PL183631B1 - Derivatives of a polypeptide constituting a growth and development factor for megacariocytes (of mgdf polypeptide) method of obtaining such derivative and pharmacological composition containing same - Google Patents

Abstract

The factors of growth and differentiation of megacaryocytes and their derivatives combined with water-soluble polymers such as polyethylene glycol are disclosed as well as the methods for their synthesis and the pharmaceutical compositions.

Description

The present invention relates to novel derivatives of a megakaryocyte growth and development factor polypeptide, hereinafter synonymously referred to as Mp1 ligand or MGDF, which stimulates megakaryocyte growth and enhances megakaryocyte differentiation or maturation, with the ultimate effect of increasing the number of platelets. The invention also provides a method for the preparation of these derivatives. The invention also relates to a pharmaceutical composition containing MGDF derivatives as an active ingredient.

At least two broad fields of research are involved in the present invention. The first relates to the development of megakaryocytes and the subsequent production of platelets, while the second relates to the polypeptide member of the growth factor receptor family, hereinafter referred to as the Mp1 receptor - and its ligands. Each of these research areas will now be discussed in outline.

A. PLATE FORMATION FROM MEGACARIOCYTES

Platelets are circulating cells that play a critical role in preventing bleeding and blood coagulation. Megakaryocytes are the cellular source of platelets and arise from the precursor cell of normal bone marrow which causes all haemopoietic (ie, hematopoietic) cell lines. This common precursor cell is known as multi-directional stem cell or PPSC.

The hierarchy of megakaryocyte progenitor cells was determined based on the time of appearance and size of megakaryocyte (MK) colonies emerging in in vitro culture systems in response to the appropriate growth factors. The most primitive cell of a megakaryocytic progenitor is the megakaryocyte of the burst-forming unit (BFU-MK). BFU-MK is believed to ultimately produce numerous colony forming unit megakaryocytes (CFU-MK), which are the more differentiated MK progenitor cells.

As MK cells undergo subsequent differentiation, they lose the ability to undergo mitosis but acquire the ability to endoreduplicate. Endoreduplication (or endomitosis) is the phenomenon of nuclear division in cells in the absence of cell division. Endoreduplication ultimately results in MK which is a polyploid. Further maturation of MK results in the acquisition of cytoplasmic organelles and membrane components that characterize the plaques.

Plates are produced from mature MKs by a poorly defined process that has been suggested to be a consequence of physical MK fragmentation or other mechanisms. Observations of the extensive membrane structures within megakaryocytes have led to a model of plaque formation in which the demarcation membrane system scratches emerging plaques within the cell substance. Another model of platelet formation is based on the observation that megakaryocytes will form long cytoplasmic outgrowths bounded by compartments depending on the size of the platelets, from which platelets are believed to escape due to blood flow pressures in the marrow and / or lungs. These cytoplasmic outgrowths were by Becker and De

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Bruyna called proplates to emphasize their assumed role of a precursor in the creation of tiles. See Becker and De Bruyn, Amer. J. Anat. 145: 183 (1976).

In the accompanying drawing, Fig. 1 shows a top view of various precursor cells associated with megakaryocyte and platelet development. The leftmost cell of the figure shows PPSC and the additional cells to the right of PPSC show BFU-Mk followed by CFU-MK. The endoreduplicated cell located immediately to the right of the PPSC is an adult megakaryocyte cell. As a result of endomitosis, this cell became polyploid. The next structure to the right includes long cytoplasmic outgrowths emerging from the polyploid nucleus of a mature megakaryocyte cell. The rightmost side of the figure shows a number of plaques generated by fragmentation of cytoplasmic processes.

Some of the prior art publications relating to the above description of megakaryocyte maturation and platelet production are summarized below:

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

2. Levin, J., Molecular Biology and Differentiation of Megakaryocytes, publ. Wile-y-Liss, Inc. 1-10 (1990).

3. Gewirtz, A.M., The Biology of Haematopoesis, Publ. Wiley-Liss, Ina .. 123-132 (1990).

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

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

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

B. ADJUSTING THE TILE CREATION

The considerable amount of data obtained in many laboratories indicates that the production of platelets is regulated by humoral factors. The complexity of this biological process was initially underestimated and it is now shown that growth factors have this capacity.

Regulation of megakaryocytes takes place at many cellular levels. A number of cytokines improve platelet production by expanding the progenitor cell pool. The second group of humoral growth factors serve as maturation factors acting on more differentiated cells to promote endoreduplication. Moreover, it turns out that there are two independent biofeedback loops regulating these processes.

Several nonspecific haemopoietic lineage growth factors exert an important influence on MK maturation. Granulocyte macrophage colony stimulating factor (GMCSF), interleukin-3 (IL-3), IL-6, IL-11, leukemia inhibitory factor (LIF) and erythropoietin (EPO) each individually promote the maturation of human MK as determined in vitro by their effect on size, MK number or ploid. The MK maturation-related effects of LIF, IL-6 and IL-11 are partially (LIF and IL-6) or completely (IL-11) additive to the effects of iL-3. Data from prior art publications suggest that a combination of cytokines may be necessary to promote MK maturation in vivo.

Some prior art publications relating to the regulation of megakaryocyte and platelet production are given below:

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., Cohen, J.L., Clin. Pharmacol. Ther., 46 (3): 250-256 (1989).

11. Gewirtz, A.M., 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: 372-281 (1993).

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

It has also been reported (see reference 16) that aplastic human serum contains a megakaryocyte colony stimulating activity different from that of IL-3, a granulocyte colony stimulating factor, and factors present in a lymphocyte medium. However, the molecule responsible for this activity has never been isolated or characterized.

16. Mazur, E.M., et al., Blood: 290-297 (1990) C. Receptor Mpl

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Leukemia Myeloproliferative Virus (MPLV) is a replication-incomplete murine retrovirus, causing acute leukemia in infected mammals. The MpLV-expressed gene was found to consist of the part of the gene encoding the retroviral envelope (or outer protein envelope) of the virus linked to a sequence related to the cytokine receptor family, including receptors for gM-CsF, G-CSF and EpO.

Expression of the MPLV gene as described above has the interesting biological property of causing murine progenitor cells of different types to directly gain growth factor independence for both proliferation and end maturation. In addition, some bone marrow cell cultures sharply transformed with MPLV contained megakaryocytes, suggesting an association between the MPLV gene and megakaryocyte growth and differentiation.

It is now recognized that the MPLV viral gene (called v-Mpl) has a homolog in mammalian cells called the cellular Mpl (or c-Mpl) gene. The cDNAs corresponding to the human c-Mpl gene were cloned using probes derived from v-Mpl. Compare pCt application publication No. WO 92/07074 (published April 30, 1992; discussed below). Sequence analysis showed that the protein encoded by the c-Mpl gene product belongs to the superfamily of the highly conserved cytokine receptor, similar to the homologous v-Mpl gene product.

This cellular gene, c-Mpl, is believed to play a functional role in haemopoiesis based on the observation that its expression has been found in bone marrow, spleen, and fetal liver in normal mice by securing the RNAse probe and RT- experiments. PCR, but not found in other tissues. In particular, c-Mpl is expressed on megakaryocytes. The human cell gene, human c-Mpl, has also been shown to be expressed in CD34 positive cells, including purified megakaryocytes and plates. CD34 is an antigen indicative of early hemopoietic progenitor cells. Furthermore, exposure of CD34 positive cells to synthetic oligodeoxynucleotides antisense to c-Mpl mRNA or information significantly inhibits the colony formation capacity of CFU-MK megakaryocyte progenitors, but has no effect on erythroid or granulomacrophage progenitors.

The above data and observations suggest that c-Mpl encodes a cell surface molecule, referred to herein as the Mpl receptor, which binds to a ligand that activates the receptor, potentially leading to the production and / or development of megakaryocytes.

PCT Application Publication No. WO 92/07074 targets the protein sequence produced by the c-Mpl gene from both human and mouse bodies. This gene product, which is believed to be a receptor, as explained above, consists of at least three essential regions or domains: the extracellular domain, the endothelial domain, and the intracellular (or cytoplasmic) domain. Together, these domains form the entire Mpl receptor. Said PCT publication also relates to a soluble form of the receptor which essentially corresponds to the extracellular domain of the mature c-Mpl protein. The intracellular domain contains a hydrophobic region which, when attached via the endothelial region to the extracellular domain of a protein, causes the entire protein to aggregate and become insoluble. On the other hand, when the extracellular domain of the c-Mpl gene product is separated from the transmembrane domain and the intracellular domain, it becomes soluble, thus the extracellular form of the protein is called the "soluble" form of the receptor.

The following is a summary of some of the prior art publications relating to the above description of the v-Mpl and c-Mpl genes and receptors:

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, I., et al., 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).

23. Methia, N., et al., Blood 82 (5): 1395-1401 (1993).

24. Wendling, F, et al., Blood 80: 246a (1993).

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D. THE REQUIREMENT OF A FACTOR CAPABLE OF STIMULATING THE PRODUCTION OF PLATES.

Recently, it has been reported that platelet transfusions are increasingly performed in medical centers in North America, Western Europe. and Japan. See Gordon, M.S. andHofcian, R., Blood 80 (2): 302-307 (1992). This increase appears to be largely due to advances in medical technology and greater access to techniques such as heart surgery and bone marrow, heart and liver transplantation. Dose intensification as a means of treating cancer and HIV-1 patients has also contributed to the great need for platelet supply.

The use of platelets carries the potential for the transmission of many blood-borne infectious diseases as well as for alloimmunization, that is, immunization with allogeneic antigens derived from a genetically different individual of the same species. Moreover, the production of cleaned wafers is an expensive endeavor and therefore the increased use of such wafers increases the overall medical costs. Consequently, there is an urgent need for new and improved methods of making plaques for human use.

Examples of previous approaches to improving wafer manufacturing are described in the following:

In US Patent 5,032,396 it is reported that interleukin-7 (IL-7) is capable of stimulating platelet production. Interleukin-7 is also known as lymphopoietin-1 and is a lymphopoietic growth factor capable of stimulating the growth of B and T cell progenitors in the bone marrow. Published PCT Application Serial Number 88/03747, filed October 19, 1988, and European Patent Application No. 88309977.2, filed October 24, 1988, disclose DNA vectors and associated processes for producing mammalian IL-7 proteins by recombinant DNA technology. IL-7 can increase platelet circulation in normal and sub-lethal irradiated mice.

US Patent 5,087,448 discloses that megakaryocytes and platelets can be stimulated to proliferate in mammals by treating them with interieukin-6. Recombinant human interleukin-6 is a glycoprotein with a molecular weight of 26,000 and numerous biological activities. The data presented in this patent shows that IL-6 has the effect of increasing megakaryocyte colony in vitro.

None of the above-cited patents mentions anything about the Mpl ligands of the present invention.

Despite the above disclosures, there remains an urgent need for new mammalian megakaryocyte and / or platelet stimulators.

E. STATE OF THE ART WITH REGARD TO CHEMICALLY MODIFIED MGDF

Proteins for use in therapy are now available in suitable forms and amounts, largely as a result of advances in recombinant DNA techniques. Chemical derivatives of such proteins can effectively block the proteolytic enzyme from making physical contact with the backbone of the protein itself and thus prevent degradation. Additional benefits may include, under certain circumstances, increasing the stability and circulation time of a therapeutic protein, and reducing immunogenicity. However, it should be noted that the effect of modifying a particular protein cannot be predicted. Review article describing protein modification and protein fusion: Francis, Focus on Growth Factors 3: 4-10 (May 1992) (published: Mediscript, Mountview Court, Friern Barnet Lane, London N20 OlD, UK).

Polyethylene glycol ("PEG" or "peg") is one such chemical used in the manufacture of protein therapeutic products.

For example, Adagen ^R , a pegylated adenosine deaminase preparation, is approved for the treatment of major complex immunodeficiency disease;

pegylated superoxide dismutase is in clinical trials for the treatment of head injury;

183,631 pegylated alpha interferon was studied in Phase I clinical trials for the treatment of viral hepatitis; reported that pegylated glucoceramidase and pegylated hemoglobin are in preclinical studies.

For certain proteins, the addition of polyethylene glycol has been shown to protect against proteolysis, see Sada, et al., J. Fermentation Bioengineering 71: 137-139 (1991), and methods are available for adding certain polyethylene glycol residues. See U.S. Patent No. 4,179,337 to Davis et al., "Non-Immunogenic Polypeptides" issued December 18, 1979; and U.S. Patent No. 4,002,531, Royer, "Modifying Enzymes with Polyethylene Głykol and Product Produced Thereby," issued Jan. 11 and 911. For the review, see Abuchowski et al., in Enzymes as Drugs. (J.S. Holcerberg and J. Roberts, eds. Pp. 367-383 (1981)).

Other water-soluble polymers for protein modification have been used, such as copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene copolymer and maleic anhydride and polyamino acids (homopolymers or random copolymers).

In the case of polyethylene glycol, a number of methods have been used to attach polyethylene glycol molecules to protein. In general, polyethylene glycol molecules are attached to the protein via a reactive group in the protein. Suitable amino groups for such attachment are amino groups, such as at lysine or N-terminus residues. For example, Royer (US Patent No. 4,002,531, supra) teaches that reductive alkylation was used to attach a polyethylene glycol molecule to the enzyme. EP 0539167, published April 28, 1993, Wright, "Peg Imidates and Protein Derivates Thereof" reports that free amino group (s) peptides and organic compounds are modified with the imidine derivative of PEG or with related water-soluble organic polymers. U.S. Patent No. 4,904,584 to Shaw, issued February 27, 1990 relates to the modification of the number of lysine residues in proteins to add a polyethylene glycol molecule by reactive amino groups.

One specific therapeutic protein that has been chemically modified is granulocyte colony stimulating factor, "G-CSF."; see European patents EP 0401 384, EP 0473, 268, and EP 0335 423.

Another example is pegylated IL-6; see EP 0442 724, entitled "Modified hIL-6," (See co-pending US Patent Application Serial No. 07 / 632,070) which discloses polyethylene glycol molecules added to IL-6. EP 0154316, published September 11, 1985 describes the reaction of a lymphokine with a polyethylene glycol aldehyde.

The ability to modify MGDF is unknown in the art since the modifiability of any particular protein is determined by the specific structural parameters of that protein. Moreover, the effect of such modification on the biological properties of each protein is unpredictable according to the prior art. Due to the many clinical uses of MGDF as set out herein, a MGDF derivative product with altered properties is desired. Such molecules may have increased half-life and / or in vivo activity as well as other properties.

Pegylation of a protein molecule produces in general a mixture of chemically modified protein molecules. For example, protein molecules with five lysine residues and a free N-terminal amino group reacted according to the above methods may result in a heterogeneous mixture of six polyethylene glycol residues, some five, some four, some three, some two, some one, and some zero. Among molecules with several such residues, the polyethylene glycol residues may not be attached at the same location in different molecules. It will often be desirable to obtain a homogeneous product containing essentially in each case one or a small number (e.g. 2-3) of modified protein molecules which differ in the number and / or location of chemical moieties such as PEG. Nevertheless, mixtures of, e.g., mono-, di and / or tri-pegylated species may be desirable or tolerable for a given therapeutic indication.

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Variability of the mixture from batch to batch would be disadvantageous in the development of a pegylated protein therapeutic product. In such a development, it is important that biological activity is predictable. For example, it was shown that in the case of non-selective coupling of superoxide dismutase with polyethylene glycol, several parts of the modified enzyme were completely inactive (P. Mc Goff et al. Chem. Pharm. Bull. 36: 3079-3091 (1988)). See See also: Rose et al., Bioconjugate Chemistry 2: 154-159 (1991), which describes the selective addition of a carbohydrazide linker group to the C-terminal carboxyl group of a protein substrate (insulin). There is no such predictability when the therapeutic protein differs in composition from batch to batch. Some of the polyethylene glycol species may not be bound as stably in some places as others, and this can cause such residues to dissociate from the protein. Of course, if such residues are randomly attached and thus randomly dissociate, the pharmacokinetics of the therapeutic protein cannot be accurately predictable.

There is also a highly desirable derivative of MGDF product which lacks a residue linking the rest of the polymer to the MGDF molecule. The problem with the above methods is that they typically require a linking residue between the protein and the polyethylene glycol molecule. These linking residues can be antigenic, which is also disadvantageous for therapeutic protein development.

A non-linkage method is described in Francis et al., In:

"Stability of protein pharmaceuticals in vivo pathways of degradation and strategies for protein stabilization" (Eds. Ahem., T., Manning, M.C.) Plenum, New York, 1991). Also: Delgado et al. “Coupling of PEG to Protein By Activation with Tresyl Chloride, Applications In Immunoaffinity Celi Preparation” in: Fisher et al., Ed., Separations Using Aqueous Phase Systems, Applications In Celi Biology and Biotechnology, Plenum Press, N.Y., N.Y. 1989, pp. 211-213 relates to the use of tresyl chloride resulting in the absence of a linking group between polyethylene glycol and protein residues. This method can be difficult to apply to make therapeutic products because the use of tresyl chloride can produce toxic by-products.

Chamow et al., Bioconjugate Chem. 5: 133-140 (1994) describe the modification of a CD4 immunoadhesin with monomethoxypolyethylene glycol aldehyde ("MePEG glycol") by reductive alkylation. The authors report that 50% of the CD4-Ig was mePEG modified by selective reaction at the alpha-amino group of the N-terminus. Id. on p. 137. The authors also report that the in vitro binding capacity of the modified CD4-Ig (to gp 120 protein) decreased to an extent that correlated with the degree of mepegylation. Ibid.

Thus, there is a need for derivatives of MGDF, and in particular, a need for pegylated MGDF. There is also a need for methods for the preparation of such derivatives.

The object of the present invention is to provide a solution that meets this need.

The object of the invention is achieved by providing the solution according to the invention.

The invention includes derivatives of a megakaryocyte growth and development factor MGDF polypeptide selected from the group of polypeptides consisting of:

- a polypeptide with the sequence of amino acids 22-353 shown in Figure 11, termed mGDf-1;

- a polypeptide with the sequence of amino acids 22-195 shown in Figure 11, termed mGdF-2;

- a polypeptide with the sequence of amino acids 22-286 shown in Figure 12, termed mGDf-3;

- a polypeptide with the sequence of amino acids 22-172 shown in Figure 11, termed mGdF-4;

- a polypeptide with the sequence of amino acids 22-177 shown in Figure 11, termed mGDf-5;

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- a polypeptide with the sequence of amino acids 22-191 shown in Figure 11, referred to as MGDF-6;

- a polypeptide with the sequence of amino acids 22-198 shown in Figure 11, referred to as MGDF-7;

- a polypeptide with the sequence of amino acids 22-265 shown in Figure 11, referred to as MGDF-8;

- a polypeptide with the sequence of amino acids 22-184 shown in Figure 11, referred to as MGDF-11;

- a polypeptide with the amino acid sequence 27-353 shown in Figure 11, referred to as MGDF-12;

- a polypeptide with the amino acid sequence 27-195 shown in Figure 11, termed mGDf-13;

- a polypeptide with the amino acid sequence 27-172 shown in Figure 11, termed MGDF-14, and

- a polypeptide with the sequence of amino acids 27-184 shown in Figure 11, referred to as MGDF-15;

wherein the MGDF polypeptide is attached to at least one water-soluble polymer.

Preferably in the derivative according to the invention the water-soluble polymer is pharmaceutically acceptable, especially selected from the group consisting of dextran, poly (N-vinyl-pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide-ethylene oxide copolymers, polyoxyethylene polyols, polyvinyl alcohols and mixtures thereof. .

A particularly preferred water-soluble polymer is polyethylene glycol, especially monomethoxylated polyethylene glycol.

Preferably in the derivative according to the invention, said polyethylene glycol is attached to the MGDF polypeptide by an acyl or alkyl bond, and in particular the polyethylene glycol residue is attached to the N-terminus of the MGDF polypeptide.

Preferably, the polyethylene glycol group has an average molecular weight of 10,000 to 50,000.

Alternatively, in the derivative of the invention, the MGDF polypeptide is covalently linked to two water-soluble polymer molecules, and preferably both indicated water-soluble polymer molecules are filtered with polyethylene glycol molecules.

A preferred group of derivatives of the invention are monopegylated MGDF polypeptides

A preferred such derivative is (MGDF polypeptide monopegylated at the α-amino group at the N-terminus of said polypeptide and in the form of a substantially homogeneous preparation.

A preferred such derivative is a polyethylene glycol monopegylated MGDF polypeptide with an average molecular weight of 5,000 to 50,000.

Another preferred such derivative is a monopegylated MGDF polypeptide with a polyethylene glycol group attached to the N-terminus of the polypeptide.

A method of producing a derivative of a megakaryocyte growth and development factor MGDF polypeptide selected from the group of polypeptides consisting of:

- a polypeptide with the sequence of amino acids 22-353 shown in Figure 11, termed mGDf-1;

- a polypeptide with the sequence of amino acids 22-195 shown in Figure 11, termed mGDf-2;

- a polypeptide with the sequence of amino acids 22-286 shown in Figure 12, referred to as MGDF-3;

- a polypeptide with the sequence of amino acids 22-172 shown in Figure 11, referred to as MGDF-4;

- a polypeptide with the sequence of amino acids 22-177 shown in Figure 11, referred to as MGDF-5;

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- a polypeptide with the sequence of amino acids 22-191 shown in Figure 11, referred to as MGDF-6;

- a polypeptide with the sequence of amino acids 22-198 shown in Figure 11, referred to as MGDF-7;

- a polypeptide with the sequence of amino acids 22-265 shown in Figure 11, referred to as MGDF-8;

- a polypeptide with the sequence of amino acids 22-184 shown in Figure 11, referred to as MGDF-11;

- a polypeptide with the amino acid sequence 27-353 shown in Figure 11, referred to as MGDF-12;

- a polypeptide with the amino acid sequence 27-195 shown in Figure 11, termed mGDf-13;

- a polypeptide with the amino acid sequence 27-172 shown in Figure 11, termed MGDF-14, and

- a polypeptide with the sequence of amino acids 27-184 shown in Figure 11, referred to as mGDf-15;

wherein the MGDF polypeptide is attached to at least one water-soluble polymer, comprising linking the water-soluble polymer to the MGDF polypeptide, the present invention comprises using a water-soluble polymer having an aldehyde reactive group and reacting the terminal aldehyde derivative group A polyethylene glycol aldehyde with the indicated MGDf polypeptide in the presence of a reducing agent, or a water-soluble polymer having a single reactive aldehyde group is used. and contacting said MGDF polypeptide with the water-soluble polymer under reductive alkylation conditions at a pH acidic enough for the α-amino group at the amino terminus of said MGDF polypeptide to be reactive, or a water-soluble polymer having a single reactive ester group is used and contacting the MGDF polypeptide with the water-soluble polymer under conditions permitting attachment of said MGDF polypeptide to the water-soluble polymer via an acyl bond, followed by isolation of the derived MGDF polypeptide linked to the at least one water-soluble polymer.

In the process of the invention, a pharmaceutically acceptable polymer is preferably used as the water-soluble polymer.

In the process of the invention, preferably the water-soluble polymer is a polymer selected from the group consisting of dextran, poly (N-vinyl-pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide-ethylene oxide copolymers, polyoxyethylene polyols and polyvinyl alcohols.

In the process according to the invention, polyethylene glycol is preferably used as the water-soluble polymer.

In the process of the invention, preferably a polyethylene glycol having a single reactive aldehyde group is used as the water-soluble polymer, and said MGDF polypeptide is contacted with the indicated polyethylene glycol molecule under reductive alkylation conditions at a pH sufficiently acidic so that the α-amino group at the amino terminus of the indicated The MGDF polypeptide may have been reactive and the monopegylated MGDF polypeptide produced is isolated.

Preferably, the MGDF-11 polypeptide having the amino acid sequence 22-184 shown in Figure 11 is used in the above reaction as MGDF polypeptide.

Preferably, an MGDF-11 polypeptide is used, produced by E. coli expression of a DNA encoding a polypeptide having amino acids 22-184 shown in Figure 11 and a Met'-Lys' sequence at its N-terminus, isolating the expressed polypeptide and the detachment of Met ' ² -Lys'1 from the isolated polypeptide.

According to the invention, polyethylene glycol with an average molecular weight of 2,000 to 100,000 is preferably used.

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The invention also encompasses a pharmaceutical composition containing a pharmaceutically active substance and a pharmaceutically acceptable diluent, adjuvant or carrier, which according to the invention comprises as pharmaceutically active substance a derivative of the megakaryocyte growth and development factor MGDF polypeptide selected from the group of polypeptides consisting of:

- a polypeptide with the sequence of amino acids 22-353 shown in Figure 11, referred to as MGDF-1;

- a polypeptide with the sequence of amino acids 22-195 shown in Figure 11, referred to as MGDF-2;

- a polypeptide with the sequence of amino acids 22-286 shown in Figure 12, referred to as MGDF-3;

- a polypeptide with the sequence of amino acids 22-172 shown in Figure 11, referred to as MGDF-4;

- a polypeptide with the sequence of amino acids 22-177 shown in Figure 11, termed mGdF-5;

- a polypeptide with the sequence of amino acids 22-191 shown in Figure 11, referred to as MGDF-6;

- a polypeptide with the amino acid sequence 22-198 shown in Figure 11, termed MgDf-7;

- a polypeptide with the sequence of amino acids 22-265 shown in Figure 11, referred to as MGDF-8;

- a polypeptide with the sequence of amino acids 22-184 shown in Figure 11, referred to as MGDF-11;

- a polypeptide with the amino acid sequence 27-353 shown in Figure 11, referred to as MGDF-12;

- a polypeptide with the amino acid sequence 27-195 shown in Figure 11, termed mGdF-13;

- a polypeptide with the amino acid sequence 27-172 shown in Figure 11, termed mGDF-14, and

- a polypeptide with the sequence of amino acids 27-184 shown in the figure

Figure 11, referred to as MGDF-15, is a combination of MGDF polypeptide with at least one water-soluble polymer.

The composition of the invention preferably comprises a monopegylated MGDF polypeptide as a derivative of MGDF.

The starting polypeptides for derivatization according to the invention are novel compounds and are the subject of application no. P-312577, filed on 03/30. 1995. Promote specifically the growth and / or development of megakaryocytes ("Ligands Mpl" or "MGDFs"). These compounds can be obtained in their native form from the body of mammals. Two exemplary Mpl ligands were isolated from dog aplastic plasma. However, it turned out that closely related Mpl ligands are present in aplastic plasma from both human and porcine organisms. In particular, the activity of each of the Mpl ligands (human, porcine and dog) is specifically inhibited by the soluble form of the Mpl receptor from the mouse body, showing that all of these Mpl ligands (as well as those from other mammalian organisms, including mice) they are closely related both on the structural and activity levels.

While the present invention relates generally to derivatives of Mpl ligands, these ligands can be derived from mammalian organisms such as dogs, pigs, humans, mice, horses, sheep and rabbits. Especially preferred are the Mpl ligands from dogs, pigs and humans. Mpl ligands can also be produced by chemical synthesis or biotechnological methods.

Two preferred canine Mpl ligands have an apparent molecular weight of about 25,000 and 31,000 as determined by gel electrophoresis (SDS-PAGE) using sodium dodecyl sulfate and polyacrylamide under non-reducing conditions. Both proteins

183,631 were purified using the same purification protocol detailed in the examples section below.

Two preferred human ligands, MGDF-1 and MGDF2, are 332 and 174 amino acids long, respectively, not including the signal 21 amino acid test peptide. These sequences and a third related molecule, MGDF-3, are shown in Figures 11 and 12.

The starting Mpl ligand proteins for derivatization according to the present invention are also characterized by one or more of the physical, biochemical, pharmacological activities described herein.

The present invention relates to chemically modified MGDF consisting of a MGDF protein molecule combined with at least one water-soluble polymer, and to methods of making and using such compositions. In particular, the present invention includes chemically modified MGDF in which the MGDF is reacted with reactive polyethylene glycol molecules to attach the PEG to the MGDF. Such addition may be accomplished by a pegylation reaction discussed in the minor text, such as acylation or alkylation. The acylation or alkylation of PEG can be performed under conditions where the main product is monopegylated or polypegylated. Half-regulation generally involves the addition of PEG to the ε-amino groups of lysine residues and may additionally involve pegylation at the N-terminus of the polypeptide. Monopegylation preferably involves the addition of PEG to the α-amino group at the N-terminus of the protein. The efficiency and homogeneity of such a monopegylation reaction can be improved by a type of reductive alkylation that selectively modifies the α-amino group of the N-terminal MGDF protein residue, thereby ensuring the selective addition of a water-soluble polymer residue to the N-terminus of the protein. This ensures substantially homogeneous production of polymer / MGDF protein conjugate molecules as well as (when polyethylene glycol is used) the production of a pegylated MGDF protein molecule with a polyethylene glycol residue directly coupled to the protein molecule.

Another aspect of the present invention provides pharmaceutical compositions containing a therapeutically effective amount of an isolated naturally occurring or recombinant Mpl ligand, which can be derivatized using a water-soluble polymer such as polyethylene glycol together with a pharmaceutically acceptable carrier, diluent or excipient. These pharmaceutical compositions can be used in methods of treating disease states or disorders characterized by a deficiency of megakaryocytes and / or platelets as well as deficiency in vivo of the Mpl ligand. They can also be used prophylactically to ameliorate expected megakaryocyte or platelet deficiencies (e.g., by surgery).

The starting Mpl ligands for derivatization according to the present invention have been found to be specifically active in lineage, increase maturation and / or proliferation of megakaryocytes as shown in the studies in Examples 2 and 4 of Application No. P-312577 dated 03.03.1995. "Specifically" is meant that polypeptides exhibit relatively greater biological activity on megakaryocytes as compared to other cell types. It is expected that those that stimulate megakaryocytes have the activity of stimulating platelet production in vivo by stimulating the maturation and differentiation of megakaryocytes.

Thus, Mpl ligands can be used in the treatment of aplastic anemia, e.g., to increase platelet production in patients with impaired platelet production (such as patients with AIDS or undergoing cancer chemotherapy). Ligand Mpl can be used to treat blood disorders such as thrombocytopenia. Ligand Mpl can be used as adjunctive therapy in patients undergoing bone marrow transplantation. Such patients may be humans or other mammals. It can be expected that an Mpl ligand from one species is also useful in another species.

The pharmaceutical composition according to the invention finds use in the treatment of the above-mentioned and other pathological conditions resulting from the deficiency of platelets. The methods of therapy may include administering, simultaneously or sequentially, an Mpl ligand, an effective amount of at least one other megakaryocyte colony stimulating factor, a cytokine (e.g., EPO), a soluble Mpl receptor, a hemopoietin, an interleukin, a growth factor, or an antibody.

Other aspects and advantages of the present invention will become apparent from considering the following detailed description of its preferred embodiments.

Brief description of the drawings

Numerous features and advantages of the present invention will become apparent from the drawings of which

Figure 1 shows a general diagram of the formation and maturation of megakaricytes and platelets

Figure 2 shows that the soluble mouse Mpl receptor almost completely inhibits the ability of irradiated dog plasma ("aplastic canine plasma" or "APK9) to induce megakaryocyte development. The megakaryocyte formation assay is described in Example 2 of Application No. P-312577 filed on 03.30.1995.

Figure 3 shows that increased activity from APK9 in chromatographic procedures ("Mpl ligands) by the relationship of lectins to the murine Mpl receptor stimulates the growth of 1A6.1 cells and that the soluble mouse Mpl receptor blocks this growth.

Figure 4 shows a general schema of the purification process for purifying 25,000 and 31,000 (25 kd and 31 kd) canine Mpl receptor species from aplastic canine plasma.

Figure 5 demonstrates the purification of Mpl ligand by reverse phase HPLC (RP-HPLC). Fraction 21 contains the highly purified Mpl ligand with a molecular weight of 31,000, fraction 22 contains a mixture of Mpl ligands with a molecular weight of 31,000 and 25,000; and fraction 23 contains a highly purified Mpl ligand with a molecular weight of 25,000.

Figure 6 compares the activity of the Mpl ligand in the fractions containing 25,000 and / or 31,000 molecular weight ligand proteins by reverse phase HPLC (C4 column).

Figure 7 shows the number of megakaryocytes derived from cultures of selected CD34 cells from peripheral blood stimulated with APK9, Mpl ligand and various other factors.

Figure 8 shows the total. the number of leukocytes obtained from cultures of selected CD34 cells from peripheral blood stimulated with APK9, Mpl ligand and various other factors.

Figure 9 shows the percentage of megakaryocytes obtained from cultures of selected CD34 cells from peripheral blood stimulated with APK9, Mpl ligand and various other factors.

Figure 10 shows that human IL-3 is not involved in the formation and growth of megakaryocytes induced by Mpl ligands.

Figure 11 shows the cDNA and putative amino acid sequence of human MGDF-1 and MGDF-2.

Figure 12 shows the cDNA and putative amino acid sequence of human MGDF-3.

Figure 13 shows a comparison between MGDF-1 and other MGDF (Mpl ligands) from dog (A) and mouse (B).

Figure 14 shows an example of acylation of MGDF using active N-hydroxysuccinimidyl (NHS) monomethoxypolyethylene glycol ester to give a polypegylated derivative.

Figure 15 shows an example of the nonspecific reductive alkylation of MGDF using mono-methoxy polyethylene glycol aldehydes to provide a polypegylated derivative.

Figure 16 shows an example of site-specific reductive alkylation of MGDF at the α-amino group of the N-terminus of the chain, using mono-methoxy polyethylene glycol aldehydes to give virtually substantially mono-pegylated.

183 631

Figure 17 shows the results of a size exclusion (SEC) HPLC analysis of MePEG-MGDF conjugates obtained with activated MePEG derivatives with a molecular weight of 20,000 (20 kDa):

A. poly-MePEG-MGDF conjugates obtained by acylating MGDF with MePEG glycol NHS ester (PEG 11);

B. poly-MePEG-MGDF conjugates obtained by alkylation of MGDF with MePEG aldehyde (PEG 20);

C. mono-MePEG-MGDF conjugates obtained by alkylation of MGDF with MePEG aldehyde (PEG 16).

Figure 18 shows the number of mouse platelets treated with recombinant human MGDF: squares = CHO 22-353 MGDF derivatives; circles - non-pegylated 22-184 MGDF produced by E. coli (i.e. 1-163 MGDF); and dots = PEGylated 22-184 MGDF produced by E. coli.

Figure 19 shows schematically the purification process for r-HuMGDF.

Figure 20 shows the effect of r-HuMGDF (E. coli 1-163) on platelet count for the murine carboplatin model. Balb / c mice were intraperitoneally injected with a single dose of carboplatin (1.25 mg / mouse) on day zero. The vehicle-alone group did not receive carboplatin. 24 hours later, the carboplatin-treated animals received subcutaneous injections with either vehicle or 100 gg / kg r-HuMGDF per day for the remaining study days. (n = 10 for each group; 5 animals were bled at each measurement time point).

Figure 21 shows the effect of r-HuMGDF (E.coli 1-163) on platelet count in irradiated mice. Balb / c mice were irradiated with a single dose of 500 rads of gamma radiation (cesium source) on day zero. The vehicle alone group was not illuminated. After 24 hours, irradiated animals were injected with either vehicle or 100 gg / kg r-HuMGDF per day until the end of the study period (n = 8 for each group); 4 animals were bled at each measuring time point).

Figure 22 shows the effect of r-HuMGDF (E.coli 1-163) on platelet count in mice subjected to simultaneous irradiation and carboplatin administration. Balb / c mice were irradiated with a single dose of 500 rads of gamma radiation (cesium source) and received a dose of carboplatin (1.25 mg / mouse) on day zero. After 24 hours, the animals thus treated subcutaneously were injected with either vehicle or a dose of 100 gg / kg r-HuMGDF per day until the end of the study (n = 8 for each group). Without HuMGDF support, most animals did not survive this study. In the control group, 1 in 8 animals survived. In the test group to which the test agent was administered, 8 out of 8 animals survived.

Figure 23 shows the effect of r-HuMGDF (E.coli 1-163) on radiation-induced thrombocytopenia in rhesus monkeys. These monkeys were irradiated (700 cGy Co-80). Subsequently, r-HuMGDF (n = 3) or human plasma albumin (n = 9) were administered subcutaneously at a dose of 25 gg / kg / day for the next 18 days starting 24 hours after irradiation. Blood cell analysis was performed on an electronic blood analysis device. Each symbol represents an average value (+/- sem).

Figure 24 shows the effect of pegylated and glycosylated r-HuMGDF on platelet count in irradiated and carboplatin-treated mice. Mice were treated with a combination of irradiation and carboplatin as described with reference to the studies depicted in Figure 22. Subcutaneous injections of said r-HuMGDF formulation (50 gg / kg / day) were applied daily throughout the study period starting 24 hours after exposure. harmful factors. Blood cell numbers were determined on the indicated days using an electronic cell count (Sysmex, Baxter).

Figure 25 shows the synthetic gene sequence for recombinant human MGDF, amino acids 1-163, containing E. coli optimized codons.

Additional aspects and advantageous features of the invention will be apparent to those skilled in the art from considering the following description detailing the practice of the invention.

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As mentioned, novel mammalian megakaryocytic factors promoting growth and / or platelet production, referred to as Mpl ligands, are described in detail in Application No. P-312577 filed on 03. 03 1995.

Generally, the term "Mpl ligands" as used in the present invention means the Mpl ligands disclosed herein as well as active fragments and variants thereof, which are described in more detail below.

In summary, exemplary Mpl ligands are characterized by one or more of the following biochemical and biological properties:

(a) such ligands Mpl are separated from aplastic dog plasma;

(b) such Mpl ligands have apparent molecular weights of approximately 25,000 or 31,000 as determined by 12-14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions;

(c) The Mpl ligands include the following amino acid sequences:

SEq ID NO: 1 for a protein of 25,000 (25 kd); or SEQ ID NO: 2 for a protein of 31,000 (31 kd);

(d) Mpl ligands further comprise the amino acid sequence of SEQ ID NO:

(especially preferred in a protein of 25,000-25 kd);

(e) Mpl ligands bind to a wheat germ lectin;

(f) Mpl ligands bind to immobilized soluble Mpl receptor from the mouse body;

(g) the activity of the Mpl ligand can be inhibited in vitro by the soluble Mpl receptor; and (h) Mpl ligands bind to the anion exchange column at about pH 8-9.

The biological activity of the preferred Mpl ligands is demonstrated by their ability to specifically stimulate the growth and development of megakaryocytes in an attempt to promote the growth of megakaryocytes of Example 2 of Application No. P-312577 filed on 03.30.1995.

The present invention broadly covers chemically modified MGDF compositions and methods for their preparation and use. The present application discloses that it is possible to modify MGDF and improve its properties.

In one aspect, the present invention relates to an MGDF product comprising a protein linked to at least one water-soluble polymer molecule.

In another aspect, the present invention relates to an MGDF derivative wherein said MGDF protein is linked to at least one polyethylene glycol molecule.

In another aspect, the present invention relates to a MGDF molecule attached to at least one polyethylene glycol molecule via an acyl or alkyl linkage.

The pegylation of MGDF can be performed by any pegylation reaction known in the art. See e.g., Focus on Growth Factors 3 (2): 4-10 (1992); EP O 154 316; EP O 401 384; and other publications cited in this pegylation text. Preferably, the pegylation is performed by an acylation or alkylation reaction with a reactive polyethylene glycol molecule (or an analogous water-soluble reactive polymer). These preferred polyethylene glycol derivatization processes will now be discussed in more detail.

I. Acylation

PEGylation by acylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with the MGDF protein. Any known or subsequently discovered reactive PEG molecule can be used to perform the pegylation of MGDF. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide ("NHS"). The term "acylation" as used herein is considered to include, without limitation, the following types of linkage between MGDF and a water-soluble polymer such as PEG: amide, carbamate, urethane, and the like. Bioconjugate Chem. 5: 133-140 (1994). The reaction conditions may be selected from any known in the art for pegylation or those subsequently developed, but conditions such as temperature, dissolution and dilution should be avoided.

The reagent and pH inactivated the modified MGDF species. Reaction conditions which will generally apply to MGDF pegylation will be described below. An exemplary reaction with the NHS monomethoxy-PEG ester is shown in Fig. 14.

Pegylation by acylation will generally result in a polypegylated MGDF product in which the ε-amino groups of the lyses are pegylated by the acyl linkage. Kozzystnie, the connection will be an amide. It is also preferred that the product obtained will be substantially only (e.g.> 95%) mono, di- or tri-glycylated. However, certain species with higher degrees of pegylation (up to a maximum number of β-amino groups in the MGDF lysis plus one α-amino group beyond the amino terminus of the MGDF) will normally be formed in amounts depending on the α-amino group of the reaction conditions used. If desired, more purified pegylated species can be separated from the mixture, especially unreacted species, by standard oasis techniques, including, but not limited to, dialysis, salting out, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis.

II. Alkylation

Peeylation by alkylation generally involves reacting PEG derivatives of the terminal protein aldehyde, such as MGDF, in the presence of a reducing agent. As with the allation discussed above, the reaction conditions are described below.

PEGylation by alkylation can also result in polypegylated MGDF. An exemplary reductive alkylation reaction with MGDF yielding a polypegylated product is shown in Fig. 15. In addition, the reaction conditions can be varied as described herein to favor pegylation essentially only at the α-amino group of the N-terminus of the MGDF species (i.e., monopngylated species). An exemplary reductive alkylation reaction with MGDF yielding a mono-pegylated product is shown in Fig. 16. In any case: moaopngylation or polypegylation, the PEG groups are preferably attached to the protein via a -CH2-NH- group. With particular reference to the group -CH2-, this type of linkage is aacgwann herein as "alkyl" linkage.

Derivatization by reductive alkylation to produce a monopegylated product utilizes the differential reactivity of the different types of penguin amino groups (in lysine and at the N-terminus) available for derivatization in MGDF. The reaction is performed at a pH (see below) which makes it possible to take advantage of the pKa differences between the ε-amino groups of lysine residues and the α-amino groups of the N-terminal residue of the protein. By such selective derivatization, it is controlled to attach a water-soluble polymer that contains a reactive group, such as an aldehyde, to the protein. The conjugation to the polymer takes place mostly at the N-terminus of the protein and there is no significant modification of other reactive groups such as the lysine side chain amino groups. In one important aspect, the present invention provides a substantially homogeneous preparation of monopolymer / MGDF protein conjugate molecules (meaning an MGDF protein to which the polymer molecule is substantially only attached (i.e. (95%) at one position). More specifically, where glycol is used. The invention also provides a pegylated MGDF protein with possibly no antigenic linking groups and with a polyethylene glycol molecule directly linked to the MGDF protein.

Thus, in a preferred aspect, the present invention relates to pegylated MGDF wherein the PEG group (s) is (are) attached via acyl or alkyl groups. As discussed above, such products may be monopegylated or polycycled (eg containing 2-6, preferably 2-5 PEG groups). PEG groups are generally attached to the protein at the α- or ε-amino acid groups of the amino acids, but it is also contemplated that the PEG groups could be attached to any amino group attached to the protein that is reactive to be attached to the PEG group. under appropriate reaction conditions.

The polymer molecules used in both the acylation and alkylation approaches can be selected from water-soluble polymers or mixtures thereof.

183 631

The selected polymer should be water soluble so that the protein to which it is attached does not precipitate in an aqueous medium, such as a physiological environment. The selected polymer should be modified to have a single reactive group such as an active acylating ester or an alkylating aldehyde, preferably that the degree of polymerization is controllable, as provided for by the present methods. A preferred reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or its mono-derivatives of C1-C10 alkoxy or aryloxy (see US Patent 5,252,714). The polymer can be branched or unbranched. Preferably, for end product formulation uses in therapy, the polymer will be pharmaceutically acceptable. The water-soluble polymer may be selected from the group consisting of, e.g., polyethylene glycol, monomethoxypolyethylene glycol, dextran, poly (N-vinylpyrrolidone) ethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g. glycerin) and polyvinyl alcohol. In the case of an acylation reaction, the selected polymer (s) should have a single reactive ester group. For the present reductive alkylation, the selected polymer (s) should have a single reactive aldehyde group. In general, the water-soluble polymer will not be selected from naturally occurring glycosyl residues as they are usually more conveniently obtained from recombinant expression systems from the mammalian body. The polymer may be of any molecular weight and may be branched or unbranched.

A particularly preferred water-soluble polymer for use in the present invention is polyethylene glycol, abbreviated PEG; According to the present invention, polyethylene glycol is contemplated to include any form of PEG that has been used to derivatize other proteins, such as mono- (C 1 -C 10) alkoxy or ary loxy-gI and ethylene co-ol.

As used in this application, MGDF is defined to include any of the different forms of MGDF described in Application No. P-312577 filed on 03. 03 1995. For example, full-length or truncated, glycosylated or unglycosylated forms are included. The following MGDF molecules are suitable for derivatization (in each case the numbering refers to the amino acids numbered according to Fig. 11):

MGDF- 1 amino acids 22-353 Fig. 11

MGDF- 2 amino acids 22-195 Fig. 11

MGDF- 4 aa 22-172 Fig. 11

MGDF-11 aa 22-184 Fig. 11

MGDF-12 aa 27-353 Fig. 11

MGDF-13 aa 27-195 Fig. 11

MGDF-14 aa 27-172 Fig. 11

MGDF-15 aa 27-189 Fig. 11

The above preferred species may be glycosylated, non-glycosylated or deglycosylated, preferably non-glycosylated. They can be obtained recombinantly in bacterial (e.g. E. coli) or mammalian (e.g. CHO) cells.

The following subgroups of chemically derivatized molecules are particularly preferred according to the present invention (in each case, they are mono- or poly-, e.g. 2-9, PEG residues attached via an acyl or alkyl group):

pegylated MGDF-11 pegylated MGDF-4 pegylated MGDF-2.

In general, chemical derivatization can be carried out under any suitable conditions used to react the biologically active substance with the activated polymer molecule. Methods for producing pegylated MGDFs will generally include the steps of: (a) reacting the MGDF polypeptide with a polyethylene glycol (such as a reactive PEG ester or aldehyde derivative) under conditions such that the MGDF is attached to one or more PEG groups, and (b) obtaining the product (s). In general, the optimal reaction conditions for the acylation reaction will be from

183,631 cases based on the known parameters and the desired result. For example, the greater the PEG: protein ratio, the greater the percentage of polypegylated product.

Reductive alkylation to produce a substantially homogeneous population of monopolymer / MGDF protein conjugate molecules will generally include the steps of: (a) reacting the MGDF protein with a reactive PEG molecule under reductive alkylation conditions at a pH suitable to allow for the selective modification of the α-amino group at the amino terminus of said protein MGDF; and (b) obtaining reaction products.

For a substantially homogeneous population of mono-polymer / MGDF protein conjugate molecules, the conditions of the reductive alkylation reaction are such as to allow the selective addition of a water-soluble polymer species to the N-terminus of the MGDF. Such reaction conditions generally ensure that there are differences in pKa between the lysine amino groups and the α-amino group at the N-terminus (pKa is the pH at which 50% of the group's amino groups are protonated and 50% are not). The pH also influences the proportion of polymer to protein used. In general, if the pH is lower, a greater excess of polymer to protein will be desired (ie, the less reactive the N-terminal α-amino group, the more polymer is needed to achieve optimal conditions). If the pH is higher, the polymer: protein ratio need not be so high (i.e. the more reactive the groups are, the less polymer molecules are needed). For the purposes of the present invention, the pH will generally be in the range

3-9, preferably 3-6.

Another important consideration is the molecular weight of the polymer. In general, the higher the molecular weight of the polymer, the smaller the number of polymer molecules that can be attached to the protein. Likewise, the branching of the polymer should be considered when optimizing these parameters. In general, the higher the molecular weight or the more branches there are, the higher the polymer: protein ratio. In general, for the pegylation reactions contemplated in the smaller application, the preferred average molecular weight is about 2,000 to 100,000 (about 2 to about 100 kDa) - "about" indicates 1,000 (1 kDa). The preferred average molecular weight is about 5,000 to about 50,000 (5 kDa to about 50 kDa), especially about 12,000 to about 25,000. The ratio of the water-soluble polymer to the MGDF protein will generally be from 1: 1 to 100: 1, preferably ( for polypegylation) 1: 1 to 20: 1 and (for monopegylation) 1: 1 to 5: 1.

Using the conditions outlined above, the reductive alkylation will selectively attach the polymer to any MGDF protein having an α-amino group at the amino terminus, and will result in a substantially homogeneous monopolymer / MGDF protein conjugate preparation. The term "monopolymer / protema MGDF conjugate" is used herein to mean a composition comprising a single polymer molecule attached to an MGDF protein molecule. The monopolymer / protein MGDF conjugate preferably will have the polymer molecule situated at the N-terminus, but not on the amine side groups of the lysine. The formulation will preferably contain greater than 90% monopolymer / MGDF protein conjugate, and more preferably greater than 95% monopolymer / MGDF protein conjugate, with residues of observable unreacted molecules (ie, polymer species without protein). The following examples provide a formulation that contains at least about 90% monopolymer / protein conjugate and about 10% unreacted protein. The monopolymer / protein conjugate is biologically active.

For the present reductive alkylation, the reducing agent should be stable in an aqueous solution and preferably be capable of reducing only the Schiff's base formed in the initial reductive alkylation process. Preferred reducing agents may be selected from the group consisting of: sodium borohydride, sodium cyanoborohydride, dimethylamine borate, trimethylamine borate, and pyridine borate. A particularly preferred reducing agent is sodium cyanoborohydride.

Other reaction parameters, such as solvent, reaction times, temperatures, etc. and methods for purifying the products, can be determined on a case-by-case basis based on published information relating to the preparation of protein derivatives with water-soluble

183,631 polymers (see publications cited in this application). Sample details are provided in the examples section below.

It is possible to choose to prepare the mixture of polymer / protein conjugate molecules by acylation and / or alkylation methods and the advantage provided in this application is that the amount of polymer / protein conjugate contained in the mixture can be selected. Thus, if desired, a mixture of a different protein with a different number of polymer molecules attached (i.e., di-, tri-, tetra-, etc.) can be prepared and combined with the monopolymer / protein conjugate material prepared using the present methods and a pre-mixed mixture available. a specific monopolymer / proteyin conjugate ratio.

The following working examples show the preparation of chemically modified MGDF and the preparation of pegylated MGDF by acylation and alkylation. Thus, other aspects of the present invention pertain to this production.

In general, conditions that can be alleviated or modulated by administration of polymer / MGDF derivatives include those described above for the MGDF molecule. However, the polymer / MGDF molecules disclosed herein may have additional effects, increased or decreased effects, or other characteristics as compared to the non-derivatized molecules.

In yet another aspect of the present invention, pharmaceutical compositions of the foregoing chemically modified MGDF molecules are provided. Such pharmaceutical compositions may contain any of the ingredients listed herein for non-derivatized molecules.

As further research is conducted, information on appropriate dosage levels for treating different conditions in different patients will emerge, and the conventional skilled person, considering the therapeutic context, age, and general health of the recipient, will be able to determine the appropriate dosage. In general, the dose will be between 0.01 gg / kg body weight (calculating the weight of the protein alone, without chemical modification) and 300 µg / kg (based on the same). A preferred dose will generally be from 5 gg / kg body weight to 100 gg / kg body weight, especially from 10 gg / kg body weight to 75 gg / kg body weight.

Conditions that can be treated with the compositions of the present invention are generally those associated with an existing megakaryocyte / platelet deficiency or an expected future megakaryocyte / platelet deficiency (e.g. due to elective surgery). Such conditions will usually result from a deficiency (temporary or permanent) of active Mpl ligand in vivo. The term generic relating to platelet deficiency is thrombocytopenia, and thus the methods and compositions of the present invention are generally available for the treatment of thrombocytopenia.

Thrombocytopenia (platelet deficiencies) can occur for a variety of reasons, including chemotherapy and other drug therapy, radiation therapy, surgery, accidental blood loss, and other specific disease states. Exemplary specific disease states that are associated with thrombocytopenia and can be treated in accordance with the present invention are: aplastic anemia, idiopathic thrombocytopenia, metastatic tumors that cause thrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi syndrome, vitamin B12 deficiency, May-Hegglin anomaly, Wiskott-Alldrich syndrome and paroxysmal nocturnal hemoglobinuria. Certain AIDS treatments also cause thrombocytopenia (eg AZT). Increasing the platelet count may have a beneficial effect in certain wound healing disorders.

Due to expected shortages of platelets, e.g. due to future operations, the Mpl ligand of the present invention could be administered several days to several hours before platelets are needed. Due to acute cases, e.g. blood loss in large amounts and in accidents, the Mpl ligand could be administered with blood or cleaned platelets.

The Mpl ligands and their derivatives according to the present invention may also be useful in stimulating certain types of cells other than megakaryocytes, if such cells are found to be an Mpl receptor. Conditions related to those cells that make ex22

Mpl receptor pressures that correspond to stimulation from the Mpl ligand are also within the scope of the present invention.

MGDF molecules and their derivatives, which are not themselves active in the activity assays set forth in this application, may be useful as modulators (e.g., inhibitors or stimulants) of receptors in vitro or in vivo.

The polypeptide derivatives of the invention may also be used alone or in combination with other cytokines, soluble Mpl receptor, haemopoietic factors, interieukins, growth factors, or antibodies to treat the above-identified conditions.

Thus, yet another aspect of the invention is therapeutic compositions for the treatment of the above-mentioned conditions. Such compositions comprise a therapeutically effective amount of an Mpl ligand polypeptide or a therapeutically effective polypeptide fragment in admixture with a pharmaceutically acceptable carrier. The carrier may be water for injection, preferably supplemented with other materials normally found in solutions for administration to mammals. Typically, the therapeutic ligand of Mpl will be administered in the form of a composition containing the purified protein in association with one or more physiologically acceptable carriers, excipients, or diluents. Neutral buffered saline or saline mixed with plasma albumin are examples of suitable carriers. Preferably, the product is formulated as a lyophilisate using suitable excipients (e.g. sucrose). Other standard carriers, diluents, and excipients may be included, if desired. Other exemplary compositions are Tris buffer, pH 8.0, and acetate buffer, pH 5.0, which in any case may further contain sorbitol.

The present compositions can be systemically administered parenterally. Alternatively, the compositions can be administered intravenously or subcutaneously. For systemic administration, the therapeutic compositions for use in the present invention may be in the form of a pyrogen-free parenterally acceptable aqueous solution. The preparation of such pharmaceutically acceptable protein solutions for reasons of pH, isotonicity, stability etc. is within the skill of the art.

The dosage regimen associated with the method of treating the above-described conditions will be determined by the treating physician taking into account various factors that modify the action of the drugs, e.g., age, condition, body weight, sex and diet of the patient, severity of infection, time of administration, and other clinical factors. In general, the daily dosage should be in the range of 0.1 to 1000 micrograms of protein or fragment thereof per kilogram of body weight.

Therapeutic methods, compositions and derivatives of the polypeptides of the present invention can also be used, alone or in combination with other cytokines, soluble Mpl receptor, haemopoietic factors, interieukins, growth factors, or antibodies in the treatment of disease states characterized by other symptoms as well as platelet deficiencies. The ligand molecule Mpl would be expected to prove useful in the treatment of some forms of thrombocytopenia in combination with general stimulants of haemopoiesis such as IL-3 or GM-CSF. Other megakaryocytic stimulation factors, i.e., megakaryocytic stimulating factors, i.e. meg-CSF, stem cell factor (SCF), leukemia inhibition factor (LIF), oncostatin M (OSM) or other molecules having megakaryocyte stimulating activity, can also be applied to the Mpl ligand. Additional exemplary cytokines or hemopoietic factors for such co-administration include ILr-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL, -11, colony stimulating factor-1 ( C5 F-1), GM-CSF, Granulocyte Colony Stimulating Factor (G-CSF), EPO, Interferon-alpha (IFN-alpha), IFN-beta or IFN-gamma. Furthermore, it may be useful to administer, simultaneously or sequentially, an effective amount of a soluble Mpl receptor from a mammal that appears to have the effect of causing the megakaryocytes to fragment into plaques once the megakaryocytes have matured. Thus, the administration of the Mpl ligand (to increase the number of mature megakaryocytes) followed by the administration of the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocytes to produce platelets) seems to be particularly

183 631 an effective way to stimulate the production of plaques. The above-indicated dose would be adjusted to compensate for such additional ingredients in the therapeutic composition. The progress of the patient's treatment can be monitored by conventional means.

The following examples are included to more fully illustrate the present invention. Moreover, these examples give preferred embodiments of the invention, but are not to be construed as limiting the scope thereof, unless otherwise indicated. Standard methods for many of these procedures described in the following examples, or suitable alternative procedures, are provided in generally recognized molecular biology textbooks such as, e.g., 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).

The methods of isolating and producing Mpl ligands and studying their properties illustrated in the accompanying drawings are described in detail in the examples of the invention application No. P-312577 dated 03. 03. 1995.

Example I.

The present example describes the syntheses of 12 different pegylated MGDF molecules, PEG9-PEG 12 and PEG 14-PEG 21. In each case, the pegylated MGDF molecule was produced by E. coli MGDF-11 (amino acids 22-184, numbered from the beginning of the signal protein or amino acids 1-163 numbered from the beginning of the mature protein). Details of all these pegylated species are summarized in Tables 1-5.

1.1. Preparation of poly-MePEG-MGDF conjugates by acylation

MGDF activated with MePEG derivatives

A cooled (4 ° C) solution of MGDF (2.5 mg / ml) in 0.1M BICINE buffer, pH 8 was added to a 10-fold molar excess of solid MePEG imidyl succinic acid propionate (20 kDa MW) (Shearwater Polymers, Inc. ). The polymer was dissolved with vigorous stirring and the mixture was then left at room temperature.

The progress of the protein modification during the reaction was monitored by HPLC size exclusion (SEC) using a Superdex 200 HR10 / 30 column (Pharmacia Biotech) eluted with 0.1 M phosphate buffer (Na) pH 6.9 at a ratio of 0.7 ml / min.

Analysis of the reaction mixture by SEC HPLC at 30 minutes showed that no free protein remained in the mixture. At this point, the protein concentration in the reaction mixture was reduced to 1 mg / ml by adding sterile water and the mixture was adjusted to pH = 4 with a few drops of acetic acid.

The MePEG-MGDF conjugate was separated from excess MePEG and other reaction by-products by ion exchange chromatography using an SP Sepharose HP ion exchange resin (Pharmacia Biotech).

The reaction mixture (2.5 mg / ml resin) was placed on the column and unreacted MePEG was eluted with 3 column volumes of the starting buffer A (20 mM sodium phosphate, pH = 7.2, 15% glycerol). The MePEG-MGDF conjugate was then eluted with 10 column volumes of buffer B (1M NaCl in buffer A) with a linear increasing gradient from 0% to 30%. The eluate was monitored at 280 nm wavelength. Fractions containing polyMePEG-MGDF conjugate were concentrated and sterile filtered.

The purified poly-MePEG-MGDF conjugate was subjected to HPLC SEC chromatographic analysis using TSK-GEL G4000SWXL and G2000SWXL gel filtration columns assembled with batteries. Proteins were detected by the absorbance of UV light at 280 nm. BIO-RAD gel filtration standards served as markers for the molecular weight of globular proteins.

As can be seen in Fig. 17A, HPLC SEC analysis reveals two primary components in the formulation (in a ratio of about 2: 1) whose elution positions correspond to the positions of the 370,900 and 155,000 globular proteins (370.9 kDa and 155.0 kDa), respectively. . See also table 2.

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PEG 9, PEG 10 and PEG 12 conjugates prepared by acglation of MGDF with succinic acid dyl esters with a molecular weight of 6,000-50,000 (6-50 kDa) MePEG were prepared in an analogous manner. The main reaction conditions used in these preparations are summarized in Table 1.

The results of the HPLC SEC analyzes of these conjugates are shown in Table 2.

1.2. Preparation of poly-MePEG-iPGDF Ooningatates are carried out by addition of MePEG aldehydes.

Preparation of poly-MePEG (20kDa) -MGDF (PEG 20) conjugate. To a chilled (4 ° C) and mixed solution of MGDF (2 mL, 2.5 mg / mL) in 100 mM sodium phosphate, pH = 5 containing 20 mM NaCNBH3, a 10-fold molar excess of moaomethoxg-polyethylene glycol aldehyde (MePEG ) with an average molecular weight of 20,000 (20 kDa) and stirring was continued all at the same temperature.

The degree of protein modification during the reaction was monitored by HPLC SEC using a Superdex 200 HR 10/30 column (Pharmacia Biotech) eluted with 0.1 M phosphate buffer, pH = 6.9, at a rate of 0.7 ml / min.

After 16 hours, SEC HPLC analysis showed that more than 90% of the original amount of protein was modified. At this time, the protein concentration in the reaction mixture was adjusted to 1 mg / ml by diluting it with sterile water and the pH was adjusted to 4 (0.5 M acetic acid).

The MePEG-MGDF conjugate was separated from excess MePEG and other reaction by-products by ion exchange chromatography using an SP Sepharose HP ion exchange resin (PCarmαaia Biotech).

The reaction mixture (2.5 mg / ml resin) was applied to the column and unreacted MePEG was eluted with the starting buffer A (20 mM sodium phosphate, pH = 7.2, 15% glycerol) in an amount equal to three column volumes. Subsequently, the MePEG-MGDF conjugate was eluted - maintaining a linear gradient from 0% to 30% at ten column volumes - with final buffer B (1M NaCl in buffer A). The eluate was monitored at 280 nm. Fractions containing the poly-MePEG-MGDF conjugate were pooled, concentrated, and sterile filtered.

The purified poly-MePEG-MGDF conjugate was analyzed by HPLC SEC using TsK-GEL G4000SWXL and G2000SWXL gel filtration columns assembled with batteries. The proteins were induced by measuring the absorbance of UV radiation at 280 nm. BIO-RAD gel filtration standards served as globulaone markers of protein molecular weight.

As can be seen in Fig. 17B, HPLC SEC analysis reveals two major components (representing 52% and 47% of the total amount) in the preparation whose elution positions correspond to the positions of the globulamgaC proteins 359,400 and 159,300, respectively (359.4 kDa and 159.3 kDa ). See also table 2.

The PEG 18, PEG 19 and PEG 21 conjugate obtained by reductive alkylation of MGDF with 6000-25000 (6-25 kDa) MePEG aldehydes was prepared analogously. The main reaction parameters used in these procedures are summarized in Table 1.

The results of the HPLC SEC analyzes performed on the tgch conjugates are shown in Table 2.

1.3. Preparation; komugotówztikolmonomotoksytp ₀ lictylenone -MGDFmiFro (^ zc of local attachment at the N-terminus of the α-amino residue

Preparation of mono-MePEG (20kDa) -MGDF (PEG 16) conjugate For chilled (4 ° C) and mixed MGDF solution (2 ml, 2.5 mg / ml) in 100 mM sodium phosphate solution, pH = 5, containing 20 mM NaCNBH3, a 5-fold molar excess of methoxg polyethylene glycol aldehyde (MePEG) with an average molecular weight of 20,000 (20 kDa) was added and the reaction mixture was continued to stir at the same temperature.

The degree of protein modification during the reservoir was monitored by SEC HPLC using a Superdnx 200 HR 10. / 30 column (PCaomαciα Biotech) eluted with 0.1 M sodium phosphate buffer, pH = 6.9, at a rate of 0.7 ml / min.

183 631

After 16 hours, SEC HPLC analysis showed that approximately 90% of the original amount of protein had been modified. At this time, the protein concentration in the reaction mixture was reduced to 1 mg / ml by dilution with sterile water, and the pH of the reaction mixture was adjusted to 4 (0.5 M acetic acid).

The mono-MePEG (20kDa) -MGDF conjugate was separated from excess MePEG and other reaction by-products by ion exchange chromatography using Sp Sepharose HP ion exchange resin (Pharmacia Biotech).

The reaction mixture (2.5 mg / ml resin) was applied to the column and the fed MePEG was eluted with the starting buffer A (20 mM sodium phosphate, pH = 7.2, 15% glycerol) in an amount equal to three column volumes. The MePEG-MGDF conjugate was then eluted - maintaining a linear gradient from 0% to 25% at ten column volumes - with final buffer B (1M NaCl in buffer A). The eluate was monitored at 280 nm. Fractions containing the poly-MePEG-MGDF conjugate were pooled, concentrated, and sterile filtered.

The homogeneity of mono-MePEG-MGDF conjugates was confirmed by sodium dodecyl sulfate-polyacrylic acid amide gel electrophoresis, using ready-made gels with a gradient of 4-20% (NOVEX). There was one distinct band corresponding to the location of the protein with a molecular weight of 46,900 (46.9 kDa).

The purified poly-MePEG-MGDF conjugate was analyzed by HPLC SEC using TSK-GEL G4000SWXL and G2000SWXL gel filtration columns assembled with batteries. Proteins were detected by measuring the absorbance of UV radiation at 280 nm. BIO-RAD gel filtration standards served as globular markers of protein molecular weight.

As can be seen in Fig. 17C, SEC HPLC analysis reveals one major formulation component whose elution position corresponds to that of a globular protein with a molecular weight of 181100 (181.1 kDa). See also table 3.

PEG 14, PEG 15 and pEg 17 mono-MePEG-MGDF conjugates obtained by reductive alkylation with MePEG aldehydes having a molecular weight of 6-25 kDa were prepared analogously. The main reaction parameters used in these procedures are summarized in Table 1.

The results of the HPLC SEC analysis of these conjugates are shown in Table 3.

Table 1

Parameters of the MGDF modification reaction

Code Reactive MePEG MGDF concentration mg / ml Reaction conditions Concentration molar [MpPEG / MGDF Type Weight molecular pH Temperature, ° c Time at 1 2 3 4 5 6 7 8 PEG 9 NHS ester 6000 2.5 8 peaceful 0.5 15 PEG 10 NHS ester 6000 2.5 8 peaceful 0.5 1 PEG 11 NHS ester 20,000 2.5 8 peaceful 0.5 10 PEG 12 NHS ester 50,000 2.5 8 peaceful 0.5 5 PEG 14 aldehyde 6000 2.5 5 4 ° C 16 5 PEG 15 aldehyde 12,000 2.5 5 4 ° C 16 5 PEG 16 aldehyde 20,000 2.5 5 4 ° C 16 5 PEG 17 aldehyde 25,000 2.5 5 4 ° C 16 10

183 631 cont. table 1

1 2 3 4 5 6 7 8 PEG 18 aldehyde 6000 5 5 4 ° C 16 10 PEG 19 aldehyde 12,000 5 5 4 ° C 16 10 PEG 20 aldehyde 20,000 5 5 4 ° C 16 10 PEG 21 aldehyde 25,000 5 5 4 ° C 16 10

T a b e 1a 2

Characteristics of poly-MePEG-MGDF according to HPLC SEC

Code MoPEG reactive Apparent molecular weight SEC Component quantity % PEG 9 NHS ester 6000 87900 52700 75 25 (side peak) PEG 10 NHS ester 6000 69200 14 (side peak) PEG 11 NHS ester 20,000 370900 155,000 68 32 PEG 12 NHS ester 50,000 865600 368,000 53 47 PEG 18 aldehyde 6000 84600 41500 60 40 PEG 19 aldehyde 12,000 218400 106700 59 41 PEG 20 aldehyde 20,000 359400 159300 52 47 PEG 21 aldehyde 25,000 450500 218400 54 46

Table 3

Apparent molecular weight of mono-MePEG-MGDF conjugates

Code MoPEG reactive Apparent molecular weight of SEC Apparent molecular weight of SDS PAGE Type Part weight PEG 14 aldehyde 6000 44500 27700 PEG 15 aldehyde 12,000 104700 38300 PEG 16 aldehyde 20,000 181100 46900 PEG 17 aldehyde 25,000 226400 55500

1.4. Prepared DtMe PEG (12 ld) a) -MGDF deconjugates by reductive alkylation of MGDF with methoxypolyethylene glycol aldehyde (PEG 22)

The following procedure produces a purified molecule referred to herein as PEG 22:

183 631

A fivefold excess of methoxy polyethylene glycol aldehyde (MePEG; i.e. OHC- (CH ₂ ) ₂ O- (CH2-CH ₂ O) n-CH3; where n is a multiple of a molecular weight of about 12,000) (Shearwater Polymers) was added to 2.5 mg / ml of MGDF solution (manufactured by E. coli, 1-163) in 100 mM sodium acetate solution, pH = -5.0, temperature 5 ° C. After stirring for 10 minutes, sufficient sodium cyanoborohydride was added to reach a concentration of 20 mM in the reaction mixture.

This mixture was stirred for 16 hours at a temperature of about 5 ° C. At the end of this period, sufficient Purified (USP) water was added to bring the MGDF concentration to 1 mg / ml. This solution was filtered through a 0.2 micron vacuum filter. Thus, 90 mg of the reaction product was obtained. Small amounts of 1.0 M phosphate monobasic solution and 1 N sodium hydroxide solution were added to the reaction mixture to reach a concentration of 10 mM phosphate and pH = 6.8.

The conjugate was purified over a cation exchange column. A 40 ml high throughput column packed with SP-Sepharose resin with a bed height of 7.5 cm was prepared. The column was equilibrated with an equilibration buffer (10 mM phosphate buffer, pH = 6.8 with 15% glycerol). The preparation to be purified was loaded onto the column at a rate of 2.2 mg / ml of resin at a rate of 0.15 column volumes (CV) per minute. The equilibration buffer was then passed through the column until the base line was reached. The column was eluted with ten column volumes with a linear gradient from buffer A (20 mM phosphate buffer, pH = 7.2, 15% glycerin) to buffer B (buffer A plus 0.3 M NaCl). The flow rate was kept at 0.15 CV per minute at all times. The eluate was monitored at 280 nm.

Fractions were analyzed by SDS-PAGE collecting fractions containing the DiPEG conjugate. The combined fractions were concentrated and filtered through a 0.2 micron filter.

An example. Biological activity of pegylated MGDF molecules

A. PEG-9 - PEG-12 and PEG-14 - PEG-21.

Platelets were counted in mice dosed with recombinant human MGDF and the results are shown in Figure 18. CHO MGDF 212-353 (1> wa <> iraty), non-pegylated E. coli MGDF 22-184 (circles) and pegylated product. the product E. coli MGDf 22-184 (dots), at the concentrations indicated above in the description of the figures, was injected subcutaneously into healthy Balb / c mice once a day for 5 days. Twenty four hours after the last injection, blood was collected for testing from small lateral tail vein incisions. Blood cell analysis was performed using a Sysmex electronic blood cell analyzer (Baxter Diagnostic, Inc. Irvine, CA). Results are presented as the mean of the measurements read for the four animals ± standard deviation of the mean. Other blood cell parameters, such as total white blood cell counts or red blood cell counts, were not affected by the current test.

Additional forms of recombinant human MGDF were tested as above. The number of platelets in mice treated with 50 gg / kg / day or 10 / gg / kg / day of the indicated form of r-HuMGDF is shown in the next table 4. The numbers reported are the mean of the readings for 4 animals with standard deviation in italics.

Table 4

Character 50 (tan / kg / day 10 pg / kg / day Mean n = 4 Deviation Mean n = 4 Deviation 1 2 3 4 5 CHO 22-353 4343 309 2571 80 E. Coli 22-184 2021 29 1439 18 PEG 9 2728 56 2369 34 PEG 10 2431 291 1556 126 PEG11 3778 59 1861 73

183 631 cont. table 4

1 2 3 4 5 PEG 12 3885 156 1740 88 PEG 14 3567 80 2020 63 PEG 15 4402 57 2834 99 PEG 16 4511 239 3215 11 PEG 17 4140 188 3113 261 PEG 18 4586 59 2931 129 PEG 19 3980 330 4189 80 PEG 20 3942 285 3054 339 PEG 21 4195 145 4002 91 baseline 939 25

Explanations for table 4:

In each case, the MGDF molecule was a pegylated E. coli product designated MGDF-11 (amino acids 22-184 counting from the beginning of the signal peptide or amino acids 1-163 counting from the beginning of the mature protein) as described in Example I above.

Name Pegylation Average molecular weight of the PEG PEG reactive molecule for synthesis PEG 9 polypegylated 6000 NHS ester from MePEG PEG 10 polypegylated 6000 NHS ester from MePEG PEG11 polypegylated 20,000 NHS ester from MePEG PEG 12 polypegylated 50,000 NHS ester from MePEG PEG 14 monopegylated 6000 aldehyde from MePEG PEG 15 monopegylated 12,000 aldehyde from MePEG PEG 16 monopegylated 20,000 aldehyde from MePEG PEG 17 monopegylated 25,000 aldehyde from MePEG PEG 18 polypegylated 6000 aldehyde from MePEG PEG 19 polypegylated 12,000 aldehyde from MePEG PEG 20 polypegylated 20,000 aldehyde from MePEG PEG 21 polypegylated 25,000 aldehyde from MePEG

The baseline number of platelets is measured in healthy animals that have not received any treatment.

As can be seen, pegylation of recombinant human MGDF does not adversely affect the ability of the molecule to increase cell count in the animals it is administered to, and may actually increase the activity of the product 22-184 produced by E. coli to a level equal to or greater than that observed in the 22-353 molecule. as a CHO derivative.

B. PEG-22

The results for PEG-22 are shown in Figure 24. Noticeably, the normalization of the platelet count at PEG-22 occurred several days earlier than with the full length MGDF aldehyde derivatives PEG-16 or PEG-17.

Studies on both the production and in vivo activity of r-HuMGDF are disclosed in Application No. P-312577 dated 03.30.1995.

183 631

Summary of sequences (1) GENERAL INFORMATION:

(i) APPLICANTS:

Bartley, Timothy D.

Bogenberger, Jakob M.

Bosselman, Robert A Hunt, Pamela Kinstler, Olaf B.

Samal, Babru B.

(ii) ENTRY TITLE: Compositions and methods for stimulating the growth and differentiation of megakaryocytes (iii) NUMBER OF SEQUENCES: 34 (iv) ADDRESS FOR CORRESPONDENCE:

(A) ADDRESS: Amgen Inc.

(B) STREET: 1840 Dehavilland Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: States of Zj ^^^ oc ^ one of North America (F) CODE: 91320-1789 (v) FORM READ BY COMPUTER:

(A) MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (vi) CURRENT APPLICATION DATE:

(A) APPLICATION NUMBER:

(B) APPLICATION DATE:

(C) CLASSIFICATION:

(viii) INFOORM ^ C ^^ A. ABOUT RIVER ^ ZZ ^ KKPATENNC ^ OWY ^:

183 631 (A) SURNAME: Cook, Robert R.

(C) NUMBER: A-290-C (2) INFORMATION ABOUT SEQ ID No .: 1:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 31 amino acids (B) TYPE: Amino acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Protein (xi) SEQ ID NO: 1:

Ala Pro Pro Ala Xaa Asp Pro Arg Leu Leu Asn Lys Met Leu Arg Asp 15 10 15

Ser His Val Leu His Xaa Arg Leu Xaa Gin Xaa Pro Asp He Tyr 20 25 30 (2) INFORMATION ABOUT SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 21 amino acids (B) TYPE: Amino acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 2:

Ala Pro Pro Ala Xaa Asp Pro Arg Leu Leu Asn Lys Met Leu Arg Asp 15 10 15

His Val Leu His cheese 20

183 631 (2) INFORMATION ABOUT SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 17 amino acids (B) TYPE: Amino acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 3:

Thr Gin Lys Glu Gin Thr Lys Ala Gin Asp Val Leu Gly Ala Val Ala Leu 15 10 15 (2) INFORMATION ABOUT SEQ ID NO: 4:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 17 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGOA: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GCNCCNCCNG CNTGYGA (2) INFORMATION ABOUT SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 21 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 5:

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GCARTGYAAC ACRTGNGART C (2) INFORMATION ABOUT SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (u) PARTICLE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 6:

Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp 15 10 15

Ser His Val Leu His 20 (2) INFORMATION ABOUT SEQ ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 21 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 7:

GTACGCGTTC TAGANNNNNN T (2) INFORMATION ON SEQ ID No .: 8:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 21 base pairs

183 631 (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) PARTICLE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 8:

AGTTTACTGA GGACTCGGAG G (2) INFORMATION ABOUT SEQ ID No .: 9:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 30 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 9:

TTCGGCCGGA TAGGCCTTTT ΤΊΤΙΤΤΤΤΤΤ (2) INFORMATION ABOUT SEQ ID NO .: 10:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 29 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 10:

TTCGGCCGGA TAGGCCTTTT ΊΤΠΤΠΤΊ

183 631 (2) INFORMATION ABOUT SEQ ID No .: 11:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 11:

TGCGACCTCC GAGTCCTCAG (2) INFORMATION ABOUT SEQ ID NO .: 12:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 23 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 12:

GAGTCCTCAG TAAACTGCTT CGT (2) INFORMATION ABOUT SEQ ID NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA

183 631 (xi) SEQUENCE DESCRIPTION: SEQ ID No .: 13:

GGAGTCAACGA AGCAGTTTAC (2) INFORMATION ABOUT SEQ ID NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 18 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 14:

CCTTTACTTC TAGGCCTG (2) INFORMATION ABOUT SEQ ID No .: 15:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 19 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (d) TOPOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 15:

GAGGTCACAA GCAGGAGGA (2) INFORMATION ABOUT SEQ ID NO .: 16:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 19 base pairs (B) TYPE: Nucleic acid (C) FORM OF THREAD: Single

183 631 (D) TOPOLOGY: Linear (ii) PARTICLE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID No .: 16:

GGZCECGEZZ GGGACGTCG (2) INFORMATION ON SEQ ID No .: 17:

(i) WANTED SEQUENCE CHANNEL:

(A) LENGTH: 19 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 17:

TCZTCCTGZT TGTGACZEC (2) INFORMATION ON SEQ ID No .: 18:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 19 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 18:

ZCAGGCAGGA TTZCGGGGA (2) INFORMATION ABOUT SEQ ID NO .: 19:

183 631 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 19:

CAACAAGTCA ACCGCCAGCC AGACACCCCG (2) INFORMATION ABOUT SEQ ID NO .: 20:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 24 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) PARTICLE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 20:

GGCCGGATAG GCCACTCNNN NNNT (2) INFORMATION ABOUT SEQ ID NO .: 21:

(i) CHARACTERISTICS OF THE SEQUENCES:

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183 631

GCARTGYAAN ACRTGNGART C (2) INFORMATION ABOUT SEQ ID NO .: 22:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 16 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 22:

TTGGTGTGCA CTTGTG (2) INFORMATION ABOUT SEQ ID NO .: 23:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY Linear (ii) TYPE OF PARTICLE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 23:

CACAAGTGCA CACCAACCCC (2) INFORMATION ABOUT SEQ ID NO .: 24:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH !: 1342 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear

183 631 (n) TYPE OF PARTICLE: cDNA (ix) FEATURES:

(A) NAME / KEY: CDS (B) LOCATION: 99 ...... 1094 (xi) SEQUENCE DESCRIPTION: SEQ ID No .: 24:

CAGGGAGCCA CGCCAGCCAA GACACCCCGG CCAGAATGGA GCTGACTGAA TTGCTCCTCG60

TGGTCATGCT TCTCCTAACT GCAAGGCTAA CGCTGTCC AGC CCG GCT CCT CCT 113 Ser Pro Ala Pro Pro 1 5

GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC 161 Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp ser His Val

15 20

CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA 209 Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr

30 35

CCT GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC 257 Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr

45 50

CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT 305 Gln Met Glu Glu Thr Lys Ala Gln Asp lie Leu Gly Ala Val Thr Leu

60 65

CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC 353 Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys 70 75 80 85

CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT 401 Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu

100

183 631

GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg

105 110 115

ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His

120 125 130

CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr

135 140 144

CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr 150 155 110 115

TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG ACT TCT GGA TTG Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu

170 175 180

TTG GAG ACA AAC TCC ACT GCC TCA GCC AGA ACT ACT GGC TCT GGG CTT Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu

185 190 195

CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG ATT CCT GGT CTG CTG AAC Leu Lys Trp Gin Gin Gly Phe Arg Ala Lys Iie Pro Gly Leu Leu Asn

200 205 210

CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC CTG AAC AGG ATA Gln Thr Ser Arg Ser Leu Asp Gin Ile Pro Gly Tyr Leu Asn Arg Ile

215 220 225

CAC GAA CTC TTG AAT GGA ACT CGT GGA CTC TTT CTT GGA CCC TCA CGC His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg 230 235 2 4 245

449

497

545

593

641

669

737

788

833

AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA TCA GAC ACA GGC 881

183 631

Arg Thr Leu Gly Ala Pro Asp He Ser Ser Gly Thr Ser Asp Thr Gly 250 255 260

TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT TCC CCA ACC CAT 929

Ser Leu Pro Pro Asn Leu Gin Pro Gly Tyr Ser Pro Ser Pro Thr His

265 270 275

CCT CCT ACT GGA CAG TAT ACG CTC TTC CCT CTT CCA CCC ACC TTG CCC 977

Pro Pro Thr Gly Gin Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro

280 285 290

ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT CCT GAC CCT TCT GCT CCA 1 (025

Thr Pro Val Val Gin Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro

295 300 305

ACG CCC ACC CCT ACC AGC CCT CTT CTA AAC ACA TCC TAC ACC CAC TCC 1073 Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr His Ser 310 315 320 325

CAG AAT CTG TCT CAG GAA GGG TAAGGTTCTC AGACACTGCC GACATCAGCA 1124 Gln Asn Leu Ser Gln Glu Gly

330

TTGTCTCGTG TACAGCTCCC TTCCCTGCAG GGCGCCCCTG GGAGACAACT GGACAAGATT1184 TCCTACTTTC TCCTGAAACC CAAAGCCCTG GTAAAAGGGA TACACAGGAC TGAAAAGGGA1244 ATCATTTTTC ACTGTACATT ATAAACCTTC AGAAGCTATT TTTTTAAGCT ATCAGCAATA1304 CTCATCAGAG CAGCTAGCTC TTTGGTCTAT TTTCTGCA 1342 (2) INFORMATION FOR SEQ ID NO: 25:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 332 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 25:

183 631

Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 15 10 15

Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30

His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45

Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp De Leu 50 55 60

Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80

Leu Gly Pro Thr Cys Leu Cheese Cheese Leu Leu Gly Gin Leu Cheese Gly Gin 85 90 95

Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110

Pro Pro Gin Gly? Ag ITr TTp Ala His Lys Asp Pro Asn Ala He Phe 115 120 122

Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140

Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala 145 150 155 160

Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn 165 170 175

Arg Thr Ser Gly Leu Leu Glu TPr Asn Pee Płir AL Ser Ala Arg Thr 180 185 190

Thr Gly Ser Gly Leu Leu Lys Trp Gin Gin Gly PPe Arg Ala Lys De

183 631

195 200 205

Pro Gly Leu Leu Asn Gin Thr Ser Arg Ser Leu Asp Gln He Pro Gly 210 215 222

Tyr Leu Asn Arg De His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe 225 230 235 220

Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp lie Ser Ser Gly 245 250 225

Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser 260 265 270

Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu 275 280 285

Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro 290 295 300

Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr 305 310 315 332

Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly 325 330 (2) INFORMATION ABOUT SEQ ID NO .: 26:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1342 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (ix) FEATURES:

(A) NAME / KEY: CDS

183 631 (B) LOCATION: 9E: .921 (xi) SEQUENCE DESCRIPTION: SEQ ID No .: 26:

CAGGGAGCCA CGCCAGCCAA GACACCCCGG CCAGAATGGA GCTGACTGAA TTGCTCCTCG60

TGGTCATGCT TCTCCTAACT GCAAGGCTAA CGCTGTCC AGC CCG GCT CCT CCT 113 Ser Pro Ala Pro Pro 1 5

GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC 161 Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val

15 20

CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA 209 Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val His Pro Leu Pro Thr

30 35

CCT GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC 257 Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr

45 50

CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT 305 Gin Met Glu Glu Thr Lys Ala Gin Asp He Leu Gly Ala Val Thr Leu

60 65

CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC 353 Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin Leu Gly Pro Thr Cys 70 75 80 85

CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT 401 Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin Val Arg Leu Leu Leu

95 100

GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG 449 Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu Pro Pro Gin Gly Arg

105

110

115

183 631

ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC 497 Thr Thr Ala His Lys Asp Pro Asn Ala He Phe Leu Ser Phe Gin His

120 125 130

TG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC 545 Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr

135 140 145

CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT CTG CCC AGC AGA ACC 593 Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr 150 155 160 165

TCT CTA GTC CTC ACA CTG AAC GAG CTC C CAAACAGGAC TTCTGGATTG 641 Ser Leu Val Leu Thr Leu Asn Glu Leu

170

TTGGAGACAA ACTTCACTGC CTCAGCCAGA ACTACTGGCT CTGGGCTTCT GAAGTGGCAG701

CAGGGATTCA GAGCCAAGAT TCCTGGTCTG CTGAACCAAA CCTCCAGGTC CCTGGACCAA761

ATCCCCGGAT ACCTGAACAG GATACACGAA CTCTTGAATG GAACTCGTGG ACTCTTTCCT821

GGACCCTCAC GCAGGACCCT AGGAGCCCCG GACATTTCCT CAGGAACATG AGACACAGGC881

TCCCTGCCAC CCAACCTCCA GCCTGGATAT TCTCCTTCCC CAACCCATCC TCCTACTGGA941

CAGTATACGC TCTTCCCTCT TCCACCCACC TTGCCCACCC CTGTGGTCCA GCTCCACCCC1001

CTGCTTCCTG ACCCTTCTGC TCCAACGCCC ACCCCTACCA GCCCTCTTCT AAACACATCC1061

TACACCCACT CCCAGAATCT GTCTCAGGAA GGGTCCGGTT CTCAGACACT GCCG ACATCA1121

GCATTGTCTC GTGTACAGCT CCCTTCCCTG CAGGGCGCCC CTGGGAGACA ACTGGACAAG1181

ATTTCCTACT TTCTCCTGAA ACCCAAAGCC CTGGTAAAAG GGATACACAG GACTGAAAAG1241

GGAATCATTT TTCACTGTAC ATTATAAACC TTCAGAAGCT ATTTTTTTAA GCTATCAGCA1301

183 631

ATACTCATCA GAGCAGCTAG CTCTTTGGTC TATTTTCTGC A (2) INFORMATION ABOUT SEQ ID No .: 27:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 174 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO .: 27:

Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 15 10 15

Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val 20 25 30

His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45

Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu 50 55 60

Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln 65 70 75 80

Leu Gly Pro Thr Cys Leu Cheese Cheese Leu Leu Gly Gln Leu Cheese Gly Gln 85 90 95

Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu 100 105 110

Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe 115 120 125

1342

Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu

183 631

130 135 140

Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala

145 150 155 160

Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu 165 170 (2) INFORMATION ABOUT SEQ ID NO .: 28:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 1164 base pairs (B) TYPE: Nucleic acid (C) THREAD FORM: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: cDNA (ix) FEATURES:

(A) NAME / KEY: CDS (B) LOCATION: 27 ... 824 (xi) SEQUENCE DESCRIPTION: SEQ ID No .: 28:

AGGGAGCCAC GCCAGCCAGA CACCCCGGCC AGAATGGAGC TGACTGAACC GCTCCTCGTG60

GTCATGCTTG TCCTAACTGC AAGGCTAACG CTGTCC AGC CCG GCT CCT CCT GCT114 Ser Pro Ala Pro Pro Ala

5

TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAT GTC CTT 166

Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu

15 20

CAC AGC AGA CTG AGC CAG TTGC CCA GAG GTT CAC CCT TTG CCT ACA CCT 210 His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro

30 35

183 631

GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG 258 Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln

45 50

ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG 306 Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu 55 60 65 70

CTG GAG GGA GTG ATTG GCA GCA CGG GGA CAA CITG GGA CCC ACT TGC CTC354 Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu

80 85

TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG 4 (0ł

Cheese Cheese Leu Leu Gly Gln Leu Cheese Gly Gln Val Arg leu Leu Leu Gly

95 100

GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC 450 Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr

105 110 115

ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG 498 Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu

120 125 130

CTC CGA GGA AAG GAC TTC TGG ATT GTT GGA GAC AAA CTT CAC TGC CTC 554 Leu Arg Gly Lys Asp Phe Trp Ile Val Gly Asp Lys Leu His Cys Leu 135 140 145 150

AGC CAG AAC TAC TGG CTC TGG GCT TCT GAA GTG GCA GCA GGG ATT CAG 559 Ser Gln Asn Tyr Trp Leu Trp Ala Ser Glu Val Ala Ala Gly Ile Gln

155 160 165

AGC CAA GAT TCC TGG TCT GCT GAA CCA AAC CTC CAG GTC CCA GGA CCA 642 Ser Gln Asp Ser Trp Ser Ala Glu Pro Asn Leu Gln Val Pro Gly Pro

170 175 180

AAT CCC CGG ATA CCT GAA CAG GAT ACA CGA ACT CTT GAA TGG AAC TCG 690 Asn Pro Arg Ile Pro Glu Gln Asp Thr Arg Thr Leu Glu Trp Asn Ser

183 631

185 190 195

TGG ACT CTT TCC TGG ACC CTC ACG CAG GAC CCT AGG AGC CCC GGA CAT 738 Trp Thr Leu Ser Trp Thr Leu Thr Gin Asp Pro Arg Ser Pro Gly His

200 205 210

TTC CTC AGG AAC ATC AGA CAC AGG CTC CCT GCC ACC CAA CCT CCA GCC 786 Phe Leu Arg Asn Ile Arg His Arg Leu Pro Ala Thr Gin Pro Pro Ala 215 220 225 230

TGG ATA TTC TCC TTC CCC AAC CCA TCC TCC TAC TGG ACA GTA TAC GCT 834

Trp De Phe Ser Phe Pro Asn Pro Ser Ser Tyr Trp Thr Val Tyr Ala

235 240 245

CTT CCC TCT TCC ACC CAC CTT GCC CAC CCC TGT GGT CCA GCT CCA CCC 882 Leu Pro Ser Ser Thr His Leu Ala His Pro Cys Gly Pro Ala Pro Pro

250 255 260

CCT GCT TCC TGACCCTTCT GCTCCAACGC CCACCCCTAC CAGCCCTCTT 931

Pro Ala Ser

265

CTAAACACAT CCTACACCCA CTCCCAGAAT CTGTCTCAGG AAGGGTAAGG TTCTCAGACA991

CTGCCCACAT CAGCATTGTC TCGTGTACAG CTCCCTTCCC TGCAGGGCAC CCCTGGGACA1051

CAACTGGACA AGATTTCCTA CTTTCTCCTG AAACCCAAAG CCCTGGTAAA AGGGATACAC1111

AGGACTGAAA AGGGAATCAT TTTTCACTGT ACATTATAAA CCTTCAGAAG CTA 1164 (2) SEQ INFORMATION ID NO .: 29:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 265 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Protein

183 631 (xi) SEQUENCE DESCRIPTION: SEQ ID No .: 29:

Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 15 10 15

Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val 20 25 30

His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu 35 40 45

Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gln Asp De Leu 50 55 60

Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 65 70 75 80

Leu Gly Pro Thr Cys Leu Cheese Cheese Leu Leu Gly Gin Leu Cheese Gly Gin 85 90 95

Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu 100 105 110

Pro Pro Ghi Gly Arg TTr Thr rMa His Lys Asp Pro Asn Ala Ile PPe 115 120 125

Leu Ser Phe Gin His Leu Leu Arg Gly Lys Asp Phe Trp Ile Val Gly 130 135 110

Asp Lys leu His Cys Leu Ser Gin Asn Tyr Trp Leu Trp Ala Ser Glu 145 150 155 160

Val Ala Ala Gly Ile Gin Ser Gin Asp Ser Trp Ser Ala Glu Pro Asn 165 170 177

Leu Gin Val Pro Gly Pro Asn Pro Arg Ile Pro Glu Gln Asp Thir Arg 180 185 190

183 631

Thr Leu Glu Trp Asn ser Trp Thr Leu Ser Trp Thr Leu Thr Gln Asp 195 200 205

Pro Arg Ser Pro Gly His Phe Leu Arg Asn De Arg His Arg Leu Pro 210 215 220

Ala Thr Gln Pro Pro Ala Trp De Phe Ser Phe Pro Asn Pro Ser Ser 225 230 235 240

Tyr Trp Thr Val Tyr Ala Leu Pro Ser Ser Thr His Leu Ala His Pro 245 250 255

Cys Gly Pro Ala Pro Pro Pro Ala Ser 260 265

Claims (24)

Patent claims CLAIMS 1. Derivative of a MGDF growth factor and development factor polypeptide selected from the group of polypeptides consisting of: - a polypeptide with the sequence of amino acids 22-353 shown in Figure 11, termed MgDf-1; - a polypeptide with the sequence of amino acids 22-195 shown in Figure 11, termed mGDf-2; - a polypeptide with the sequence of amino acids 22-286 shown in Figure 12, termed mGDf-3; - a polypeptide with the sequence of amino acids 22-172 shown in Figure 11, referred to as MGDF-4; - a polypeptide with the sequence of amino acids 22-177 shown in Figure 11, referred to as MGDF-5; - a polypeptide with the sequence of amino acids 22-191 shown in Figure 11, referred to as MGDF-6; - a polypeptide with the sequence of amino acids 22-198 shown in Figure 11, referred to as mGdF-7; - a polypeptide with the sequence of amino acids 22-265 shown in Figure 11, referred to as MGDF-8; - a polypeptide with the sequence of amino acids 22-184 shown in Figure 11, referred to as MGDF-11; - a polypeptide with the amino acid sequence 27-353 shown in Figure 11, referred to as MGDF-12; - a polypeptide with the amino acid sequence 27-195 shown in Figure 11, referred to as MGDF-13; - a polypeptide with the amino acid sequence 27-172 shown in Figure 11, termed MGDF-14, and - a polypeptide with the amino acid sequence 27-184 shown in Figure 11, referred to as mGdF-1, wherein the MGDF polypeptide is attached to at least one water-soluble polymer. 2. A derivative according to claim 1 The pharmaceutical composition of claim 1, wherein the water-soluble polymer is pharmaceutically acceptable. 3. The derivative according to claim 1 The method of claim 1, wherein the water-soluble polymer is selected from the group consisting of dextran, poly (N-vinyl-pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide-ethylene oxide copolymers, polyoxyethylene polyols, polyvinyl alcohols and mixtures thereof . 4. A derivative according to claim 1 The process of claim 1, wherein the water-soluble polymer is polyethylene glycol. 5. The derivative according to claim 1 4. The process of claim 4, wherein said polyethylene glycol is monomethoxylated polyethylene glycol. 6. The derivative according to claim 1 The method of claim 4, wherein said polyethylene glycol is attached to the MGDF polypeptide by an acyl or alkyl bond. 183 631 7. The derivative according to claim 1 6. The method of claim 6, wherein the polyethylene glycol group is attached to the N-terminus of the MGDF polypeptide. 8. The derivative according to claim 1 6. The process of claim 6, wherein the polyethylene glycol group has an average molecular weight of 10,000 to 50,000. 9. The derivative according to claim 1 The method of claim 1, wherein the MGDF polypeptide is covalently linked to two water-soluble polymer molecules. 10. The derivative according to claim 1 9. The method of claim 9, wherein both said water-soluble polymer molecules are polyethylene glycol molecules. 11. The derivative according to claim 1 The polypeptide of claim 1 which is a monopegylated MGDF polypeptide. 12. A derivative according to claim 1 The polypeptide of claim 11, which is a monopegylated MGDF polypeptide at the α-amino group at the N-terminus of said polypeptide and is substantially homogeneous. 13. Derivative according to resources. The polypeptide of claim 12, which is a monopegylated polyethylene glycol MGDF polypeptide with an average molecular weight of 5,000 to 50,000. 14. A derivative according to claim 1 14. The polypeptide of claim 11, which is a monopegylated MGDF polypeptide with a polyethylene glycol group attached to the N-terminus of said polypeptide. 15. A method of producing a derivative of a megakaryocyte growth and development factor MGDF polypeptide selected from the group of polypeptides consisting of: - a polypeptide with the sequence of amino acids 22-353 shown in Figure 11, referred to as mGdF-1; - a polypeptide with the sequence of amino acids 22-195 shown in Figure 11, termed mGdF-2; - a polypeptide with the sequence of amino acids 22-286 shown in Figure 12, referred to as MGDF-3; - a polypeptide with the sequence of amino acids 22-172 shown in Figure 11, termed mGDf-4; - a polypeptide with the sequence of amino acids 22-177 shown in Figure 11, termed mGdF-5; - a polypeptide with the sequence of amino acids 22-191 shown in Figure 11, referred to as mGdF-6; - a polypeptide with the sequence and amino acids 22-198 shown in Figure 11, referred to as mGdF-7; - a polypeptide with the sequence of amino acids 22-265 shown in Figure 11, referred to as mGdF-8; the polypeptide with the sequence of amino acids 22-184 shown in Figure 11, referred to as M.G.DF-11; - a polypeptide with the amino acid sequence 27-353 shown in Figure 11, termed mGdF-12; - a polypeptide with the amino acid sequence 27-195 shown in Figure 11, referred to as MGDF-1.3; - a polypeptide with the amino acid sequence 27-172 shown in Figure 11, termed MGDF-14, and - a polypeptide with the sequence of amino acids 27-184 shown in Figure 11, referred to as MGDF-15; wherein the MGDF polypeptide is attached to at least one water-soluble polymer, comprising attaching the water-soluble polymer to the MGDF polypeptide, wherein the water-soluble polymer having an aldehyde reactive group is used and the aldehyde terminal group reaction of the polyethylene glycol aldehyde derivative is carried out with the indicated MGDF polypeptide in the presence of a reducing agent, or a water-soluble polymer having a single reactive aldehyde group is used and contacting the indicated MGDF polypeptide with the water-soluble polymer under reductive alkylation conditions at a sufficiently acidic pH that the α-a4 group is The 183 631 min at the amino terminus of the indicated MGDF polypeptide may be reactive, or a water-soluble polymer having a single reactive ester group is used and the MGDF polypeptide is contacted with the water-soluble polymer under conditions that allow the indicated MGDF polypeptide to be attached to the water-soluble polymer by an acyl bond, and then isolating the derivatized MGDF polypeptide linked to at least one water-soluble polymer. 16. The method according to p. The process of claim 15, wherein the water-soluble polymer is a pharmaceutically acceptable polymer. 17. The method according to p. The method of claim 15, wherein the water-soluble polymer is a polymer selected from the group consisting of dextran, poly (N-vinyl-pyrrolidone), polyethylene glycols, polypropylene glycol homopolymers, polypropylene oxide-ethylene oxide copolymers, polyoxyethylene polyols and polyvinyl alcohols. 18. The method according to p. The process of claim 15, wherein the water-soluble polymer is polyethylene glycol. 19. The method according to p. The method of claim 15, wherein the water-soluble polymer is a polyethylene glycol having a single reactive aldehyde group, and contacting said MGDF polypeptide with a molecule of said polyethylene glycol under reductive alkylation conditions at a pH sufficiently acidic that the α-amino group at the amino terminus of The MGDF polypeptide may have been reactive and the monopegylated MGDF polypeptide produced is isolated. 20. The method according to p. The method of 19, wherein the MGDF polypeptide is MGDF-11 having a sequence of amino acids 22-184 shown in Figure 11. 21. The method according to p. 20, characterized in that the MGDF-11 polypeptide is produced by E. coli expression of a DNA encoding a polypeptide containing amino acids 22-184 shown in Figure 11 and a Met-Lys sequence at its N-terminus, isolating the expressed polypeptide and cleavage Met ² -Lys ¹ from the isolated polypeptide. 22. The method according to p. The process according to claim 18 or 19, characterized in that the polyethylene glycol has an average molecular weight of 2,000 to 100,000. 23. A pharmaceutical composition containing a pharmaceutically active substance and a pharmaceutically acceptable diluent, adjuvant or carrier, characterized in that the pharmaceutically active substance is a derivative of a megakaryocyte growth and development factor polypeptide - MGDF, selected from the group of polypeptides consisting of: - a polypeptide with the sequence of amino acids 22-353 shown in Figure 11, termed mGDf-1; - a polypeptide with the sequence of amino acids 22-195 shown in Figure 11, termed mGDf-2; - a polypeptide with the sequence of amino acids 22-286 shown in Figure 12, termed mGdF-3; - a polypeptide with the sequence of amino acids 22-172 shown in Figure 11, termed mGdF-4; - a polypeptide with the sequence of amino acids 22-177 shown in Figure 11, termed mGDf-5; - a polypeptide with the sequence of amino acids 22-191 shown in Figure 11, referred to as mGDf-6; - a polypeptide with the sequence of amino acids 22-198 shown in Figure 11, referred to as mGDf-7; - a polypeptide with the sequence of amino acids 22-265 shown in Figure 11, referred to as mGdF-8; - a polypeptide with the sequence of amino acids 22-184 shown in Figure 11, referred to as mGdF-1 1; 183 631 - a polypeptide with the amino acid sequence 27-353 shown in Figure 11, referred to as MGDF-12; - a polypeptide with the sequence of amino acids 27-195 shown in Figure 11, referred to as MGDF-13; - a polypeptide with the amino acid sequence 27-172 shown in Figure 11, termed MGDF-14, and - a polypeptide with the amino acid sequence 27-184 shown in Figure 11, referred to as MGDf-15, which is a combination of the MGDF polypeptide with at least one water-soluble polymer. 24. The composition of claim 24 The method of claim 23, wherein the derivative of MGDF comprises a monopegylated MGDF polypeptide.