including fusion: Focus on Growth Factors 3:4-10 (May, 1992) (published by Mediscript, Mountview Court, Friern Barnet Lane, London N20, OLD UK).

Polyethylene glycol ("PEG", "peg" or " PEG) is one of the chemicals used for the preparation of pharmaceutical forms of therapeutic proteins. For example, Adagen-R, the drug peyrovani the adenozindezaminazy recommended for the treatment of severe combined immunodeficiency; peyrovani superoxide dismutase undergoing clinical trials for treatment of head injuries; peyrovani alpha-interferon has passed phase 1 clinical trials for the treatment of hepatitis; peyrovani glucocerebrosidase and peyrovani hemoglobin are under ERISA (Sada et at., J. Fermentation Bioengineering 71: 137:139 (1991)), and known methods of attachment of polyethylene glycol. Cm. U.S. patent N 4,179,337, Davis et al., "Non-immunogenic polypeptides", issued December 18, 1979, and U.S. Patent N 4,002,531, Royer Modifying enzymes polyethylene glycol and the products of this process", issued on 11 January 1977. As an overview, see Abuchowski et al., in Enzymes as Drugs (J. S. Holcerberg and J. Roberts, eds.pp. 367-383 (1981)).

For modification of proteins used by other water-soluble polymers such as a copolymer of polietilenglikol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, copolymer ethylene/maleic anhydride and polyaminoamide (as homopolymers and random copolymers).

A number of approaches have been used to attach molecules of polyethylene glycol to the protein. Usually the molecules of the polyethylene glycol is attached to the protein by one of the active groups of the protein. Suitable for this accession are amino groups, for example amino groups of lysine residues or the amino group of the N-end. For example, Royer (U.S. Patent N. 4,002,531) used restorative alkylation to attach molecules of polyethylene glycol to the enzyme. In the European patent N 0539167 "Emiliania with a free amino group (or groups), modified imidate derivatives Page or other water-soluble organic polymers. In U.S. Patent No 4,904,584, issued February 27, 1990, the Show describes the modification of lysine residues in proteins to attach molecules of polyethylene glycol through a reactive amino group.

One of therapeutic protein, which was chemically modificirowan, it is granulocyte colony-stimulating factor, G-CSF". Cm.EP N 0401384, EP N 0473268 and EP N 0335423.

Another example is peyrovani IL-6 (see European patent N 0442724, entitled "Modified hlL-6", as well as related application U. S. 07/632,070, which describes a molecule of polyethylene glycol is attached to IL-6. In the European patent N 0154316, published on September 11, 1985, describes the interaction of lymphokine with aldehyde peg.

The possibility of modifying MGDF is unknown, since it depends on the specific structural parameters of each specific protein. In addition, the unpredictable impact of such modifications on the biological properties of individual protein. Due to the many clinical applications MGDF described in this application, it would be desirable to obtain a derivative of this protein with the modified t other properties.

Peyrovani protein usually leads to the formation of a mixture of molecules of chemically modified protein. For example, protein molecules with five lysine residues and a free amino group at the N-end when peyrovani by the described method can form a heterogeneous mixture in which some of the protein molecules will have six attached molecules Page, some five, some four or three or two or one, or none at all. This polietilenglikolya groups can be attached to different molecules of protein in different areas.

It is often desirable to obtain a homogeneous product containing essentially only one or a small number (2-3) of modified forms of the protein, different number and/or localization polietilenglikolya groups. However, under certain therapeutic indications may be desirable or suitable mixtures of mono-, di-and/or three-peyrovani forms.

The volatility of the mixture from batch to batch is a disadvantage when developing therapeutic paglialunga protein. In this case, important predictability of biological activity. For example, it is shown that under non-selective conjugation of superoxide dismutase with polyethylene glycol individual fractions of modifica Chemistry 2: 154-159 (1991), which shows selective attachment of the linker group carboxylicacid to-end carboxyl group of the protein substrate (insulin). If therapeutic protein is not constant in composition from batch to batch, the predictability of its properties unattainable. Individual molecules of polyethylene glycol in certain areas can be attached weaker than others, which can lead to dissociation of Page and protein. Obviously, if the molecules of Page randomly attached to the protein and dissociate accidentally, the pharmacokinetics of therapeutic protein to predict accurately is impossible.

Also it is desirable to obtain such derived MGDF, in which there was no connecting link between the polymer and protein. One of the problems associated with the use of the above methods is that usually need some kind of connecting link between a protein and a molecule of polyethylene glycol. This link may be an antigen that is also a drawback in the development of therapeutic protein drug.

Methods without the participation of the connecting groups described Fransis et al., in kN. "Stability of protein pharmaceuticals: the path of degradation in vivo and strategy one hundred and with trail-chloride, use in immunoaffinity cell preparations and Fisher et al., edited Separation using systems with the aqueous phase. Applications in cell biology and biotechnology. Plenum Press, N. Y., 1989, pages 211-213, which describes the use of trail-chloride, which leads to the absence of connecting groups between peg and protein. This method is difficult to apply to obtain therapeutic drugs, because the use of trail-chloride leads to the formation of toxic by-products.

Chamow et al.,. Bioconjugate Chem. 5: 133-140 (1994) described a modification of the CD immunoadhesin aldehyde mono-methoxy poly (ethylene glycol) ("MePEG) via reductive alkylation. The authors report that 50% of CD4-lg was modified MePEG in selective reactions at the alpha-amino group of N-Terminus (see CIT.source, page 137). The authors also report that the ability of the modified CD4-lg contact in vivo protein gp 120 decreased depending on the degree of binding MePEG.

Thus, there is a need for derivatives MGDF, in particular in peyrovani MGDF. There is also a need to develop methods for obtaining such derivatives.

In the present invention claimed new polypeptides, specifically Wuxi from other proteins (i.e., proteins mammals in the case of Mpl - ligand, obtained from relevant sources). These proteins can be isolated from cells that produce factors naturally or due to the induction of other factors. They can also be obtained by the methods of genetic engineering. In addition, the Mpl - ligand can be synthesized by chemical methods or obtained by combining the above approaches.

Claimed in the present invention Mpl-ligand can be obtained in the native form of the cells and tissues of mammals. In the Examples section of this application describes two Mpl ligand isolated from aplastic plasma of dogs. However, as described in other Examples of the present application, closely related Mpl-ligand present in aplastic plasma of humans and pigs. It is noteworthy that the activity of human, porcine and canine Mpl-ligand specifically inhibited soluble form of the murine Mpl receptor, indicating a strong similarity of these Mpl-ligand both in terms of structure and function.

Mpl-ligand humans, pigs and other mammals can be isolated from natural sources using the methods described in this application (see Example 10). Thus, and rabbit. Especially preferred are the Mpl-ligand dogs, pigs and humans.

In addition, the genes encoding the Mpl-ligand man, were cloned from libraries of embryonic kidneys and liver. These genes prosecution, as described in the Examples section of this application. It is shown that two polypeptide sequences person have activity in cell tests (see Example 4). They differ in length, but are identical in a large part of their amino acid sequences. The same areas have homology with erythropoietin. Mpl-ligand are indicated and as factors of growth and development of megakaryocytes (MGDFs); all references to the Mpl-ligand have the same attitude and to MGDFs, and Vice versa. "Polypeptide MGDF" means a polypeptide having the ability to specifically stimulate or inhibit the growth and/or development of megakaryocytes. Examples of such polypeptides described in this application.

Described in the present invention Mpl-ligand show a specific activity in relation to megakaryocytes the germ blood, increasing maturation and/or proliferation of megakaryocytes, as shown in Examples 2 and 4 below. The term "specific" means that the biological activegroonga types. Those factors, which have a stimulating effect on megakaryocytes, have in vivo activity, stimulating the formation of platelets. This occurs through the stimulation of the maturation and differentiation of megakaryocytes.

Two preferred Mpl ligand of canine origin have a molecular weight of about 25 KD and 31 KD, as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) in non conditions. Both proteins were purified by the same methodology described in the Examples section.

Two preferred ligand of human origin, MGDF-1 and MGDF-2, are composed 332 and 173 amino acids and do not include a 21-amino acid of the intended signal peptide.

Another aspect of the present invention are processes for isolation and purification of these Mpl-ligand or fragments from natural sources, preferably whole blood, serum or plasma of mammals. As the source material is the most preferred aplastic blood, serum or plasma. They can be obtained using irradiation procedures mammals, for example, a dose of about 400-800 happy with cobalt-60, which makes daimere 1. In the case of a person aplastic blood, serum or plasma can be obtained from patients undergoing radiotherapy, for example, with cancer.

Then aplastic blood, serum or plasma are subjected to a cleaning process. Claimed in the present invention the cleaning process consists of several key steps: affinity chromatography with pectin and affinity chromatography with Mpl receptor. Each of these stages provides approximately 300-500-fold purification of 25 KD and 31 KD protein from aplastic plasma of the dog. Other standard methods of protein purification may be used together with the above for further purification Mpl-ligand, as described below.

Another aspect of the present invention are polynucleotide encoding a protein Mpl-ligand mammals. Such DNA sequences may include selected DNA sequences that direct the expression of proteins Mpl-ligand mammals, as described next. These DNA sequences may also include 5' and 3' non-coding sequence mammals, langeroudi coding sequences Mpl-ligand. In addition, DNA sequences can encode aminoterminal the hydrated or partial chemical synthesis. Codons can be optimized for expression in a particular host (e.g. E. coli or CHO cells).

The present invention also stated recombinant DNA molecules, each of which is a vector DNA, linked with DNA sequences encoding the Mpl-ligand mammals. In these recombinant DNA molecules Mpl - ligand is functionally linked with a regulatory sequence that provides replication and expression of Mpl-ligand in certain cells of the host. Cell owners (e.g., bacteria, insect, mammalian, yeast or plants) transformed with such DNA molecules with the purpose of expression of recombinant protein Mpl-ligand, are also subject of this invention.

DNA molecules and transformed cells, as claimed in the present invention, can be used to produce recombinant protein Mpl-ligand mammals or its fragments. The cell line transformed with DNA sequences encoding the Mpl ligand or fragment (or the above-mentioned recombinant DNA molecules) in functional Association with appropriate regulatory sequences, cultivated in sootvetstvuetopredelennyj as host cells for expression of the protein. Preferred to obtain the Mpl-ligand are mammalian cell lines (e.g., CHO cells) and bacterial cells (e.g. E. coli).

To obtain the Mpl-ligand in cells of E. coli preferably, N is the end of the expressed protein was the remains of Met and Lys, as in this case, the output of the expression product is usually higher. Human MGDF consists of 165 amino acids (i.e., Met-2 Lys-1 [1-163] MGDF (when counting from the first amino acid of the Mature protein). After purification of the product, expressed in such bacterial cells like E. coli, terminal Met-Lys residues can be removed by processing any dipeptidase (for example, cathepsin S).

Downregulation of protein Mpl-ligand is then isolated from the host cells, cell lysate or culture medium by one of the usual methods. The conditioned medium can be subjected to the same cleaning steps (or their modifications) Mpl-ligand, and aplastic plasma.

Another object of the present invention are recombinant Mpl-ligand. These proteins, essentially free from other biological structures of animal origin, in particular proteins. Claimed in the present invention Mpl-ligands are characterized by one or Bo in the application.

The present invention also stated chemically modified MGDF, consisting of actual protein MGDF connected to at least one water-soluble polymer, and methods for obtaining and using such compositions. In particular, the claimed such chemically modified MGDF, which by reaction with ethylene glycol to MGDF attached PEG. Such connection can be achieved such as described below with reactions peyrovani as acylation or alkylation. Acylation or alkylation with Peg may be conducted under such conditions that the final product will be monopegylated or paliperidone. When paliperidone usually attaching Page to psilonerou lysine residues, this also may peyrovani N-Terminus of the protein. When monopedigree preferably occurs accession Page to the alpha-amino group of N-Terminus of the protein. The yield and homogeneity of the reaction product of montegiove can be increased by means of such a reducing alkylation, in which the selectively modified alpha-amino group of the N-end MGDF protein, thereby providing selective attachment of the water-soluble polymer to the N-end of the protein. This Podpolkovnik Page) drugs paglialunga MGDF protein, in which the glycol is directly connected with protein.

The present invention also claimed pharmaceutical preparations (compositions) containing a therapeutically effective amount of purified natural or recombinant Mpl-ligand, which can be modified in such a water-soluble polymer, such as polyethylene glycol, together with a pharmaceutically acceptable carrier, diluent or buffer. Data pharmaceutical drugs can be used to treat such medical conditions or disorders that are characterized by lack of megakaryocytes and/or platelets, as well as the lack of Mpl-ligand in vivo. They can also be used prophylactically to compensate for the expected loss of megakaryocytes or platelets (e.g., during surgery).

Thus, as claimed in the present invention Mpl - ligand can be used to treat aplastic anemia, for example, to enhance the formation of platelets for patients (for example, AIDS patients or in patients undergoing cancer chemotherapy). Mpl - ligand can be used for the treatment of blood disorders like thrombocytopenia. Mpl-ligand can be castledawson. Mpl-ligand, obtained from one species can be applied to the patients of another species.

Another object of the present invention is a method of treatment of other pathological conditions characterized by lack of platelets, by introducing the patient a therapeutically effective amount of the above pharmaceutical drug. Such therapeutic methods may involve the introduction simultaneously with the Mpl-ligand or sequentially an effective amount of at least one megakaryocyte colony-stimulating factor, cytokine (e.g., EPO), soluble Mpl receptor, hematopoietin, interleukin, growth factor or antibodies.

The present invention also stated antibodies (polyclonal, monoclonal, humanized or recombinant), and antibody fragments obtained (i.e., reactive) against the Mpl - ligand mammals or its fragment. Accordingly, in the present invention includes cells able to secrete these antibodies (i.e. hybridoma in the case of monoclonal antibodies, methods of producing such antibodies and their use in diagnostic and therapeutic purposes.

Drought to be used antibodies specifically recognizes the Mpl-ligand, as in "sandwich" form, in and of themselves. A similar approach can be used to determine whether the introduction of patient Mpl-ligand and/or identifying in the patient a deficiency of platelets. Reagents for the production of such tests can be collected in a set that includes positive and negative control antibodies and other standard components such sets.

Other aspects and advantages of the claimed invention will become apparent from the subsequent detailed description of the variants of its implementation.

Numerous advantages of the present invention will be more apparent upon consideration of the following drawings:

Fig.1 reflects the development and maturation of megakaryocytes and platelets.

Fig.2 shows that soluble murine Mpl receptor is almost completely suppresses the ability of the plasma irradiated dogs ("aplastic dog" or "ARC") to induce the development of megakaryocytes. Test the development of megakaryocytes described in example 2.

Fig. 3 shows that the activity ARC enriched by affinity chromatography with lectin and affinity chromatography with Mpl - receptor ("Mpl ligand"), stimulates R is surface cleaning 25 KD and 31 KD forms Mpl-receptor dogs from aplastic plasma of the dog.

Fig.5 shows the purification of Mpl-ligand back-phase chromatography high resolution (RP-HPLC). In fraction 21 contains highly purified 31 KD Mpl - ligand; fraction 22 contains a mixture of 31 KD and 25 KD Mpl-ligand; fraction 23 contains highly purified 25 KD Mpl-ligand.

Fig. 6 shows a comparison of the activities of the Mpl-ligand in the fractions obtained RP-HPLC (C4 column) and containing 25 KD and/or 31 KD protein Mpl-ligand.

Fig. 7 represents the number of megakaryocytes formed in cultures of CD34-positive peripheral blood cells stimulated APK9, Mpl-ligand, and various other factors.

Fig. 8 reflects the total number of lymphocytes formed in cultures of CD34-positive peripheral blood cells stimulated APK9, Mpl-ligand, and various other factors.

Fig. 9 indicates the percentage of megakaryocytes formed in cultures of CD34-positive peripheral blood cells stimulated APK9, Mpl-ligand, and various other factors.

Fig. 10 shows that IL-3 persons not involved in induced Mpl-ligand development of megakaryocytes.

Fig. 11 represents the cDNA sequence and predicted amino acid sequence of MGDF-1 and outermost MGDF-3 people.

Fig. 13 shows a comparison of MGDF-1 and MGDFs (Mpl-ligand) dog (a) and mouse (B) origin.

Fig. 14 shows an example of acylation MGDF using N-hydroxysuccinimidyl (NHS) active esters of monomethoxy-glycol to obtain paliparang product.

Fig. 15 shows an example of nonspecific reductive alkylation MGDF using aldehydes, monomethoxy-glycol to obtain paliparang product.

Fig. 16 shows an example of site-specific reductive alkylation MGDF on the alpha amino group of the N - terminal residue using the aldehydes of the mono - methoxypolyethyleneglycol to obtain essentially montegiove product.

Fig. 17 shows HPLC analysis on the basis of the exceptions in size (SEC HPLC) conjugates MePEG-MGDF obtained with the use of activated derivatives of MePEG (mol. weight of 20 KD:

A. poly-MePEG-MGDF-conjugate, obtained by acylation of MGDF NHS-ester MePEG (PEG11);

B. poly-MePEG-MGDF-conjugate, obtained by alkylation MGDF MePEG aldehyde (PEG 20);

Century poly-MePEG-MGDF-conjugate, obtained by alkylation MGDF aldehyde MePEG (PEG 16).

Fig. 18 shows the number of the th in CHO cells; unfilled circles - nefariously MGDF 22-184 obtained in E. coli (i.e., MGDF 1-163); full circles - peyrovani MGDF 22-184 obtained in E. coli.

Fig.19 shows the purification scheme r-HuMGDF.

Fig.20 on mice shows the effect of r-HuMGDF (E. coli 1-163) on the platelet count. On day 0 mice Batb/c administered intraperitoneally once was administered carboplatin (1.25 mg/mouse). Control group mice were injected solvent instead carboplatin. After 24 hours the animals treated with carboplatinum, was subcutaneously injected solvent or 100 µg/kg r-HuMGDF. Injections were performed daily until the end of the experiment (n=10 for each group; at each time point, blood was taken from five animals).

Fig.21 shows the effect of r-HuMGDF (E. coli 1-163) on platelet counts in irradiated mice. On day 0 mice Balb/c mice received a single dose of 500 rad of gamma radiation (cesium source). A control group of mice to radiation were not exposed. After 24 hours, irradiated animals was subcutaneously injected solvent or 100 µg/kg r-HuMGDF. Injections were performed daily until the end of the experiment (n=8 for each group; at each time point, blood was taken from five animals).

Fig. 22 shows the effect of r-HuMGDF (E. coli 1-163) on the platelet count in mice, under the zu 500 rad of gamma radiation (cesium source) and a single dose of carboplatin (1.25 mg/mouse). After 24 hours the treated animal was subcutaneously injected solvent or 100 µg/kg r-HuMGDF. Injections were performed daily until the end of the experiment (n= 8 for each group). Most of the animals not treated with r-HuMGDF, did not survive. In the control group survived 1 animal out of eight. In the experimental group survived 8 animals from 8.

Fig. 23 shows the effect of r-HuMGDF (E. coli 1-163) on radiation-induced thrombocytopenia in Makarov RH. Monkeys received a dose of 700 cGy, 80. Within 18 days 24 hours after irradiation, the animals were injected subcutaneously r-HuMGDF (n= 3) or serum albumin human (n=9) (each at a dose of 25 mg/kg/day). Analysis of blood cells was measured with an electronic analyzer. Each icon represents the mean value (+/- mean deviation).

Fig. 24 shows the effect peyrovani and glycosylated r-HuMGDF on platelet counts in mice treated with carboplatin and irradiation. Mice were subjected to the combined effect of carboplatin and irradiation, as described in the experiment shown in Fig.22. Subcutaneous injection of the specified drug r-HuMGDF (50 mg/kg/day) produced daily during the whole period of the experiment, starting from 24 hours after the initial treatment. Counting will lnstitut sequence synthetic recombinant human MGDF (amino acids 1-163), with the codons for improved expression in E. coli.

Additional aspects and advantages of the invention will become more clear for professionals working in this research area, from the following description that reveals the feasibility of the use of the invention.

New factors that enhance the growth of megakaryocytes mammals, and/or factors produced by platelets, labeled Mpl-ligand and obtained according to the present invention, represent a homogeneous protein, virtually free of other protein compounds. Preferably, the target proteins were 90% free from other proteins, more preferably 95%, and very preferably, the target proteins were approximately 98% and more free from other proteins. These proteins can be produced in large quantities using recombinant technology so that the final product is a high degree of purity of the active Mpl-ligand, which can successfully be used in therapy. Alternatively, such proteins can be obtained in a homogeneous form of aplastic blood of mammals, plasma or serum, as well as from clerigo active fragments can be chemically synthesized. In General, the term "Mpl-ligand", as used in the present invention, refers to themselves as the Mpl-ligand, and their active fragments and variants, which are described in more detail below.

Two preferred Mpl ligand dogs have a molecular weight of about 25 KD and 31 KD, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) in non conditions. Both protein purified according to one technique, which is described in more detail in the following examples. For example, both the Mpl - ligand bind the lectin wheat germ and immobilized Mpl - receptor. Protein of 25 KD has the following amino acid sequence:

Ala-Pro-Pro-Ala-Xaa-Sap-Pro-Arg-Leu-Leu-Asn-Lys-Met-Leu - Arg-Asp-Ser-His-Val-Leu-His-Xaa-Arg-Leu-Xaa-Gln-Xaa-Pro - Asp-Ile-Tyr (SEQ ID N0:1).

Protein size 31 KD has the following amino acid sequence: Ala-Pro-Pro-Ala-Xaa-Asp-Pro-Arg-Leu-Leu-Asn-Lys-Met-Leu - Arg-Asp-Ser-His-Val-Leu-His (SEQ ID N0:2).

Amino acids "Xaa" in sequences of SEQ ID N0: 1 and 2 are not precisely defined, but presumably are cysteine, serine, threonine, or (less likely) by tryptophan. The above sequence shows that the ligand size 31 KD contains at least a portion of the ligand raslabliatsia and in particular the fact that that both proteins are active in studies of the properties of Mpl-ligand, described in the present invention, allow us to conclude that both proteins are very closely related both in structure and in function. It is likely that protein size form 31 KD differs from the protein of 25 KD structure of the C-terminal sequence, as well as other character glycosylation and/or other character splicing of the gene encoding the proteins. In addition to the above-described sequences in the process of sequencing the 25-KD band until the final stage of purification (using obraniakowi HPLC) was determined by another sequence. It was shown that this sequence is associated with a band of 25 KD in non conditions, but not in reducing conditions, suggesting that it is the result of splitting into two fragments (for example, using protease) 25-KD protein, and these fragments are held together by a disulfide bond. This sequence has the following structure: Thr-Gln-Lys - Glu-Gln-Thr-Lys-Ala-Gln-Asp-Val-Leu-Gly-Ala - Val-Ala-Leu (SEQ ID N0:3).

Although localization sequence SEQ ID N0:3 within the sequence of the 25-KD protein neizvedannostyu is located near 114-th amino acid protein of 25 KD. It should be noted that the likely (but not proven) localization sequence SEQ ID N0: 3 and within the protein size 31 KD, despite the fact that it again starts near the amino acids 114. Information about the sequence described in more detail in Example 7.

Thanks to the original experiments on the purification of the ligands of the dog, described above, was cloned gene that encodes the ligand. The result was determined the complete amino acid sequence of the specified ligand dogs, which is shown in Fig.13A. Based on the molecular mass predicted that the ligands dogs size 25 KD and 31 KD are the C-terminal progressirovanii forms of the ligand full length, whose sequence is shown in Fig.13A. In addition to that received Mpl - ligand mouse having the sequence shown in Fig.13B. Such purified ligands can also be characterized according to the specific activity that is manifested in the study of human megakaryocytes (Example 2), which is at least 5.7 billion megakaryocytic units/ml Megakaryocytes unit defined as the amount of material, from which you can get as many megakaryocytes as from about 1, such purified ligands can be characterized according to the specific activity shown in the study Mpl-dependent growth of cells (Example 4), which is at least 6.9 billion units of cell growth/mg a Unit of cell growth is defined as the amount of ligand required for growth 200 cells A.1 in the study described in Example 4. In Table 1 (see end of description) are more specific indicators of activity of purified Mpl-ligand dogs obtained according to this invention.

Summarizing the above information, as examples can be characterized some of Mpl-ligand on the basis of one or more biochemical and biological properties:

(a) such Mpl-ligand isolated from aplastic plasma;

(b) such Mpl-ligands have a molecular weight of about 25 KD and 31 KD, as determined by the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) in non conditions;

(C) Mpl-ligand contain the following amino acid sequences: SEQ ID N0:1 in the case of protein mol. mass of 25 KD; or SEQ ID N0:2 in the case of protein mol. weight of 31 KD;

(g) the Mpl-ligand optionally contain aminokislotna the n wheat germ;

(e) Mpl-ligand bind immobilized soluble Mpl receptor mouse;

(g) the activity of Mpl-ligand can also be inhibited in vitro soluble Mpl receptor and

(C) Mpl-ligand contact with the anion-exchange column at pH of about 8-9.

The biological activity are preferred Mpl-ligand, obtained according to the present invention, is confirmed by their ability to specifically stimulate the growth and development of megakaryocytes in the study enhance the growth of cells, which is described in Example 2. In the specified experiment Mpl-ligands stimulate the differentiation of CD34+ cells in human peripheral blood (ie, CD34-cells, secreted by immunoadsorption) during 8 days of cultivation. The megakaryocytes identify staining using specific antibodies to platelets and counted under a microscope. In addition, the Mpl-ligand stimulates the growth factor-dependent cell line A.1. In the absence of Mpl-ligand cells die. The number of cells in the culture containing the Mpl-ligand, determined after 2 days. Mpl-ligand, described above, have a specific activity are shown in Table 1 (see above).

Sources of Mpl-ligand are aplastic blood of mammals, Plaza body fluids of mammals, cells derived from such liquids, etc. Clearing of native Mpl - ligand in the allocation of the above sources is based on two main procedures:

(a) affinity chromatography using lectin, mainly agglutinin wheat germ; and

(b) affinity chromatography with immobilized Mpl receptor.

Additional procedures may include further purification of the protein. These include, for example, ion exchange chromatography, gelfiltration chromatography and back-phase chromatography.

The method of purification, in fact, used to allocate the Mpl-ligand from aplastic plasma dogs, includes the following stages (see Example 7):

(a) lectin-affinity chromatography (especially preferred chromatography using agglutinin from wheat germ);

(b) affinity chromatography using soluble Mpl receptor (Mpl-X) (particularly preferably using immobilized Mpl-X mouse);

(C) ion exchange chromatography (cation or anion exchange chromatography, in particular when using column Mono Q);

(g) gelfiltration chromatography under conditions causing dissociation (prepisovanie C4-column).

Homogeneous Mpl-ligand mammals, including ligand person, can be obtained from aplastic blood, serum, plasma or other sources of Mpl - ligand person, such as cells or tissues when using the above methods of cleaning. The necessary cleaning steps do not necessarily follow in the order given, however, the sequence of the respective steps is preferred. Methods of culturing cells or tissues, which can be a source of Mpl-ligand, known to experts in this field of research and can be applied, for example, to increase the amount of starting material.

Mpl-ligand, or one or more peptide fragments can be obtained using recombinant DNA technology. To obtain the DNA sequence of a particular Mpl-ligand, ligand-containing material is concentrated and is usually treated with a protease, such as trypsin. Resulting from enzymatic cleavage fragments is isolated and is sequenced using a suitable method. Alternatively, as shown in the examples below, the intact purified protein can be sequenced directly, to the extent that DOS used to obtain trypticase fragments, according to the following method. Synthesize oligonucleotide samples, using the genetic code to predict all possible sequences of genes encoding amino acid sequence of the sequenced fragments (or fragment). Preferably, the quality of the samples used some degenerate sequence. Gene Mpl-ligand identify when the use of these samples for screening genomic libraries mammals or other sources. In contrast, mRNA from cells, which is the source of Mpl-ligand, can be used to obtain a cDNA library, which sceneroot using samples to identify a cDNA that encodes a polypeptide Mpl-ligand. Moreover, for capacity cDNA sequences can be used polymerase chain reaction (PCR), which involves the appropriate primers.

Using appropriate probes for screening genomic libraries, get a DNA clone. To obtain a clone containing a full-sized copy of the gene Mpl-ligand samples on the basis of the obtained DNA sequences can be used for re-screening libraries and hybridization with a full-sized posledovatelnogo genomic clone of human rights in the expression vector, later transfer it into COS cells, obtaining a cDNA library from these transfected COS cells and screening cDNA Mpl-ligand hybridization. After identification of the full cDNA sequence, she or any of its sections, that encodes an active fragment of Mpl-ligand, can be embedded in any of a large number of well-known expression vectors to obtain a system of expression of Mpl-ligand, or one or more fragments.

This use of recombinant technology preferably receive DNA sequences that determine the synthesis of polypeptides Mpl-ligand. The present invention also relates to the specified sequences that are free from DNA sequences encoding other proteins (i.e., isolated sequences), and provide for the expression of polypeptides Mpl - ligand activity Mpl-ligand (i.e., polypeptides that cause growth and/or development of megakaryocytes). These DNA sequences include those sequences that encode all or any portion of Mpl-ligand, and those sequences that hybridize, preferably in hard hybridization conditions with the cDNA sequences [see Maniatis et al.) - Rev. Yat in 4 x SSC at 62-67oC, followed by cleaning in 0.1 x SSC at 62-67oC. Alternatively, hybridization in hard conditions hold for 45 - 55% formamide, 4 x SSC at 40-45oC. DNA sequences, which hybridize to the sequences of Mpl-ligand under mild conditions and encode peptides Mpl-ligand, having the biological properties of Mpl-ligand, also encode new polypeptides Mpl-ligand, as claimed in the present invention. Typically, hybridization under mild conditions is carried out in 4 x SSC at 40-45oC or 30-40% formamide at 40 - 45oC. for Example, a DNA sequence, which has areas of significant homology (for example, glycosylation or disulfide bonds) with sequences Mpl-ligand and encodes a protein having one or more types of biological activity of Mpl-ligand, explicitly encodes the polypeptide Mpl-ligand, even if such a DNA sequence and will not gibridizatsiya sequence (sequences) Mpl-ligand in tough conditions.

Allelic variants (with the natural changes of bases in the genetic structures of organisms that comprise this population, which do not necessarily lead to replacement of amino acids) DNA-consequently the same as analogues and derivatives of these sequences. Similarly, DNA sequences that encode polypeptides Mpl-ligand, but differ in some codons due to the degeneracy of the genetic code, as well as different variants of the DNA sequence of Mpl-ligand, which is due to point mutations or induced modifications to enhance the activity, the longer half-life or enhance the yield of the polypeptide encoded by such modified sequences, are also included in the scope of the present invention.

In the result of the cloning process described below in Example 11 determine the cDNA sequence and the amino acid sequence of proteins of human MGDF-1, MGDF-2 and MGDF-3, which is shown here in the application materials. MGDF-1 presents the amino acid 22-353 (Fig.11) and contains 332 amino acids. MGDF-2 is a split plot MGDF-1 and contains amino acids 22-195, as shown in Fig.11. MGDF-2, respectively, consists of 174 amino acids. MGDF-3 presents the amino acid 22-289 (Fig. 12) and contains 268 amino acids. When describing each of MGDF mean that the sequence of the signal peptide (amino acids 1-21 of Fig.11 and 12) is also the and the development of megakaryocytes, preferably the removal of the signal peptide. In General, data MGDFs can be summarized as follows:

MGDF-1 amino acids 22-353 Fig.11

MGDF-2 amino acids 22-195 Fig.11

MGDF-3 amino acids 22-289 Fig.12

In the experiments described above MGDF-1 and MGDF-2 are active, while MGDF-3 is inactive.

Based on given here the information about the activity, it can be assumed that human MGDF is expressed in vivo as relatively inactive or less active polypeptide is a precursor containing various C-terminal amino acids. When the removal of C-terminal amino acids (signal peptide) protestirovanny form (s) of the polypeptide becomes active or becomes more active. Considering the above assumption, it is believed that the activity of MGDF-1 necessary processing (e.g., cleavage

the protease). The fact that MGDF-1, from which is derived a specific area (i.e., MGDF-2) is active, confirms this assumption.

Conditioned medium of 293 cells human kidney (Invitrogen), transfected with the gene MGDF-1 shows the corresponding activity in experiments on cells described Ni. Presumably this can be explained by the ability of cells 293 to processional polypeptide MGDF-1, possibly by removal of a certain area, so that the molecule actually active, is protestirovanny form, while the 32D cells are not capable of processing MGDF-1.

From the point of view of the above hypotheses, various active molecules can result in removal of certain sections from the sequence shown here as a sequence of MGDF-1 (Fig.11). To the conservative features characteristic of cytokines, such as erythropoietin (EPO), are the four alpha-helix and four cysteine. When studying the arrangement of molecules of cysteine (evolutionary conservative and functionally essential elements) found that Cys 172 in the sequence MGDF-1 is the terminal compared with molecules of cysteine in other sequences. Accordingly, the preferred option MGDF-1 are those which are formed by the removal from MGDF-1 C-terminal sites, starting with amino acid at position 173 and to the amino acid at position 353 (in combination with the removal of the signal peptide). Preferably, h is around 90-I - 172-I amino acids, starting from the C-terminal end of the sequence. As noted, the length of the signal peptide is a 21-amino acid, however, the signal peptide can contain 23 amino acids based on sequence MGDF-1. Accordingly, the polypeptides, and related stated, but starting with the 24th amino acids (see Fig.11 or 12), is also subject to review.

Below are some particularly preferred options MGDF-1, which can be active (i.e., the ability to enhance the growth of megakaryocytes and/or platelets, or to inhibit/stimulate the activity of natural receptor):

MGDF-4 amino acids 22-172 Fig.11

MGDF-5 amino acids 22-177 Fig.11

MGDF-6 amino acids 22-191 Fig.11

MGDF-7 amino acids 22-198 Fig.11

MGDF-8 amino acids 22-265 Fig.11

MGDF-11 amino acids 22-184 Fig.11

In some clones amino acids 133-136 in the sequence MGDF-1 was absent, so the sequence corresponding to the above, but in which these amino acids are lost (and the number of C-terminal amino acids decreased by 4), can also be active.

In one clone, with a termination codon at position 192, instead of a Thr residue at position 191 is detected residue Ala, as this is position 191 is Ala.

MGDF-3 was formed by the removal sequence, referred to here as the IVS-5 (intermediate sequence-5), because this sequence spiceroads within the fifth exon. As the 5'-end of the IVS-5 is part of the codon, deletion of this sequence leads to a shift of the reading frame in the remaining sequence MGDF, which occurs at position 160 in the direction towards the end of the molecule MGDF-3.

For the MGDF-3 is not detected biological activity by transfection of 293 cells and testing the corresponding air-conditioned environment in experiments on cells as described in Example 4. Obviously, unlike MGDF-1, 293 cells are not able to processional MGDF-3 with the formation of the active form. However, based on theory of detachment described above with respect MGDF-1, the removal of C-terminal amino acids (for example, in the position 40-102) from MGDF-3 can provide its activity. Preferably the removal of 50 - 90 amino acids. Two particularly preferred options MGDF-2 are the following:

MGDF-9 22-179 Fig.12

MGDF-10 22-190 Fig.12

All of these Mpl-ligand, including those mentioned above MGDFs, on the N-terminal region may be a methionine residue, especially in the case, when it is obtained and by using known methods of chemical synthesis. Such techniques are well known to specialists in this field of research. Synthetic polypeptides Mpl-ligand, having primary, secondary, tertiary structure and conformational properties of polypeptides Mpl-ligand obtained in other ways, can have similar biological properties. So, they can replace biologically active and immunologically active natural, purified polypeptides Mpl-ligand in therapeutic and immunological applications.

Modifications of peptides or DNA sequences encoding the Mpl-ligand, can be obtained by any specialist in this field of research using known techniques. Such modifications sequences Mpl-ligand can include rearrangements, insertions or deletions selective amino acid residues in the coding sequence. Methods for mutagenesis with the goal of obtaining such rearrangements, insertions or deletions are known to experts in this field of research [see, for example U.S. Patent No. 4 518 584]. Preferred peptides can be obtained by proteolytic enzymes or by directed chemical synthesis. Such options Mpl-ligand and polynucleotides VA Mpl - ligand may include modifications in the glycosylation site (for example, serine, threonine or aspartic). The absence of glycosylation or only partial glycosylation are the result of the replacement or deletion of the amino acid asparagine-linked glycosylation site or any other site, modified by addition of O - linked carbohydrate. Asparagine-linked glycosylation site contains a sequence of Tripeptide, which is specifically recognized by the relevant cellular enzyme glycosylation. This Tripeptide sequence can be a sequence Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid except Pro. Various substitutions or deletions of amino acids in one or both of the first or third positions of amino acids in the recognized site of glycosylation (and/or amino acid deletion at the second position) lead to the absence of glycosylation on the modified Tripeptide sequence. The expression of such modified nucleotide sequences leads to the formation of products that are not glycosylated at this site.

Additional analogs/derivatives MGDF

Other analogs or derivatives of the sequences MGDF (Mpl-ligand), which can save MGDF (Mi using these methods. Such modified products are also included in the scope of this invention. More precisely, the present invention encompasses compositions based on chemically modified MGDF, as well as methods of making and using such compositions. This description shows that it is possible to modify MGDF with increased activity. One aspect of the invention is to obtain MGDF-product containing MGDF protein associated with at least one water-soluble polymer. On the other hand, the invention relates to MGDF - product, despite the fact that specified MGDF protein is associated with at least one molecule of polyethylene glycol. In addition, the invention relates to MGDF-protein associated with at least one molecule poliatilenglikola by acyl or alkyl of communication.

Peyrovani MGDF can be carried out using any reactions, known from the prior art, see, for example, Focus on Growth Factors 3(2): 4-10 (1992); EP 0154316; EP 0401384 and other publications cited here, related to peyrovani. Preferably, peyrovani was carried out by acylation reaction or an alkylation reaction with a reactive molecule of polyethylene glycol (or molecule similar water-soluble porownanie

Connection with glycol by acylation basically consists in carrying out the reaction between the active ester of polyethylene glycol (PEG) and MGDF protein. Any known or subsequently received reactive PEG molecule can be used to peyrovani MGDF. Preferred activated ester of PEG is PEG esterified with the formation of N - hydroxysuccinimide ("NHS"). Thus, in the context of the present invention, the term "acylation" is deemed to include without limitation the following types of relationships MGDF with a water-soluble polymer (PEG: amide, urethane, urethane and the like. Cm. Bioconjugate Chem. 5: 133-140 (1994). Reaction conditions attaching PEG can be selected from any known or discovered later, however, in any case, you should avoid the use of such temperatures, solvents or pH values, which are able to inactivate MGDF-products subject to modification. Reaction conditions typically used to attach PEG to MGDF, described below. For example, the reaction with NHS-ester monomethoxy-PEG shown in Fig.14.

Attaching PEG by acylation typically leads to the formation of MGDF-product associated with multiple PEG molecules, and amino Epsilon-lysine connection is the train preferably, to the obtained product, essentially, was associated only (>95%) with one, two or three molecules of PEG. However, some products with a higher degree of peyrovani (in response entered the maximum number of amino groups Epsilon-lysine and one alpha-amino group on aminoterminal plot MGDF) in the norm can be formed, and their number depends on the specific reaction conditions. If necessary, the reaction mixture can be isolated purified peyrovani products and unreacted compounds using standard methods of treatment, including dialysis, vysalivaniya, ultrafiltration, ion exchange chromatography, gelfiltration chromatography and electrophoresis.

Alkylation

Connection with PEG by alkylation generally involves the reaction between a terminal aldehyde derivative of PEG and protein, such as MGDF, in the presence of a reducing agent. As in the case of acylation, detailed reaction conditions for the alkylation are described below.

Connection with PEG by alkylation may also lead to the formation of MGDF-product associated with multiple PEG molecules. For example, the reaction of alkylation MGDF in vosstanavlivaya Fig. 15. In addition, you can modify the reaction conditions so that the joining PEG will occur only after the alpha-amino group of the N-terminal part of the molecules MGDF (i.e., will be formed MGDF-products associated with a single PEG molecule). For example, the reaction of alkylation MGDF in reducing conditions, which leads to the formation of MGDF-product associated with a single PEG molecule, shown in Fig. 16. When monopedigree", as with "paliperidone", PEG-groups preferably are connected with the protein via a-CH2-NH-group. Due to the presence of the group-CH2-, this type of communication is called "alkyl" link.

Deriving MGDF, are connected to a single PEG molecule, by reductive alkylation based on the different reactivity of different types of primary amino groups (lysine in contrast to the N-terminal amino groups) suitable for obtaining the derivatives of MGDF. The reaction is carried out at this pH (see below), which allows the use of the pKa differences between the remnants of the Epsilon-amino group of lysine and the alpha - amino groups of N-terminal site of the protein. Thanks to this approach, is controlled by attaching to protein water-soluble polymer containing reactive ability, the x protein, and a significant modification of other reactive groups, such as side groups of the amino groups of lysine, does not occur. One important aspect of the present invention is to obtain essentially homogeneous drug conjugated to monopolisers MGDF protein (i.e., MGDF protein, to which a polymer molecule attached to almost only one (>95%) certain place). More precisely, if glycol is used, the present invention also relates to peyrovani MGDF-protein, no longer possible antigenic groups and containing a molecule of polyethylene glycol, directly associated with MGDF protein.

Thus, a preferred aspect of the invention is to obtain paglialunga MGDF, and the PEG group(s) attached to the protein molecule by acyl or alkyl groups. As noted above, such products can be monopolizirovany (e.g., containing 2 to 6, preferably 2-5-PEG-groups). The PEG groups are attached to a protein mainly through alpha or Epsilon-amino groups, but it does not exclude the possibility that the PEG groups can be attached to any amino group of the protein, which possesses sufficient reactivity to spezialisierung and alkylation, can be selected from water-soluble polymers or mixtures thereof. In the case of water-soluble polymer protein to which it is attached, will not precipitate in the aquatic environment, such as a physiological fluid. The selected polymer should be modified so that it contains one reactive group, such as an active ester in the case of acylation or aldehyde in the case of alkylation. Preferably, the degree of polymerization could be monitored, as described in the present invention. Preferred reactive PEG aldehyde is Propionaldehyde of polyethylene glycol, which is stable in water, or mono C1-C1-alkoxy or aryloxy its derivatives (see U.S. Patent N 5,252, 714). The polymer may be linear or branched. When used for therapeutic purposes of the final product preferably, the polymer was pharmaceutically acceptable. Water-soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, monometoksipolietilenglikolya, dextran, poly(N-vinyl pyrrolidone) polyethylene glycol, homopolymers of propylene, a copolymer of polypropylene oxide/ethyleneoxide, polyoxyethylene polyols (e.g. glycerol) and p is klono-able ester group. For alkylation reactions in the reducing conditions of the polymer(s) is chosen so that it(they) was(and is) a single reactive aldehyde group. In General, water-soluble polymer should not choose from natural helicoiling residues, because these residues usually more convenient to attach to a protein by expression in mammalian cells. The polymer may be of any molecular weight.

Particularly preferably used as the water-soluble polymer polyethylene glycol (PEG, PEG). As noted in the present description, to obtain the derivatives of various proteins can be used in any form of PEG, for example, mono-(C1-C10) alkoxy or aryloxy-glycol.

As noted earlier, under MGDF also understand any of the forms described here. For example, it may be glycosylated or deglycosylated form MGDF, as well as proteins with full-length or formed as a result of splitting the original product. Below is preferred MGDF-proteins on the basis of which can be obtained by a variety of derivatives (in each case, the number of amino acids correspond to those in Fig. 11):

MGDF-1 amino acids 22-353 Fig.11

MGDF-2 aminolysis.11

MGDF-13 amino acids 27-195 Fig.11

MGDF-14 amino acids 27-172 Fig.11

MGDF-15 amino acids 27-184 Fig.11

These MGDF-proteins can be deglycosylated, deglycosylation, preferably, deglycosylation. They can be obtained in recombinant bacterial (e.g. E. coli) or in mammalian cells (e.g. CHO).

The following are the most preferred derivatives MGDF obtained by chemical means (in each case, they are mono or paliperidone, i.e., for example, contain 2-4 PEG molecules attached via acyl or alkyl groups):

peyrovani MGDF-11

peyrovani MGDF-4

peyrovani MGDF-2

In General, deriving MGDF by chemical may occur in any suitable conditions for entry into the reaction of biologically active compounds with an activated polymer molecule. Methods of obtaining paglialunga MGDF typically include the following stages: (a) passing the reaction between the polypeptide MGDF and polyethylene glycol (for example, a reactive ester or aldehyde derivative of PEG) under conditions when MGDF connects to one or more PEG groups, and (b) isolation of the product (s) reactions. In General, the optimal reaction uslovima, the larger the ratio of PEG: protein, the greater the yield (in percent) paliparang product.

Reductive alkylation to obtain essentially homogeneous conjugate monopoliser/MGDF protein typically involves the following stages: (a) passing the reaction between MGDF protein with a reactive PEG under conditions of reductive alkylation at a pH of providing selective modification of the alpha-amino group at the amino-terminal region MGDF protein; and (b) isolation of the product (s) reactions.

To obtain essentially homogeneous conjugate monopoliser/MGDF protein reaction conditions, reductive alkylation should provide selective binding of water-soluble polymer with N-terminal stretch of MGDF. These conditions are usually based on the pKa differences between the amino groups of lysine and the alpha amino group at the N-terminal region (pKa is the pH at which 50% of the amino groups protonated and 50% - no). the pH also affects the used ratio of the polymer and protein. In General, if a lower pH is required excess polymer in relation to the protein (i.e., the less reactive N-terminal alpha-amino group, the more polymer is needed for doselect (more reactive groups, so fewer polymer molecules). For the purposes of the present invention the pH is usually 3-9, preferably 3-6.

Another important aspect is the molecular weight of the polymer. In General, the more they say. the weight of the polymer, the smaller the number of molecules able to connect with the protein. To optimize these parameters should take into account the degree of branching of the polymer. Typically, when a greater mole. mass (greater degree of branching) above, the ratio of polymer : protein. For the reactions considered here the connection PEG to protein mol. weight PEG average about 2 KD - 100 KD (the term "about" is +/-1 KD). Preferably, the average mol. weight of PEG was about 5 KD to 50 KD, particularly preferably about 12 KD to 25 KD. The ratio of the water-soluble polymer : MGDF protein, mainly is 1: 1-100:1, preferably (paliperidone) 1:1 to 20: 1 and (for montegiove) 1:1-5:1.

When using the above conditions, reductive alkylation will provide for selective adherence of the polymer to any MGDF-protein with the alpha amino group at the amino-terminal region, and to provide essentially homogeneous drug monopoliser, conjugating PEG-protein, associated with one molecule of the polymer. This conjugate is preferably will contain a polymer molecule, located on the N-terminal region, but not on the lateral amino groups of lysine. The resulting preparation, preferably more than 90% consists of monopoliser conjugated with MGDF protein, and preferably more than 95%, while the molecules remain, unreacted (protein molecules that interact with not enough polymer). The following examples shows how to obtain the drug, at least 90% consisting of a conjugate of monopoliser and protein and 10% consisting of unreacted protein. This conjugate is biologically active.

For rehabilitation of alkylation, the reducing agent should be stable in aqueous solution and preferably be able to restore only the Schiff's bases formed in the initial stages of rehabilitation alkylation. Preferred reducing agents can be selected from the group consisting of borohydride sodium, cyanoborohydride sodium, dimethylaminoborane, trimethylaminuria, pyridinone. Especially preferred regenerating agent having the action, temperature and so on, as well as the degree of purification of the products can be determined from case to case on the basis of published information on obtaining derivatives of proteins when using water-soluble polymers described in the present description reference). More details on these reactions described in the examples below.

To obtain a mixture of a protein conjugated with a polymer may be selected method of acylation and/or alkylation, and the advantage of the approach described here is that it is possible to prepare a mixture with a specified relative contents monopoliser/protein. Thus, if necessary, receive a mixture of various proteins associated with different number of polymer molecules (di-, tri-, Tetra -, and so on), in combination with the conjugate monopoliser/protein, obtained using the methods outlined here, and this mixture is characterized by a given content conjugate monopoliser/protein.

The following examples disclosed is a method of obtaining chemically modified MGDF and MGDF connected to the PEG via acylation or alkylation. Accordingly, other aspects of the invention relate to these compounds.

the polymer. However, these conjugates may have a modified activity, as increased or reduced, as well as other properties compared to unmodified molecules.

Another aspect of the present invention relates to pharmaceutical compositions based on chemically modified MGDF protein. Such pharmaceutical compositions may contain any of the unmodified MGDF protein.

As additional research information appears appropriate doses of these drugs for the treatment of various diseases in different patients, and the average expert in the field of research, taking into account medical conditions, age, and General condition of the patient will be able to choose the appropriate dose. In General, the dose may be from 0.01 μg/kg body weight (calculated on the weight of the protein, without chemical modification), 300 µg/kg body weight (calculated in the same way). The preferred dosage, in General, may range from 5 μg/kg body weight to 100 mg/kg of body weight, especially preferred dose of 10 mg/kg of body weight up to 75 µg/kg

The present invention relates also to a method for MGDF-polypeptides (polypeptides MGDF-ligand) or actuator expression to obtain the expression system, the Mpl-ligand. Selected cells are the owners of transferout data vector and cultured. The method, as claimed in the present invention, therefore, involves culturing a suitable cell or cell line, transfected with the DNA sequence encoding or expressing the polypeptide Mpl-ligand under the control of known regulatory sequences. Regulatory sequences are fragments of the promoter, terminator and other suitable sequences that direct/control the expression of the protein in an appropriate cell hosts. Then the expression product is isolated and purified from the culture medium (or from the cells in the case of intracellular expression) by any suitable method known to the expert in this field of research. In addition, to obtain the claimed polypeptides/polynucleotides can be used the methods described in U.S. Patent N 5,272,071.

Suitable cells or cell lines can be mammalian cells, such as cells Chinese hamster ovary (CHO) or T. Methods selection of suitable host cells, their transformation and cultivation, amplification, screening, isolation and purification of the target product known level of the 9 446. Other suitable cell lines mammals include cell line monkey COS-1, COS-7 and CV-1. The following examples are mammalian cells, including cell lines primates and rodents, in particular, transformed cell lines. Can also be used in normal diploid cells, cell strains derived in vitro culture of primary tissue, primary explants. Cells can be genetically defective in selective marker or contain a dominant selective functioning gene. Other suitable cell lines mammals include (but are not limited to these) HeLa cells, L-929 mouse T-lines derived from Swiss mice, Balb/c or NIH, as well as cells of hamster BHK or HaK.

Suitable cells-owners for the purposes of the present invention are bacterial cells. For example, specialists in biotechnology is well known for the various strains of E. coli (e.g., HB101, DH5-alfa, DH10, and MC1061), which can be used as host cells. In addition, when carrying out the inventive method can be applied to various strains of B. subtilis, Pseudomonas spp., Bacillus spp., Streptomyces spp. and similar to them.

For the expression of the claimed polyp is Noah area of research. If necessary for the implementation of the claimed method as host cells can be used insect cells, see, e.g. Miller et al., Genetic Engineering, 8: 277-298 (1986) and described in this description of the link.

The present invention also relates to recombinant molecules or vectors for use in the method of expression of new proteins Mpl-ligand. These vectors contain DNA sequences Mpl-ligand, alone or in combination with other sequences encode polypeptides Mpl-ligand (with signal peptide and without it), as claimed in the present invention, or active fragments. Alternatively, the vectors containing the modified sequence, as described above, also included in the scope of the invention and is used to obtain polypeptides Mpl-ligand. The vector used for implementing the method that contains the selected regulatory sequence functionally linked to a DNA coding sequences obtained according to the present invention, and capable of directing the expression of such sequences in the cells of the host.

For protein expression in COS-cells are especially preferred is the R, the CHO cells, is the vector pEMC2B1. Described herein are vectors for protein expression in mammalian cells can be obtained by known methods. Components of vectors, for example, replicons, the selective marker genes, enhancers, promoters, etc. can be obtained from natural sources or synthesized using known approaches, see, for example, Kaufman et al., J. Mol. Biol. 159: 511-521 (1982); and Kaufman, Proc. Natl. Acad. Sci. USA 82: 689-693 (1985). Alternatively, the DNA vector may contain a gene or part of the genome of bovine papilloma virus [Lusky et al., 36: 391-401 (1984)] and replicated in cell lines, such as cells, C127 mouse as a stable episomal element. The transfer of these vectors into appropriate cell hosts leads to expression in these cells polypeptides Mpl-ligand.

For the purposes of the present invention can be used, and other suitable vectors designed for expression of proteins in mammalian cells, insect, yeast, fungi and bacteria.

Painful conditions, which are subject to correction by using the claimed compositions and methods of their use, in General, are the States with the failure of megakaryocytes/platelets, or the occurrence of such a failure of writing the result of the failure (chronic or transient) active Mpl ligand in vivo. The usual name of a deficiency of platelets thrombocytopenia is, therefore, the claimed compositions and methods of their use are related to the treatment of thrombocytopenia.

Thrombocytopenia (lack of platelets) may be due to various causes, including chemotherapy, drug therapy, radiation therapy, surgery, accidental hemorrhage and other diseases. Examples of painful conditions in which there is a thrombocytopenia that may be corrected in accordance with the present invention, are aplastic anemia, idiopathic thrombocytopenia, metastatic tumors, leading to thrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi syndrome, a deficiency of vitamin B12, a deficiency of folic acid, the anomaly may-Hegglin syndrome Wiskott-Aldrich and paroxysmal nocturnal hemoglobinuria. In addition to thrombocytopenia results in the treatment of AIDS certain drugs (such as AZT). Certain violations in wound healing can also be caused by an increase in the number of platelets.

When the expected failure of platelets, for example in the future hirumi, of several days to several hours before needed platelets. In the case of acute conditions, for example, when a sudden and large blood loss, Mpl - ligand can be introduced together with the replacement of blood or purified platelets.

Declared Mpl-ligand can be used to stimulate certain types of cells, different from megakaryocytes, if it is shown that these cells Express Mpl-receptor. Condition associated with cells expressing Mpl - receptor responsible for the effect of stimulation by the Mpl - ligand, are also considered in the scope of the present invention.

MGDF-molecules, which themselves are not active in the experiments described here, may be useful as modulators (e.g., stimulants or inhibitors) Mpl receptor in vitro.

For the treatment of the above diseases claimed polypeptides can be used alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies.

Accordingly, another aspect of the present invention lies in p is automatically effective amount of the polypeptide Mpl-ligand or an active fragment in complex with a pharmaceutically acceptable carrier. The media can be a water for injection, preferably supplemented by other components to obtain solutions that can be introduced mammals. When therapeutic use of Mpl-ligand can be applied in the form of a composition containing purified protein, conjugated with one or more physiologically acceptable carriers, solvents or diluents. Neutral buffer solution or the solution is mixed with serum albumin, are examples of suitable carriers. Preferably, the product prepared in lyophilized form using appropriate solvents (e.g., sucrose). Can be used and other conventional carriers, diluents or solvents, such as Tris-buffer (pH 8.0) and acetate buffer (pH 5.0), which sometimes contain sorbitol. The claimed compositions can be administered parenterally. Alternatively, the composition can be applied intravenously or subcutaneously. The systems use for the purposes of the present invention, therapeutic compositions can be prepared in pyrogen-free form in aqueous solution suitable for parenteral administration. The methods of preparation of such pharmaceutically suitable is the illusion in this research area.

Dosage and mode of administration in the implementation of the method of treatment of the above diseases are selected practicing physician considering various factors which modify the action of drugs, such as age, condition of the patient, the nature of his power, body weight, gender, severity of any infection, time of administration and other clinical factors. In General, daily should be used at about 0.1-1000 μg Mpl protein-ligand or its fragment per 1 kg of body weight.

Compositions and polypeptides, as claimed in the present invention, when carrying out treatments can be used alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies in korrigirovanija of diseases characterized by symptoms other than platelet deficiency. It is expected that the Mpl-ligand will be really useful in the treatment of some forms of thrombocytopenia in combination with such a powerful stimulant hematopoesis as IL-3 or GM-CSF. In addition, together with the Mpl-ligand can be applied and other factors stimulation of megakaryocytes, for example, Meg-CSF (meg-CSF), stem cell factor (SCF), leukemia-ingibirujut is sustained fashion examples of cytokines or hematopoietic factors for the specified combination therapy are alpha, IL-1, beta IL-1, IL-2, IL-3, IL-4, II-5, IL - 6, II-11, colony stimulating factor-1 (CSF-1), GM-CSF, granulocyte colony-stimulating factor (G-CSF), EPO, alpha - interferon (alpha-IFN, IFN) beta-interferon or gamma-IFN. It may be appropriate simultaneous or sequential introduction together with the above compounds soluble Mpl receptor mammals, which apparently contributes to the destruction of megakaryocytes with the formation of platelets, as only the Mature megakaryocytes. Thus, the introduction of Mpl-ligand (to increase the number of Mature megakaryocytes) followed by the application of soluble Mpl receptor (for inaktivirovanie ligand and destruction of Mature megakaryocytes with the formation of platelets), seems to be effective to stimulate the formation of platelets. Doses above can be skorrigirovanna depending on what additional components are included in the pharmaceutical composition. The effectiveness of the treatment is controlled in an appropriate manner. In addition, the claimed new polypeptides used to generate antibodies. Thus, the scope of the invention includes antibodies that interact with the Mpl-ligand, obtained according to the present izaberete the YMI, chimeric, single-chain and/or bespecifically etc., the antibody Fragment may be any fragment that interacts with the Mpl-ligand, such as Fab, Fab', etc. In the scope of the present invention also includes hybridoma obtained using the Mpl-ligand or its fragment as antigen for immunization of animals, with subsequent fusion of animal cells (e.g., spleen cells) with a particular tumor cell line. In the merger carried out by known methods, receive an immortal cell lines that produce antibodies. Methods of obtaining hybridomas and antibodies directed to a polypeptide Mpl-ligand or its individual parts, also included in the scope of the present invention.

Antibodies can be used in therapy, for example for inhibiting the binding of Mpl-ligand to the receptor. Antibodies associated with the corresponding label, can find application in diagnostics in vivo and in vitro for the detection of Mpl-ligand in body fluids.

The following Examples more fully disclose the invention. In addition, they reflect the preferred embodiments of the invention without limiting its scope. Standard methods for the NCD is, is slojeny in the well-known manuals of molecular biology, for example, Sambrook et al., Molecular Cloning, Second edition, Cold Spring Harbor Laboratory Press (1987) and Ausubel et al., (Eds), Current Protocols in Molecular Biology, Greene associates/Wiley Interscience, New York (1990).

Example 1. Aplastic plasma dogs

Heparinised aplastic plasma of the dog ("ARC") or normal plasma dog ("NK9) was obtained as described in the following publications, with the difference that the dose recipients was 450 glad:

1. Mazur.E. and South, K. Exp.Hematol. 13:1164-1172 (1985).

2. Arriada, M., South, K., Cohen, J. L, and Mazur, E. M. Blood 69:486-492 (1987).

3. Mazur.E., Basilico, D., Newton, J. L., Cohen, J. L, Garland, C., Sohl, P. A., and Narendran, A. Blood 76:1771-1782 (1990).

Example 2. The definition of megakaryocytes man

ARC and fractionated ARK were tested for ability to stimulate the development of megakaryocytes man from CD34+ precursor cells. CD34+ cells were obtained from peripheral blood cells as described (Hokom, M. N., Choi.E., Nichol, J. L, Hornkohl,A., Arakawa.T., and Hunt, P. Molecular Biology of Haematopoiesis 3: 15-31,1994), and incubated in the culture medium of the following composition: Wednesday Dulbecco in modification Actions (IMDM; GIBCO, Grand Island, NY) with 1% of mixture of glutamine-penicillin - streptomycin (Irvine Scientific, Santa Ana, CA), 10% heparinized free thrombosis is denzin, guanine, citizen, uridine, thymidine, 2-detoxication, 2-deoxyadenosine, 2 - deoxyguanosine (each 10 μg/ml, Sigma); human recombinant insulin (10 μg/ml), human transferrin (300 μg/ml), soybean lipids (1%, Boehringer Mannheim, Indianapolis, IN); human recombinant basic fibroblast growth factor (2 ng/ml, Genzyme, Cambridge, MA); human recombinant epidermal growth factor (15 ng/ml), growth factor, derived from platelets (10 ng/ml, Amgen,Inc., Thousand Oaks, CA).

CD34+ cells were seeded at a concentration of 200,000/ml of culture medium in a final volume of 15 μl in cell tablets Terasaki (Vanguard, Inc., Neptune, NJ). Cells were incubated for 8 days in a humid atmosphere with 5% CO2at 37oC, then recorded directly in tablets of 1% glutaraldehyde and incubated with a mixture of monoclonal antibodies (anti-GPIb, anti GPIIb, (Bio design) and anti-GPIb (Dako, Carpinteria, CA). The immune reaction was revealed with the help of a streptavidin-beta - galactosidase (HistoMark, Kirkegaard and Perry). The megakaryocytes identified as blue colonies were counted on an inverted phase microscope with magnification of 1000x. The results were represented as the average number of megakaryocytes per well +/- standard error definitions (SEM). In some cases, the data appeared to the development of megakaryocytes, was normalized with respect to the used in the experience of positive control - APK9. One unit was defined as the amount of material that resulted in the formation of the same number of megakaryocytes, and 1 μl of standard APK9. The activity was attributed to the Mpl-ligand if it could be blocked 5-10 ál/ml MPL-X (soluble Mpl receptor).

It is shown that APK9 contains the factor(s) that stimulates the development of human megakaryocytes in the system. CD34+ cells incubated with 10% NK9 for 8 days showed negligible number painted in blue megakaryocytes, whereas the same cells incubated for 8 days with 10% APK9, gave a very large number of painted megakaryocytes.

In Fig.2 shows that increasing the concentration of Mpl-X added to the culture system of human megakaryocytes, increasingly block the development of megakaryocytes. At concentrations of Mpl-X is greater than 5 µg/ml comes complete suppression. In this experience of CD34+ cells stimulated with 5% APK9. This indicates the fact that the activity of interacting with Mpl-X (presumably Mpl-ligand), necessary for the development of megakaryocytes from human and APK9 is actually Mpl-ligand.

Have been received and others who s the man. APK9 (135 ml) was diluted 6 times environment Suits and put on affinity column Mpl-X. Unbound material was collected and concentrated to the original volume before taking the test. Bound peroxidase material was suirable 10 ml of 1 M NaCl, 20% of the collected pool was subjected to diafiltration and then concentrated in 4 times. CB34+ cells, which were incubated in the medium without additives APK9, did not start the megakaryocytes. Cells incubated with 5% APK9 (the same sample that was applied on the column), given 48 +/- 8 megakaryocytes per cell. Cells incubated with unbound material (concentration 10%), did not form megakaryocytes. Cells incubated with 10% of the eluate gave a score of 120 +/- 44 megakaryocytes per cell. Activity applied to the column sample and activity in the eluate is almost completely suppressed 5 μg/ml Mpl-X.

Example 3. Transfection of Mpl-receptor mouse or human in the murine cell line

A. Mpl-receptor mouse

Full-size cDNA Mpl-receptor mouse was subcloned in the expression vector containing a transcriptional promoter, derived from the LTR sarcoma virus of mice, Malone. 5 μg of this construct and 1 μg of plasmid pWLNeo, carrier selective marker (Stratagene), were co-elektroporcelany cells IL-3-dependent mispoke electroporation, then transferred to selective medium containing 800 μg/ml of Geneticin (G418, Sigma) and 1 ng/ml IL-3. Then the surviving cells were divided into two pools at 200,000 cells in each and pokasivali for analysis. Six populations of cells were tested for surface expression of Mpl - receptor method of flow cytofluorimetry (FACS) using polyclonal rabbit used for preparing antipeptide serum. One of the populations was selected for sorting duct using the same serum. Single-cell clones of the original cell lines were obtained by growing cells in medium with 10% APK9 and Geneticin. After selection in the presence of APK9 within 35 days the cells were maintained by culturing in the presence of 1 ng/ml murine IL-3. One of the subclones, 1A6.1, was used to perform the basic work.

B. Mpl-receptor human

Full sequence Mpl-receptor human (Vigon.l., et al. , PNAS 89: 5640 - 5644 (1992)) were subcloned in the expression vector containing a transcriptional promoter sarcoma virus of mice, Malone (the same vector as in the case of mouse receptor). Six μg of this construct and 6 µg amphotropic retroviral packaging construct (Landau, N. R., Littman, D. R. , J. Virology 66: 5110-5113 (1992)) were transferiria is(Stratagene). The same cells were retrospectively after 2 days of cultivation and again retransfusion 4 days later. The day after the last transfection of 293 cells was cocultivated cells IL-3-dependent mouse line (32D, clone 23; Greenberger et al., PNAS 80: 2831- 2936 (1983)). After 24 hours of cocultivation 32D cells were separated and fractionally in the BSA gradient (Path-o-cyte; Miles Inc.). Then the cells were pokasivali in the presence of 1 ng/ml murine IL-3 and selected for ability to grow in pristutstvii 20% ARK. Cells were sorted running cytofluorimetry (FACS) on a surface receptor expression using a polyclonal rabbit used for preparing antipeptide serum. These cells are then used in the experiments.

Example 4. The definition of Mpl-ligand with cells 1A6.1

Cells 1A6.1 washed from the present in the environment of IL-3 and seeded (1000 cells/ 15 μl final volume/cell) in tablets Terasaki in the environment of alpha-MEM (Gibco) with 10% fetal calf serum (FCS), Geneticin (800 μg/ml), 1% penicillin-streptomycin (Gibco) in 1:1 serial dilutions of test samples. After 48 hours of cultivation microscopically determined the number of viable cells in the cell. Per unit activity took such activity, which led alleles included in the definition of 5-10 µg/ml Mpl-X. The activity of Mpl-ligand in the average units APK9 was 4400 +/- 539 units/ml aplastic plasma. Unless specifically stated, the units of activity Mpl-ligand was determined in experiments using cells 1A6.1.

Define cells, transfitsirovannykh genome Mpl-receptor of the person (Example 3B), were exactly the same as with cells 1A6.1.

Example 5. Mpl-ligand is present in aplastic plasma or serum of mice, dogs, pigs and humans

Mpl-ligand is present in aplastic plasma or serum of mice, dogs, pigs and humans (table 2). Plasma was collected from mice BDF1 before irradiation and after 12 days after irradiation dose of 500 rad. Plasma was tested in the test cell 1A6.1, and was discovered activity of 2000 units/ml, which is almost completely inhibited Mpl-X (10 µg/ml). Plasma irradiated mice was also positive in the test for the human megakaryocytes, and its activity was 1833 IU/ml In dogs plasma collected before exposure and 10 days after irradiation at a dose of 450 rads. Plasma was tested in the test cell 1A6.1 and in the test on megakaryocyte person. In both definitions was detected activity, which completely inhibited Mpl-X (10 µg/ml). In swine plasma collected before obah person. In both definitions activity detected ligand (suppressed Mpl-X at a concentration of 10 μg/ml), comparable to those found in aplastic plasma of the dog. Were also obtained serum from aplastic patients. This material was obtained from patients undergoing bone marrow transplantation. Serum of six patients were tested in the test cell 1A6.1 and activity detected 903 u/ml, 88% of which was due to the Mpl-ligand (suppressed 10 μg/ml Mpl-X). Serum 14 plastic patients were also tested in the test for the human megakaryocytes. In General, they showed a significant activity, 941 IU/ml, which was completely suppressed 10 μg/ml Mpl-X. the Data obtained with the use of murine IL-3, is enabled to show the specificity of the test on the cells 1A6.1. Although this recombinant cytokine induces the growth of this cell line, its activity is not blocked 10 μg/ml Mpl-X.

Example 6. Mpl-ligand stimulates cell growth 1A6.1 and the development of megakaryocytes man

Mpl-ligand (enriched in at least 100000 times after chromatography on pectin and affinity chromatography; see Example 7) stimulates cell growth 1A6.1 and the development of megakaryocytes man from 34+ cells of peripheral blood in zavisimosti definitions can be completely blocked Mpl-X.

Applicants it was also shown that fractionated using flow cytofluorimetry (FACS) 34+ cells in peripheral blood during the incubation with the Mpl-ligand (in this case, 100 u/ml within 9 days) develop into phenotypically normal, Mature megakaryocytes. Thus it is shown that purified Mpl - ligand has the same effect on megakaryocytes that and untreated APK9. In addition, in this experiment used the purified CD34+ cells (100% CD34+), and not enriched, in which the proportion of CD34+ cells is usually 30 - 50%.

Example 7. Cleaning Mpl-ligand dogs

1. Summary

Were purified proteins (25 KD and 31 KD), which showed activity attributed to Mpl ligand-receptor. Proteins isolated from plasma of irradiated dogs under the scheme, including affinity chromatography on agglutinin wheat germ (WGA) affinity chromatography on Mpl-receptor uninominal chromatography, gel - filtration chromatography and C4 HPLC with reversed phase, see Fig.46, which presents this purification scheme.

Mpl-ligands with molecular weight of 25 KD and 31 KD were purified essentially to homogeneity and shown that they consist of amino acid sequences described in this isoleucinol from irradiated dogs (see Example 1) was thawed overnight at 4oC; thawing of plasma in large bottles was carried out at room temperature for several hours, and then in a cold room. Insoluble material was removed by centrifugation for 6 hours at 11,000 g. Plasma was diluted phosphate buffer solution, pH 7.3, containing 0.01% of sodium azide (PBS/azide), and filtered through a 1.2 μm filter. The lightening was usually resulted in a twofold dilution of the starting material.

B. Affine chromatography on agglutinin wheat germ

All operations were performed at 4oC. the Clarified plasma (two parties) was applied on a column with immobilized agglutinin wheat germ (1 liter, 10 x 12 cm, EY Laboratories), balanced PBS/azide. After applying the sample, unbound material is washed off the column with PBS/azide and the column was washed with 0.5 M NaCl in 20 mm TrisHCl, pH 8. The activity of Mpl-ligand, contacting WGA-column, suirable 0.35 M N-acetylglucosamine (GleNAc), 0.5 M NaCl, 20 mm Tris-HCl, pH 8. The activity of Mpl-ligand was detected in the flow-through or wash fractions.

C. Affinity chromatography on Mpl-X receptor

Used soluble murine Mpl receptor (Mpl-X) corresponded to a extracellular house is atoron on affinity column, suirvey with WGA-column material was concentrated using membrane ultrafilter (cutoff molecular weight of 10,000, YM-10, Amicon) and the concentration of NaCl made up to 0.2 m of subsequent breeding. The concentrated material with WGA-column was applied to a 20-ml column with m - Mpl-X (soluble murine Mpl-X receptor)/tiobraid - enabled Sepharose (2.6 x 4.2 cm, 1.5 mg m-Npl-X per ml of resin) at a flow rate of 0.9 ml/min the Column was washed in PBS/azide at a flow rate of 1.5 ml/min, and then with high salt buffer (405 ml) consisting of 10 mm Tris-HCl, 1 M NaCl, 1 mm CHAPS, pH 8.0. Bound peroxidase Mpl-ligand was suirable with a column buffer of the following composition: 20 mm CAPS, 1 M NaCl, 5 mm CHAPS, pH of 10.5. Collected the appropriate faction. To neutralize the pH of each fraction was added Tris.

SDS-PAGE and the absorption at 280 nm showed the presence in the profile of elution from the affinity column with Mpl-X-early receptor protein peak in fractions 1-4 in that the main activity of Mpl-ligand was swirbul after faction 5.

, Mono-Q-uninominal chromatography

The most purified fractions from multiple affinity columns with Mpl-X receptor were pooled, concentrated and were dialyzed against 20 mm Tris-HCl, 5 mm CHAPS, pH 8.7 to a final volume of 58.5 ml Concentration ect was applied at a flow rate of 0.5 ml/min column on Mono-Q HR 5/5 (Pharmacia), balanced 20 mm Tris-HCl, 5 mm CHAPS, pH of 8.7. The elution was performed by a linear gradient to 0.36 M NaCl in the same buffer for 27 minutes. Then the column for 6 minutes, washed with gradient to 0.54 m NaCl and a final wash was performed with 0.9 M NaCl. Collected fractions of 1 ml

The profile of elution from the column Mono-Q showed that the flow-through and wash fractions do not contain Mpl-ligand, and in General any significant amount of protein. A significant activity of Mpl-ligand was lirowaus in fractions 5-7 at the initial stages of the NaCl gradient. "Shoulder" activity was observed in fractions 8-10, followed by the main peak, represented by fractions 11-15. In the active fractions there was a clear strip of 25 KD (SDS-PAGE in non conditions). The intensity of the band directly corresponded to the activity of Mpl-ligand in the fractions. This band was absent in fractions 3 and 4 (no activity). It was expressed in fractions 5 and 6 (the peak of activity in tests on cells 1A6.1) and similarly intensively coloured band was present in fractions 11-14 (peak activity in tests on cells 1A6.1). The band was weak in the United fractions 15 and 16, which corresponded to a significantly lower activity of fraction 16.

D. Experiments on elution from gel

oC during the night. The next day, samples were centrifuged, selected aliquots, and the samples were subjected to diafiltration against environment Iscove with the addition of BSA as a protein carrier. Diafiltration samples were used for further definitions.

The results show the presence of two peaks of activity of Mpl-ligand. One peak corresponds to the region of the gel 25 KD, and another peak of activity Mpl-ligand corresponds to the region of 31 KD.

E. Gel Filtration on Superdex 200

Fractions 13-16 with uninominal column Mono Q and two equivalent fractions of a second fractionation on Mono Q were combined and concentrated using membrane ultrafilter (Method-10, Amicon). Sodium dodecyl sulphate (SDS) was added to a final concentration of 0.1% and the sample was applied to a column of Superdex 200 HR 10/30 (Pharmacia). The column was balanced 50 MMTris, 0,1% SDS, pH 7.5 at a flow rate of 0.3 ml/min and room temperature. Fractions were collected every was libnautilus in fractions 42-46. Analysis of the fractions by the method of SDS-PAGE showed the presence of clear bands 25 KD in the active fractions.

J. C4 chromatography high-resolution reversed-phase

Faction 43-46 with column Superdex 200 was combined with fraction 42 and concentrated using a membrane ultrafilter (Microcon, Amicon). The concentrated mixture was applied to a 1 x 100 mm C4 column with reversed phase (SynChropack RP-4). The column was balanced with a solution of 0.04% TFA in water (buffer); buffer had the following composition: a 0.035% TFA in 80% acetonitrile. After application of the sample spent a linear gradient of buffer B to 45% for 4 minutes. Followed by a linear gradient to 75% buffer b for 40 minutes. The flow rate was set to 75 ál/min cleaning Results faction 42 shown in Fig. 5. Clear peaks of activity of Mpl-ligand was observed in the fractions 21-23. These fractions were analyzed by electrophoresis on 14% polyacrylamide gel in reducing and non conditions. Fraction 21 consisted of a single strip 31 KD; fraction 23 consisted of a single broad band of 25 KD; and fraction 22 contained both strips - 25 KD and 31 KD. Other significant bands could not be seen. Interestingly, preliminary experiments on elution from the gel gave reason to associate the activity of Mpl-who was lodales in all fractions in non-gels, but it was not possible to detect in reducing gels.

H. Analysis of N-terminal sequences of 25 KD and 31 KD Mpl-ligand

Analysis of N-terminal sequencing was performed on containing the active fractions C4 RP-HPLC. Set the sequence data of proteins above. In addition to the main sequence, the respective band 25 KD (at least 90% of the sample), sequencing revealed two minor sequences that were associated with minor additional strip, described earlier in section "C". Comparison with known sequences showed that minor sequence represent a heavy chain and Kappa chain immunoglobulin dogs. If necessary, these minor contamination can be removed by an additional treatment step, preferably, an additional gel filtration.

I. comparison of the activity of Mpl-ligand from purified fractions C4

The data presented in Fig.6, show that the activity present in fractions 22 and 23 chromatography C4 RP-HPLC, essentially equivalent. Fraction 22 contains a mixture of strips 25 KD and 31 KD, and the fraction 23 - only strip 25 KD. Aliquots of each fraction rassadina cell fraction 23 - 6000 cells per cell). The diluted fractions were incubated with increasing concentrations of Mpl-X. Fractions were equally sensitive to inhibition Mpl-X, the activity of both fractions is completely blocked Mpl-X concentration 7-1,4 µg/ml, This indicates that the active protein(proteins) of each fraction are a variety of Mpl - ligand with the same biological activity.

Example 8. Comparison of the actions of Mpl-ligand on the development of megakaryocytes with the effects of other factors

It is shown that the number of recombinant factors and organic compounds, such as phorbol-myristyl-acetate (PMA), affect the growth and development of megakaryocytes. Studied the impact of these factors on CD34-positive cells in peripheral blood. Recombinant interleukin 3 (IL-3, 1 ng/ml), growth factor stem cells (SCF, 50 ng/ml), Interleukin 6 (IL-6, 25 ng/ml), erythropoietin (EPO, 1 u/ml), factor inhibiting leukemia (LIF, 10 ng/ml) and granulocyte-macrophagecolony colony-stimulating factor (GM-CSF, 25 ng/ml, Amgen, Inc.); interleukin 11 (IL-11, 25 ng/ml, R+D Systems, minneapolis, MN); phorbol-myristyl-acetate (PMA, 10-10M, Sigma) was added to

the cultures as indicated. Mpl-ligand used in a concentration of 275 units per ml, APK9 use the and equal to one for individual use. After 8 days of cultivation, the cells were fixed in cells and stained for megakaryocytes (n=6 cells for each option) or counted the total number of cells (n=3 cells for each option). Data presented are average +/- standard error.

In Fig.7 shows that APK9 and Mpl-ligand resulted in the formation of the largest number of megakaryocytes per cell. IL-3 has also led to the development of megakaryocytes, especially in combination with SCF, IL - 6, IL-11, EPO had a minor effect on the number formed of megakaryocytes in the individual impact or in combination with IL-3. PMA, LIF and GM-CSF had a weak impact. In Fig. 8 shows the same experiment, showing the total number of cells in cells ("cells"). APR9 and Mpl-ligand weakly changed this value, and II-3 and SCF showed a moderate impact. The combination of SCF and IL-3 had the strongest effect. The data shown in Fig.7 and Fig.8 were used to calculate the percentage of megakaryocytes per cell, as shown in Fig.9. It is obvious that the factor leading to the formation of the highest percentage of megakaryocytes in the cell is the Mpl-ligand, the active ingredient APK9. This indicates the specificity of Mpl - ligand towards megakaryocytes.

Example 10. Analysis of Mpl-ligand pigs

1. Summary

Proteins from plasma irradiated pigs with the activity of Mpl-ligand were characterized using WGA-affinity chromatography, affinity chromatography on Mpl-receptor ionoobmennoi chromatography and C4 chromatography high-pressure reversed phase (HPLC). The activity is also characterized by the elution of slices SDS - polyacrylamide gel (see tab. A).

Example 11. Cloning Mpl-ligand man (human MGDF)

Below are two of the used approach:

1. The first example of cloning

A. obtaining a sample MGDF man

The number of degenerate PCR primers is Oh human DNA used different pairs of primers. After 40 cycles of amplification using the sense primer 5' GCN CCN CCN HCN TGY GA 3' (SEQ ID N0: 4) encoding the first five amino acids of the protein dog (SEQ ID N0: 1) and antisense primer: 5' GCA RTG YAA CAC RTG NGA RTC 3' (SEQ ID N0: 5) encoding amino acids with 16 to 21 of the sequence SEQ ID N0: 1, the PCR product was dispersed in the gel, a 2.0% agarose in TBE buffer. The band corresponding to fragment 63 base pairs was cut from the gel and reamplification with the same set of primers. The PCR product was cloned in the vector PCR II (Invitrogen, San Diego). The number of colonies was proskenion using DNA sequencing. Plasmid DNA encoding the peptide-like protein MGDF dogs, used to obtain radioactive samples in subsequent screening crnkovich libraries. Amino acid sequence encoded by the fragment of the gene was as follows: Ala-Pro-Pro-Ala-Cys-Asp-Leu-Arg-Val-Leu-Ser - Lys-Leu-Leu-Arg-Asp-Ser-His-Val-Leu-His (SEQ ID N0: 6).

Fragment agarose gel containing human MGDF, used to obtain samples through the "hot" PCR. A typical PCR reaction mixture of 100 μl contained the following components:

Matrix DNA - 2-3 ál

5' Primer (SEQ ID N0:4) - 1 µl, 20 PM

3 Primer (SEQ ID N0:5) - 1 µl, 20 PM

10 x buffer 10 ál

dATP (0.1 mm) - 2 ál

ál

Taq DNA polymerase and 0.5 μl, 2.5 units

Water - 77 ál

A total volume of 100 µl

The conditions of amplification were as follows: initial heating 94oC, 2 min; annealing - 53oC, 30 sec; capacity - 72oC, 30 sec; denaturation - 94oC, 30 sec.

Was performed 40 cycles of amplification on the instrument Perkin Elmer GeneAmp System 9600.

The product was purified on column (push column (Stratagene, San Diego). In the scintillation counter was calculated 1 µl of sample. Those samples that contained 1-3 million pulses per ml, was added to the hybridization mixture.

B. Constructing libraries of embryonic liver

The company Clontech laboratories was purchased by the poly+PHK fetal human liver. About 4 μg PHK used for synthesis of cDNA, in which the priming served random hexamer, 5' GTA CGC GTT HUNDRED GAN NNN NNT 3' (SEQ ID N0: 7), coupled with the oligonucleotide, containing a Xba restriction site 1.

To obtain dunatoi cDNA was used Protocol (Gibco - BRL. Adapter Eco RI-Bstxl (Invitrogen, San Diego) ligated with dunatoi cDNA and then restrictively enzyme Xba 1.

The selection of cDNA size was carried out on a column of Sephacryl S500 (Life Technologies, Inc. ). The cDNA molecules having a size greater than 400 base pairs, ligated with a vector to Express what was sformirovali competent cells 1E.coli ODH10 and the resulting library was divided into 7 pools under 100,000 cDNA clones in each.

C. Screening of libraries in phage lambda

Library embryonic human liver in phage lambda gt11 with titer 650 million plaque-forming units/ml was purchased from the company Clontech. About 2 million plaques were Procreditbank with a break of the obtained PCR (see above). Hybridization was carried out for 15 hours at 56oC in 6 x SSC, 5x denhardt's solution, 0.1% of SDS, 100 μg/ml ednonachalie DNA salmon sperm.

Conducted numerous rounds of screening. DNA from individual plaques amplified and identifying true-positive plaques this DNA hybridized with an internal primer 5' AGT TTA CTG AGG ACT CGG AGG 3' (SEQ ID N0: 8), encoding amino acids from 7-th to 13-th sequence SEQ ID N0: 6.

, 3'-Praimirovanie rapid amplification of cDNA ends (RACE)

Polyadenylated RNA from human embryonic human embryonic kidney and liver was purchased from Clonetech. One microgram PHK were subjected to reverse transcription using as primers the oligonucleotide 5' TTC GGC CGG ATA GGC CTT TTT TTT TTT TTT 3' (SEQ ID N0: 9).

Kit for cDNA synthesis Gibco-BRL (Life Technologies,Inc. Cat.# 18267-013) used to obtain the first strands of cDNA. The total volume was 30 ál. The reaction was stopped by adding 500 mm EDTA the quality matrix of 0.5 µl of cDNA. Primer SEQ ID N0: 9 and competitive oligonucleotide 5' TTC GGC CGG ATA GGC CTT TTT TTT TTT TT-P 3' (SEQ ID N0: 10) was used as the antisense primer, whereas the oligonucleotide 5' GAC TGC CTC CGA GTC CTC AD 3' (SEQ ID N0: 11) encoding amino acids from 5-th to 11-th sequence SEQ ID N0: 6, used as a sense primer. Forty cycles of amplification were carried out according to the following Protocol: 94oC, 30 sec; 65oC, 30 sec; 72oC, 30 sec; after the initial 2-minute incubation at 94oC. were used For amplification device Perkin Elmer GeneAmp System 9600.

Pinning ("nesting") was performed using sense primer 5' GAG TCC TCA GTA AAC TGC TTC GT 3' (SEQ ID N0: 12) encoding amino acids with 8-th to 14-th sequence SEQ ID N0: 6, with the sequence SEQ ID N0: 9, SEQ ID N0: 10 served antimyeloma primers. It was held forty cycles of amplification with annealing at 65oC. the PCR Products were fractionally electrophoresis in 0.8% of agarose gel and photographed in UV light. Visible bands in the region of 0.8 - 1.2 kilobase.

Then the PCR products were cloned in the vector PCR II (Invitrogen). Selected individual colonies and plasmids were isolated using Qiagen kits (Cat. # 12143 and 12145). Dontcache sequencing was performed using the vector PRA D. The growth of the DNA chain using 3' and 5'- primers

To obtain the full sequence of the gene MGDF was conducted capacity chain DNA with 3'- and 5'- primers using as a matrix of different rulow library embryonic liver. For amplification of the 5'-primer cDNA was used as a matrix of about 20 ng of cDNA from each pool. Used MGDF-specific antisense primer 5' GGA GTC ACG AAG CAG TT AC 3' (SEQ ID N0: 13) encoding amino acids from the 12-th to 17-th sequence SEQ ID N0: 6, and 5' vector v19.8 sense primer 5' CCT TTA CTT HUNDRED GGC CTG 3" (SEQ ID N0: 14). Amplification was performed for 30 cycles with annealing at 53oC. Fixation was carried out for 30 cycles with antimuslim primer 5' GAG GTC ACA AGC AGG AGG A-3' (SEQ ID N0: 15) (encodes amino acids 1 to 6 of the sequence SEQ ID N0: 6) and the vector primer SEQ ID N0: 14.

To build the circuit with 3' ends of cDNA MGDF used antisense vector primer 5' GGC ATA GTC CGG GTC GAC G 3' (SEQ ID N0: 16) and MGDF-specific primer 5' TTC TCC TGC TTG TGA CCT 3' (SEQ ID N0: 17), encoding amino acids 1 to 6 of the sequence SEQ ID N0: 6. Amplification was performed for 30 cycles with annealing at 58oC.

Amplification pinning (nesting amplification") was performed in technologey in pools 1, 7 and 8, which are then cloned in the vector PCR II. Plasmid DNA from individual colonies was purified and sequenced.

That is, the Allocation of full-length clones MGDF man

Many of the original clones had lost part of the amino-end of MGDF, as part of a sequence MGDF was used for priming and docking. Primer 5' CCA GGA AGG ATT CAG GGG 3' (SEQ ID N0: 18), the sequence of which was obtained in experiments on capacity 5'-primer, as described above, was used as a sense primer. Vector primer SEQ ID N0: 16 served antimuslim primer. Spent 35 cycles of amplification with annealing at 58oC. MGDF-specific primer 5' CAA CAA TGC GAC CGC CAG CCA GAC ACC CCG 3' (SEQ ID N0: 19) containing the restriction site Sal 1, and a vector primer (SEQ ID N0: 15) used for fixing (nesting) for 35 cycles. The PCR product was cloned in the vector PCR II and sequenced.

II. The second example of cloning

A. Cloning of N-terminal cDNA MGDF dogs

Degenerate oligonucleotide primers were designed based on the N-terminal amino acid sequence MGDF dogs, described in the previous sections. These primers were used in polymerase chain reaction (PCR) for amplification of posledovatelnostyakh by Chomzynski and Sacchi (Biochem. 162: 156-159 (1987)). First strand cDNA was obtained with a random primer-adaptor 5' GGC CGG ATA GGC CAC TCN NNN NNT 3' (SEQ ID N0: 20) using reverse transcriptase Mo-MuLV and used as template in subsequent polymerase chain reactions. PCR was performed in a volume of 0.5 µl with ~ 50 ng of cDNA using A primer 5' GCN CCN CCN GCN TGY GA 3' (SEQ ID N0: 4), primer semantic chain encoding amino acids 1-6 of the sequence SEQ ID N0: 1 and primer 5' GCA RTG NAG NAC RTG NGA RTC 3' (SEQ ID N0: 5) or primer With 5' GCA RTG YAA NAC RTG NGA RTC 3' (SEQ ID N0: 21), which are primers antisense chain, encoding amino acids 16-21 sequence SEQ ID N0: 1, with three additional nucleotides at the 5'end to enhance stability during hybridization. PCR with Taq polymerase was carried out for 35-45 cycles up until the strip product did not become clearly visible during electrophoresis in agarose gels. For the first two cycles of PCR annealing was carried out at 37oC for two minutes, for subsequent cycles of PCR annealing was carried out at 50oC for one minute. Each reaction was observed multiple lanes of product. Fragments of the gel containing the band of the expected size (66 base pairs) were removed using a Pasteur pipette and were reamplification Stela. Three clones were sequenced and shown that they encode in one frame is read expected sequence MGDF dogs, residues 1-21. Thus was obtained a unique cDNA MGDF dogs, representing the area of the third nucleotide of codon 6 to the third nucleotide of codon 15. One of these clones served as a matrix for obtaining labeled cDNA samples MGDF dogs.

B. Construction of cDNA-OIC libraries from fetal human liver

PHK was isolated from human fetal liver (International Institute for the Advancement of Medicine, Exton, PA) by lysis fabric 5.5 M guanidinoacetate and purification by centrifugation in CsTFA (Pharmacia). Polyadenylation PHK were obtained using oligo (dT)25 dynabeads (Dynal, according to the manufacturer's instructions). Dantcathy cDNA was received from the specified PHK using plasmid system Superscript for cDNA synthesis (Life Technologies, Inc. with one exception, which consists in using a different adapter: 5' TTG GTG TGC ACT TGT G 3' (SEQ ID N0: 22) and 5' CAC AAG TGC ACA ACC CC 3' (SEQ ID N0: 23). After selection of the size of this cDNA was directionally built on sites Bst XI NotI in the vector for expression in mammalian cells pBCB (pBCB obtained on the basis of plasmids Rc/CMV, Invitrogen, consisting of a skeleton pUC19, CMV realnogo strain 10V (Life Technologies, Inc.).

C. Screening for MGDF cDNA-OIC library embryonic human liver

The library of human embryonic liver, fixed in the form of replicas on the filters hybridized with radioactive PCR product of cDNA N-end MGDF (5x SSPE, 2x denhardt's solution, 0.05% sodium pyrophosphate and 0.5% SDS, 100 μg/ml lysate of yeast tRNA, and 100 μg/ml denatured DNA salmon sperm) while 64oC for 18 hours. Filters were washed at 64oC in 5x SSPE and 0.5% SDS and exposed to x-ray film overnight. Were isolated and analysed by two different clone, gibridizatsiya with the specified breakdown.

G. Expression of cDNA-type clones MGDF man

Purified DNA crnkovich clones MGDF was transfusional cell line 293 EBNA (Invitrogen). of 1.5 μg DNA was mixed with 7.5 μl of Lipofectamine (Life Technologies, Inc. ) in 100 μl of medium DMEM without serum. After 20 minutes incubation at room temperature the mixture of DNA-Lipofectamine was added to 5 x 105cells/well (24 - hole tablets Greiner) in 400 μl of DMEM, 1% serum (Fetal Clone II), and incubated at 37oC 6 hours. Then the cells were added to 500 µl of DMEM with 20% serum (Fetal Clone II). After 72 hours was collected, air-conditioned environment and centrifuged through a 0,22 μm centrifuge villanov MGDF man

Using the above strategy for cloning were obtained from the cDNA clones of the person shown in Fig.11 (MGDF-1 and MGDF-2; SEQ ID NOS: 24, 25, 26, 27) and Fig.12 (MGDF-3: SEQ ID NOS: 28, 29). Each is represented in the drawings, the sequence contains a signal sequence of amino acids 1-21, so that in each case the Mature protein begins with amino acids 22.

The results of determining the activity using one described in Example 4A cell tests are presented below in Tables 3 and 4. In Table 3 the conditioned medium of 293 cells, transfected with each of the constructs were harvested after two days of cultivation and tested in the test cell 1A6.1 (32D/mur-MPL+) +/-10 μg/ml mur-MPL-x In Table 4 conditioned medium of 293 cells, transfected with each of the constructs were harvested after four days of cultivation and tested in the test cell 32D/mur-MPL+ (Example 3A) and cells 32D/hu-MPL+ (Example 3B). As can be seen from these data, MGDF-1 and MGDF-2 people, but not MGDF-3, is active against cell lines expressing Mpl human and mouse. Cell line expressing the receptor Mpl person, more sensitive to MGDF-1 and MGDF-2 person than cell line expressing Mpl - receptor mouse.

Presented in almost completely suppressed by soluble human mpl receptor (hu-MPL-X). Hu-MPL-X is present in the conditioned medium of CHO cells producing the protein. This conditioned medium was concentrated to 120 times and then added to the cultures to a concentration of 6.6%. Air-conditioned environment control CHO cells in this definition did not show any effect. The determination was performed as described in Example 4B, except that viable cells was determined after three days of cultivation.

The definition of megakaryocytes man

MGDF-1 and MGDF-2, but not MGDF-3 induced the formation of megakaryocytes from CD34-positive peripheral blood cells. The experiment presented in Table 6, were performed as described in Example 2 with the exception that CD34-positive cells in peripheral blood was collected without elutriation and culture used after seven days of growth. Conditioned medium from each 293 EBNA MGDF construct was used at a final concentration of 20% +/-30 mg/ml mur-MPL-x Control plasma APK9 used at a final concentration of 6%.

Example 12.

This example describes the synthesis of 12 different peyrovani molecules MGDF, namely PEG - PEG and PEG - PEG. In each case, the molecule MGDF, which was combined with Peg, represented the acids 1-63, numbered from the beginning of the Mature peptide). Detailed information about all peyrovani the compounds are given below in tables 7 to 10.

12.1. Obtaining poly-MePEG-MGDF by acylation with MGDF activated MePEG-derived

Obtaining poly-MePEG (20 KD) - MGDF-conjugate (PEG)

Chilled (4oC) solution of MGDF (2.5 mg/ml) in 0.1 M BICINE buffer, pH 8, add to 10-fold molar excess of solid succinimidylester MePEG (mol. mass of 20 KD) (Shearwater Polymers, Inc.). The polymer is dissolved with gentle stirring, and then the reaction was performed at room temperature.

The degree of modification of the protein in the process of the reaction is controlled by using HPLC, given the size of the molecules (size exclusion SEC), using column Superdex 200 HR 10/30 (Pharmacia Biotech). The elution is conducted with 0.1 M sodium phosphate buffer at pH 6.9 and flow rate of 0.7 ml/min

Study of the reaction mixture by SEC HPLC after 30 minutes shows that it is full of protein. At this point in time the protein concentration in the reaction mixture is reduced to 1 mg/ml by adding sterile water, and the pH of the mixture was adjusted to 4 with a few drops of 0.5 M acetic acid.

MePEG-MGDF-conjugate is separated from the excess MGDF and other side reaction products PU the Reaction mixture was applied to a column (2.5 mg/ml ionoobmennoi resin) and unreacted MePEG elute the starting buffer And (3 volume of the column, 20 mm sodium phosphate, pH 7,2, 15% glycerol). Then elute MePEG-MGDF-conjugate using a linear gradient (from 0% to 30%) of the destination buffer (10 column volumes) (1 M NaCl in buffer A). The eluate tested at 280 nm. The fractions containing MePEG-MGDF-conjugate are pooled, concentrated and sterilized by filtration.

Purified poly-MePEG-MGDF-conjugate analyzed by SEC HPLC using gel-filtration columns TSK-GEL 64000SWXL and 62000SWXL combined in series. Proteins detected by absorption in the UV at 280 nm. As markers of molecular weight of globular proteins using gel-filtration standards BIO-RAD.

As can be seen in Fig.17A, with the help of SEC HPLC in drug identified two main components (in approximate ratio of 2 to 1), and their point of elution correlate with globular proteins mol. mass 370.9 KD and 155.0 KD, respectively (see also below Table.8).

Conjugates PEG9, PEG10 and PEG12 obtained by acylation with MGDF succinyldicholine esters MePEGs mol. mass 6-50 KD, exploring the same way. The main parameters of the reactions used to obtain the corresponding drugs are shown in Table 7.

The research results obtained conjugates using SE MePEG in reducing conditions

Obtaining poly-MePEG (20 KD)-MGDF-conjugate (PEG20)

To a chilled (4oC) mixed solution MGDF (2 ml, 2.5 mg/ml) in 100 mm sodium phosphate, pH 5, containing 20 mm NaCNBH3add 10-fold molar excess of monomethoxy - polietilenglikoli aldehyde (MePEG) (average mol. mass of 20 KD), and stirring the reaction mixture continued at the same temperature.

The degree of modification of the protein in the reaction are controlled by SEC HPLC using column Superdex HR 10/30 (Pharmacia Biotech). The elution is conducted with 0.1 M sodium phosphate buffer at pH 6.9 and flow rate of 0.7 ml/min

After 16 h the study of the reaction mixture by the method of SEC HPLC reveal lost that more than 90% of the initial amount of modified protein. At this time, the concentration of protein in the reaction mixture set at 1 mg/ml, sterile diluting it with water, and the pH adjusted to 4 by addition of 0.5 M acetic acid.

MePEG-MGDF-conjugate is separated from the excess of MePEG and other side reaction products by ionoobmennoi chromatography using SP Sepharose HP (Pharmacia Biotech) as ionoobmennoi resin.

The reaction mixture was applied to a column (2.5 mg/ml resin), and unreacted MePEG elute the starting buffer And (3 volume of the column) of the destination buffer (10 column volumes) (1 M NaCl in buffer A). The eluate tested at 280 nm. The fractions containing poly-MePEG - MGDF-conjugate are pooled, concentrated and sterilized by filtration.

Purified poly-MePEG-MGDF-conjugate analyzed by SEC HPLC using gel-filtration columns TSK-GEL 64000SWXL and 62000 SWXL, combined in series. Proteins detected by absorption in the UV at 280 nm. As markers of molecular weight of globular proteins using gel - filtration standards BIO-RAD.

As can be seen in Fig.17B, with the help of SEC HPLC in drug revealed two main components (52% and 47% of total protein), and their point of elution correlate with globular proteins mol. mass 359.4 KD and 159.3 KD, respectively (see also below Table.8).

Conjugates PEG18, PEG19 and PEG21 obtained by reductive alkylation MGDF with aldehydes MePEG mol. mass 6-25 KD, exploring the same way. The main parameters of the reactions used to obtain the corresponding drugs are shown in Table 7.

The research results obtained conjugates with SEC HPLC are shown in Table 8.

12.3. Obtaining conjugates MGDF with monometoksipolietilenglikolya by its connection with alpha aminoethanol on the N-term is Astaro MGDF (2 ml, 2.5 mg/ml) in 100 mm sodium phosphate, pH 5, containing 20 mm NaCNBH3add a 5-fold molar excess of monomethoxy-polietilenglikoli aldehyde (MePEG) (average mol. mass of 20 KD), and stirring the reaction mixture continued at the same temperature.

The degree of modification of the protein in the reaction are controlled by SEC HPLC using column Superdex 200 HR 10/30 (Pharmacia Biotech). The elution is conducted with 0.1 M sodium phosphate buffer at pH 6.9 and flow rate of 0.7 ml/min

After 16 h the study of the reaction mixture by the method of SEC HPLC shows that more than 90% of the initial amount of modified protein. At this time, the concentration of protein in the reaction mixture is reduced to 1 mg/ml, sterile diluting it with water, and the pH adjusted to 4 by addition of 0.5 M acetic acid.

Mono-MePEG(20 KD)-MGDF-conjugate is separated from the excess of MePEG and other side reaction products by ionoobmennoi chromatography using SP Sepharose HP (Pharmacia Biotech) as ionoobmennoi resin.

The reaction mixture was applied to a column (2.5 mg/ml resin) and unreacted MePEG elute the starting buffer And (3 volume of the column, 20 mm sodium phosphate, pH 7,2, 15% glycerol).

Then MePEG-MGDF-conjugate elute with a linear is, operasie poly-MePEG-MGDF-conjugate are pooled, concentrated and sterilized by filtration.

The homogeneity of the mono-MePEG-MGDF-conjugate confirm gelelectrophoresis in polyacrylamide gel in the presence of SDS using 4-20% gradient gel company NOVEX. Identify one major band corresponding to the protein mol. mass 46,9 KD.

Purified poly-MePEG-MGDF-conjugate analyzed by SEC HPLC using gel-filtration columns TSK-GEL 64000SWXL and 62000 SWXL, combined in series. Proteins detected by absorption in the UV at 280 nm. As markers of molecular weight of globular proteins using gel - filtration standards BIO-RAD.

As can be seen in Fig.17C, with the help of SEC HPLC in drug revealed one main component, and its point of elution corresponds to a globular protein mol. mass 181.1 KD (see also PL.9).

Mono-MePEG-MGDF-conjugates PEG14, PEG15 and PEG17 obtained by reductive alkylation MGDF with aldehydes MePEG mol. mass 6-25 KD, exploring the same way. The main parameters of the reactions used to obtain the corresponding drugs are shown in Table 7.

The research results obtained conjugates with SEC HPLC shows what letreguilly (PEG20) in reducing conditions

The following method yields a purified substance called here PEG22.

5-fold excess of the aldehyde methoxypolyethyleneglycol (MePEG, for example, OHC-(CH2)2-O-(CH2-CH2O)n-CH3; where n is repeated many times, so they say. weight was about 12 KD) (Shearwater Polymers) are added to 2.5 mg/ml solution MGDF (obtained in E. coli, 1-163) in 100 mm sodium acetate, pH 5, cooled to 5oC. After 10 minutes of mixing, add the appropriate amount of cyanoborohydride sodium (Aldrich) to its concentration in the reaction mixture consisted of 20 mm.

The resulting mixture is stirred for 16 hours at a temperature of 5oC. after the specified period of time the concentration of MGDF set at the level of 1 mg/ml by adding sufficiently purified water. The mixture is then vacuum filtered through a filter with pore size 0.2 μm. In this procedure receive 90 mg of the reaction product. To the mixture containing the reaction product, add a small number of solutions of 1.0 M monobasic phosphate and 1 N sodium hydroxide to obtain a solution containing 10 mm phosphate at pH of 6.8.

The conjugate is purified on a cation-exchange column. Prepare 40 ml Colo is appropriate buffer (10 mm phosphate, pH of 6.8, 15% glycerol). Column load of 2.2 mg/ml of resin at a flow rate of 0.15 column volumes (CV) per minute. Then the column was washed with equilibrating buffer until reaching the zero point of reference. Column elute with a linear gradient from buffer A (10 column volumes of 20 mm phosphate, pH 7,2, 15% glycerol) to buffer B (buffer a and 0.3 M NaCl). The flow rate is maintained at a level of 0.15 CV. The eluate tested at 280 nm.

Polyacrylamide gels containing SDS, separated into fractions, and those that contain conjugate DiPEG are pooled and filtered through a cell with a pore size of 0.2 μm.

Example 13. Biological activity peyrovani molecules MGDF

A. PEG-9 to PEG-12, PEG-14 - PEG-21

The number of platelets in mice treated with recombinant human MGDF, reflected in Fig.18. Obtained in CHO cells MGDF (22-353) (open stars), not-peyrovani MGDF obtained in E. coli (22-184) (empty circles), and peyrovani MGDF obtained in E. coli (22-184) (filled circles) at concentrations indicated in the description of the drawings above, was administered subcutaneously to normal Balb/c mice once a day for 5 days. 24 hours after the last injection took control samples of blood from a small lateral incision caudal vein. Analyses of blood cells providere animals +/- standard deviation. This treatment had no effect on other parameters of blood cells, such as the total number of red and white blood cells.

Other forms of recombinant human MGDF tested as described above. The number of platelets in mice treated with 50 mg/kg/day or 10 mg/kg/day specified form r-HuMGDF, are given in Table 10. The values presented are the average of four animals, and standard errors are given in italics.

B. PEG-22

The results obtained with PEG-22, shown in Fig.24. It is noteworthy that the normalization of the platelet count with PEG-22 came a few days earlier than with a full size obtained in CHO PEG-MGDF-16 or PEG-17.

Example 14. Expression of recombinant human MGDF (1 - 163) in E. coli

For the expression of r-HuMGDF in E. coli using the optimal codons of E. coli was chemically synthesized sequence encoding the first 163 amino acids of the Mature protein. In addition, the DNA sequence encoding amino acids Methionine and Lysine were added to the 5'-end of the gene. Thus, the protein r-HuMGDF, consisting of 165 amino acids, began with Met-Lys. The sequence of this gene is shown in Fig.25.

Synthesis gene r-HuMGDF (1-163) was performed in several ethanolamide (length of 60-70 base pairs), representing fragments adjacent to the gene. During this synthesis, the codons for the amino acids Methionine and Lysine were placed on the 5'-end of the Mature gene and the stop codon was placed at the 3'end of the gene. In addition, the recognition sites of the restriction enzymes Xbal and Hindlll were placed in close proximity to the 5' and 3' ends of the gene, respectively, and synthetic binding site of ribosomes was placed at an appropriate distance above from the initial Methionine. Secondly, there were shibatani oligonucleotides, complementary to each fragment of the gene. Thirdly, these individual synthetic gene fragments were amplified in polymerase chain reactions. Fourth, amplificatoare fragments were subcloned into an appropriate vector. Fifth, verify the sequence of the subcloned fragments. Sixth, the individual fragments are ligated together and was subcloned into an appropriate vector, restoring full gene r-HuMGDF (1-163). Finally, verify the sequence of the gene restored.

Synthetic fragment r-HuMGDF gene is flanked by restriction sites Xbal and HindIII 5' and 3' ends, respectively, contained the binding site of the ribosome, the initiating ATG codon, the sequence code is kotinurmi the expression vector pAMG11. Vector pAMG11 is nicocodine plasmid with the beginning of replication derived from the pR100. Expressing plasmid pAMG11 can be obtained from plasmid pCFM1656 (deposited 24 February 1994 in the American collection of cell cultures, ADS # 69576) through a series of directed substitutions of bases in the overlapping oligonucleotides in a polymerase chain reaction. Starting with Bglll site (base pair plasmid # 180) directly in the 5' direction of the promoter replication plasmids PcopB and forth in the direction of replicative genes plasmid of base-pair substitutions were such (see tab. B).

Expression of r-HuMGDF cloned in pAMG11, directed synthetic lactoseintolerant promoter, similar to the Ps4, which has the following sequence:

5'GACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCTAA- - ATTCTTGATTAATATTCTCAATTGTGAGCTCACAATTTATCGAT3' (SEQ ID N0: 34).

The promoter Ps4 is suppressed by the lactose repressor (LacI), the lac gene product of E. coli.

Then the plasmid pAMG11-r-HiMGDF transformed cells of E. coli strain K-12, with laclq-allele. Allele laclq is a mutation of the lacI promoter, which enhances the expression of LacI, which leads to a more strict control of protein expression from the promoter of the Ps4. Thus in the cells of this strain in the absence of lactose Ps4 is reduced and begins transcription r-HuMGDF with the Ps4. Ispolzovanie in this example, the cells of the host E. coli deposited under number ANNC # 69717 November 30, 1994

Cells of E. coli ATCC # 69717 transformed with plasmid pAMG11-r-HuMGDF and were grown according to the following Protocol. The strain of E. coli was inoculable in Luria broth and incubated at 30oC for approximately 12 hours. The cells are then aseptically transferred into the fermenter, which contained a medium of the following composition: 20 g/l yeast extract; 3.4 g/l citric acid; 15 g/l K2HPO4; 15 ml Dow P2000; 5 g/l glucose; 1 g/l MgSO47H2O. the First phase of the cultivation process (batch phase) lasted up until the culture reached an optical density of 5.0 +/- 1.0 at 600 nm. Then began the phase of feeding (fed-batch) with the filing of the first fueling environment (700 g/l glucose; 6,75 g/l MgSO47H2O). The rate of recharge was adjusted every two hours according to the set schedule. Feeding a second feed medium (129 g/l trypticase patina; 258 g/l yeast extract) was started when the culture optical density of 20-25 at 600 nm. Feeding a second feed medium was maintained at a constant level, whereas the supply of the first fueling environment continued to change. The temperature during the entire process fermenter is being the level of dissolved oxygen is maintained, changing the stirring speed and the feed rate of air and oxygen in the fermenter. When the optical density of the culture reached 57-63 at 600 nm, began filing third fueling environment. Third fueling environment (300 g/l lactose) was injected into the fermenter at a constant speed DC; adding the first fueling environment at this moment stopped, and the feed speed of the second feed medium was set on a different constant level. The fermentation is continued for approximately ten hours from the start of adding a third fueling environment. At the end of fermentation, the culture was cooled to 15 +/-5oC. the Cells were collected by centrifugation. The resulting paste was packaged and stored at a temperature below -60oC.

Purification of recombinant MGDF obtained in E. coli, as described above, was carried out as follows. 1800 g of cell paste is suspended in approximately 18 liters of 10 mm EDTA and passed through a high-pressure homogenizer at 15,000 psi. A destroyed suspension of cells was centrifuged and the precipitate resuspendable in 10 liters of 10 mm EDTA. The suspension was centrifuged and the resulting 200 g sediment was solubilizers in 2 liters of buffer of the following composition: 10 mm Tris, 8M guanidine hydrochloride, 10 mm diti 3M urea; 30% glycerol, and 3 mm tsistamin; 1 mm cysteine, pH of 10.5. The diluted solution was slowly stirred at room temperature for 16 hours and the pH was brought to 6.8. Then the solution was brought to pH was osvetleni and put on 2-liter column of CM Sepharose, equilibrated with 10 mm sodium phosphate, 1.5 M urea, 15% glycerol, pH of 6.8. After loading the column was washed with 10 mm sodium phosphate, 15% glycerol, pH of 7.2. MGDF was suirable gradient of NaCl from 0 to 0.5 M, 10 mm sodium phosphate, pH 7,2.

The eluate was concentrated and was replaced with a buffer of 10 mm sodium phosphate, pH 6.5 using a membrane with a cutoff molecular weight of 10,000. A concentrated solution (about 2 mg/ml) were treated with cathepsin C (molar ratio of 500: 1) for 90 minutes at a suitable temperature. Then the solution was applied to a 1.2-liter column of SP Sepharose High Performance, balanced 10 mm sodium phosphate, 15% glycerol, pH of 7.2. MGDF was suirable gradient of NaCl from 0.1 to 0.25 M, 10 mm sodium phosphate, pH 7,2.

The eluate from the column of SP Sepharose High Performance) was added ammonium sulfate to a concentration of 0.6 M and Then the eluate was applied to a 1.6-liter column of Phenyl Toyopearl, equilibrated with 10 mm sodium phosphate, 0.6 M ammonium sulfate, pH of 7.2. Peak MGDF was suirable gradient of ammonium sulfate from 0.6 to 0 M, 10 mm phosphate Neemrana with a cutoff molecular weight of 10,000.

Example 15. Biological properties of r-HuMGDF (E. coli 1-163) in vivo

Biological efficacy of r-HuMGDF (E. coli 1-163), obtained as described above in Example 14, was evaluated in rodents. Normal female Balb/c mice for five consecutive days was administered subcutaneously increasing doses of r-HuMGDF. Doses ranged from 15 ág/kg/day to 1500 mg/kg/day. 24 hours after the last injection count of blood cells using electronic counter cells (Sysmex, Baxter). There has been a linear increase in the number of platelets parallel to the logarithmic increase of the concentration of the cytokine. In this system, at the dose of 1500 mg/kg/day, the number of platelets was increased up to 300% compared to the original. Other indicators of blood cells, such as the number of red and white cells or hematocrit did not change during this process.

Rats were injected subcutaneously r-HuMGDF (E. coli 1-163) at doses of 300 mg/kg/day for 6 days, then they took platelets and determined the ability of the latter to aggregate in response to ADP. It is shown that platelets treated animals are virtually indistinguishable from platelets control animals sensitivity to agonist of platelet ADP.

Evaluated the ability of r-HuMGDF to Bolotin, chemotherapeutic agent, can cause profound thrombocytopenia. In the beginning of the experiment, mice Balb/c mice were injected subcutaneously with 1.25 mg carboplatin. After 24 hours, started daily injections of 100 µg/kg/day r-HuMGDF (E. coli 1-163) or control solvent. For the ninth day the platelet count fell to 15% from normal mice treated with a single solvent, but remained within the normal range of mice treated with r-HuMGDF (see Fig.20). In experiments with irradiation, mice received a single dose of 500 rad of gamma radiation (cesium source). This sublethal dose resulted in a decrease in the number of platelets 90% of the 11-th day. The platelet count did not return to normal until the 21st day. With the introduction of irradiated mice with 1-th to 20-th day 100 mg/kg/day r-HuMGDF (E. coli 1-163) reduction in the number of platelets was less sharp and return to their normal number faster than in mice treated with a single solvent (Fig. 21). To test r-HuMGDF on models of acute and prolonged thrombocytopenia, was applied combination carboplatin and irradiation (Fig.22). In this case, the platelet count decreased to extremely low levels (3-5% of normal) and most animals (7/8) had not experienced such treatment. However, when the animal is first degree korrelirovalisj, return to the original number of platelets was faster and all treated with r - HuMGDF animals (8/8) survived.

r-HuMGDF tested also on rhesus monkeys. Normal makaka RH was administered subcutaneously drug r-HuMGDF at doses of 2.5 or 25 mg/kg/day for ten days (days 0-9). In the group treated with low dose, platelet count increased to 400% of normal by the 12th day, and in the group treated with the high dose, the increase amounted to 700% by the 12th day. After injection of the platelet count returned to normal for 25-30 th day. This treatment does not affect the number of white and red blood cells.

r-HuMGDF was also tested in a Primate model of acute thrombocytopenia (Fig.23). The animals that were irradiated (700 rad, a cesium source) resulted in a decrease in the number of platelets to 1-2% of normal to the 15-th day. 35-40 day the platelet count returned to normal. In irradiated animals, which were injected r-HuMGDF (25 mg/kg/day), platelet count decreased only up to 10% from the norm and on average did not fall below 20,000/ml, the value at which to start the transfusion of platelets for people suffering from thrombocytopenia. Return to the normal number of platelets also occurred more rapidly in animals treated with r-HuMGDF, and it was on the 20th de is th about that r-HuMGDF (E. coli 1-163) is a powerful therapeutic agent that is able to significantly influence occurring in the clinic thrombocytopenia.

Example 16. Method of obtaining r-HuMGDF (1-332) in cell culture CHO

Glycosylated r-HuMGDF 1-332 is produced transfitsirovannykh CHO cells expressing the cDNA MGDF 1-332 under the control of a suitable promoter and linked to a gene coding for amplificatory selective marker DHFR. A suitable promoter for expression of MGDF in CHO cells is the promoter SR - alfa, see Mol.Cell.Biol. 8: 466 - 472 (1988) and the patent WO 91/13160 (1991). A suitable vector for the expression of MGDF in cells is pDSR-alfa2, see WO 90/14363 (1990). Cell line CHO can produce secretory MGDF in the amount of 10-20 mg/l in standard culture medium, but the level of production can be increased from 25 to 100 mg/L. When products MGDF in a typical cell line culture may be "dispersed" passages in suspension or culture bottles (in the case of substrate - dependent growth) using culture medium consisting of equal amounts of medium Needle in the modification Dulbecco (DM) and environment ham F12 (DMEM/F12, Gibco) with the addition of from 5 to 10% fetal calf serum (FBS) or cialisbuynow fetal calf serum is about pressure. Wednesday should be additionally added a non-essential amino acids (NEAA's) and glutamine. Suspension cultures can be accrued within concentrations of 1-4 105cells/ml (seeding concentration) to approximately 1 million cells/ml At the maximum concentration of the cells that produce the passage of culture by dilution to large volumes and achieve seed concentration.

Upon receipt MGDF in roller bottles, suitable volume and density of cells in suspension culture should be selected using spinnery vials with magnetic stirring, placed in a temperature-controlled conditions (37+/-1oC), or by using automatic controlled system with a bioreactor. Roller bottles (e.g., vials company Falcon area of 850 cm2must be seeded with cells from sowing doses from 1.5 to 3 107cells per vial in such a quantity of growth medium (DMEM/F12 with 5-10% FBC, 1xNEAA and 1x glutamine), which would provide education merged monolayer on the 3-4th day of cultivation (150-300 ml). Growth medium should be buffered with sodium bicarbonate to a pH of 6.9-7.2 and balanced by the carbon dioxide to the partial pressure of 60-90 mm Hg. The bottles due and rotation speed of about one revolution per minute for 3-4 days. Upon reaching monolayer mallernee culture should be transferred to serum-free production environment. This old growth environment is drained or sucked off, the culture was washed with isotonic buffer such as phosphate buffer solution Dulbecco (D-PBS), 50-100 ml per vial, then add the appropriate amount of bicarbonate buffered serum-free medium DMEM/F12 (1: 1) (200-300 ml per vial) with the addition of NEAA, glutamine and copper sulfate (1-20 μm) to minimize covalent aggregation. The vials are filled with a gas mixture of 10% CO2in the air) and incubated for 6 +/-1 days at 37oC on a roller apparatus (~ 1 rpm) or until such time when the metabolism of the cells, the glucose level in the environment drops below 0.5 g/l and/or the pH value falls below the 6.6. Air-conditioned environment is drained or sucked off from the flasks and replaced with fresh serum-free production medium of the above composition for additional fees. This can last up until the cell can no longer tolerate serum-free conditions and will not start to slide with roller bottles.

The collected air-conditioned environment is cleaned by filtration through filters with pore size of 0.45 or 0.2 μm (Sar time at this temperature or immediately concentrate and cialiswhat against buffer with low ionic strength using tangential ultrafiltration system (for example, Filtron YM-50). Ultrafiltration and diafiltration be carried out at 4oC to minimize degradation of the protein. To chromatographic purification, air-conditioned environment must be Valitova against buffer solution (for example, 10 mm potassium phosphate, pH 6,8).

The quality of the product contained in air-conditioned environment, it is best to monitor using SDS-PAGE in non conditions with subsequent Western blot testing, which can detect the amount of the aggregate, monomer and proteoliticeski degraded MGDF in samples.

Another method to obtain MGDF in CHO cells consists in the adaptation of cell lines expressing MGDF, to serum-free medium, such as Gibco S-SFM II. Cells can be adapted by serial passages in medium containing minimum serum or does not contain it at all. If the cell line will grow steadily in this environment and to produce adequate quantities of secreted MGDF, cultivation may continue by serial passages in increasing volumes, the cells are then transferred into a suitable production vessel (automatic controlled bioreactor) and the culture allowed to proliferate until the maximum of temperature, oxygen, destruction of cells). In the optimal point of production (which is determined experimentally by measuring the quantity and quality of the product) the culture is harvested from the bioreactor, the cells are removed from the air conditioned environment micron depth filter or submicron tangential microfiltration. In the case of deep micron filtration medium should be clarified in subsequent sub-micron final filtration for concentration and dialysis as described above.

Despite the fact that the claimed invention described above, both generally and in relation to specific embodiments, it should be understood that modifications of the invention based on the above descriptions may be reproduced by any specialist in this field of research. Thus, the stated claims in fact covers all variants of the invention, included in its scope.

In addition, publications and other information sources provided to assess the level of technology and a better understanding of the description of the invention, in particular for a more detailed coverage of practical issues that are included in the application materials as references.

1. Polypeptide growth factor and differe the selected FRDM-7 from 22 to 198 amino acids, shown in the graphical part and the sequence FRDM-2 comprising amino acids 22 to 195 a given sequence of FRDM-7, the sequence FRDM-4 that includes amino acids 22 to 172 a given sequence of FRDM-7, the sequence FRDM-5, which includes amino acids 22 to 177 given sequence FRDM-7, the sequence FRDM-6, which includes amino acids 22 to 191 given sequence FRDM-7 and the sequence FRDM-8, which includes amino acids 22 to 184 a given sequence of FRDM-7.

2. Selected polynucleotide encoding FRDM under item 1.

3. Selected polynucleotide under item 2, which represents the DNA sequence.

4. The DNA sequence under item 3, which is a cDNA sequence.

5. The cDNA sequence for p. 4, a nucleotide sequence of 1, or the nucleotide sequence 2 shown in the graphics part.

6. Strain cell culture 293 EBNA, stably transformed or transfetsirovannyh the DNA sequence under item 4 and expressing the specified polypeptide under item 1.

7. A method of obtaining a polypeptide FRDM, the conclusion is in a suitable nutrient medium and the allocation of the specified polypeptide.

8. Derived FRDM representing polypeptide FRDM connected with at least one water-soluble polymer having a mol.m. 2 - 100 KD, the molecular ratio of polymer:polypeptide 1:1 to 20:1 by education acyl or amide bond.

9. Derived FRDM under item 8, characterized in that the polypeptide is chosen from the group comprising sequence FRDM-1 22-353 amino acids shown in the graphics part, and the sequence FRDM-11, which includes amino acids 22 to 184 a given sequence of FRDM-1, the sequence FRDM-12, comprising amino acids 27 to 353 a given sequence of FRDM-1, the sequence FRDM-13, comprising amino acids 27 to 195 a given sequence of FRDM-1, the sequence FRDM-14, comprising amino acids 27 to 172 a given sequence of FRDM-1, and the sequence FRDM-15, comprising amino acids 27 to 184 a given sequence of FRDM-1.

10. Derived FRDM under item 8, characterized in that the polypeptide FRDM is produced in a bacterial cell in the form of a recombinant polypeptide.

11. Derived FRDM under item 8, characterized in that the water-soluble Panny water-soluble polymer selected from the group containing dextran, poly(N-vinyl-pyrrolidin), glycols, homopolymers of polypropylenglycol, copolymers of propylene oxide/ethylene oxide, polyoxyethylene polyols, polyvinyl alcohols, and mixtures thereof.

13. Derived FRDM under item 8, characterized in that the water-soluble polymer is a polyethylene glycol.

14. Derived FRDM on p. 13, characterized in that the polyethylene glycol is a monometoksipolietilenglikolya.

15. Derived FRDM on p. 13, characterized in that polietilenglikolja group connected with the N-terminal part of the polypeptide.

16. Derived FRDM on p. 13, characterized in that polietilenglikolja group has an average mol.m. 10-50 KD.

17. Derived FRDM under item 18, which is a polypeptide FRDM, covalently linked to two water-soluble polymer molecules.

18. Derived FRDM under item 17, characterized in that the said water-soluble polymer molecules are molecules of polyethylene glycol.

19. The method of obtaining derivatives FRDM under item 8 by connecting with a water-soluble polymer having a single reactive aldehyde group, zakluchalsya alkylation at a sufficiently acidic pH, to the alpha-amino group on aminoterminal the area specified polypeptide FRDM entered into the reaction, and produce the specified polypeptide FRDM connected with at least one water-soluble polymer.

20. The method of obtaining derivatives FRDM under item 8 by connecting with a water-soluble polymer having a single reactive ester group, namely that contact the specified polypeptide FRDM with a water-soluble polymer under conditions where the polypeptide FRDM connected with a water-soluble polymer through an acyl link and allocate the specified polypeptide FRDM connected with at least one water-soluble polymer.

21. The method according to p. 19 or 20, characterized in that the polymer is pharmaceutically acceptable.

22. The method according to p. 19 or 20, characterized in that the water-soluble polymer selected from the group consisting of dextran, poly(N-vinyl-pyrrolidone), polyethylene glycols, homopolymers of polypropylenglycol, copolymers of propylene oxide/ethylene oxide, polyoxyethylene polyols and polyvinyl alcohols.

23. The method according to p. 19 or 20, characterized in that the water-soluble polymer is polite the>25. The method according to p. 19, wherein the polypeptide FRDM get the removal of Met-2-Lys-1 polypeptide, obtained by expression in a cell of E. coli DNA sequence that encodes a polypeptide containing amino acids 22-184 of the sequence FRDM-1 and the sequence Met-Lys at the N-terminal region.

26. The method according to p. 19, wherein the polypeptide FRDM produced by expression in a cell of E. coli DNA sequence that encodes a polypeptide containing amino acids 22-184 of the sequence FRDM-1 and the sequence Met-Lys at the N-terminal region, highlighting expressed polypeptide and off sequence Met-2-Lys-1 from the selected polypeptide.

27. Derived polypeptide FRDM representing polypeptide FRDM connected acyl communication with one molecule of polyethylene glycol having a mol.m. 2-100 KD.

28. Derived polypeptide FRDM on p. 27, characterized in that the polypeptide FRDM selected from the group consisting of FRDM-1, MDM-2, MDM-4, FRDM-5, FRDM-6, FRDM-7, FRDM-8, PDM-11, PDM-12, FRDM-13, FRDM-14, FRDM-15, and FRDM-3 having the sequence of amino acids 22 to 286 a given sequence of FRDM-1.

29. Pengelola through the alpha amino group at the N-terminal region.

30. Derived FRDM on p. 29, wherein the polyethylene glycol has an average mol.m. 5-50 KD.

31. Derived polypeptide FRDM on p. 27, characterized in that polietilenglikolja group connected with the N-terminal part of the polypeptide.

32. Derived polypeptide FRDM under item 27, wherein the polypeptide FRDM is produced by E. coli.

33. Pharmaceutical composition, enhance growth and/or development of metacritical containing the derived polypeptide FRDM under item 8 and a pharmaceutically acceptable diluent, adjuvant or carrier.

34. Pharmaceutical composition, enhance growth and/or development of materialto containing the derived polypeptide FRDM on p. 27 and a pharmaceutically acceptable diluent, adjuvant or carrier.

35. A method of treating a patient with deficiency of the polypeptide FRDM, which includes the introduction of an effective amount of the polypeptide FRDM under item 1 or a derivative FRDM under item 8.

36. A method of treating a patient with thrombocytopenia, which includes the introduction of an effective amount of the polypeptide FRDM under item 1 or a derivative FRDM under item 8.

37. The method according to p. 36, wherein the disease is in the group, enabling the of Riem drugs or radiation.

38. The way to increase the number of Mature megakaryocytes in patients who are in need, including the introduction of an effective amount of the polypeptide FRDM under item 1 or a derivative FRDM under item 8.

39. The way to increase the platelet count in a patient who needs it, including the introduction of an effective amount of the polypeptide FRDM under item 1 or a derivative FRDM under item 8.

Priority points:

31.05.94 on PP.1-3, 35-39;

12.10.94 on PP.4-34.

 

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