MXPA97003134A - Methods and equipment using macrof stimulating protein - Google Patents

Abstract

The present invention relates to a method for stimulating megakaryocyte maturation and thrombocyte production using macrophage stimulating protein ("MSP"). In the methods, an effective amount of MSP can be administered in vivo, or alternatively, it can be used to stimulate the maturation of megakaryocytes and produce thrombocytes in vitro. Methods for treating thrombocytopenia in a mammal with MSP are also provided. We also provide equipment and articles of manufacture that include M

Description

METHODS AND EQUIPMENT USING MACROPHAGO STIMULATING PROTEIN FIELD OF THE INVENTION The invention relates generally to methods for stimulating megakaryocytopoiesis and thrombocytopoiesis. More particularly, the invention relates to methods that stimulate the maturation of megakaryocytes and the production of thrombocytes using macrophage-stimulating protein. The invention also relates to methods of treating certain hematopoietic conditions, such as thrombocytopenia, and to equipment containing macrophage-stimulating protein.

BACKGROUND OF THE INVENTION 1. Hepatocyte Growth Factor Hepatocyte growth factor ("HGF") functions as a growth factor for tissues and particular cell types. HGF was initially identified as a mitogen for hepatocytes [Michalopoulos et al., Cancer Res., 44: 4414-4419 (1984); Russel et al., J. Cell. Physiol., 119: 183-192 (1984); Nakamura et al, Biochem. REF: 24573 Biophys. Res. Comm., 122: 1450-1459 (1984)]. Nakamura et al., Supra, reported the purification of HGF from serum of partially hepatectomized rats. Subsequently, HGF was purified from rat platelets, and its subunit structure was determined [Nakamura et al., Proc. Nati Acad. Sci. USA, 83: 6489-6493 (1986); Nakamura FEBS Letters, 24 ^: 311-316 (1987)]. Purification of human HGF (-'huHGF ") from human plasma was described for the first time by Gohda et al., J. Clin. Invest., 8JL: 414-419 (1988). Both rat HGF and huHGF have have been cloned molecularly, including the cloning and sequencing of a variant found in nature lacking 5 amino acids designated "delta5 HGF" [Miyaza et al., Biochem. Biophys. Res. Comm .., 163: 967-973 (1989), Nakamura et al., Nature, 342: 440-443 (1989), Seki et al, Biochem Biophys, Res. Commun., 172: 321-327 (1990), Tashiro et al., Proc Nati. Acad Sci. USA, 82: 3200-3204 (1990), Okaj ima et al, Eur. J. Biochem., 193: 375-381 (1990).] The mature form of huHGF, which corresponds to the purified primary form of human serum, is a disulfide-linked heterodimer derived by proteolytic cleavage of human prohormone between amino acids R494 and V495. This process of cleavage generates a molecule composed of a subunit a of 440 amino acids (Mr of 69 kDa) and a ß subunit of 234 amino acids (Mr of 34 kDa). The nucleotide sequence of the huHGF cDNA reveals that both the a chain and the b chain are contained in a single open reading frame that codes for the pre-proprecursor protein. In the predicted primary structure of the mature huHGF, an S-S bridge is formed between chains between Cys 487 of the a chain and the Cys 604 of the β chain [see Nakamura et al., Nature, supra]. The N-terminus of the a chain is preferred by 54 amino acids, starting with a methionine group. This segment includes a characteristic hydrophobic leader sequence (signal) of 31 residues and the prosequence.

The a chain starts at amino acid (aa) 55, and contains four kringle domains. The kringle domain 1 extends from approximately er "to aa 128 to approximately aa 206, the kringle domain 2 is between approximately aa 211 and approximately aa 288, the kringle domain 3 ^ is defined as that which is extends from about aa 303 to about aa 383, and the kringle domain 4 extends from about aa 391 to about aa 464 of the a chain. The definition of the different kringle domains is based on their homology with the kringle-like domains of other proteins (prothrombin, plasminogen), therefore, the above limits are only approximate. Since the function of those kringles has not yet been determined. The ß chain of huHGF shows a high homology with the catalytic domain of serine proteases (38% homology with the serine protease domain of plasminogen). Nevertheless, two of the three residues that form the catalytic triad of serine proteases are not conserved in huHGF. Therefore, despite its domain similar to that of serine protease, huHGF seems to have no proteolytic activity, and the precise role of the β chain remains unknown. HGF contains four putative glycosylation sites, which are located at positions 294 and 402 of chain a and positions 566 and 653 of the β chain. In a portion of the cDNA isolated from the human leukocytes, the deletion of the frame of 15 base pairs was observed. The transient expression of the cDNA sequence of the COS-1 cells revealed that the encoded HGF molecule (delta 5 HGF) lacking 5 amino acids in the kringle 1 domain was fully functional. [Seki et al., Supra]. A variant of the huHGF found in nature has been identified, which corresponds to an alternative divided form of the huHGF transcript that contains the sequences coding for the N-terminal finger and the first two kringle domains of huHGF [Chan et al. ., Science, 25 ^: 1382-1385 (1991); Miyazawa et al., Eur. J. Biochem. , 197: 15-22 (1991)]. It has been proposed that this variant, degned as HGF / NK2, is a competitive antagonist of mature huHGF. Comparisons of the amino acid sequences of rat HGF with those of huHGF have revealed that two sequences are highly conserved and have the same structural characteristics. The length of the four kringle domains in the rat HGF is exactly the same as in huHGF. In addition, the cysteine residues are located in exactly the same positions, an indication of similar three-dimensional structures [Okajima et al., Supra; Tashiro et al., Supra). The HGF receptor has been identified as the product of the c-Met proto-oncogene [Bottaro et al., Science, 251: 802-804 (1991); Naldini et al., Oncogene, 6: 501-504 (1991)]. The receptor is known as P190MET and comprises a protein tyrosine kinase bound to the heterodimeric membrane of 190 kDa (a chain of 50 kDa bound by disulfide and a β chain of 145 kDa) [Park et al., Proc. Nati Acad. Sci. USA. 84: 6379-6383 (1987)]. The binding activity of HGF to its receptor is transmitted by a functional domain located in the N-terminal portion of the molecule, including the first two kringles [Matsuraoto et al., Biochem. Biophys. Beef.

Commun. , LL81: 691-699 (1991); Hartmann et al., Proc. Nati Acad. Sci., 89: 11574-11578 (1992); Lokker et al., EMBO J., L L: 2503-2510 (1992); Lokker and Godo ski, J. Biol. Chem., 268: 17145-117150 (1991)]. The c-Met protein remains phosphorylated at the tyrosine residues of the β-subunit of 145 kDa after the binding of HGF. Both HGF receptor HGF genes have been plotted for the long arm of chromosome 7, within the qll.l-q21.1 region [Dean et al., Nature, 31 ^: 385-388 (1985); Weidner et al., Proc. Nati Acad. Sci. USA, 88: 7001-7005 (1991); Saccone et al., Genomics, 13: 912-914 (1992)]. It has been observed that HGF levels are increased in plasma in patients with hepatic deficiency [Gohda et al., Supra] and in plasma [Lindroos et al., Hepatol., 13: 734-750 (1991)] or serum [Asami et al., J ^ Biochem. , 109: 8-13 (1991)] of animals with experimentally induced liver damage. The kinetics of this response is rapid, and precedes the first round of DNA synthesis during liver regeneration. HGF has also been shown to be a mitogen for certain cell types, including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells, and cells of epithelial origin [Matsu oto et al., Biochem. Biophys. Res. Commun. , 176: 45-51 (1991); Iga a et al., Biochem. Biophys. Res. Commun., .174: 831-838 (1991); Han et al., Biochem., 30: 9768-9780 (1991); Rubin et al., Proc. Nati Acad. Sci.

USA, 88: 415-419 (1991)]. HGF can also act as a "scattering factor" an activity that promotes the dissociation of vascular epithelial and endothelial cells in vitro [Stocker et al., Nature, 3_27: 239-242 (1987); Eidner et al., J. Cell Biol., 1U: 2097-2108 (1990); Naldini et al., EMBO J., 10: 2867-2878 (1991); Giordano et al., Proc. Nati Acad. Sci. USA, 90: 649-653 (1993)]. In addition, HGF has recently been described as an epithelial morphogen [Montesano et al., Cell, 67: 901-908 (1991)]. Therefore, it has been postulated that HGF is important in tumor invasion and embryonic development. Chronic activation of c-Met / HGF receptor has been observed in certain malignant conditions [Cooper et al., EMBO J., 5: 2623 (1986); Giordano et al., Nature, 339: 155 (1989)]. HGF and HGF variants are further described in US Patents Nos. 5,227,158, 5,316,921, and 5,328,837. 2. Macrophage Stimulating Protein A protein related to HGF has recently been identified. The protein known as HGF-like [Han et al., Supra; Degen et al., Biochemistry, 30: 9781 (1991); Shi amoto et al., FEBS, 333-61-66 (1993)] or macrophage stimulating protein ("MSP") [Leonard et al., US Patent No. 5,219,991; Skeel et al., J. Exp. Med., 173: 1227-1234 (1991); Leonard et al., Exp. Cell. Res., 114: 117-126 (1978); Yoshimura et al., J. Biol. Chem., 268: 15461-15468 (1993)] which shares with the HGF the total structure of four kringles. The cDNA that codes for the MSP. The protein appears to contain a domain structure similar to that of HGF with four kringles domains followed by a serine protease domain. MSP is a heterodimer that includes a 53 kDa α chain and a 25 kDa β chain. The MSP, however, is secreted as a single precursor chain [Yoshimura et al., Supra]. Like the precursor of HGF [Naldini et al., EMBO J., 11: 4825-4833 (1992)], it is currently believed that the maturation of MSP in a biologically active α-β heterodimer is obtained by serum-dependent proteolytic cleavage [Wang et al., J. Biol. Chem., 2-d: 3436- 3440; Wang et al., J. Biol. Chem., 269: 14027-14031 (1994)]. Wang et al., J. Biol. Chem. 269.13806-13810 (1994) report that certain proteases, such as serocalycerin, Factor Xlla, nerve growth factor gamma and epidermal growth factor binding protein, are cleaved and activated. -MSP to the heterodimer a-β. It has been found that MSP binds and activates a receptor comprising a transmembrane heterodimeric glycoprotein known as "pl85RON" or "RON" [Gaudino et al., EMBO J., 13: 3524-3532 (1994); Wang et al., Science, 266: 117 (Oct. 7, 1994)]. This glycoprotein has two chains linked by disulfide bonds: ß (150 kDa) and a (35 kDa). pl85RON is synthesized as a single chain precursor (prl70RON), which is subsequently converted to a mature, heterodimeric form by proteolytic cleavage. Unlike the single-chain, unprocessed precursor protein, the heterodimeric form of the protein is released to the surface of the cell. The sequence of the protein encoding RON was derived from a cDNA cloned from a human keratinocyte cDNA library described by Ronsin et al., Oncogene, 3: 1195-1202 (1993). The RON cDNA codes for a protein of 1,400 amino acids that shares a total similarity to the HGF receptor structurally and has a sequence identity of approximately 63% in the catalytic domain.Both genes of the MSP and the RON receptor have been mapped to chromosome 3p2.1 [Han et al., Supra; Ronsin et al., Supra]. The RON receptor is expressed mainly in cells of epithelial origin and in monocytes. P185RON also possesses intrinsic tyrosine kinase activity that is stimulated by MSP and a fusion protein of MSP, MSP-NK2 [Gaudino et al., Supra]. Such tyrosine kinase activity is not stimulated, however, by HGF [Id.]. This lacks the cross-reactivity that has been further demonstrated by the inability of MSP to bind and activate the receptor. The majority of mRNA encoding the MSP is expressed in the liver. This is also expressed, at lower levels, in the lung, adrenal gland and placenta. To date, the physiological roles of MSP in the body are not fully understood. Serum MSP is not increased for a period of 24 hours in response to intravenous lipopolysaccharides, indicating that MSP is probably not an acute phase protein [Wang et al., J. Leuk. Biol., 54: 289-295 (1993)]. Yoshimura et al., Supra, have reported that MSP stimulates a chemotactic response to C5a in macrophages. Leonard et al., US Patent No. 5,219,991, have described the use of highly purified MSP for the treatment of pathogenic infections. 3. Production of Megakaryocytes and Thrombocytes Undifferentiated pluripotent cells found primarily in the bone marrow of mammals have the potential to give rise to different types of blood cells circulating in the peripheral blood [Dexter et al., Ann. Rev. Cell Biol. 3: 423-441 (1987)]. The undifferentiated pluripotent cells differentiate into several cell lineages through the stages of maturation, thus giving rise to compromised blood cell types. A differentiated cell lineage in the bone marrow is the megakaryocytic lineage. The regulation of megakaryocytopoiesis of thrombocyte production has been reviewed by Mazur, Exp. Hemat. , 15: 248 (1987) and Hoffman, Blood, 74: 1196-1212 (1989). The three classes of megakaryocytic progenitor cells have been identified: (1) individual megakaryocytes that form bursts (BFU-MK); (2) individual megakaryocytes that form colonies (CFU-MK); and low density megakaryocyte progenitor cells (LD-CFU-MK). The maturation of megakaryocytes has also been separated in stages based on standard morphological criteria. The first recognizable member of the cells is the megakaryoblast. The intermediate form of the cells is known as promegakaryocyte or basophilic megakaryocyte. The final shape of the cells is known as mature megakaryocyte (acidophilus, granular or producer of platelets). The megakaryocyte extends the filaments of the cytoplasm to the sinusoidal spaces where they separate and fragment into individual thrombocytes or "platelets" [Willians et al., Hematology, lst Ed., McGraw-Hill, Inc., New York, New York (1972 )]. It is believed that megakaryocytopoiesis involves several regulatory factors "[Willians et al., Br. J. Haematol., 52: 173 (1982); Williams et al., J. Cell. Phys., 110: 101 (1982)]. It is believed that the first stage of megakaryocytopoiesis is mitotic, involving mainly cell proliferation and the initiation of colonies from CFU-MK but not affected by the platelet count [Burnstein et al., J. Cell. Phys., 1 ^ 9: 333 (1981), Kimura et al., Exp. Haematol., L3: 1048 (1985).] The last stage of maturation is mainly non-mitotic, involving nuclear polyploidization and cytoplasmic maturation [Odell et al., Blood, 4 ^ 3: 765 (1976); Ebbe et al., Blood, 32: 787 (1968).] Thrombocytes generally circulate in the blood and play an important role in the coagulation of the blood and the body's response to damage. The decrease in circulating levels of thrombocytes in the blood can result in several conditions and pathological therapies., for example, may result from the damaged production of thrombocytes by the bone marrow, sequestration of thrombocytes in the spleen, and eased destruction of thrombocytes by radiation or chemical therapy. Patients who receive large volumes of blood products administered quickly can also develop thrombocytogenesis due to blood thinning. Thrombopoietic conditions are best described in Schafner, "Thrombocytogenesis and Platelet Function Diseases," Internal Medicine, 3rd Ed., Huttin et al., Eds (1990). Certain cytokines and growth factors have been identified as inducers of thrombocyte production and the growth of megakaryocytes. It has been reported that cytokines have stimulatory activity in megakaryocytes (MK-CSA) uding interleukin 3 (IL-3) [Willians et al., Leukemia Res., 9: 1487-1491 (1985)], interleukin 6 (IL-6) [Bruno et al., Exp. Hemat. , 17: 1038-1043 (1989)], granulocyte macrophage colony stimulating factor (GM-CSF) [Ishibashi et al., Blood, 75: 1433-1438 (1990)], interleukin 11 (IL-11) [ Xu et al., Blood, 83: 2023-2030 (1994)], erythropoietin (Epo) [Sakaguchi et al., Exp. Hemat., 15: 1023-1034 (1987)], and interleukin 12 (IL-12) [Waldburger et al., Exp. Hemat, 22: 479a (1994) (suppl.)]. It has been reported that other cytokines modulate the development of platelets when combined with growth factors that possess established MK-CSA. These cytokines include IL-la [Gordon et al., Blood, 80: 302-307 (1992)] and Leukemia Inhibitory Factor (LIF) [Metcalf et al., "Actions of the Leukemia Inhibitory Factor in the Formation of Megakaryocytes and Platelets ", Ciba Foundation Symposium]. It has also been reported that the cMpl ligand is a stimulator of megakaryocytopoiesis and thrombopoiesis [de Sauvage et al., Nature, 369: 533-538 (1994); Lok et al., Nature, 369: 565-56 (1994); Kaushansky et al., Nature, 36: 568"571 (1994)].

In addition, a reported synthetic protein that stimulates thrombopoiesis is PIXY 321 [van de Ven et al., Exp.

Hematol., 2JD: 743-751 (1992); Willians et al., Blood, 82: 366a (1993) (suppl.); Collins et al., Blood, 82: 366a (1993) (suppl)]. PIXY 321 is a fusion protein composed of GM-CSF and IL-3 linked by a synthetic peptide chain [Willians et al., Cancer, 67: 2705-2707 (1991)]. It is believed that IL-3 primarily affects the (primary) phase of differentiation of the thrombocytopoietic process [Moore et al., Blood, 78: 1 (1991); Sonoda et al., Proc. Nati Acad. Sci. E. U. A., 85: 4360 (1988)]. IL-6 has been shown to induce proliferation of megakaryocyte progenitor cells as well as maturation in murine and primate models, although some investigators have observed that IL-6 only affects cell maturation. The interaction between IL-3 and IL-6 is commonly non-nuclear, some reports indicate that MK-CSA of IL-3 mediated by 11-6 since it was found that antibodies against Murine IL-6 abrogates MK-CSA from murine IL-3 [Lotem et al., Blood, 74: 1545-1551 (1989)]. Other investigators have reported that neutralizing antibodies of 11-6 alone do not decrease the MK-CSA of serum prepared from aplastic patients, and there is a hypothesis of a less crucial role for IL-6 in megakaryocytopoiesis. However, a number of studies have demonstrated a stimulatory effect of IL-6 when administered to lethally irradiated animals [Burnstein et al., Blood, 80: 420-428 (1992); Herodin et al., Blood, £ 0: 68-74 (1992)]. In patients receiving high doses of chemotherapy for metastatic sarcoma and lung cancer, IL-6 has reduced the decline in platelet count and accelerated the recovery of baseline levels [Demetri et al., Blood, 2: 367a (1993 ) (suppl.); Crawford et al., Blood, 8_2: 367a (1993) (suppl.)]. It has been reported that recipients of autologous bone marrow transplants treated with IL-6 experience a relatively rapid return of circulating platelets and a short period of dependence on platelet transfusion equal to that of controls [Fay et al., Blood, 82: 431a (1993) (suppl.)]. An object of the present invention is to identify a molecule capable of stimulating thrombocyte production in vivo and in vitro. Another object of the invention is to identify a molecule capable of stimulating the maturation of megakaryocytes in vivo and in vitro.

Another object of the invention is to provide the molecule in a pharmaceutically acceptable carrier for use in the treatment of physiological conditions characterized by existing or anticipated low levels of circulating thrombocytes. Another object of the invention is to provide the molecule of an article of manufacture or equipment that can be used for purposes of stimulating the production and maturation of megakaryocytes. These and other objects of the invention will be apparent to those skilled in the art upon consideration of the application as a whole.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, in one embodiment of the invention, a method for stimulating thrombocyte production using macrophage stimulating protein is provided. In another embodiment of the invention, a method is provided for stimulating megakaryocyte maturation. In a further embodiment of the invention, an article of manufacture and equipment including macrophage stimulating protein is provided. The macrophage-stimulating protein is provided in a container that has a label which identifies the macrophage-stimulating protein as an active agent to stimulate the maturation of the megakaryocyte in the production of thrombocytes. Reduced levels of thrombocytes in the blood can endanger the health of individuals. As discussed in the Background of the Invention, dangerously low levels of thrombocytes in the blood can result in a variety of pathological conditions, as well as chemotherapies and irradiation. Applicants have surprisingly found that the macrophage-stimulating protein is useful for stimulating the maturation of megakaryocytes and the production of thrombocytes. The macrophage stimulating protein of the present invention can be used in vitro and in vivo. For in vivo use, the macrophage stimulating protein can be administered as a curative therapy for those individuals suffering from haematopoietic conditions such as thrombocytopenia. The macrophage stimulating protein can also be administered as a prophylactic therapy for individuals who are experiencing, or are about to undergo, radiation and / or chemical therapies.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES 1. Definitions As used herein, the terms "macrophage stimulating protein" and "MSP" refer to a growth factor, growth factor which typically has a structure comprising four kringle domains. The terms "macrophage stimulating protein" and "MSP" refer to the mature, pre, prepro, and pro form of the protein, whether purified from a natural source, chemically synthesized or recombinantly produced. The macrophage stimulating protein can be in the form of a single chain or in a heterodimeric form. The present definition specifically includes the macrophage stimulating protein encoded by the sequence published by Yoshimura et al., J. Biol. Chem., 2_68: 15461-15468 (1993) (available from EMBL / GenBank / DDBJ under accession number) L11924; the nucleotide and amino acid sequences are also provided herein in SEQUENCE LISTING as SEQ ID NO: 1 and SEQ ID NO: 2, respectively). The fusion protein referred to herein as "MSP-NKs" comprises the N-terminal region (the first kringle domains) of the macrophage-stimulating protein fused to the C-terminal region of the heavy chain of human IgG-gamma is also included specifically here in the present definition. Fragments of the macrophage-stimulating protein may have the same activities described herein for the macrophage-stimulating protein, and the use of fragments with such activity is considered within the scope of the present invention. The terms "amino acid" and "amino acids" refer to all the L-a-amino acids found in nature. This definition means that it includes norleucine, ornithine, and homocysteine. The amino acids were identified by the designations of a single letter or three letters: Asp D aspartic acid lie I isoleucine Thr Threonine Leu L leucine Ser S serine Tyr Y tyrosine Glu E glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly G glycine Lys K lysine Wing A alanine Arg R arginine Cys C cysteine Trp W tryptophan Val Valine Gln Q glutamine Met M methionine Asn N arparagine The term "megakaryocyte maturation" refers to a process that involves the differentiation of megakaryoblasts and promegakaryocytes or basophilic megakaryocytes into platelet-producing cells, mature. The maturation of the megakaryocyte is typically accompanied by cellular changes, such as ploid increase and demarcation of membranes, and can be observed and quantified, for example, by ploid analysis and microscopic analysis. The term "thrombocytopenia" refers to a physiological condition typically ccterized by a thrombocyte level of less than 150 x 109 / liter of blood. The terms "treat", "treatment", and "therapy" refer to curative therapy, prophylactic therapy, and preventive therapy. The term "mammal" refers to any animal classified as a mammal, including humans, cows, horses, dogs and cats. In a preferred embodiment of the invention, the mammal is a human. 2. Methods and Compositions of the Invention The present invention provides methods for stimulating the maturation of megakaryocytes and the production of thrombocytes using macrophage stimulating protein, referred to hereinbefore as "MSP". The MSP useful in the practice of the present invention can be prepared in numerous ways. For example, MSP can be prepared using an isolated or purified form of the MSP. Methods of isolation and purification of MSP from natural sources are known in the art and are described, for example, by Skeel et al., J. Exp. Med., 173: 1227-1234 (1991) and Leonard et al. , US Patent No. 5,219,991. Such isolation and purification method can be used to obtain the serum or plasma MSP. Alternatively, MSP can be chemically synthesized and prepared using the recombinant DNA techniques known in the art and described in greater detail in Examples 1, 2 and 3 below. The MSP can be human or any non-human species. For example, a mammal can be administered the MSP of a different mammalian species (for example, the mouse can be treated post human MSP). Substantial homology (an amino acid identity of 81%) exists between mouse MSP and human MSP, and thus it is expected that the MSP of different mammalian species may be employed. Preferably, however, the mammal is treated with MSP homologs (eg, humans are treated with human MSP) to avoid potential immune reactions against MSP. The present invention includes methods for stimulating megakaryocyte maturation and thrombocyte production in vivo and in vitro. According to the method of the invention for stimulating megakaryocyte maturation in vitro, bone marrow cells or samples of cells suspected of containing megakaryoblasts, promegakaryocytes and / or basophilic megakaryocytes were provided and placed in a cell culture medium. The cells were then cultured in the presence of an effective amount of MSP. Suitable tissue culture media are well known to those skilled in the art and include, but are not limited to, Minimum Essential Medium ("MEM"), RPMI-1640, and Dulbecco's Modified Eagle Medium ("DMEM"). These tissue culture media are commercially available from Sigma Chemical Company (St. Louis, MO) and GIBCO (Grand Island, NY). The cells were then cultured in the cell culture medium under conditions sufficient for the cells to remain viable and grow. Cells can be cultured in a variety of ways, including culture in a clot, agar, or liquid culture. The cells were cultured in the presence of an effective amount of MSP. The amount of MSP may vary, but preferably it is in the range of about 10 ng / ml to about 100 ng / ml. The MSP can of course be added to the culture in a given dose determined empirically by those skilled in the art without undue experimentation. The concentration of MSP in the culture will depend on several factors, such as the conditions under which the cells and the MSP are cultured.

The specific temperature and the duration of the incubation, as well as other culture conditions, may vary depending on such factors, for example, the concentration of the MSP, and the type of cells and the medium. Those skilled in the art will be able to determine optimal operating and culture conditions without undue experimentation.

The maturation of the megakaryocytes in the culture can be determined by several assays known in the art, such as those described in Willians et al., Leukemia.

Res., 9: 1487-1491 (1985); Bruno et al., Exp. Hematol., 17: 1038-1043 (1989); Ishibashi et al., Blood, 25: 1433-1438 (1990); Xu et al., Blood, 83: 2023-2030 (1994); Sakaguchi et al., Exp. Hematol., 15: 1023-1034 (1987)]. It was contemplated that the use of MSP to stimulate in vitro megakaryocyte maturation will be useful in a variety of ways. For example, megakaryocytes cultured in vitro in the presence of MSP can be infused in a mammal suffering from reduced levels of platelet-forming cells. According to the method of the invention for stimulating the megakaryocyte maturation and the production of thrombocytes in a mammal, an effective amount of MSP is administered to the mammal. It was contemplated that the MSP may be administered at the time of, or after, administering to the mammal a therapy, such as high doses of radiation or chemotherapy, that may adversely affect the levels of thrombocytes in the blood. MSP can also be administered prophylactically to prevent the decrease in thrombocyte levels in the blood. The MSP is preferably administered to the mammal in a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Particularly suitable compositions for the clinical administration of the MSP used for the practice of this invention include sterile aqueous solutions or sterile hydratable powders such as lyophilized protein. Typically, an appropriate amount of a pharmaceutically acceptable salt is also used in the formulation to make it isotonic formulation. Examples of pharmaceutically acceptable carrier include saline, Ringer's solution and dextrose solution. It will be apparent to those skilled in the art that certain carriers may be more preferable depending on, for example, the route of administration and concentration of the MSP that is administered. The MSP is preferably administered to the mammal by injection (eg, intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion that accelerates its release into the bloodstream in an effective manner. The effective doses and schedules for administering the MSP can be determined empirically, and make such determinations even within the skill of the art. The escalation of doses between species can be effected in a manner known in the art, for example, as described in Mordenti et al., Pharmaceut. Res. 8_: 1351 (1991). It should be understood by those skilled in the art that the dose of MSP that must be administered will vary depending on, for example, the mammal receiving the MSP, the nature of a medical condition or therapy believed to be responsible for the decrease in levels of thrombocytes, the degree of damage to the tissues producing blood cells, the route of administration and the identity of any other drugs that are administered to the mammal. It should also be understood that it may be necessary to give more than one dose of MSP. Generally, multiple doses of MSP will be required for administration. The administration of the MSP could be continued until the levels of acceptable thrombocytes in the mammal are achieved. The invention also provides a method for treating thrombocytopenia in a mammal. In the method, it is first diagnosed that the mammal suffers from thrombocytopenia. Making the diagnosis is within the experience of the technique. Those skilled in the art will also appreciate that different levels of thrombocytes can guarantee a diagnosis of thrombocytopenia for different mammalian species. The diagnosis is usually made in humans when thrombocyte levels fall below approximately 150 x 109 thrombocytes / liter of blood. Thrombocytopenia can be the result of a condition of production, decrease or destruction of thrombocytes or thrombocyte producing cells. To treat thrombocytopenia, the MSP is administered to the mammal according to the modes and administration schedules described above. In the methods mentioned above, the MSP can be administered alternatively in combination with one or more biological or chemically active agents. Preferably, such agents have megakaryocytopoietic or thrombocytopoietic activity. It is currently believed, for example, that MSP can be administered in combination with the cMpl ligand or trompoietin [de Sauvage et al., Supra.; Lok et al., Supra; Kaushansky et al., Supra], IL-3, GM-CSF, or LIF to stimulate thrombocyte production. An expert in medical techniques can determine the appropriate dose of each agent useful herein, generally reducing the normal dose when the MSP is combined with any of those agents. The MSP can be administered in the same formulation as the other agents or the administration of the MSP and the other agents can occur separately. The other agents are administered in the modes, routes and programs appropriate for the particular agent. The maturation of the megakaryocyte and the production of thrombocytes can be measured or verified in several ways. For example, maturation and thrombocyte production can be measured using in vitro assays. Trials of megakaryocyte progenitors are known in the art and can be carried out, for example, by culturing the cells in methyl cellulose according to that described by Tanaka et al., Br. J. Haematol., 13: 18 (1989). Simple cell growth assays can also be performed such as those described by Williams et al., Cell Tisue Kinetics, 15: 483 (1982); Banu et al., Br. J. Haematol., 7j3: 313 (1990); Oon et al., Leukemia Res., 10: 403 (1986); Sparrow et al., Leukemia Res., 11:31 (1987). The maturation and production of thrombocytes can also be verified by the analysis of peripheral blood or in the bone marrow. Thrombocyte levels in circulating blood can be determined by cell count analysis. The cell count analysis can be carried out counting the viable cells by the exclusion of trypan blue. The cells can also be examined morphologically. For example, bone marrow samples can be obtained from the mammal and prepared for the microscope using standard histological techniques known in the art. By staining the cells, one can observe the size, cellular characteristics, and number of megakaryocytes in the marrow sample. The invention also provides an article of manufacture and equipment containing useful materials for stimulating megakaryocyte maturation and thrombocyte production. The article of manufacture comprises a container with a label. Suitable containers include, for example, bottles, hull, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container contains a composition which is effective to stimulate the maturation of the megakaryocyte and the production of the thrombocyte. The active agent in the composition is the MSP. The label on the container indicates that the composition is used to stimulate megakaryocyte maturation and thrombocyte production, and may also indicate guidelines for its use either in vivo or in vitro, such as those described above. The equipment of the invention comprises the container described above and a second container comprising a buffer. This may also include other desirable materials from a commercial and user's point of view, including shock absorbers, diluents, filters, needle, syringes, and package inserts with instructions for use. The invention will be understood more fully by reference to the following examples. They, however, should not be construed as limiting the scope of the invention. All the literature cited will be incorporated here as a reference.

Examples Example 1: MSP Recombinant Production The cDNA encoding the full-length MSP can be constructed by joining the cDNAs encoding amino acids 1-340 of the MSP (5 'MSP clone) and 341-711 (clone 3 'MSP) (using the numbering system reported by Yoshimura et al., Supra]. These cDNAs can be isolated by PCR amplification (as described in U.S. Patent No. 4,683,195, issued July 28, 1987 and in Current Protocols in Molecular Biology, Ausubel et al, eds., Green Publishing Associates and Wiley-Interscience 1991). , Volume 2, Chapter 15) of the human liver cDNA. Total human liver RNA (10 ug) was used as a standard for inverted transfection (RT) using a mixture of random hexamer and oligodT, using M-MLV-RT (commercially available from BRL, UK). To obtain the 5 'MSP clone, a PCR reaction was performed in a volume of 100 ul with a content of 10 ul of RT reaction mixture, using 1 U of Vent DNA polymerase (commercially available from New England Biolabs), and 50 pmol of each of the forward primers CAGTGCAGCCTCCAGCCAGAA (SEQ ID NO: 3) and the reverse primer CTGTACAACGCCGGATCTGGTAG (SEQ ID NO: 4). After 30 cycles of denaturation, (95 ° C, 1 min), annealing (55 ° C, 45 seconds), and extension (72 ° C, 2 min), 2 ul of the PCR reaction was reamplified with the primer to nested forward AGGACGAATCACCATGGGGTGGCTCCCACTCCTGCTGCTTCTGACT (SEQ ID NO: 5) and nested reverse primer CCGGAATTCGAACTTCTGCCGGAACCCCGAC (SEQ ID NO: 6). To obtain the clone 3 'MSP, they used the forward forward CCGGAATTCGAACTTCTGCCGGAACCCCGAC (SEQ ID NO: 7) and the reverse primer ACGGAATTCCCAAGGCATATGGCATCAAGGCT (SEQ ID NO: 8). The PCR products were digested with EcoRI restriction enzyme (commercially available from New England Biolabs), purified and cloned into the vector pRK7 (EP 278, 776, published on 8/17/88). The sequence of the amplified inserts of the separated PCR reactions can then be determined by dideoxynucleotide sequencing. The cDNA can be expressed and purified using the techniques described by Yoshimura et al., Supra.

Example 2: Recombinant Production of the MSP / NK2 Fusion Protein A partial cDNA containing the 268 N-terminal amino acids of the MSP (using the numbering system reported by Yoshimura et al., Supra) was fused to a heavy chain sequence of the IgG-gammal [Bennett et al., J Biol. Chem., 266: 23060-23067 (1991)] This can be achieved by using the synthetic complementary oligonucleotide GATCCGCAGATCGAGCGAGAATTCTGTACCTGCCGCGGTGCGAGACG (SEQ 'ID NO: 9), and GTGACCGTCTCGCACCGCGGCAGGTCACAGAATTCTCGCTCGATCTGCG (SEQ ID NO: 10) to ligate the MSP sequences through the unique BamH1 site in the MSP to the BstEII site in the heavy chain cDNA of human IgG-gam a [Id. ] The resulting constructs contain the coding sequences of amino acids 1-268 of the MSP, the binding sequences encoding the amino acids Glu, Thr, Val, and Thr, followed by the coding sequences of amino acids 216-443 of the heavy chain of the human IgG-gammal.

The cDNA encoding the MSP / NK2 can be inserted into the EBV-based expression plasmid pCIS.EBON [US Patent No. 5,328,837], and inserted into the 293 cells [human embryonic kidney cell line, Graham et al., J. Gen. Virol., 36: 59 (1977)] using the procedure described by Cachianes et al., Biotechniques, 5: 255-259 (1993). For the purification of MSP / NK2, the serum-free conditioned medium of the 293 cells expressing MSP / NK2 was sterilized by filtration, and citrate buffer (pH 6) was added to give a final concentration of 100 mM citrate. All purification procedures were carried out at 4 ° C. The filtered medium is then loaded onto a HiTrap ™ protein A column [Pharmacia LKB, Piscataway, NJ] equilibrated with 100 mM citrate., pH 6. The bound protein was eluted in 100 mM citrate, pH 6, 3.5 M MgCl2, 2% glycerol (volume / volume). Each fraction was exchanged by buffer immediately by passing it through a PD-10 column (Sephadex G-25) pre-equilibrated with phosphate buffered saline. The fraction then gathered and concentrated. The protein concentration can be determined by human anti-Fc ELISA (see, for example, US Pat. Nos. 5,316,921 and 5,328,837) and by total hydrolysis of amino acids. The NH2-terminal sequence of the purified MSP / NK2 can be confirmed by sequencing the protein. The purity and integrity of the protein can be evaluated with silver staining of SDS-PAGE gels as well as by western blotting using an antibody directed against the human Fc region of IgGl.

Example 3: Effect of the MSP on the Maturation of Human Megacariocytic Cell Lineages Human egacariocytic cell lineages were analyzed in vitro to evaluate their response to various concentrations of conditioned medium containing MSP and recombinant huHFG, prepared as described in sections A and B, respectively, below.

A. Preparing the Conditioned Medium Containing MSP The cDNAs encoding the MSP were prepared as described in Example 1. The sequence of the MSP is identical to the human MSP sequence as reported by Yoshimura et al., Supra. The cDNAs were inserted into the expression plasmid pCIS.EBON (identified in Example 2) and populations of 293 cells (identified in Example 2) containing these plasmids were established according to that described by Cachianes et al., Biotechniques, supra. The control medium 293 cells and MSP transfectants were tested before being used with 5% PCS for 1 hour at 37 ° C to allow the pro-MSP to be processed to the form of two mature chains [Wang et al. al., J. Biol. Chem., 263: 3436-3440 (1994)]. Such conditioned medium from cells transfected with either a vector alone or with a vector containing the MSP was collected for 48 hours and used at a 1:10 dilution in the assay described below.

B. Preparation of Recombinant huHGF Recombinant huHGF was prepared essentially as described in US Patent No. 5,227,158. A huHGF cDNA clone (HLC3) isolated from a human leukocyte library according to that described by Seki et al., Supra, was cloned into the expression vector, pSVI6B5 (ATCC Deposit No. 68,151). The complete amino acid sequence of human leukocyte HGF is shown in U.S. Patent No. 5,227,158, SEQ ID NO: 2. CHO-dhfr cells "[Urlaub et al., Proc. Nati. Acad. Sci. USA, 22 = 4216-4220 (1980)] were co-transfected with the hHGF expression vector based on pSVl6B5 described above and with the vector of selection dhfr, pDFll [Simonsen et al., Proc. Nati Acad. Sci. USA, 80: 2495-2499 (1983)], using the general procedure of Graham et al., Virology, 52_: 456-467 (1973). The last plasmid codes for DHFR, thereby conferring resistance to methotrexate to the transfected cells and allowing the selection of the huHGF expression transformants. Transformed dhfr cells were selected for growth in medium deficient in glycine, hypoxanthine and thymidine.The colonies that emerged on this selection medium were isolated using cotton plugs and propagated in the same medium for several generations. cells, the cells were amplified and selected with increasing amounts of methotrexate using standard techniques.Clones that could grow on selective medium, and therefore incorporated the plasmid containing transfected DHFR, were selected for the presence of secreted HGF. of HGF in the middle of these clones was tested with a mitogenic assay described in Nakamura et al., Proc. Nati, Acad. Sci. USA, 80: 7229-7233 (1983) .The activity of HGF in culture media also can be measured by the incorporation of 125I-labeled deoxyuridine in rat hepatocytes in primary culture as described by Nakamura et al., Nature, 342: 440-443 (1989). The huHGF was then purified essentially as described by Nakamura et al., Proc. Nati Acad. Sci., Supra.

C. Cell Culture Assays The human megakaryocyte cell lineages "DAMI" (obtained from S. Greenberg, Brigham and Women's Hospital, Boston, MA) and "CMK" (obtained from T. Sato, Chiba University, Japan) were used in the trials. Both cell lineages were maintained in culture according to that described by Greenberg et al., Blood, 22: 1968-17 (1988) and Komatsu et al., Blood, 2i: 42 (1989). Cells were cultured in 24-well culture plates (Corning, Corning, NY) at 2 x 10 5 cells / ml in RPMI-1640 medium containing 5% platelet-poor plasma.

("PPP") (prepared as described in Avraha et al., Blood, 29: 365-371 (1992)) for 5 days in a humidified atmosphere with 5% C02. The cells were cultured with the medium conditioned with MSP (described in Section A above), recombinant huHGF (described in Section B above) at three different concentrations or conditioned medium control of 293 cells and with or without phorbol-12-myristate 13-acetate ("PMA"). The PMA was used as a positive control. The PMA was prepared by dissolving PMA (Sigma, St. Louis, MO) in dimethyl sulfoxide (DMSO) and stored at -80 ° C.

Just before use, the PMA was diluted in RPMI-1640 culture medium. The diluted PMA was then incubated with the cells at a concentration of 10 mg / ml. After incubation the cells were washed twice with Hank's Balanced Salt Solution ("HBSS") and resuspended in Core Isolation Medium ("NIM", 0.2% BSA, 0.4% Ninidet p40, and 10 M HEPES). pH 7.4 in HBSS) plus 54 units of Worthington / l of Rnasa A (Biolab, New England, MA) at 2 x 106 cells / ml. The DNA content, or ploid, of the cultured cells was examined by staining the cells with an equal volume of NIM (described above) containing 25 ug / ml of propidium iodide (Sigma, St. Louis, MO). The samples were then stored in the dark at 4 ° C and analyzed on the same day using a fluorescein-activated cell sorting scan (Becton Dickinson, Mountain View, CA) and the CellFit ™ program. The results are shown below in Table 1. The results were expressed as the mean ± SEM of the data obtained from three experiments carried out in duplicate.

TABLE 1 Effect of HGF and MSP on the ploid of CMK cells The results of the experiments revealed that the treatment with MSP over a range of concentrations increased the maturation of the CMK cells according to what was taught by ploid. In contrast, the huHGF over a concentration range did not affect the CMK ploid.

D. Northern blot analysis of the DAMI and CMK cells Total cellular RNA was extracted from CMK and DAMI cells by a guanidine isothiocyanate method, and 20 ug of RNA was subjected to electrophoresis on 1% agarose gel with 2.2 mol / L formaldehyde. After transfer to the nylon filters (Hybond-N), the filters were hybridized with complementary DNA inserts labeled "" "" "nte. This hybridization was carried out at 37 ° C in the presence of 50% formaldehyde, 3 x sodium chloride and sodium citrate (SSC), 0.5% sodium dodecyl sulfate.

(SDS), 10% dextran sulfate, and 100 ug / ml denatured salmon sperm DNA. The filters were washed at 60 ° C for 2 hours in 0.2 x SSC and 0.5% SDS. The membranes were then exposed to Kodak Xomat films (Eastman Kodak, Rochester, NY) for 48 hours. Specific messenger RNA (mRNA) transcripts were detected with the partial human cDNA probe by RON, consisting of the kinase domain. The results of the spot analysis showed that the 5.0 kb and 2.0 kb specific transcripts of the RON gene were constitutively expressed in CMK and DAMI cells (data not shown).

Example 4: Effect of the MSP on the Secretion of Cytokine by Megacariocytes of the Human Medulla A. In vitro assays Using specific cytokine assays, the supernatants of the DAMI cells and the cultured human megakaryocytes, treated with MSP conditioned medium or recombinant huHGF, were tested to examine the synthesis and secretion of IL-6, IL-lβ and GM-CSF. The bone marrow was aspirated from healthy donors under sterile conditions in condom-free heparin using standard techniques. The primary bone marrow megakaryocytes were isolated with immunogenic beads coated with a cocktail of monoclonal antibodies to the GpII / IIIa surface (M753, Dako.Carpenteria, CA), as described in Tanaka et al., Brit. J. Haematol ., 73: 18-24 (1989). The purity of the isolated marrow megakaryocytes was then measured by flow cytometry (using the method of Tanaka et al., Supra) and determined to be 95-98%. Isolated primary marrow megakaryocytes (10 5 cells / ml) and DAMI cells (10 6 cells / ml) (obtained from S. Greenberg, Brigham &; Women's Hospital, Boston, MA) were cultured in RPMI-1640 medium containing 1% PPP (identified in Example 3, Section C) in the presence or absence of MSP-conditioned medium (prepared as described in Example 3 , Section A), recombinant huHGF (prepared as described in Example 3, Section B), or conditioned cell control medium 293 for 24 hours under the conditions described by Avraham et al., Supra. Cultures were maintained in duplicate for each test culture. Poor platelet plasma was used in the assays to avoid the presence of TGF-β or other factors derived from platelets that could be present in relatively high amounts in the serum. The supernatants of the cell cultures were obtained and tested for immunoreactive cytokines. Immunoassays for interleukin-1-β-human, human GM-CSF, and human interleukin-6 were obtained from R & D Systems, Minneapolis, MN and were used according to the manufacturer's instructions. A standard curve was made with a cytokine positive control in each assay. It was determined that the lower limit of detection was 0.35 pg / ml for the IL-6, 1.5 pg / ml for GM-CSF, and 0.3 pg / ml for IL-lβ. The results of the test, reported in Table 2 below, were expressed as the mean ± SEM of the data obtained from the tests carried out in duplicate. Statistical significance was determined using the Student's T test.

TABLE 2 EFFECT OF THE MSP AND THE HGF ON SECRETION OF CYTOKINE BY DAMI CELLS AND MEGACARCOCYTES OF PRIMARY BONE MEDULA * Statistically significant compared to the conditioned control medium. As shown in Table 2, the addition of MSP produced an increase in the secretion of IL-6 in all the cultures examined on all other treatments. A similar increase was observed with the DAMI cells; Approximately 34 pg / ml of IL-6 was detected in the cultures of untreated cultures compared to the 90 pg / ml of IL-6 detected in the cultures treated with MSP. To a lesser degree, treatment with MSP increased the secretion of GM-CSF and IL-lβ over all other treatments for megakaryocytes of primary bone marrow and over a control for DAMI cells. Secretion of IL-1β by human megakaryocytes was also evaluated using an ELISA for specific IL-1β. The IL-lβ protein was detected in supernatants of unstimulated megakaryocytes. The MSP stimulated modestly in the secretion of IL-1β but does not seem to be significant. No effect on the secretion of GM-CSF by MSP or huHGF was observed.

B. Analysis of megakaryocyte RNA Total cellular RNA was extracted from isolated primary bone marrow megakaryocytes. RNA was extracted by the guanidine isothiocyanate procedure, followed by ultracentrifugation through a CsCl buffer. The total RNA was run on an agarose gel with 1.2% formaldehyde, and the intact RNA was visualized by staining with ethidium bromide. The reverse transcription (RT) of RNA was carried out using 2 ug of the total RNA of each sample. PCR assays, using RON primers; were carried out with forward and reverse primers. The PCR products were analyzed on a 2% agarose gel (BRL, Bethesda, MD). The amplified DNA bands were visualized with a UV transluminator. The amplified DNA could be detected in the expected size by staining with ethidium bromide of the gel. Internal reaction standards were performed for PCR controls. Those standards included RNA with and without primers, RNA without primers, and RNA with actin primers. All primer patterns and total RNA preparations were analyzed to exclude contamination by cellular DNA. The results of the RNA analysis showed that the bone marrow megakaryocytes expressed mRNA specific for RON (data not shown).

Example 5: Effect of MSP on murine megakaryopopoiesis To examine the effect of MSP on murine megakaryocytic differentiation, single-cell megakaryocyte growth assays were performed according to the methods described in Oon et al., Leukemia Research, 10: 403-412 (1986) and Sparrow et al. al., Leukemia Research, 11: 31-36 (1987). Populations of a cell were prepared from bone marrow of the femur of normal C57B1 / 6 mice (obtained from Jackson Laboratories) by rinsing the bones with DMEM containing 10% FCS. The populations of immature megakaryocytes were obtained in fractions of 1.07-1.085 g / cm-3 from a suspension of simple bone marrow cells separated by Percoll gradient (Pharmacia). The fractionated cells were cultured at 10 5 cells / ml in DMEM-10 & of FCS for 5 days at 37 ° C in a 10% C02 modified incubator in the presence of titrated doses of MSP-conditioned medium (prepared as described in Example 3, Section A) and IL-6 (obtained from R & amp; amp;; D Systems, Minneapolis, MN). The cultures were dried and stained for acetylcholinesterase according to that described by Williams et al., Cell Tisue Kinetics, 15: 483-494 (1982); Banu et al., British J. Haemat., 25: 313-318 (1990); Oon et al., Supra; Sparrow et al., Supra. Only murine megakaryocytes were recorded as number of cells positive for acetylcholinesterase by bone marrow cell cultures of 5 x 104 fractionated. The growth of immature megakaryocytes was quantified by the number of simple large megakaryocytes that were detected by simple microscopy. The results are shown in Table 3. The reported results are the mean ± of EM for triplicate cultures from three experiments.

TABLE 3 Effect of MSP on immature megakaryocytes The addition of MSP to the cultures of immature megakaryocytic populations showed a greater growth response with an increase in the detectable number of megakaryocytes positive to acetylcholinesterase. Neutralization assays were also conducted using a method of indirect immune complex depletion [Sparrow et al., Supra]. Neutralizing monoclonal antibodies to IL-6 were obtained from the Genetics Institute, Boston, MA. Suboptimal levels of growth factors (MSP and huHGF) and optimal antibody levels (1:10 dilution) were selected to provide an excess of antibody. The different growth factors and antibodies were mixed and incubated at 4 ° C for 2 hours. For controls, normal rabbit serum (obtained from Sigma, St. Louis, MO) was used. Immune complexes were precipitated by adding 50 ul of Protein A-sepharose CL-4BMR (Pharmacia LKB, Piscataway, NJ). Culture supernatants were harvested and assayed for the stimulatory activity of the residual megakaryocyte in the growth test of the immature murine megakaryocyte described ab The results of the neutralization assays showed the increased growth of immature murine megakaryocytes in response to MSP (as well as IL-6) were neutralized by a monoclonal antibody directed against IL-6. See Table 3. The Table also shows that a non-immune serum added to IL-6 or MSP had no significant effect on IL-6 or MSP alone. Although it is not completely understood if by some theory, the results suggest that MSP can function in the regulation of the megakaryocyte maturation inducing the cytokine secretion of the megakaryocyte or the induction of IL-6 by accessory cells.

SEQUENCE LIST (1. GENERAL INFORMATION: (i) APPLICANT: Genentech, Inc. New Ingland Deaconess Hospital Corp. (ii) TITLE OF THE INVENTION: Methods and Equipment that Use Macrophage Stimulating Protein (iii) SEQUENCE NUMBER: 10 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP : 94080 (v) LEGIBLE FORM IN COMPUTER: (A) TYPE OF MEDIA: 1.44 Mb flexible disk, 3.5 inches (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: WinPatin (Genentech) (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (viii) INFORMATION FROM THE MANDATORY / AGENT (A) NAME: Marschang, Diane L. (B) REGISTRATION NUMBER: 35,600 (C) REFERENCE NUMBER / FILE: P0912PCT (ix) INFORMATION BY TELECOMMUNICATION: (A) TELEPHONE: 415 / 225-5416 (B) TELEFAX: 415 / 952-9881 (C) TELEX: 910 / 371-7168 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 2232 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: GAATTCCACC ATGGGGTGGC TCCCACTCCT GCTGCTTCTG ACTCAATGCT 50 TAGGGGTCCC TGGGCAGCGC TCGCCATTGA ATGACTTCCA AGTGCTCCGG 100 GGCACAGAGC TACAGCACCT GCTACATGCG GTGGTGCCCG GGCCTTGGCA 150 GGAGGATGTG GCAGATGCTG AAGAGTGTGC TGGTCGCTGT GGGCCCTTAA 200 TGGACTGCCG GGCCTTCCAC TACAACGTGA GCAGCCATGG TTGCCAACTG 250 CTGCCATGGA CTCAACACTC GCCCCACACG AGGCTGCGGC GTTCTGGGCG 300 CTGTGACCTC TTCCAGAAGA AAGACTACGT ACGGACCTGC ATCATGAACA 350 ATGGGGTTGG GTACCGGGGC ACCATGGCCA CGACCGTGGG TGGCCTGCCC 400 TGCCAGGCTT GGAGCCACAA GTTCCCGAAT GATCACAAGT ACACGCCCAC 450 TCTCCGGAAT GGCCTGGAAG AGAACTTCTG CCGTAACCCT GATGGCGACC 500 CCGGAGGTCC TTGGTGCTAC ACAACAGACC CTGCTGTGCG CTTCCAGAGC 550 TGCGGCATCA AATCCTGCCG GGAGGCCGCG TGTGTCTGGT GCAATGGCGA 600 GGAATACCGC GGCGCGGTAG ACCGCACGGA GTCAGGGCGC GAGTGCCAGC 650 GCTGGGATCT TCAGCACCCG CACCAGCACC CCTTCGAGC C GGGCAAGTTC 700 CTCGACCAAG GTCTGGACGA CAACTATTGC CGGAATCCTG ACGGCTCCGA 750 GCGGCCATGG TGCTACACTA CGGATCCGCA GATCGAGCGA GAGTTCTGTG 800 ACCTCCCCCG CTGCGGGTCC GAGGCACAGC CCCGCCAAGA GGCCACAACT 850 GTCAGCTGCT TCCGCGGGAA GGGTGAGGGC TACCGGGGCA CAGCCAATAC 900 CACCACTGCG GGCGTACCTT GCCAGCGTTG GGACGCGCAA ATCCCTCATC 950 AGCACCGATT TACGCCAGAA AAATACGCGT GCAAAGACCT TCGGGAGAAC 1000 TTCTGCCGGA ACCCCGACGG CTCAGAGGCG CCCTGGTGCT TCACACTGCG 1050 GCCCGGCATG CGCGCGGCCT TTTGCTACCA GATCCGGCGT TGTACAGACG 1100 ACGTGCGGCC CCAGGACTGC TACCACGGCG CAGGGGAGCA GTACCGCGGC 1150 ACGGTCAGCA AGACCCGCAA GGGTGTCCAG TGCCAGCGCT GGTCCGCTGA 1200 GACGCCGCAC AAGCCGCAGT TCACGTTTAC CTCCGAACCG CATGCACAAC 1250 TGGAGGAGAA CTTCTGCCGG AACCCAGATG GGGATAGCCA TGGGCCCTGG 1300 TGCTACACGA TGGACCCAAG GACCCCATTC GACTACTGTG CCCTGCGACG 1350 CTGCGCTGAT GACCAGCCGC CATCAATCCT GGACCCCCCA GACCAGGTGC 1400 AGTTTGAGAA GTGTGGCAAG AGGGTGGATC GGCTGGATCA GCGGCGTTCC 1450 AAGCTGCGCG TGGTTGGGGG CCATCCGGGC AACTCACCCT GGACAGTCAG 1500 CTTGCGGAAT CGGCAGGGCC AGCATTTCTG CGGGGGGTCT CTAGTGAAGG 1550 AGCAGTGGAT ACTGACTGCC CGGCAGTGCT TCTCCTCCTG CCATATGCCT 1600 CTCACGGGCT ATGAGGTATG GTTGGGCACC CTGTTCCAGA ACCCACAGCA 1650 TGGAGAGCCA AGCCTACAGC GGGTCCCAGT AGCCAAGATG GTGTGTGGGC 1700 CCTCAGGCTC CCAGCTTGTC CTGCTCAAGC TGGAGAGATC TGTGACCCTG 1750 AACCAGCGCG TGGCCCTGAT CTGCCTGCCC CCTGAATGGT ATGTGGTGCC 1800 TCCAGGGACC AAGTGTGAGA TTGCAGGCTG GGGTGAGACC AAAGGTACGG 1850 GTAATGACAC AGTCCTAAAT GTGGCCTTGC TGAATGTCAT CTCCAACCAG 1900 GAGTGTAACA TCAAGCACCG AGGACGTGTG CGTGAGAGTG AGATGTGCAC 1950 TGAGGGACTG TTGGCCCCTG TGGGGGCCTG TGAGGGTGAC TACGGGGGCC 2000 CACTTGCCTG CTTTACCCAC AACTGCTGGG TCCTGGAAGG AATTATAATC 2050 CCCAACCGAG TATGCGCAAG GTCCCGCTGG CCAGCTGTCT TCACGCGTGT 2100 CTCTGTGTTT GTGGACTGGA TTCACAAGGT CATGAGACTG GGTTAGGCCC 2150 AGCCTTGATG CCATATGCCT TGGGGAGGAC AAAACTTCTT GTCAGACATA 2200 AAGCCATGTT TCCTCTTTAT GCCTGTCTCG AG 2232 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 711 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Mßt Gly Trp Leu Pro Leu Leu Leu Leu Leu Thr Gln Cys Leu Gly 1 5 10 15 Val Pro Gly Gln Arg Ser Pro Leu Asn Asp Phß Gln Val Leu Arg 20 25 30 Gly Thr Glu Leu Gln His Leu Leu His Wing Val Val Pro Gly Pro 35 40 45 Trp Gln Glu Asp Val Wing Asp Wing Glu Glu Cys Wing Gly Arg Cys 50 55 60 Gly Pro Leu Met Asp Cys Arg Ala Phe His Tyr Asn Val Ser Ser 65 70 75 His Gly Cys Gln Leu Leu Pro Trp Thr Gln His Ser Pro His Thr 80 85 90 Arg Leu Arg Arg Ser Gly Arg Cys Asp Leu Phß Gln Lys Lys Asp 95 100 105 Tyr Val Arg Thr Cys lie Met Asn Asn Gly Val Gly Tyr Arg Gly 110 115 120 Thr Mßt Wing Thr Thr Val Gly Gly Leu Pro Cys Gln Wing Trp Ser 125 130 135 His Lys Phß Pro Asn Asp His Lys Tyr Thr Pro Thr Leu Arg Asn 140 145 150 Gly Leu Glu Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Pro Gly 155 160 165 Gly Pro Trp Cyß Tyr Thr Thr Asp Pro Wing Val Arg Phß Gln Ser 170 170 180 Cys Gly Zlß Lys Ser Cys Arg Glu Ala Wing Cys Val Trp Cys Asn 185 190 195 Gly Glu Glu Tyr Arg Gly Wing Val Asp Arg Thr Glu Ser Gly Arg 200 205 210 Glu Cys Gln Arg Trp Asp Leu Gln His Pro His Gln His Pro Phe 215 220 225 Glu Pro Gly Lys Phe Leu Asp Gln Gly Leu Asp Asp Asn Tyr Cys 230 235 240 Arg Asn Pro Asp Gly Ser Glu Arg Pro Trp Cys Tyr Thr Thr Asp 245 250 255 Pro Gln lie Glu Arg Glu Phe Cys Asp Leu Pro Arg Cys Gly Ser 260 265 270 Glu Ala Gln Pro Arg Gln Glu Ala Thr Thr Val Ser Cys Phe Arg 275 280 285 Gly Lyß Gly Glu Gly Tyr Arg Gly Thr Wing Asn Thr Thr Thr Wing 290 295 300 Gly Val Pro Cys Gln Arg Trp Asp Wing Gln He Pro His Gln Kis 305 310 315 Arg Phe Thr Pro Glu Lys Tyr Ala Cys Lys Asp Leu Arg Glu Asn 320 325 330 Phe Cys Arg Asn Pro Asp Gly Ser Glu Pro Wing Trp Cys Phß Thr 335 340 345 Leu Arg Pro Gly Met Arg Wing Wing Phe Cys Tyr Gln He Arg Arg 350 355 360 Cys Thr Asp Asp Val Arg Pro Gln Asp Cys Tyr His Gly Wing Gly 365 370 375 Glu sln Tyr Arg Gly Thr Val Ser Lys Thr Arg Lys Gly Val Gln 380 385 390 Cys Gln Arg Trp be Wing Glu Thr Pro His Lys Pro Gln Phe Thr 395 400 405 Phß Thr Ser Glu Pro His Wing Gln Leu Glu Glu Asn Phe Cys Arg 410 415 420 Asn Pro Asp Gly Asp Ser His Gly Pro Trp Cys Tyr Thr Met Asp 425 430 435 Pro Arg Thr Pro Phe Asp Tyr Cys Ala Leu Arg Arg Cys Ala Asp 440 445 450 Asp Gln Pro Pro Ser He Leu Asp Pro Pro Asp Gln Val Gln Phe 455 460 465 Glu Lys Cys Gly Lys Arg Val Asp Arg Leu Asp Gln Arg Arg Ser 470 475 480 Lys Leu Arg Val Val Gly Gly His Pro Gly Asn Ser Pro Trp Thr 485 490 495 Val Ser Leu Arg Asn Arg Gln Gly Gln His Phe Cys Gly Gly Ser 500 505 510 Leu Val Lys Glu Gln Trp lie Leu Thr Wing Arg Gln Cys Phß Ser 515 520 525 Ser Cys His Mßt Pro Leu Thr Gly Tyr Glu Val Trp Leu Gly ThrLeu Phe Gln Asn Pro Gln His Gly Glu Pro Ser Leu Gln Arg Val 545 550 555 Pro Val Wing Lys Met Val Cys Gly Pro Ser Gly Ser Gln Leu Val 560 565 570 Leu Leu Lys Leu Glu Arg Ser Val Thr Leu Asn Gln Arg Val Ala 575 580 585 Leu Ilß Cys Leu Pro Pro Glu Trp Tyr Val Val Pro Pro Gly Thr 590 595 600 Lys Cys Glu Zle Wing Gly Trp Gly Glu Thr Lys Gly Thr Gly Asn 605 610 615 Asp Thr Val Leu Asn Val Ala Leu Leu Asn Val? Le Ser Asn Gln 620 625 630 Glu Cys Asn Zle Lys His Arg Gly Arg Val Arg Glu Ser Glu Met 635 640 645 Cys Thr Glu Gly Leu Leu Wing Pro Val Gly Wing Cys Glu Gly Asp 650 655 660 Tyr Gly Gly Pro Leu Wing Cys Phe Thr His Asn Cys Trp Val Leu 665 670 675 Glu Gly Zle Zlß Zlß Pro Asn Arg Val Cys Wing Arg Ser Arg Trp 680 685 690 Pro Ala Val Phe Thr Arg Val Ser Val Phe Val Asp Trp? Le His 695 700 705 Lys Val Met Arg Leu Gly 710 711 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 21 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 CAGTGCAGCC TCCAGCCAGA A 21 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 23 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: CTGTACAACG CCGGATCTGG TAG_23_(2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 47 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: AGGACGAATG CACCATGGGG TGGCTCCCAC TCCTGCTGCT TCTGACT 47 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 31 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CCGGAATTCG AACTTCTGCC GGAACCCCGA C 31 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 31 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 CCGGAATTCG AACTTCTGCC GGAACCCCGA C 31 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 32 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: ACGGAATTCC CAAGGCATAT GGCATCAAGG CT 32 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 47 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: GATCCGCAGA TCGAGCGAGA ATTCTGTACC TGCCGCGGTG CGAGACG 47 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 49 base pairs (B) TYPE: Nucleic Acid (C) HEBRA: Simple (D) TOPOLOGY: Linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: GTGACCGTCT CGCACCGCGG CAGGTCACAG AATTCTCGCT CGATCTGCG 49 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (12)

1. A method for stimulating thrombocyte production in a mammal, characterized in that it comprises administering to the mammal an effective amount of macrophage stimulating protein in a pharmaceutically acceptable carrier.

2. The method according to claim 1, characterized in that the macrophage stimulating protein is a human macrophage stimulating protein.

3. The method according to claim 1, characterized in that the macrophage stimulating protein that is administered to the mammal is administered after the chemotherapy.

4. The method according to claim 1, characterized in that the macrophage stimulating protein that is administered to the mammal is administered before the chemotherapy.

5. The method according to claim 1, characterized in that the pharmaceutically acceptable carrier is sterile saline.

6. A method for treating thrombocytopenia in a mammal, characterized in that it comprises administering to a mammal who is diagnosed as having thrombocytopenia an effective amount of macrophage-stimulating protein in a pharmaceutically acceptable carrier.

7. A method to stimulate in vitro megakaryocyte maturation, characterized in that it comprises culturing a cellular sample suspected of containing megakaryoblasts, promegacarioblasts and / or basophilic megakaryocytes in the presence of an effective amount of macrophage-stimulating protein.

8. The method according to claim 7, characterized in that the amount of macrophage stimulating protein is from about 10 ng / ml to about 100 ng / ml.

9. An article of manufacture, characterized in that it comprises: a container; a label on the container; and a composition contained within the container; wherein the composition is effective to stimulate megakaryocyte maturation and thrombocyte production, the label and the container indicate that the composition can be used to stimulate megakaryocyte maturation and thrombocyte production, and that the active agent in such composition It comprises macrophage stimulating protein.

10. The article of manufacture according to claim 9, characterized in that it further comprises instructions for administering the macrophage stimulating protein to a mammal.

11. The article of manufacture according to claim 9, characterized in that it further comprises instructions for using the macrophage stimulating protein in an in vitro cell culture.

12. A kit, characterized in that it comprises: a first container, a label in such container, and a composition contained within such container; wherein the composition is effective to stimulate megakaryocyte maturation and thrombocyte production, the label and the container indicate that the composition can be used to stimulate megakaryocyte maturation and thrombocyte production, and that the active agent in such composition comprises macrophage stimulating protein; and a second container comprising a damper.