HK1009151B

HK1009151B - Mpl ligand analogs

Info

Publication number: HK1009151B
Application number: HK98109942.3A
Authority: HK
Inventors: S‧G‧艾利奥特
Original assignee: 安姆根有限公司
Priority date: 1995-02-15
Filing date: 1996-02-13
Publication date: 2005-01-07

Description

MPL ligand analogues

Technical Field

The present invention relates to mpl ligand analogs having at least one altered O-or N-type glycosylation site. The invention also relates to DNA sequences encoding these mpl ligand analogs and recombinant plasmids and host cells expressing the analogs.

Background

MGDF, or megakaryocyte growth and development factor, is a newly cloned cytokine that appears to be a major regulator of circulating platelet levels. See the following documents: bartley, T.D., et al, Cell 77 Vol 1117-1124 (1994); lok, S.et al, Nature 369 (Nature) volume 565 and 568 (1994); de Sauvage, F.J., et al, Nature 369 Vol.533-; miyazake, H.et al, Experimental hematology 22, pp.838 (1994); kuter, D.J. et al PNAS USA, 91 Vol 11104-. Bartleg, T.D. et al, cell 77, 1117-page 1124 (1994) also refer to MGDF as Thrombopoietin (TPO), mpl ligand and megakaryopoietin. Herein, "mpl ligand" generally refers to all polypeptides that activate the mpl receptor, including TPO and MGDF. The mpl receptor is a cell surface protein that, when activated, causes the production and/or development of megakaryocytes and platelets. See WO 92/07074.

An "mpl ligand analog" is a polypeptide that differs from the native sequence by a difference that affects the number, location, and type of glycosylation sites. These polypeptides are an aspect of the invention. The mature natural human mpl ligand protein has a total of 332 amino acids. The sequence of the protein (ligated to a leader sequence of 21 amino acids in length) and the corresponding cDNA are shown in FIG. 1 (SEQ ID NO: 1 and 2).

Recombinant mpl ligands produced by Chinese Hamster Ovary (CHO) cells and e.coli cells are biologically active and are capable of specifically stimulating or increasing megakaryocytes and/or platelets in cats, mice and monkeys. See, for example, Hunt.P., et al, Blood 84, Vol.10, 390A (1994). The cleaved human mpl ligand molecule (compared to the 332 amino acid protein encoded by human cDNA) has at least 151 amino acids (starting at amino acid position 22 in fig. 1) and remains biologically active in vivo. FIG. 2 (SEQ ID NOS: 3 and 4) shows an example of a cleaved mpl ligand molecule, the mature form of which has 174 amino acids and is biologically active. FIG. 2 shows that this 174 amino acid protein is linked to a 21 amino acid N-terminal leader sequence. This molecule was used in the examples below to create certain mpl ligand analogs. Other analogs are based on amino acids 1-199, 1-191, and 1-183 in FIG. 1. It is also possible to remove the 6 amino acids from the N-terminus of the mature human mpl ligand protein while still retaining biological activity. Thus, biological activity appears to be dependent upon amino acids 7 through 151 (including amino acids 7 and 151) of the amino acid sequence of the mature human mpl ligand in FIG. 1.

In general, many cell surface proteins and secreted proteins produced by eukaryotic cells carry one or more oligosaccharide genes. This modification, known as glycosylation, can significantly affect the physical properties of the protein, and is also important for protein stability, secretion, and subcellular localization. Proper glycosylation is essential for biological activity. In fact, when certain genes of eukaryotes are expressed in bacteria (e.g., E.coli), the resulting proteins are not glycosylated and thus have little or no activity due to the lack of cellular processes for glycosylation of proteins.

Glycosylation occurs at specific positions or sites of the polypeptide backbone, and there are generally two types: the O-type oligosaccharides are linked to a serine (Ser) or threonine (Thr) residue, while the N-type oligosaccharides (chains) are linked to an asparagine (Asn) residue. These amino acid residues are part of the Asn-X-Ser/Thr sequence, where X can be any amino acid other than proline, preferably one of the 19 natural amino acids other than proline. The structure of the N-type and O-type oligosaccharides and sugar residues in each type is different, and the common sugar in the two types is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is often a terminal residue of both N-and O-type oligosaccharides and, due to its negative charge, may make the glycoprotein acidic.

As used herein, "glycosylation" sites "refer to amino acid residues that are structurally capable of attachment to a sugar residue, and these sites may or may not be actually attached to the sugar residue. As mentioned above, the O-type site is a Ser or Thr residue, and the N-type site is Asn-X-Ser or Asn-X-Thr, where X is any amino acid other than Pro (preferably one of the 19 natural amino acids other than Pro). Whether a site is glycosylated with a sugar chain depends on the host cell expressing the molecule, the amino acids in adjacent sites, and other factors.

As used herein, the number of "chains" attached to an mpl ligand analog refers to the average number of carbohydrate (i.e., glycosyl) chains attached to an mpl ligand expressed by a particular host cell. It should be noted that the glycosylation sites of the natural and corresponding recombinant mpl ligands are generally the same, and the number of chains may vary depending on whether the particular host cell used for recombinant expression attaches a sugar chain to this site (as compared to the ligand of natural origin). Herein, when comparing recombinant and natural mpl ligand analogs, the same number of amino acids are compared, regardless of whether the natural source actually produced an mpl ligand molecule of that length. Thus, "native" refers to the sequence used in a particular species (e.g., human) and not to the length of the molecule actually expressed in such a natural source.

Naturally occurring mpl ligands are mono-glycosylated molecules. The glycosylation pattern of the natural mpl ligand is related to two key regions in the mpl ligand. The first about 151 amino acid sequences of mature human mpl ligand, corresponding to the active site of the molecule, exhibit considerable homology to the cytokine Erythropoietin (EPO) which stimulates erythropoiesis, and are referred to as the "EPO-like" region of human mpl ligand. The remaining amino acids of the mature protein constitute a so-called "N-type carbohydrate" region, since they include at least most of the natural N-type glycosylation sites. There are six N-type glycosylation sites in human mpl ligands, all within the N-type glycosylation region. Both regions have O-type glycosylation sites. There are about 12-14O-type glycosylated chains in the whole molecule. Experimental data on recombinant expression of human mpl ligand DNA in CHO cells indicate that there are at least two O-type sites glycosylated in the EPO-like region, position 1 (Ser) and position 37 (Thr).

Glycoproteins such as mpl ligands can be separated into different charged forms by techniques such as isoelectric focusing (IEF). For example, several groups have reported IEF studies of crude and partially purified erythropoietin preparations. (Lukowsky et al, J.Biochem., vol.50, 909 (1972); Shelton et al, medical biochemistry, biochem. Med., vol.12, vol.45 (1975); Fuhr et al, Biochem. Biophys. Res. Comm., vol.98, vol.930 (1981)).

Despite the above knowledge regarding glycosylation of mpl ligand molecules, there remains a need for mpl ligand molecules with different glycosylation patterns that maintain or enhance biological activity.

Thus, it is an object of the present invention to provide novel glycosylated mpl ligand molecules, i.e., mpl ligand analogs. It is another object of the invention to provide pharmaceutical compositions containing these molecules and methods of treating diseases treatable with mpl ligands using mpl ligand analogs of the invention.

Summary of The Invention

In one embodiment, the present invention relates to mpl ligand analogs comprising an amino acid sequence comprising at least one added, at least one deleted, and/or at least one added and at least one deleted glycosylation site in combination as compared to the corresponding natural sequence mpl ligand. The addition or deletion of glycosylation sites can lead to the following results; the number of carbohydrate chains of the analog is increased or decreased and the sialic acid content is increased or decreased compared to the mpl ligand of the corresponding native sequence, especially human mpl ligand. For example, an analog may comprise a deletion of one or more N-or O-type sites, or an addition of one or more N-or O-type sites at the same or different positions.

In the above embodiments, another aspect of the present invention relates to mpl ligand analogs that include amino acid sequences in which one or more N-or O-type glycosylation sites are replaced by one or more non-naturally occurring sites. Thus, one N-type site may be replaced by a different N-type site; an N-type site may be substituted with an O-type site; one O-type site may be substituted by a different O-type site; an O-type site may be substituted with an N-type site.

Combinations of any of the above variations are also included in the invention.

The invention also includes DNA sequences encoding these mpl ligand analogs and recombinant plasmids and host cells expressing the analogs.

In each of the above cases, the change in glycosylation site results in a change in the number, content, location or type (N-to-O-) of sugar chains in the resulting mpl ligand analog and maintains the biological activity of the mpl ligand, i.e., the analog still activates the mpl receptor. Activation of the mpl receptor implies increased megakaryocytopoiesis, thus leading to increased platelets in vivo.

Brief Description of Drawings

FIG. 1 shows the DNA and amino acid sequences of a natural human mpl ligand, including the signal peptide (-21 to-1 amino acids) and the mature amino acid sequence (1-332).

FIG. 2 shows the DNA and amino acid sequence of mpl ligand corresponding to amino acids 1-174 of mature human mpl ligand linked to a 21 amino acid signal peptide. XbaI and SalI cloning sites were introduced at the 5 'and 3' ends of the flanking sequences of the coding region, respectively.

FIG. 3 shows the Western blot results of E.coli and CHO-expressed mpl ligand. MK represents Met-Lys, which is added to the N-terminus of mpl ligand for expression in E.coli and cleaved with a dipeptidase such as cathepsin C. The molecule from which MK is removed is called desMK. The figures indicate the glycosidase neuraminidase and O-glycanase treatments.

FIG. 4 shows the E.coli and CHO expressed mpl ligand activity (expressed in platelet counts) in normal mice. The data obtained show that the activity of the glycosylated mpl ligand (CHO material) is higher than that of the non-glycosylated (E.coli). This may be due to the longer half-life of the glycosylated ligand. For example, CHO 332 represents amino acids 1-332 of human mpl ligand expressed by CHO cells (FIG. 1).

FIG. 5 shows the results of Western blot analysis of COS cell supernatants expressing recombinant human mpl ligand and analogs 4, 6, 7, 9, 10, and 11. The construction of the analogs is described in example 4. Analogues 4, 7, 10 contain at least one additional carbohydrate chain because they move slower in the gel. The analog numbers correspond to the analog numbers in table 1 (e.g., 11 corresponds to analog N11). The control in table 1 is N1.

FIG. 6 shows the results of Western blot analysis of COS cell supernatants expressing recombinant human mpl ligand and analogs 4, 5, 13, 14, and 15. The construction of the analogs is described in example 4. Analogues 4, 13, 14 and 15 contain at least one additional carbohydrate chain because they move slower in the gel.

FIG. 7 shows the results of Western blot analysis of COS cell supernatants expressing human mpl ligand and the indicated mpl ligand analogs after treatment with N-glycanase. The results indicate that the analogs have different glycosylation patterns.

FIG. 8 shows the results of in vivo analysis of human megakaryocyte growth with mpl ligand analogs. Group a and group D are positive and negative controls, respectively. Group A wells received COS-1 conditioned media with 37.5pg wild-type (i.e., native sequence) mpl ligand 1-174, and showed extensive megakaryocyte growth. Group D received 1.5. mu.l COS-1 mock conditioned medium and no growth was seen. Panel B and C are mpl ligand 1-174 analogs 7 and 10, respectively. Group B received 9.0pg mpl ligand COS-1 conditioned media, while group C received 27pg, both of which showed good megakaryocyte growth.

FIG. 9 shows the results of Western blot analysis of CHO mpl ligands 1-174 and analogs N4 and N15 (see Table 1). The slower movement in the gel confirmed that analog N4(4B) contained one additional oligosaccharide, while analog N15(15-8) contained two additional oligosaccharides.

FIG. 10 shows the Western blot results of mpl ligand analogs produced by CHO cells with and without N-glycanase treatment. The presence of N-type oligosaccharides was confirmed by the slower movement in the gel after treatment with N-glycanase.

Figure 11 shows the results of platelet counts for mice treated with different types of mpl ligands at different doses. The data obtained demonstrate that increasing the amount of N-and/or O-type carbohydrates leads to an increase in vivo activity.

FIG. 12 shows the results of Western blot analysis of COS-produced mpl ligands 1-174 and analogs N10, N15, N33, N39, N31, N35, and N40. The increased number of N-type glycosylation sites is also indicated in the figure. The graph shows that increasing the number of N-type sites also increases the number of N-type carbohydrates, thereby reducing the mobility of the mpl ligand.

FIG. 13 shows the results of Westenr blot analysis of COS-generated mpl ligands 1-174 and analogs N15, N29, N30 and N38, with the number of N-type sugar chains also indicated.

Detailed Description

The present invention provides mpl ligands with different glycosylation sites compared to the native mpl ligand with the corresponding sequence. Preferably, the resulting molecule contains additional glycosylation sites that are occupied by sugar chains when expressed in mammalian cells (e.g., COS, CHO, and human cells).

In a first embodiment, the present invention relates to mpl ligand analogs comprising an amino acid sequence that is at least one additional, at least one absent, and/or at least both one and one absent glycosylation site as compared to the corresponding natural sequence mpl ligand. The addition or deletion of glycosylation sites results in an increase or decrease in the number of carbohydrate chains, an increase or decrease in sialic acid content, as compared to the corresponding native sequence mpl ligand, especially as compared to human mpl ligands. Deletion of one site and addition of another site at the same time does not result in alteration of the number of sites, but the position and/or type of sites is altered. The present invention also includes such analogs with simultaneous modification.

In the above embodiments, another aspect of the present invention relates to mpl ligand analogs that include amino acid sequences in which one or more N-or O-type glycosylation sites are replaced by one or more non-naturally occurring sites. Thus, one N-type site may be replaced by a different N-type site; an N-type site may be substituted with an O-type site; one O-type site may be substituted by a different O-type site; and/or one O-type site may be substituted with one N-type site. Substitution of one site for another at substantially the same position may increase the glycosylation efficiency of that site, or cause other effects. For example, evidence herein suggests that substitution of a threonine residue for a serine residue may increase the glycosylation efficiency of an O-type site.

In this context, mpl ligands include natural mpl ligands, truncations of natural mpl ligands, and certain non-natural polypeptides having the exact same amino acid sequence and glycosylation as the natural mpl ligand, which have the biological activity of specifically stimulating the growth, development, and/or production of megakaryocytes and/or platelets. Mpl ligand analogs based on at least amino acids 7-151 through amino acids 1-332 of figure 1 are preferred.

In a preferred embodiment, the mpl ligand is the expression product of an exogenous gene transfected into a eukaryotic host cell; i.e., in a preferred embodiment the mpl ligand is a "recombinant mpl ligand", preferred eukaryotic cells are mammalian cells, more preferably CHO cells. The production of recombinant mpl ligands is facilitated according to the methods described herein and in the publications cited herein with respect to the cloning and expression of mpl ligands.

According to FIG. 1, some other preferred mpl ligand molecules have the following amino acid sequence:

mpl ligand 1-332 amino acids 1-332 of FIG. 1

mpl ligand 1-199 amino acids 1-199 of FIG. 1

mpl ligand 1-191 amino acids 1-191 of figure 1

mpl ligand 1-183 amino acids 1-183 of figure 1

mpl ligand 1-174 amino acids 1-174 of FIG. 1

mpl ligand 1-163 amino acids 1-163 of FIG. 1

mpl ligand 1-153 amino acids 1-153 of FIG. 1

mpl ligand 1-152 amino acids 1-152 of FIG. 1

mpl ligand 1-151 amino acids 1-151 of FIG. 1

mpl ligand 7-332 amino acids 7-332 of FIG. 1

mpl ligand 7-199 amino acids 7-199 of FIG. 1

mpl ligand 7-191 amino acids 7-191 of figure 1

mpl ligand 7-183 amino acids 7-183 of FIG. 1

mpl ligand 7-174 amino acids 7-174 of FIG. 1

mpl ligand 7-163 amino acids 7-163 of FIG. 1

mpl ligand 7-153 amino acids 7-153 of FIG. 1

mpl ligand 7-152 amino acids 7-152 of FIG. 1

mpl ligand 7-151 amino acids 7-151 of FIG. 1

For example, it should be noted that mpl ligands 1-183, 1-191, 7-183, and 7-191 contain one or two additional naturally occurring glycosylation sites at their C-terminus as compared to the shorter sequences. In all of the above examples, the N-terminus may further comprise methionine-lysine.

The in vitro specific activity referred to herein is the determination of relative in vitro specific activity, not absolute in vitro specific activity. For purposes of this application, specific activity is used only to compare the relative activities of mpl ligand analogs determined by the same assay under the same conditions, including the same internal standard, and the same analysis is performed on the data used to calculate specific activity.

The term "mpl ligand analog" as used herein refers to one or more changes in the amino acid sequence of the mpl ligand that result in a change in the type (N-or O-type, which affects the amount of carbohydrate attached), number, or position of the carbohydrate attachment site. In a preferred embodiment, the change in glycosylation site results in a change in the number of sugar chains attached to the mpl ligand molecule. In a more preferred embodiment, the alteration of the glycosylation site adds at least one (typically 1-6, preferably 1-5, more preferably 2-4) sugar chain, most preferably the chain is added via an N-bond. In another more preferred embodiment, the mpl ligand analog retains at least the same in vivo biological activity, and may have substantially higher in vivo activity, as compared to a naturally-occurring-sequence mpl ligand (e.g., human mpl ligand), based on the results of the biological activity assay. These assays include measuring megakaryocyte or platelet production.

These mpl ligand analogs are prepared by preferably adding, deleting or replacing amino acid residues by site-directed mutagenesis to increase, delete or alter glycosylation sites. "alteration" refers to deletion of one site with addition of another site at the same or a different position from the deletion site. However, as will be appreciated by those skilled in the art, other methods may also be used to produce genes encoding the same amino acid sequences, and such methods are also included herein. The resulting analogs have fewer or more (preferably more) carbohydrate chains attached as compared to the native human/recombinant mpl ligand.

An important object of the present invention is to add one or more carbohydrate (i.e., sugar) chains to the mpl ligand. Mpl ligand analogs containing more carbohydrate chains than the corresponding naturally occurring amino acid sequence (e.g., 1-332 or 1-174, etc.) incorporate glycosylation sites that do not disrupt their secondary or tertiary structure, thereby substantially not reducing their biological activity. As used herein, a "naturally occurring" mpl ligand refers to an amino acid sequence that is the same number of amino acids as the relevant ligand, even though a specific length of the mpl ligand species is not actually expressed in the natural species. It is highly advantageous that the mpl ligand analogs have up to 6 additional N-or O-glycosylation sites, which allows for the addition of 1 to 6 additional N-or O-carbohydrate chains (or both chains).

For example, a substitution of proline at position 30 with asparagine and a substitution of valine at position 32 with threonine results in an asparagine-glutamic acid-threonine sequence that can serve as a new site for N-glycosylation (see analog N4 in table 1 below).

Analogs having two or more N-type chains can also be constructed by combining mutations; for example, analogs N4 and N10 described in table 1 can be combined to produce an analog having two additional carbohydrate-adding sites (i.e., analog N15 in table 1). Analogs with three or more additional chains can be constructed in the same manner. As will be appreciated by those skilled in the art, the present invention encompasses a variety of other mpl ligand analogs with different glycosylation sites (distinguished by number, type, location of sites). The mpl ligand analogs of the present invention are more preferably based on mpl ligands containing human amino acid sequences under a variety of conditions (see FIGS. 1 and 2); however, mpl ligand sequences based on other species (e.g., dog, pig, monkey, mouse, or rat) are also included herein.

Insertion of amino acids to create glycosylation sites is also contemplated. For example, the following shows that Glu at position 57 is substituted by Thr, while Asn is inserted between Met and 56 at position 55:

-Gln-Met-Glu-Glu-Thr-

54 55 56 57 58

→

-Gln-Met-Asn-Glu-Thr-Thr-

54 55 55′56 57 58

this adds a new glycosylation site (amino acids 55', 56 and 57). See analog N23 below.

Analogs of the invention also include analogs in which one or more amino acids extend at the carboxy terminus of the mpl ligand, with the carboxy-terminal protuberance providing at least one additional carbohydrate site. The carboxy terminus of the mpl ligand varies depending on the particular form of mpl ligand used (e.g., mpl ligand of amino acids 1-332 or mpl ligand of amino acids 1-163). The addition of an amino acid containing one or more N-or O-type glycosylation sites at the carboxy terminus enables the addition of an additional carbohydrate site at the carboxy terminus of the mpl ligand.

Tables 1 and 6 list representative mpl ligand analogs that contain additional sites for the addition of N-type sugar chains, the human mpl ligand polypeptide chains of these analogs being based on human amino acid sequences with Asn-X-Ser or Asn-X-Thr sequences at different positions in the polypeptide chains to create N-type sites. Tables 4 and 7 list analogs to which at least one additional N-type sugar chain was added, as evidenced by migration of these glycoproteins in SDS gel (see example 6 and fig. 3, 5, 6, 7, 9, 10, 12, and 13). Note that some truncations (i.e. N1, N16, N17 and N31) are also included in these tables and are not "analogs" as defined herein. They are listed in the table to show how different truncates were made.

The invention also includes DNA sequences encoding the mpl ligands disclosed herein, preferably encoding analogs containing additional sites for the addition of N-type strands. Examples 4 and 14 disclose methods for introducing changes into mpl ligand DNA sequences to create, delete, and/or alter carbohydrate attachment sites.

These mpl ligand analogs can be the expression product of a foreign DNA sequence, i.e., a sequence produced by recombinant DNA techniques, which can be the product of chemical synthesis, or can be produced by a combinatorial approach. Exogenous DNA sequences include cDNA, genomic DNA or chemically synthesized DNA encoding mpl ligand analogs. The invention also provides recombinant DNA plasmids and eukaryotic host cells for expressing these analogs. Expression vectors include any vector capable of expressing a cloned DNA sequence in a eukaryotic host cell, particularly vectors for expression in COS and CHO cells. Examples of such vectors include the pDSR α and pDSR α 2 plasmids, see molecular cell biology (mol.cell.biol.)8, pp 466-472 (1988); WO 91/13160(1991) and WO 90/14363 (1990). Culture of COS and CHO host cells expressing mpl ligand analogs was performed using standard methods known to those skilled in the art.

Altering the number, type, location or amount of carbohydrate chains attached to the mpl ligand may result in some advantageous properties, such as increased solubility, increased resistance to proteases, decreased immunogenicity, increased half-life in serum and increased or altered biological activity.

Conditioned media from COS cells expressing mpl ligand analog N2-N15(N1 is human mpl ligand 1-174, see FIG. 2) were analyzed to determine in vitro biological activity, and the results are shown in Table 4.

Conditioned media from COS cells expressing mpl ligand analog/truncate N15-N40 were analyzed to determine their in vitro biological activity, and the results are shown in Table 7.

The results of the various forms of in vivo bioactivity are presented in figure 11 (see example 13).

Another embodiment of the invention relates to mammalian (e.g., Chinese hamster ovary, CHO) host cells that preferentially synthesize mpl ligand or mpl ligand analogs having more sialic acid per molecule, e.g., having a sialic acid content that exceeds that of naturally or recombinantly produced mpl ligand 1-332, 1-199, 1-191, 1-183, 1-174, 1-163, 1-153, 1-152, or 1-151 of eukaryotic cells. The in vitro activity of analogs N4 and N15 and full-length and different length analogs expressed in CHO cells is shown in table 5.

The sialic acid content of mpl ligand molecules may affect their biological activity in vivo. For example, tetrakistentaculated (four-branched) N-type oligosaccharides typically provide four possible sialic acid attachment sites, while di-and tri-tentaculated oligosaccharides (which may replace tetrakistentaculated oligosaccharides at the site of aspartic acid attachment) typically have only a maximum of two or three sialic acid attachments. O-type oligosaccharides typically provide two sites for attachment of sialic acid. Thus, if the N-type oligosaccharide is tetrakistentacular, substitution of the mpl ligand molecule for the O-type carbohydrate with the N-type carbohydrate may provide two additional sialic acids per chain. Mammalian cell cultures were screened for cells that normally added a tetratentacular chain to the recombinant mpl ligand to maximize the number of sialic acid attachment sites.

Dihydrofolate reductase deficient Chinese Hamster Ovary (CHO) cells are host cells commonly used to produce recombinant glycoproteins, including recombinant mpl ligands.

The invention also includes compositions comprising a therapeutically effective amount of an mpl ligand analog according to the invention, which compositions further include suitable diluents, adjuvants and/or carriers useful in mpl ligand therapy. As used herein, a "therapeutically effective amount" refers to an amount that provides a therapeutic effect under a given condition and regimen.

The composition can be administered systemically in an parenteral manner. The composition can be administered intravenously or subcutaneously. For systemic administration, the therapeutic compositions of the present invention may be in the form of pyrogen-free, parenterally acceptable aqueous solutions. It is within the skill of the art to prepare such pharmaceutically acceptable protein solutions while taking into account pH, isotonicity, stability and other aspects. The choice of a particular route depends on the disease to be treated. Where mpl ligand or mpl ligand analogue is used, it is preferably provided as part of a formulation comprising a suitable carrier (such as human serum albumin), a suitable diluent (such as a buffer solution) and/or a suitable adjuvant. The dosage required is an amount sufficient to raise the platelet level in a patient and will vary depending on the severity of the condition to be treated, the method of administration and other conditions.

The disease treated with the methods and compositions of the invention is typically one that already exhibits megakaryocyte platelet deficiency or is expected to develop megakaryocyte/platelet deficiency (e.g., due to planned surgery). These diseases are often due to a lack of (transient or permanent) active mpl ligands in vivo. The general term for platelet deficiency is thrombocytopenia, and thus the methods and compositions of the present invention are generally useful for treating thrombocytopenia.

Thrombocytopenia (platelet deficiency) can result from a variety of causes, including chemotherapy, bone marrow transplantation and various medications, radiation therapy, surgery, accidental blood loss, and other specific diseases. Representative specific diseases associated with thrombocytopenia and treated in accordance with the present invention include: the traditional method is used for monitoring.

The analogs of the invention may also be modified to increase activity, stability, half-life, and the like. For example, pegylation (pegylation) (multiple or one) can be added to the mpl ligand analog through an amino or carbohydrate group on the protein. Fatty acids or other polymers may also be attached to the protein or carbohydrate groups.

More specifically, the following embodiments are not to be construed as being included:

1) an mpl ligand analog, wherein

(a) The mpl ligand analogs have the ability to specifically stimulate or increase the biological activity of megakaryocytes or platelets,

(b) the mpl ligand analog is a product of expression of exogenous DNA in CHO or COS cells, and

(c) the mpl ligand has at least one added N-type glycosylation site selected from the following ligands of amino acid sequences 1-174 or 1-199 in FIG. 1:

[Asn²⁵]an mpl ligand;

[Asn³⁰，Thr³²]an mpl ligand;

[Asn⁸²，Ala⁸³]an mpl ligand;

[Asn¹²⁰，Thr¹²²]an mpl ligand;

[Asn⁵³，Thr⁵⁵]an mpl ligand;

[Asn⁵⁸，Thr⁶⁰]an mpl ligand;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²]an mpl ligand;

[Asn⁵⁴，Ser⁵⁶]an mpl ligand;

[Asn⁵²，Thr⁵⁴]an mpl ligand;

[Asn⁸¹，Thr⁸³]an mpl ligand;

[Thr¹⁶³，Asn¹⁶⁴]an mpl ligand;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²，Asn⁵⁵⁽ⁱ⁾，Thr⁵⁷]an mpl ligand;

[Asn³⁰，Thr³²，Asn^55′(i)，Thr⁵⁷，Thr¹⁶³，Asn¹⁶⁴]an mpl ligand;

[Asn⁵⁵，Thr⁵⁷]an mpl ligand;

[Asn⁵⁶]an mpl ligand;

[Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]an mpl ligand; and

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²，Asn⁵⁵，Thr⁵⁷，Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]mpl ligand.

2) An analog of the above 1) which is

[Asn²⁵]mpl ligand 1-174;

[Asn³⁰，Thr³²]mpl ligand 1-174;

[Asn⁸²，Ala⁸³]mpl ligand 1-174;

[Asn¹²⁰，Thr¹²²]mpl ligand 1-174;

[Asn⁵³，Thr⁵⁵]mpl ligand 1-174;

[Asn⁵⁸，Thr⁶⁰]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²]mpl ligand 1-174;

[Asn⁵⁴，Ser⁵⁶]mpl ligand 1-174;

[Asn⁵²，Thr⁵⁴]mpl ligand 1-174;

[Asn⁸¹，Thr⁸³]mpl ligand 1-174;

[Thr¹⁶³，Asn¹⁶⁴]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²，Asn⁵⁵⁽ⁱ⁾，Thr⁵⁷]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn^55′(i)，Thr⁵⁷，Thr¹⁶³，Asn¹⁶⁴]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn^55′(i)，Thr⁵⁷，Thr¹⁶³，Asn¹⁶⁴]mpl ligand 1-199;

[Asn⁵⁵，Thr⁵⁷]mpl ligand 1-174;

[Asn⁵⁶]mpl ligand 1-174;

[Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²，Asn⁵⁵，Thr⁵⁷，Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]mpl ligand 1-174; or

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²，Asn⁵⁵，Thr⁵⁷，Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]mpl ligand 1-199.

3) A DNA sequence encoding the mpl ligand analog of 2) above.

4) A CHO or COS cell transfected with the DNA sequence of 3) above in such a manner that the host cell can express the mpl ligand analog.

5) A pharmaceutical composition comprising a therapeutically effective amount of an mpl ligand analog of 1) above and a pharmaceutically acceptable diluent, adjuvant or carrier.

6) A DNA sequence encoding the mpl ligand analog of 2) above.

7) A CHO or COS cell transfected with the DNA sequence of 6) above in such a manner that the host cell can express the mpl ligand analog.

8) A pharmaceutical composition comprising a therapeutically effective amount of the mpl ligand analog of 2) above, and a pharmaceutically acceptable diluent, adjuvant or carrier.

The following examples are provided to fully illustrate the present invention and should not be construed as limiting the scope thereof. The mpl ligand standard used in the activity assays in the examples was a recombinant mpl ligand standard that refolded to an active conformation after expression in E.coli and purified. Thus, only the relative specific activity was determined.

Example 1

Construction of Mpl ligand 1-174

The human mpl ligand gene encoding amino acids 1-174 (starting sequence S-P-A-P-P-A..) in FIG. 2 was generated from ｃA human fetal liver cDNA library by Polymerase Chain Reaction (PCR) (Bartley et al, Cell 77, 1117-1124 (1994)). The 5' PCR primers encoded the amino terminus of the human mpl ligand, the XbaI site, and the optimized Kozak sequence. The 3' primer included a stop codon and a SalI restriction site. The amplified DNA fragment was digested with XbaI and SalI, and then ligated to pDSRa 2 which had been excised with XbaI and SalI. The resulting plasmid pDSR alpha 2mpl ligand 1-174 was expressed in mammalian cells. The resulting gene sequences (including the signal peptide) are shown in FIG. 2.

The plasmid DNA containing mpl ligand 1-174 was digested with XbaI and SalI restriction enzymes, the resulting DNA fragment was subjected to agarose gel electrophoresis, and the 605-nucleotide mpl ligand 1-174DNA fragment was subjected to GeneClean^TMThe kit was isolated from the gel according to the method provided by the manufacturer (BIO 101, Inc.). The plasmid pDSR α 2 described in WO 90/14363 was also digested with XbaI and SalI restriction enzymes, and the vector fragment was recovered. The two fragments were ligated to give pDSR α 2(mpl ligand 1-174).

Example 2

Expression and purification of Mpl ligand 1-174 in CHO cells

Transfection of dihydrofolate reductase Deficiency (DHFR) with pDSR alpha 2-mpl ligand 1-174^-) Chinese Hamster Ovary (CHO) cells of (i). One day before transfection, 1X 10 cells were added⁶CHO DHFR^-The traditional method is used for monitoring.

Example 1

Construction of Mpl ligand 1-174

The human mpl ligand gene encoding amino acids 1-174 (starting sequence S-P-A-P-P-A …) in FIG. 2 was generated from ｃA human fetal liver cDNA library by Polymerase Chain Reaction (PCR) (Bartley et al, Cell 77 Vol. 1117-1124 (1994)). The 5' PCR primers encoded the amino terminus of the human mpl ligand, the XbaI site, and the optimized Kozak sequence. The 3' primer included a stop codon and a SalI restriction site. The amplified DNA fragment was digested with XbaI and SalI, and then ligated to pDSRa 2 which had been excised with XbaI and SalI. The resulting plasmid pDSR alpha 2mpl ligand 1-174 was expressed in mammalian cells. The resulting gene sequences (including the signal peptide) are shown in FIG. 2.

Example 2

Expression and purification of Mpl ligand 1-174 in CHO cells

Transfection of dihydrofolate reductase Deficiency (DHFR) with pDSR alpha 2-mpl ligand 1-174^-) Chinese Hamster Ovary (CHO) cells of (i). One day before transfection, 1X 10 cells were added⁶CHO DHFR^-Cells were seeded in 100mm tissue culture dishes at CHO D^-Growth in medium (DMEM, 10% fetal bovine serum, 1% penicillin/streptomycin/glutamine, 1% non-essential amino acids (Gibco) and 1% HT supplements (Gibco)). Four transfections were performed. At each transfection, plasmid DNA (50. mu.g) was digested into a linear form with PvuI and buffer H (Boehringer Mannheim). Each time a mammalian cell transfection kit (Special Media) is used, DNA is precipitated andadd dropwise to the plate. After 24 hours of culture in a tissue culture incubator, fresh CHO D was added^-The medium displaces the medium therein. After 24 hours, the cells were transferred to 96-well tissue culture plates containing 100 microliters of CHO selection medium (D-MEM, 5% dialyzed fetal bovine serum, 1% penicillin/streptomycin/glutamine, 1% nonessential amino acids (Gibco)) per well and transformants were selected. Media was changed weekly until colonies appeared. After two weeks, mpl ligand expression was screened using the 32D cell proliferation assay described below (see example 9). Expression in excess of 1X 10⁵The clones in unit/ml were amplified and frozen at low temperature. One clone was expanded for spinner flask production, yielding approximately 8 liters of conditioned media.

Plasmid pDSR α 2 containing mpl ligand 1-174 cDNA was transfected into DHFR deficient CHO cells as described above. Two liters of spinner flask serum free CHO cell conditioned medium (50% D-MEM, 50% HAMS-F12, 1% penicillin/streptomycin/glutamine, 1% nonessential amino acids (Gibco)) with CHO cells expressing mpl ligand 1-174 was concentrated 15-fold using a 2 liter Amicon model 2000 stir chamber and a membrane (YM10, Amicon) that withheld 10000 daltons. 45 ml of concentrated conditioned medium were then directly loaded onto a 4ml hu-MPL-X affinity chromatography column using Pharmacia FPLC at a flow rate of 0.4 ml/min. The construction of this affinity column was carried out according to the manufacturer's recommended method: each ml of Pharmacia CNBr activated Sepharose resin was coupled to 1.5-2.5 mg of Mpl-X (soluble extracellular domain of Mpl receptor). After loading, 10ml of phosphate buffered saline (PBS; 10mM NaPO) was used₄pH6.8/150mM NaCl) and then washed with 24ml of 10mM Tris, pH8.0/1M NaCl. Mpl ligand (1-174) was applied with 40ml of 20mM CAPS (3- [ cyclohexylamine)]-1 propane sulfonic acid) pH10.5/1M NaCl/5mM CHAPS (3- [ (3-Cholamidopropyl) dimethyl ammonium]-1-propanesulfonic acid) was eluted, and collected at 6ml portions. Fraction 2 produced a single band pattern in a 14% SDS gel. It was concentrated and dialyzed against 0.9% NaCl salt solution, and it was biologically active both in vivo and in vitro. Other mpl ligands expressed by CHO cells were purified in a similar manner.

Example 3

In vivo biological Activity of recombinant human Mpl ligand

Platelets were counted in mice treated with different types of mpl ligands. The CHO-derived Mpl ligands 1-332, 1-174, 1-163 and 1-153 were generated and purified by Mpl receptor affinity chromatography. Coli-derived Met-Lys-mpl ligand 1-332, Met-Lys-mpl ligand 1-174, Met-Lys-mpl ligand 1-163 and Met-Lys-mpl ligand 1-153 were also produced and purified by conventional chromatography.

FIG. 4 shows the results of platelet counts of mice treated with different types of CHO cell-derived (solid line) or E.coli-derived (dashed line) recombinant human mpl ligand. Normal female Balb/C mice were injected subcutaneously with mpl ligand at the indicated concentrations for 5 consecutive days. 24 hours after the last injection, a small opening was cut in the side of the tail vein, and a blood sample was collected. Blood cells were analyzed with a Sysmex electronic blood cell analyzer (Baxter Diagnostics, inc. Irvine, CA). Data shown are mean, mean +/-standard deviation of 4 animal determinations. Other blood cell parameters (such as total white blood cells or red blood cells) were not affected by these treatments (data not shown).

The results show that the activity of mpl ligand expressed by CHO cells in vivo is higher than that of the same mpl ligand produced by E.coli. As described in example 6, the various mpl ligands expressed by CHO cells contain N and/or O-type carbohydrates, whereas the mpl ligands expressed by E.coli do not. This indicates that carbohydrates increase the in vivo activity of mpl ligands. The increased in vivo activity of carbohydrates may be due to an increased circulatory half-life, stability, or both.

Example 4

Construction of Mpl ligand analog N2-N15

The method for creating additional glycosylation sites on the mpl ligand is as follows.

The following oligonucleotide primers were synthesized for in vitro mutagenesis to prepare the analogs N2-N14 (see Table 1 for the structures of these analogs):

N2 - CCCATGTCAATCACAGCAGACT SEQ ID NO.：5

N3 - CTTCACAGCAACCTGAGCCAGT SEQ ID NO.：6

N4 - CAGTGCAACGAGACCCACCCTTTG SEQ ID NO.：7

N5 - GCCTACAAATGTCACGCTGCCTGCT SEQ ID NO.：8

N6 - CCCACTTGTAACTCATCCCTC SEQ ID NO.：9

N7 - CAACTGAACGCCACTTGTCTCTCA SEQ ID NO.：10

N8 - ACTTGTCTCAACTCCACCCTGGGGGA SEQ ID NO.：11

N9 - CTCCTGGGGAACCTTTCTGGA SEQ ID NO.：12

N10 - GACCACAAATCACACCGATCCCAAT SEQ ID NO.：13

N11 - ACCCTTTGTCTACAAATGTCACGCTGCCTGCT SEQ ID NO.：14

N12 - TCTCTCAAACCTCACGGGGGAGCTT SEQ ID NO.：15

N13 - TGGAAAAATCAGACGGAGGAGAC SEQ ID NO.：16

N14 - TGGAGGAGAACAAGACACAGGACAT SEQ ID NO.：17

to construct m13mp18 mpl ligand 1-174, the gene in FIG. 2 was introduced into XbaI and SalI restriction enzyme digested m13mp18 DNA. Single-stranded DNA was recovered from the supernatant of m13mp18(mpl ligand 1-174) infected E.coli RZ1032 strain. See Kunkel et al, Methods in enzymology 154, 367 (1987) and Messing, Methods in enzymology 101, 20 (1983). IntoFor in vitro mutagenesis, approximately 0.5. mu.g of single stranded DNA and 0.125 picomoles of either of the above synthetic primers was combined with 6. mu.l buffer (250mM Tris pH 7.8, 50mM MgCl)₂50mM dithiothreitol and 1% bovine serum albumin (BSA-Pharmacia)). The primers were previously activated with ATP and T4 polynucleotide kinase. When the primer was annealed to the template, the reaction volume was adjusted to 10. mu.l with water, and the mixture was heated to 65 ℃ for 5 minutes, followed by cooling to room temperature. For the extension reaction, 2.5. mu.l each of dTTP, dATP, dGTP and dCTP and 1ml of ATP (each at a concentration of 10. mu.M) were added, followed by 1. mu.l (1 unit) of E.coli DNA polymerase (Klenow fragment) and 1. mu.l (1 unit) of T4 DNA ligase. The mixture was allowed to stand overnight at 14 ℃ and E.coli JM109 (Yanisch-Perron et al, Gene 33, 103 (1985)) was transformed as described (Messing, supra).

Mutant clones were identified by differential hybridization. Plaques on nutrient agar were transferred to Gene Screen filters (New England Nuclear). The filters were placed in a model 1800 UV Cross-linker and the DNA was cross-linked to the filters by irradiation using an automatic cross-linking system (Stratagene). The filters were then incubated in 6 XSSC (0.9M NaCl/0.09M sodium citrate) containing 1% SDS at 60 ℃ for 1 hour. Upon hybridization, the above oligonucleotide primers (8 pmoles) were replaced with T4 Polynucleotide kinase and γ³²P-labeled ATP was end-labeled and incubated with filters overnight in 6 XSSC containing 0.5% SDS and 125. mu.g/ml herring sperm DNA. The hybridization temperature is selected based on the estimated melting point of the oligonucleotide. Generally, the hybridization temperature is about 10 ℃ lower than the melting point. The following day, the membrane was washed 2 times with 6 XSSC/1% SDS at the hybridization temperature, followed by two more washes with 6 XSSC at the hybridization temperature, followed by autoradiography. If necessary, the filters are washed in 6 XSSC at higher temperatures until the hybridization signal of plaques containing the wild-type mpl ligand cDNA sequence is almost or completely undetectable. Clones that gave a positive hybridization signal under these conditions were identified and re-transfected with JM109 to isolate purified clones. Dideoxy chain termination sequence analysis indicated that a mutation occurred.

Double-stranded m13 mpl ligand 1-174DNA with the desired changes was recovered from JM109 transfected cells using the QIAGEN kit (Chatsworth CA.) following the manufacturer's protocol. The DNA was digested with XbaI and SalI, and a 605bp mpl ligand DNA fragment was isolated. pDSR α 2 was digested with XbaI and SalI. Vector fragments were isolated and ligated to the mpl ligand fragments described above. The recombinant plasmid was identified by restriction analysis. The resulting plasmid (designated mpl ligand 1-174-NX, where NX is the number of analogs) contains DNA encoding an analog of the mpl ligand with an altered amino acid residue at the indicated position. The resulting plasmid was then sequenced to confirm the presence of the desired mutation.

The analog N15 was constructed with two additional N-type glycosylation sites at positions 30 and 120. PDSR α 2mpl ligand 174-N4 with Asn30 and Thr32 mutations was digested with XbaI and PstI restriction enzymes and a DNA fragment of approximately 385 nucleotides was isolated. PDSR α 2mpl ligand 1-174-N10 containing Asn120 and Thr122 mutations was digested with PstI and SalI restriction enzymes and a DNA fragment of about 220 nucleotides was isolated. pDSR α 2 was digested with XbaI and SalI. Vector fragments were isolated and ligated to the mpl ligand fragments described above. This produced PDSR α 2mpl ligand 174-N15 with Asn30, Thr32, Asn120 and Thr122 substitutions.

These general methods were used to construct mpl ligand analogs shown in table 1. DNA sequence changes in each analog are shown; otherwise, the sequence of the oligonucleotide primer used for mutagenesis has a sequence complementary to the human mpl ligand.

TABLE 1

Analog/analog combination

Amino acid substitution sequence Change of class number

N1 (1-174)；Pro¹⁷⁵→Gly³³²Deletion CCA → TGA (stop codon)

N2 Leu²²→Asn²² CCT→AAT

N3 Arg²⁵→Asn²⁵ AGA→AAC

N4 Pro³⁰，Val³²→Asn³⁰，Thr³² CCA，GTT→AAC，ACC

N5 Pro³⁸，Leu⁴⁰→Asn³⁸，Thr⁴⁰ CCT，CTG→AAT，ACG

N6 Leu⁸⁶→Asn⁸⁶ CTC→AAC

N7 Gly⁸²，Pro⁸³→Asn⁸²，Ala⁸³ GGA，CCC→AAC，GCC

N8 Ser⁸⁷，Leu⁸⁹→Asn⁸⁷，Thr⁸⁹ TCA，CTC→AAC，ACC

N9 Gln⁹²→Asn⁹² CAG→AAC

N10 Ala¹²⁰，Lys¹²²→Asn¹²⁰，Thr¹²² GCT，AAG→AAT，ACC

N11 Pro³⁶，Pro³⁸，Leu⁴⁰→ CCT，CCT，CTG→

Ser³⁶，Asn³⁸，Thr⁴⁰ TCT，AAT，ACG

N12 Ser⁸⁸Leu⁹⁰→Asn⁸⁸，Thr⁹⁰ TCC，CTG→AAC，ACG

N13 Thr⁵³，Met⁵⁵→Asn⁵³，Thr⁵⁵ ACC，ATG→AAT，ACG

N14 Thr⁵⁸，Ala⁶⁰→Asn⁵⁸，Thr⁶⁰ ACC，GCA→AAC，ACA

N15 Pro³⁰，Val³²，Ala¹²⁰，Lys¹²²→ CCA，GTT，GCT，AAG→

Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²² AAC，ACC，AAT，ACC

Note that: analogs N2-N15 herein refer to analogs 2-15. And, herein referred to as [ Asn²²]An mpl ligand refers to the substitution of asparagine for amino acid 22 of the particular mpl ligand being studied, with a preferred mpl ligand having at least the human sequence of amino acids 7-151 of figure 1 (including the preferred human mpl ligand sequence described above). Thus, substitution of asparagine residues for the leucine residue at position 22 of mpl ligand 1-174 (human sequence) results in mpl ligand analogs that can be substituted with [ Asn²²]mpl ligand 1-174.

The plasmid constructed by inserting mpl ligand DNA into pDSR α 2 is called pDSR α 21-174-NX, where NX is the number of the analogue. The expression vector pDSR α 2 is described in general in WO 90/14363 (1990). Digestion of pDSR α 2 with XbaI and SalI yielded the pDSR α 2mpl ligand 1-174-NX plasmid. The vector fragment was isolated and ligated to a fragment of approximately 605bp containing the desired sequence.

Example 5

Expression of Mpl ligand and Mpl ligand N1-N15 in COS cells

cDNA clones of human mpl ligand and mpl ligand analogs described in Table 1 were transferred to COS-1 cells (ATCC number CRL-1650) by electroporation. Culture from semi-confluentCOS-1 cells were collected in petri dishes, washed with medium (Dulbecco's modified essential medium containing 10% fetal bovine serum and 1% L-glutamine/penicillin/streptomycin (Irvine Scientific)), and resuspended to a concentration of 6X 10⁶Cells/ml. 0.5ml cells were transferred to a 0.2cm Electroporation cell (Bio-Rad) and electroporated with 50. mu.g of plasmid DNA encoding mpl ligand analogue using a BTX Electroporation System electronic cell Manipulator 600(BTX Electroporation System electric cell Manipulator 600) operating at a low voltage of 130 volts and 650. mu.F. The electroporated cells were plated in 10ml medium onto 100mm tissue culture dishes. The original medium was replaced with 10ml of fresh medium 12 to 24 hours after plating. Conditioned media was collected 3 to 5 days after electroporation.

Example 6

Identification of Mpl ligand and Mpl ligand N1-N15

A. Identification of carbohydrate addition

COS cell supernatants containing approximately 30-60ng mpl ligand or mpl ligand analog were immunoprecipitated overnight at room temperature with rabbit anti-mpl ligand polyclonal antibody, and COS cells were transfected with mpl ligand analog cDNA as described in example 5. In some cases where the expression level is low, the maximum volume for immunoprecipitation is about 8-9 ml. The antibody is directed against purified mpl ligand 1-163 expressed in E.coli. Mu.l of 1: 1 protein A-Sepharose in Phosphate Buffered Saline (PBS) containing 0.1% sodium azide was added to the immunoprecipitates and incubated at room temperature for 1 hour. Samples were centrifuged, washed with PBS, and resuspended in SDS sample buffer (0.125M Tris-HCl pH 6.8/4% SDS/20% glycerol/10% β -mercaptoethanol/0.001% bromophenol blue). The samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and then Western analyzed with mouse monoclonal antibodies (see, e.g., Burnette et al, Biochemical analysis (anal. biochem.112, pp. 195-203 (1981); Elliott et al, Gene 79, pp. 167-180 (1989)), using antibodies generated using synthetic mpl ligand peptides (e.g., corresponding to amino acid residues 47-62 in FIG. 1) as antigens.

FIG. 5 shows that the proteins in the supernatant of COS cells transfected with the analogs N4, N7, and N10 DNA were larger than the mpl ligand 174(N1) of the human sequence. FIG. 6 shows that the proteins in the supernatant of N13, N14 and N4DNA transfected COS cells are also larger than the mpl ligand of the human sequence. This increase indicates the presence of an additional N-type carbohydrate chain. N15 contains two additional N-type glycosylation sites. FIG. 6 shows that the analogs contain more material than only 1 additional N-type glycosylation. The size of the proteins was estimated from their mobility on SDS-PAGE versus protein standards of known molecular weight. The estimated sizes of the larger bands calculated from fig. 6 are listed in table 2. This result indicates that N15 contains 2 additional N-type sugar chains. Western blot analysis of other selected analogs is also shown in figure 6.

TABLE 2

Estimation of N-type carbohydrates

The number of N-type (1-174) (Da) (Da) chains (increased over the native sequence) with the potential for molecular weight variation of Mpl ligand analogs (@4 KDa/site)
	N1 Natural 2350000N 42870052001N 72720037001N 102720037001N 132670032001N 142870052001N 1533500 10000 2

Experiments were performed to confirm that the mpl ligand analogue increase was due to the addition of N-type carbohydrates. COS cell conditioned media containing mpl ligand were immunoprecipitated as described above and washed with PBS. Then 10. mu.l of 0.5% SDS was added to each tube and each sample was boiled for 3 minutes. The following ingredients were then added: 10.8 μ l of 0.5M NaPO₄pH8.6, 5ml of 7.5% octylphenol polyoxyethylene ether and 3. mu.l of 250 units/ml N-glycanase (Genzyme). N-glycanase treatment removes N-type carbohydrates. Samples were incubated at 37 ℃ for 6 hours. SDS-PAGE sample buffer was added to stop the reaction and SDS-PAGEWestern analysis (12% acrylamide) was performed as described above using monoclonal antibodies against the mpl ligand and an anti-mouse ECL Western detection kit (Amersham). The N-type chain analysis of human mpl ligand and mpl ligand analogs using this method is shown in FIG. 7. The mobility on Western blots after N4, N7 and N10 treatment with N-glycanase decreased to the same as N1. As expected, N1 had no effect on motility after treatment with N-glycanase, since N1 had no N-type glycosylation sites. These results indicate that the increase seen is due to the addition of N-type carbohydrate.

Analysis of O-type carbohydrates on mpl ligands

To analyze the effect of O-type carbohydrates on human mpl ligands, several proteins were purified from CHO cell conditioned media as described above. Each protein was treated with O-glycanase (glycopeptide. alpha. -N-acetylgalactosamine, Oxford GlycoSs). + -. The O-glycanase removes O-type carbohydrates from the glycoprotein. Each protein form expressed in E.coli was used as an unglycosylated control. To analyze the effect of O-type carbohydrates on molecular weight differences, it was necessary to first remove all N-type carbohydrates. Since the full-length version of mpl ligand 1-332 contained N-type carbohydrates, the full-length samples expressed by CHO cells were treated with N-glycanase (peptide-N4- (N-acetyl-. beta. -glucosamino) asparaginase) as described above for the mpl ligand analogs expressed by COS cells, except that the N-glycanase treatment was incubated overnight here.

Before O-glycanase treatment of the full-length (1-332) mpl ligand, the pH of the sample was adjusted to between pH6.0 and pH7.0 using 1/15 volumes of 100mM acetic acid (pH 2.2). 1 microgram of protein was denatured by boiling in SDS for 3 minutes and then incubated with 1U/ml neuraminidase (sialidase, from Arthrobacter Ureacins, Boehringer Mannheim), 1mM calcium acetate (pH6.8) and 20mM sodium phosphate (pH6.8) solution for 60 minutes at 37 ℃.

Followed by O-glycanase treatment: the final volume was made 100. mu.l by adding 5mU of enzyme and incubated overnight at 37 ℃. Proteins (0.2. mu.g/lane) were separated by SDS-PAGE (15% acrylamide). Then transferred to a 0.2 μm nitrocellulose membrane, incubated overnight with polyclonal antibodies against mpl ligand, and mpl ligand protein was visualized using an anti-rabbit ECL Western detection kit (Amersham).

Figure 3 shows Westenr blot results for 4 different forms of human mpl ligand. Lanes 1-3 are full-length mpl ligand 1-332, lanes 4-6 are mpl ligand 1-174, lanes 7-9 are mpl ligand 1-163, and lanes 10-12 are mpl ligand 1-153. The neuraminidase and O-glycosidic treated ligands are shown in lanes 2, 5, 8 and 11, and their molecular weights are reduced to the same as the unglycosylated ligands in lanes 3, 6, 9 and 12. In each case motility was increased to the same extent as the unglycosylated form expressed in E.coli. These results indicate that the larger bands in lanes 1, 4, 7 and 10 are due to O-type carbohydrates. The molecular weight of each band was estimated by comparison with the mobility of proteins of known molecular weight.

As can be seen from the estimated molecular weights of the different proteins listed in Table 3, the apparent change in mobility can be attributed to multiple O-type carbohydrates, with mpl ligands 1-332 containing up to 14O-type carbohydrate chains (estimated 950 daltons/chain), mpl ligands 1-174 containing 9 chains, mpl ligands 1-163 containing 4 chains, and mpl ligands 1-153 containing 2 chains. Samples in lane 2 were full-length mpl ligand 1-332. The protein was apparently degraded, probably due to the prolonged incubation time of the isoglycosidase at 37 ℃. Thus, the unglycosylated form of E.coli expression in lane 3 was used to calculate the approximate molecular weight of the O-type carbohydrate added to the CHO, cell-expressed mpl ligand 1-332.

These results are consistent with the presence of carbohydrates for all mpl ligand forms expressed by CHO being tested. The presence of O-type carbohydrates in the mpl ligands 1-332, 1-174, and 1-163 expressed by CHO cells was confirmed by direct analysis of the monosaccharide composition. Sialic acid, GalNAc, and Gal are released from glycoproteins by acid hydrolysis. The monosaccharide is detected by high-pressure anion exchange chromatography and pulse current. These three sugars were detected for each mpl ligand form. This result indicates the presence of sialic acid containing O-type carbohydrates. This result is consistent with the in vivo results in FIG. 4, where the form of mpl ligand expressed by CHO cells is more active in vivo than the corresponding form expressed in E.coli. The presence of carbohydrates therefore increases the in vivo activity of mpl ligands.

TABLE 3

Calculation of O-type carbohydrate

Mpl Complex O-glycanase treatment the number of O-type chains with potential molecular weight variation (+/-) (Da) (@950 Da/chain)
	1-332-542001360014 "Escherichia coli variant 406001-174-2460086009" + 160001-163-1840039004 "+ 145001-153-1520023002" + 12900

Example 7

ELISA detection of Mpl ligand

Production of polyclonal antibodies: new Zealand white rabbits were hyperimmunized with recombinant human mpl ligand 1-163 produced by E.coli for up to three months. Antisera from six rabbits showing high antibody titers were collected and affinity purified for antibodies specific for the mpl ligand.

Affinity purification: recombinant human mpl ligand 1-163 was covalently attached to Actigel-ALD (Sterogene Bioseparations, Inc.) according to the manufacturer's instructions. A sample of rabbit antiserum was added to the mpl ligand affinity gel and the mixture was placed on a stirrer and gently agitated overnight at 4-8 ℃. Unbound serum proteins were eluted from the gel bed with PBS and specifically bound antibodies against the mpl ligand were eluted with immunopurified mild antigen/antibody Elution Buffer (ImmunoPure GentleAg/Ab Elution Buffer, Pierce Chemical Co.). The recovered antibody was dialyzed against PBS, and after several changes of the dialysate, the antibody solution was concentrated using an Amicon Stirred Cell Ultrafiltration Unit (Amicon Stirred Cell Ultrafiltration Unit), and the resulting antibody concentrate was an antibody specific against mpl ligand, which was then used for well coating and enzyme conjugate preparation.

ELISA reagents: immulon 4 Removawell Strips (Dynatech laboratories, Inc.) were coated with affinity purified antibodies to the mpl ligand from rabbit. The affinity purified antibody was diluted with 0.1M sodium bicarbonate (freshly prepared, pH about 8.2) to a concentration of 2.5. mu.g/ml. Mu.l of antibody was added to each well and the enzyme plates were incubated in a closed wet box at room temperature for 24 hours. Then, 200. mu.l of a blocking solution containing 1% fetal bovine serum and 5% sucrose in TEN solution (TEN solution: 50mM Tris 7.4/10mM EDTA/150mM NaCl) was added to each well, and the enzyme-linked plate was further incubated in a closed wet box at room temperature for 24 hours. The coating and blocking solutions in the wells were discarded. The recoating/sealing step includes: add 300. mu.l of SuperBlock (SuperBlock) blocking buffer (Pierce Chemical Co.) in PBS to each well. The solution was left at room temperature for about 5 minutes, discarded and air dried at room temperature for 24 hours. The coated enzyme-linked plates were stored in sealed plastic bags at 4-8 ℃ for mpl ligand ELISA.

Antibodies to mpl ligand affinity purified from the collected rabbit antiserum were covalently linked to horseradish peroxidase (HRPO) and used as signal-producing antibodies. Affinity purified antibodies were treated with iminothiolane hydrochloride (iminothiolane HCl Fluka Chemical Corp.). In addition, HRPO was treated with 6-maleimidocaproic acid-N-succinimidyl ester (Fluka chemical Corp.). The two activated proteins are mixed so that they are covalently bound. The reaction mixture was then subjected to column chromatography using FPLC Superose 6(Pharmacia) to isolate antibodies of the desired molecular weight (i.e.about 200 kD): HRPO conjugates. Fractions containing the desired conjugate were pooled, concentrated with Centricon 30(Amicon Division, W.R. Grace & Co.) and stored at-20 ℃ as a 50% solution in glycerol. The anti-mpl ligand antibody: HRPO concentrate was diluted with PBS containing 2% fetal bovine serum for ELISA. The final concentration of the conjugate used for ELISA was 250-500 ng/ml.

Recombinant human mpl ligand 1-163 produced in E.coli was used as a standard. The mpl ligand was diluted with TEN buffer containing 2% fetal bovine serum (Sigma Chemical Co.), 0.05% thimerosal and stored. Standards were prepared containing mpl ligand at 1.0, 0.5, 0.25, 0.125 and 0.062ng/ml, respectively.

And (3) detection: mu.l of mpl ligand standard or sample is added to the plate well, which is then incubated in a closed wet box at room temperature for 18-24 hours. The contents and residual liquid of the wells were discarded and washed once with a wash solution (TEN buffer containing 0.05% tween 20). Antibodies against mpl ligand were added to each well: HRPO conjugate solution (100. mu.l) was then incubated in a closed wet box at room temperature for 2 hours. The contents of each well were discarded and washed 4 times with TEN buffer containing 0.05% Tween 20.

For color reaction, 100. mu.l of TMB/peroxide substrate solution (Kirkegaard & Perry solution A and B1: 1 mixed) was added and incubated at room temperature for 20 minutes. The reaction was stopped by adding 100. mu.l of stop solution (0.5N sulfuric acid). The absorbance was read at 450nm on a microplate detector. The concentration of mpl ligand in the sample is calculated according to a standard curve generated by a curve fitting program.

Example 8

mpl ligand 1-174 analogs in short-term liquid culture

Biological activity in megakaryocyte assay

Mpl ligand 1-174 analogs were prepared as described above and tested for their ability to stimulate growth of liquid cultured megakaryocytes. CD34 isolated from a human leukocyte extraction cell (Nichol et al, Stem Cells 12 vol 494-⁵Perml was inoculated onto medium (IMDM/1% Procaine penicillin Glutamine/1% non-essential amino acids/1% MEM sodium pyruvate/1% MEM vitamins/10% deionized BSA/10% normal human AB plasma/10. mu.M. alpha. -thiacylglycerol/20. mu.g/ml L-asparagine). In addition, 1.5. mu.l of COS-1 conditioned medium containing mpl ligand (1-174) or mpl ligand 1-174 analog was added to each well. The final volume on a Terasaki type micro tissue culture plate (Vangard International) was 15. mu.l. Cells in 5% CO₂The wet cassette of (1) was incubated at 37 ℃ for 8 days, directly immobilized on culture wells with 1% glutaraldehyde, and then incubated with a monoclonal antibody cocktail containing anti-GPIb, anti-GPIIb (Biodesign), and anti-GPIb (Dako, Carpinteria, Calif.). The immunoreactions were visualized with a streptavidin-beta-galactosidase detection system (HistoMark, Kirkegaard and Perry). Megakaryocytes are represented in dark color (blue in the actual picture) in figure 8.

In FIG. 8, part A and part D are positive and negative controls, respectively. Group A wells received 37.5pg wild-type mpl ligand 1-174 COS-1 conditioned medium and significant megakaryocyte growth was observed. Group D wells received 1.5. mu.l COS-1 mock conditioned medium and no growth was seen. In FIG. 8, moieties B and C are mpl ligand 1-174 analogs N7 and N10, respectively. Group B wells received 9.0pg mpl of COS-1 conditioned medium as the ligand, while group C wells received 27pg, both of which showed good megakaryocyte growth.

This experiment shows that the mpl ligand analogs tested are capable of stimulating the growth of human megakaryocytes in vitro.

Example 9

Biological Activity of Mpl ligand 1-174 analogs in vitro cell proliferation assays

Mpl ligand 1-174 analogs were prepared as described above and tested for their ability to stimulate 32D-mpl cell proliferation. To construct 32D-mpl cells, the full-length human mpl receptor sequence (Vigon, I., et al, PNAS 89 Vol. 5640-5644(1992)) was subcloned into an expression vector containing the Moloney mouse sarcoma virus transcription promoter. Mu.l of this construct and 6. mu.l of an amphotropic retrovirus packaged construct (Landau, N.R., Littman, D.R., "J.Virol. Journal of Virology 66 vol. 5110. pp. 5113 (1992)) were transfected into 3X 10 using the CaPO4 mammalian transfection kit (Stratagene)⁶293 cells. These cells were transfected again after 2 and 4 days. The day after the last transfection, 293 cells were co-incubated with an IL-3 dependent murine cell line (32D, clone 23; Greenberger et al, PNAS80 Vol 2931-2936 (1983)). After 24 hours, 32D cells were recovered on a BSA gradient (Path-o-cell; Miles Inc.) and banded. Cells were expanded in 1ng/ml murine IL-3 and then selected for growth in 20% APK9 serum (Bartley et al, Cell 77, 1117-1124 (1994)). FACS was performed with polyclonal rabbit anti-peptide (MPL) serum and cells were sorted according to receptor expressed on the cell surface. These cytokine-dependent murine 32D-mpl cells respond to mpl ligand. 32D-MPL cells were grown in MEM medium containing 10% fetal clone II serum (Hyclone Laboratories) and 1.0ng/mlmu IL3 to a cell density of 1X 10⁶Cells/ml. Cells were harvested by centrifugation (approximately 500 XG), washed twice with growth medium lacking muIL3, and resuspended at 1X 10⁵Cells/ml.

A mpl ligand standard curve containing 12 points was prepared with mpl ligand 1-163 ranging from 5000-1 pg/ml. 100 μ l of standard mpl ligand or test sample at each dilution was added to 100 μ l of 96-well microtissue culture plates (10000 cells/well) resuspended in cells in appropriate wells at 37 ℃ in 10% CO₂And (3) incubating in a humidified incubator. After 48 hours, 40. mu.l of MTS reagent (aqueous phase nonradioactive cell proliferation kit, Promega) was added to each well and after 14-18 hours the optical density was read at 490nM on a cell plate reader. The in vitro activity of each sample was calculated from the dose response curve of the sample. One unit is defined as the amount of ligand per sample mpl required to produce 50% of the maximal stimulation. Specific activity was calculated as biological activity in units/ml divided by the concentration of mpl ligand in ng/ml determined by mpl ligand ELISA.

The specific biological activities of the mpl ligand analogs transfected and expressed in COS cells are shown in Table 4. The effect of amino acid substitutions on carbohydrate addition was also summarized. The in vitro activity of purified human sequence mpl ligand was determined as 200-300 units/ng as described above. It is also clear from table 4 that mpl ligand analogues containing additional N-type carbohydrates (e.g. N4 and N10) are expressed as well as natural sequence mpl ligands, even though they contain additional carbohydrate chains (as determined in part a of example 6). Both analogs also retain full in vitro biological activity. Thus, mpl ligand analogs containing N-type carbohydrates can be normally expressed in mammalian cells and they have normal or elevated in vitro biological activity.

TABLE 4

Number of Mpl ligand form sequence N-type strands Elisa (ng/ml) in vitro Activity specific Activity (amino acid length) (units/m) (units/ng) (a) (b) (c) (d)

Simulated NCNE 0 < 0.08 < 10 < 125N1(174)Natural NA 255375215N 255375215 (174) Natural 255375215 NA NAN 255375215 (174) N220 NA NAN 255375215 (174) N255375215 NA 255375215N 255375215 (174) N30T 255375215N 255375215 (174) N30T 32124 NA NAN 255375215 (174) 255375215 < 10 < 8N 255375215 (174) N255375215 < 10 < 22N 255375215 (174) N82A 830 to 255375215N 255375215 (174) N82A 830 to 255375215N 255375215 (174) 255375215 (174) 255375215 (174) S36N38T 255375215 NA < 0.625 < 10N 255375215 (174) 255375215 < 10 < 8N 255375215 (174) N53T 550 to 255375215N 255375215 (174) N58T 600 to 255375215N 72 (174) N30T32N120T 1220 to 255375215 (174)

Attention is paid to

(a) The number of additional N-type chains was estimated based on the mobility of the analog polypeptide in SDS gels as described in example 6.

(b) The amount of mpl ligand analogue in the CHO cell supernatant was determined by ELISA assay as described in this example.

(c) In vitro activity was calculated from stimulation of thymidine uptake by 32D cells that depend on mpl ligand growth.

(d) Ratio of the in vitro activity of mpl ligand analogue measured according to proliferation assay to the amount of mpl ligand analogue measured according to mpl ligand ELISA.

N.A. not obtained

Example 10

Expression and purification of Mpl ligands 1-174, N4 and N15 in CHO cells

pDSRa 2 containing mpl ligand 1-174, N4 and N15 cDNAs was transfected into DHFR-deficient CHO cells using the following modifications of the method described in example 2.

One transfection per analogue was performed. Three weeks after transfection, mpl ligand expression was screened using mpl ligand ELISA. Three expression clones in various forms were cryopreserved at low temperature. The highest expressing clones for each analog were grown in roller bottle production scale-up. N4 produced 7.4 liters of conditioned medium (50% D-MEM, 50% HAMS-F12, 1% penicillin/streptomycin/glutamine, 1% nonessential amino acids (Gibco)), and N15 produced 4.6 liters of conditioned medium.

Serum-free conditioned media from spinner flasks inoculated with CHO cells expressing mpl ligand 1-174(2.9L), N4(7.4L), N15(4.4L) were concentrated 12, 19 and 12 fold respectively using S1Y10 (10000 Dalton molecular weight cut off) Amicon spiral ultrafiltration columns. 150 ml of concentrated conditioned medium was loaded directly onto a 3.3ml hu-Mpl-X (receptor) affinity column at a flow rate of 0.3 ml/min. This affinity column was constructed by coupling 1.0-1.5 mg of Mpl-X (soluble Mpl receptor extracellular domain) per ml of Pharmacia CNBr activated Sepharose resin according to the manufacturer's recommended protocol. After loading, 30ml of phosphoric acid was addedSalt buffer (PBS; 10mM NaPO)₄pH6.8/150mM NaCl) and then 60ml of 10mM Tris, pH8.0/1M NaCl/1mM CHAPS. The Mpl ligand 1-174 was dosed with 30ml of 20mM MCPS (3- [ cyclohexylamine)]-1 propanesulfonic acid) pH10.5/1M NaCl/1mM CHAPS (3- [ 3-cholamidopropyl) dimethylamine]-1-propanesulfonic acid).

Each fraction was neutralized by adding 0.6ml of 1M Tris pH7.0. SDS-PAGE analysis indicated that 1-174mpl ligand had significant "bleeding" when eluted with 10mM Tris, pH8.0/1M NaCl 1mM CHAPS. The eluted fractions were analyzed by SDS-PAGE. Fractions containing mpl ligand 1-174 were collected. This affinity purification was repeated once adjusted as follows: 0.5ml/min loading and elution removed 10mM Tris, pH8.0/1M NaCl/1mM HAPS elution.

All fractions containing a single mpl ligand band were concentrated using YM10 (molecular weight cut off 10000 daltons) membranes in a 50ml stirred chamber on a centricon apparatus. The 0.5ml concentrate was loaded directly onto a Pharmacia Superdex 200 HR 10/30 gel filtration column equilibrated in PBS at a flow rate of 0.25ml/min, and 0.25ml of each fraction was collected. All fractions eluted containing a single mpl ligand band (analyzed by SDS-PAGE) were pooled.

Other forms of mpl ligand expressed by CHO cells (N4 and N15) were purified in a similar manner (combining two affinity purifications, separating on a Superdex 200 gel filtration column).

Example 11

Assay for carbohydrate addition in N4 and N15 expressed by CHO cells

To determine whether the mpl ligand form expressed in CHO cells contained N-type carbohydrates, conditioned media was analyzed by SDS-PAGE Western blotting as described in example 6, using the following modifications.

CHO D-conditioned medium from spinner flasks was used. Samples were loaded into a Centricon-10 centrifuge concentrator (Amicon, Beverly, Mass.) and centrifuged at 6000 RPM for 1 hour on a Beckman J2-HS centrifuge using a fixed angle rotor (JA 20.1). A concentrated sample containing approximately 100ng of mpl ligand analogue was loaded onto the SDS PAGE gel with the same volume of SDS sample buffer (as described in example 6). The E.coli-expressed carbohydrate-free mpl ligand MK1-174 was also loaded simultaneously. Figure 9 shows the difference in motility, which is consistent with the expected carbohydrate number. The most rapidly moving species are Met-Lys (1-174) E.coli mpl ligand, followed by mpl ligand 1-174(CHO), N4(CHO) and N15(CHO) in that order. See fig. 9. The most likely explanation for the molecular increase relative to the unglycosylated mpl ligand is that there is an additional O-type carbohydrate on mpl ligand 1-174(CHO), an additional O-type carbohydrate and one additional N-type oligosaccharide on N4(CHO), and an additional O-type carbohydrate and two additional N-type oligosaccharides on N15 (CHO).

To confirm that the increase in molecular weight was indeed due to the addition of N-type carbohydrate chains, the samples were treated with N-glycanase as described in example 6 to remove all N-type carbohydrates. Approximately 100ng of mpl ligand analog was contained in each sample purified from conditioned media.

The mobilities of N4(CHO) and N15(CHO) were reduced to the same extent as mpl ligand 1-174(CHO) after N-glycanase treatment. Treatment of mpl ligand MK1-174 (E.coli) or mpl ligand 1-174(CHO) with N-glycanase did not affect motility since neither form would be expected to contain any N-type carbohydrates. Comparison of the absence of N-glycanase treatment showed that the size difference of N4 corresponds to the size of one N-type carbohydrate chain, whereas the size difference of N15 corresponds to the size of two carbohydrate chains. Thus, when these two mpl ligand forms are expressed in CHO cells, an increase in their N-type glycosylation sites results in an increase in N-type carbohydrates. See fig. 10.

Example 12

In vitro biological Activity of Mpl ligand analogs produced in CHO cells

Pure Mpl ligand and analogues expressed and purified from CHO cells or E.coli cells were analyzed for in vitro biological activity using the factor-dependent cell line 32D-Mpl and the assay described in example 9, the activity being calculated from the curve obtained with reference to the Mpl ligand 1-332 produced by CHO cells as standard. The specific in vitro biological activities of several forms of mpl ligands are shown in table 5. It is evident from the table that mpl ligand analogs containing additional carbohydrates expressed in CHO cells have in vitro biological activity.

TABLE 5

In vitro Activity of MPL ligand

Number of N-type chains of MPL ligand form in vitro Activity

U/mg×10E6

MK174(E.coli) 0 13

1-163(CHO) 0 86

1-174(CHO) 0 85

N4(CHO) 1 60

N15(CHO) 2 92

1-332(CHO) 6 41

Example 13

In vivo biological Activity of Mpl ligand analogs

The platelet count of mice treated with several forms of mpl ligand was measured and the results are shown in FIG. 11. The CHO-derived mpl ligands 1-332, 1-174, N4 and N15 were purified by mpl receptor affinity chromatography. E.coli-derived Met-Lys-mpl ligand 1-174 was purified by conventional chromatography. Each of the indicated concentrations was given to normal female Balb/c mice subcutaneously once daily for 5 consecutive days. 24 hours after the last injection, blood samples were collected by cutting a small opening in the side of the tail vein. Blood cell analysis was performed with a Sysmex blood cell electron analyzer (Baxter Diagnostics, Inc. Irvine, Calif.). Data are presented as mean and +/-standard deviation of mean for 4 animal assays. Other blood cell parameters, such as white blood cell count or red blood cell count, were not affected by these treatments (data not shown).

All forms of mpl ligand stimulate an increase in platelet count. However, the activity varies in different forms. The relative in vivo activity was mpl ligand MK1-174 (E.coli) < mpl ligand 1-174(CHO) < N4(CHO) < mpl ligand 1-332(CHO) < N15 (CHO). These results indicate that the addition of non-naturally occurring N-type carbohydrates leads to an increase in vivo activity, and that increasing the amount of carbohydrates proportionally increases in vivo activity.

Example 14

Construction of Mpl ligand analogs and truncates N16-N40 by overlapping PCR

The analogs N16 to N40 (see Table 6 for structures of these analogs) were constructed by overlap PCR (polymerase chain reaction) using a method modified according to the method described by Cheng et al, PNAS 91, 5695 (1994). Typically 1 to 2 mutations are introduced into each construct.

The following oligonucleotide primers were synthesized for the preparation of the analogs N16-N40:

5′F CCCTCTAGACCACCATGGAACTGACTGAATTGCTCCTC SEQ ID NO.：18

3′R(1-174)CCCGTCGACTCAGAGCTCGTTCAGTGTG SEQ ID NO.：19

N16 - 3′R CCCGTCGACTCACTCCAACAATCCAGAAG SEQ ID No.：20

N17 - 3′R CCCGTCGACTTATCTGGCTGAGGCAGTGA SEQ ID NO.：21

N18 - F CACGTCCTTAACAGCAGCCTGAGCCAGTG SEQ ID NO.：22

N18 - R CACTGGCTCAGGCTGCTGTTAAGGACGTG SEQ ID NO.：23

N19 - F CCCTTTGCCTAACGGTTCCCTGCTGCCTGCTGT SEQ ID NO.：24

N19 - R ACAGCAGGCAGCAGGGAACCGTTAGGCAAAGGG SEQ ID NO.：25

N20 - F TGCCTACACCTAACCTGTCGCCTGCTGTGGA SEQ ID NO.：26

N20 - R TCCACAGCAGGCGACAGGTTAGGTGTAGGCA SEQ ID NO.：27

N21 - F GGAAAACCAATATGTCGGAGACCAAGGCACA SEQ ID NO.：28

N21 - R TGTGCCTTGGTCTCCGACATATTGGTTTTCC SEQ ID NO.：29

N22 - F TGGGAGAATGGAACACCACGATGGAGGAGACC SEQ ID NO.：30

N22 - R GGTCTCCTCCATCGTGGTGTTCCATTCTCCCA SEQ ID NO.：31

N23 - F AAAACCCAGATGAACGAGACGACCAAGGCACA SEQ ID NO.：32

N23 - R TGTGCCTTGGTCGTCTCGTTCATCTGGGTTTT SEQ ID NO.：33

N24 - F CCCAGATGGAGAACACCTCGGCACAGGACAT SEQ ID NO.：34

N24 - R ATGTCCTGTGCCGAGGTGTTCTCCATCTGGG SEQ ID NO.：35

N25 - F CACGGGGACAAAACGGAACCACTTGCCTCTCA SEQ ID NO.：36

N25 - R TGAGAGGCAAGTGGTTCCGTTTTGTCCCCGTG SEQ ID NO.：37

N26 - F CAGGGCAGGAACACATCTCACAAGGATCCCA SEQ ID NO.：38

N26 - R TGGGATCCTTGTGAGATGTGTTCCTGCCCTG SEQ ID NO.：39

N27 - F GGGCAGGACCAACGCTAGCAAGGATCCCAAT SEQ ID NO.：40

N27 - R ATTGGGATCCTTGCTAGCGTTGGTCCTGCCC SEQ ID NO.：41

N29 - F pair1 CAGTGCAACGAGTCCCACCCTTGG SEQ ID NO.：42

N29 - R pair1 CAAAGGGTGGGACTCGTTGCACTG SEQ ID NO.：43

N29 - F pair2 GACCACAAATCACTCCGATCCCAA SEQ ID NO.：44

N29 - R pair2 TTGGGATCGGAGTGATTTGTGGTC SEQ ID NO.：45

N30 - F GTCCCCACCAACACCTCTCTAGTCCTC SEQ ID NO.：46

N30 - R GAGGACTAGAGAGGTGTTGGTGGGGAC SEQ ID NO.：47

N31 - 3′ R CCCGTCGACTCACTTCAGAAGCCCAGAGCCAGT SEQ ID NO.：48

N36(1) - F GAAAACCCAGAACGAGACCACCAAGGCACAG SEQ ID NO.：49

N36(1) - R CTGTGCCTTGGTGGTCTCGTTCTGGGTTTTC SEQ ID NO.：50

N36(2) - F CACCAAGGCACAGGACATTCTGGGAG SEQ ID NO.：51

N36(2) - R CTCCCAGAATGTCCTGTGCCTTGGTG SEQ ID NO.：52

N37 - F GAAAACCCAGATGAACGAGACCAAGGCACAG SEQ ID NO.：53

N37 - R CTGTGCCTTGGTCTCGTTCATCTGGGTTTTC SEQ ID NO.：54

N38 - F GTCCCCACCAACACCACTCTAGTCCTC SEQ ID NO.：55

N38 - R GAGGACTAGAGTGGTGTTGGTGGGGAC SEQ ID NO.：56

f is positive direction

R is reverse

The introduction of a new glycosylation site into the construct is carried out in two sequential steps. In the first step two reactions were performed with 4 different oligonucleotides. These oligonucleotides include a 5 'forward primer, a reverse mutagenic primer, a forward mutagenic primer (usually complementary to a reverse mutagenic primer) and a reverse 3' primer. The reverse 3' primer contains the sequence of a stop codon and a SalI restriction site. Stop codons were introduced at positions 175, 184, 192 and 200. Thus, forms having lengths of 1-174, 1-183(N16), 1-191(N17), and 1-199(N31) are available. PCR1 uses template DNA (pDSR α 2 containing mpl ligand 1-174 sequence or full-length mpl ligand 1-332 sequence), a 5' forward primer and a reverse mutagenesis primer. PCR2 used template DNA, 3' reverse primer and forward mutagenesis primer. Two PCR reactions were then performed and the amplified DNA fragments were separated by agarose gel electrophoresis. Agarose patches containing DNA fragments of appropriate size were cut from the gel.

The DNA fragments from PCR1 and PCR2 were pooled and a 3 rd PCR reaction was performed using the 5 'forward primer and the 3' reverse primer. Thus, a full-length DNA fragment containing the mpl ligand inserted with the desired mutation is amplified.

The amplified fragments were separated by agarose gel electrophoresis and DNA fragments of appropriate size were isolated by Geneclean^TMThe kit was purified using the method provided by the manufacturer (Bio 101, Inc.). The purified DNA was digested with XbaI and SalI, and then with Geneclean^TMAnd (5) purifying the kit. This fragment was ligated to pDSR α 2 excised with XbaI and SalI. The ligated DNA was precipitated with 2 volumes of ethanol containing 0.3M NaOAc (pH5.2) in the presence of the vector tRNA and transformed into E.coli. Clones were examined by restriction analysis and agarose gel electrophoresis to identify clones containing DNA inserts of the appropriate size. Purified plasmid DNA was then prepared and the mpl ligand insert sequenced to confirm the presence of the desired mutation and to make sure that no additional amino acid changes were introduced.

In some cases, two or more mutations are present simultaneously, as can be seen in N29, N33, N34, N35, N39 and N40. This can be done by introducing a new substitution into the DNA which already contains an alteration. For example, N33 was obtained by introducing a change in N23 to N15. In this case, the above process is repeated using the N23 mutagenic primer and the N15 template DNA.

Another approach may be to introduce two changes simultaneously into the template DNA. The template DNA may contain the native sequence or a sequence encoding an mpl ligand that already contains the alterations. In this case, step 1 comprises 3 PCR reactions and 6 oligonucleotides. These oligonucleotides include a 5 'forward primer, 2 pairs of forward and reverse mutagenic primers and a reverse 3' primer. Each pair of primers is complementary to each other and contains a predetermined sequence introducing a new glycosylation site.

PCR1 includes template DNA, 5' forward primer and reverse mutagenesis primer in pair 1. PCR2 includes template DNA, forward mutagenic primer in pair 1 and reverse mutagenic primer in pair 2. Wherein the 2 nd primer is located 3' to the 1 st primer. PCR3 includes template DNA, forward mutagenesis primer in pair 2 and reverse 3' primer.

The DNA fragments of each PCR reaction were separated by agarose gel electrophoresis as described above and the gel was excised. The 3 DNA fragments were then pooled together and PCR amplified using 5 'forward and 3' reverse primers.

A DNA fragment encoding the entire target gene and having a sequence of two novel glycosylation sites was purified as described above, digested with XbaI and SalI, and ligated to XbaI and SalI-cleaved pDSRa 2.

Multiple mutations can also be combined by performing a PCR reaction using a template that already contains the mutation. For example, introduction of changes of N36 and N38 into N15 template DNA yielded N39. This can be done by using a set of primers (N36(2)) different from the N36(N36(1)) primers. See primers listed above. Both sets of primers introduced the same mutation.

Longer mpl ligand forms can also be produced. Thus, N40 can be generated in a similar manner to N39, except that the 3' reverse primer of PCR3 (step 1) and the PCR primer in step 2 are the primers used to generate N31. This primer introduced a stop codon at position 200, followed downstream by a SalI restriction site. In addition, the template DNA used for PCR3 contained sequences encoding the full-length mpl ligand (1-332).

A typical PCR reaction mixture includes: forward and reverse primers (5 pm/. mu.l) 4. mu.l each, 1. mu.l template (50ng), 10. mu.l 5 XP buffer (100mM N-tris (hydroxymethyl) methylglycine) pH 8.7/25% glycerol/425 mM KOAc), 10. mu.l dNTP stock (1 mM each of dATP, dTTP, dCTP, dGTP), 0.8. mu.l rtTh polymerase (Perkin Elmer; 2.5U/. beta.l) and 2. mu.l Vent polymerase (NEB; 0.01U/. mu.l of a freshly diluted solution in 1 XLP buffer 1: 100). Water was added to make the final volume 50. mu.l. The ingredients were added in the order listed and 1. mu.l of 50mM MgOAc was added to initiate PCR when the temperature rose above 60 ℃ at cycle 1. The reaction conditions are as follows: the first 2 cycles: 94 ℃, 10 seconds/45 ℃, 1 minute/68 ℃, 5 minutes; another 25 cycles were performed: 94 ℃, 10 sec/55 ℃, 1 min/68 ℃, 5 min.

These general procedures were used to construct the mpl ligand analogs and truncations N16 through N40 listed in table 6. Various forms of DNA sequence variations have also been listed.

TABLE 6

MPL ligand analogs with sites for N-type carbohydrate chain incorporation

Analogue/sequence variation

Substitution of amino acids with class number

N16 (1-183)；Tnr¹⁸⁴→Gly³³²Deletion ACA → TGA (stop codon)

N17 (1-191)；Thr¹⁹²→Gly³³²Deletion ACT → TAA (stop codon)

N18 His²³，Arg²⁵→Asn²³，Ser²⁵ CAC，AGA→AAC，AGC

N19 Thr³⁷，Pro³⁸，Val³⁹→ ACA，CCT，GTC

Asn³⁷，Gly³⁸，Ser³⁹ →AAC，GGT，TCC

N20 Val³⁹，Leu⁴¹→Asn³⁹，Ser⁴¹ GTC，CTG→AAC，TCG

N21 Gln⁵⁴，Glu⁵⁶→Asn⁵⁴，Ser⁵⁶ CAG，GAG→AAT，TCG

N22 Lys⁵²，Gln⁵⁴→Asn⁵²，Thr⁵⁴ AAA，CAG→AAC，ACG

N23 Glu⁵⁷→Asn^55′(i)，Thr⁵⁷ GAG→AAC(i)，ACG

N24 Glu⁵⁷，Lys⁵⁹→Asn⁵⁷，Ser⁵⁹ GAG，AAG→AAC，TCG

N25 Leu⁸¹，Pro⁸³→Asn⁸¹，Thr⁸³ CTG，CCC→AAC，ACC

N26 Thr¹¹⁸，Ala¹²⁰→Asn¹¹⁸，Ser¹²⁰ ACC，GCT→AAC，TCT

N27 Thr¹¹⁹，His¹²¹→Asn¹¹⁹，Ser¹²¹ ACA，CAC→AAC，AGC

N29 Pro³⁰，Val³²，Ala¹²⁰，Lys¹²²→ CCA，GTT，GCT，AAG→

Asn³⁰，Ser³²，Asn¹²⁰，Ser¹²² AAC，TCC，AAT，TCC

N30 Ser¹⁶³，Arg¹⁶⁴→Thr¹⁶³，Asn¹⁶⁴ AGC，AGA→ACC，AAC

N31 (1-199)；Trp²⁰⁰→Gly³³²Deletion TGG → TGA (stop codon)

N33 Pro30，Val³²，Glu⁵⁷，Ala¹²⁰，Lys¹²² CCA，GTT，GAG，GCT，AAG

→ →

Asn³⁰，Thr³²，Asn^55′(i)，Thr⁵⁷ AAC，TCC，AAC(i)，ACG，

Asn¹²⁰，Thr¹²² AAT，TCC

N34 Pro³⁰，Val³²，Glu⁵⁷，Ser¹⁶³，Arg¹⁶⁴ CCA，GTT，GAG，AGC，AGA

→ →

Asn³⁰，Thr³²，ASn^55′(i)，Thr⁵⁷， AAC，TCC，AAC(i)，ACG，

Thr¹⁶³，ASn¹⁶⁴ ACC，AAC

N35 N4+N23+N30+N31(1-199) --

N36 Met⁵⁵，Glu⁵⁷→Asn⁵⁵，Thr⁵⁷ ATG，GAG→AAC，ACC

N37 Glu⁵⁶→Asn⁵⁶ GAG→AAC

N38 Ser¹⁶³，Arg¹⁶⁴，Ser¹⁶⁶ AGC，AGA，TCT

→Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶ →ACC，AAC，ACT

N39 N4+N10+N36+N38(1-174) --

N40 N4+N10+N36+N38+N31(1-199) --

In the above table, "(i)" indicates the inserted amino acid. For example, Glu⁵⁷→Asn^55′(i)，Thr⁵⁷(analog 23 in Table 6) shows that Glu at position 57 is substituted with Thr, and, in addition, an Asn is inserted between Met at positions 55 and 56. Asn numbering is 55' so that the following amino acids retain their original numbering.

Examples including all variations in the previous embodiments are indicated with a "+" connection specific analog number. See analogs N35, N39, and N40. The amino acid chain lengths of these analogs are indicated in parentheses. Thus, analog N35 includes all of the changes made in analogs N4, N24, N30, and N31. The changes indicated in N31 indicate that the analog N35 is 199 amino acids long. All analogs in table 6 are 174 amino acids in length, with the exception of the different lengths indicated (or the total length increases the number of amino acids inserted when there are amino acid insertions).

Example 15

Identification of Mpl ligand analogs and truncates N16 to N40

Determination of expression level and in vitro biological Activity of mpl ligand analogs

Electroporation of N16 to N40 (example 5) or CaPO₄COS cells are transfected by the method (mammalian cell transfection kit; specially prepared culture medium). Collecting cell-free culture medium after 3-4 days, subpackaging into small parts, and storing at-70 deg.C. Expression levels were determined by ELISA assay as described in example 7. The supernatant was assayed for biological activity according to the modified assay of example 9. However, the method of example 9 was slightly modified. Activity was calculated using a standard curve prepared using mpl ligand 1-332 expressed by purified CHO cells as a standard.

The results are shown in Table 7. As can be seen from Table 7, most mpl ligand analogs are expressed and secreted. Some analogs appear to have increased secretion. Biological assays on these samples showed that the specific activity of most samples was comparable to the unmodified form. Some analogs contain multiple N-type carbohydrate chains (see below). This indicates that the addition of carbohydrate results in increased secretion of the analogue, while the in vitro activity is normal.

TABLE 7

Elisa in vitro Activity specific Activity of the N-type chain of the Mpl ligand form sequence number (amino acid length) (ng/ml) (Unit/ng) (a) (b) (c) (d)

N1(174) Natural 0283991143N 15(174) N30T32N120T 1220-2457003156N 16(183) 1-1830859276 NAN17(191) 1-191 NA < 0.311 NAN18(174) N23S250252.5N19 (174) N37G38S39 NA < 0.3 NA NAN20(174) N39S41 NA < 0.3 < 10 NAN21(174) N54S 560-1304380146N 22(174) N52T 540-12856428N 23(174) N55' (i) T57111105996N 24(174) N57S5905.345886N25 (174) N81T83 NA 0.22123559N 26(174) N118S120NA 0.996106N 27(174) N119S12104.533875N29 (174) N30S32N120S 1220-2151627108N 30(174) T163N 1640-112815592122N 31(199) 1-199 at least 115619000122N 33(174) 4+10+ 2337810057129N 34(174) 4+23+30 at least 211213536120N 35(199) 34+ 314 or 4-plus 1721311276N 36(174) N55T 570-1485808121N 37(174) N561324504141N 38(174) T163N164T 0-1253904156N 39(174) N4+ N10+ N36+ N383 to 412717661139N 40(199) N4+ N10+ N36+ N38+ N31 at least 513419735147

Attention is paid to

(a) The number of N-type chains added was estimated based on the mobility of the analog polypeptide in SDS gels.

(b) The amount of mpl ligand analogue in the supernatant of COS cells was determined by EIA.

(c) In vitro activity was determined by measuring proliferation of 32D-MPL cells grown in dependence on MPL ligand.

(d) The ratio of the in vitro activity of the mpl ligand analog measured by the proliferation assay to the amount of mpl ligand analog measured by the mpl ligand ELISA.

i-insertion of

NA: is not obtained

B. Determination of carbohydrate addition.

The analogs listed in table 6 were tested to determine if N-type carbohydrate was added using the method described in example 6.

Certain analogs (N21, N22, N30, N33 and N36) were also tested in an improved manner. This is necessary because the monoclonal antibodies used to perform the Western blot were generated using a peptide comprising amino acid residues 47 to 62, and some of the analogs listed in Table 6 (e.g., N21) contain certain substitutions that affect their immunoreactivity with this antibody. Therefore, to analyze these analogs, supernatants were immunoprecipitated with mouse monoclonal antibodies generated from mpl ligand 1-163 expressed by E.coli cells.

Typically, 50ng of mpl ligand analog is immunoprecipitated with 15. mu.g of antibody. Western blotting of the immunoprecipitates was carried out as described in example 6, but the immunoprecipitated bands were developed with rabbit anti-mpl ligand polyclonal antibody (usually 1. mu.g/ml; produced with mpl ligand 1-163 expressed by E.coli cells) and anti-rabbit ECL kit (Amersham) incubated with the blots. The results of the various tests are shown in Table 7. The size of some analogs increased, indicating the presence of N-type carbohydrates (N21, N22, N23, N29, N30, N31, N33, N34, N35, N36, N38, N39, and N40). Some of these analogues have more than 1N-type chain, such as N29, N33, N34, N35, N39 and N40. These analogs are secreted at normal or higher levels and their in vitro biological activity is comparable to mpl ligand 1-174. This indicates that multifunctional N-type glycosylation sites can be introduced into the mpl ligand without adversely affecting its expression or biological activity.

To confirm that multiple oligosaccharide chains can be added to the mpl ligand, Western blot analysis of each analog expressed in COS cells was performed as described in example 6. FIG. 12 shows that the mobility of the analogs decreases with increasing addition of N-type glycosylation sites. The analogs with 4 new positions in the figure are N39 and N40. The most N-type site analogs move the slowest. This result is seen with mpl ligands in both the 1-174 and 1-199 formats. This suggests that at least 4 analogs can be combined together to produce new analogs with multiple N-type carbohydrate chains.

Example 16

Asn-X-Ser-containing glycosylation site and polypeptide containing the same

Comparison of glycosylation sites of Asn-X-Thr

N-type glycosylation sites include Asn-X-Thr or Asn-X-Ser, where X can be any of the 20 natural amino acids other than Pro. We wanted to determine whether the third position is preferably Ser or Thr. Thus, two sets of analogues, both containing mpl ligand glycosylation, Ser or Thr at position 3 of sequon, respectively, were tested to determine whether the percentage occupancy of N-type glycosylation sites was affected. N15 contains 2 Asn-X-Thr sites, while N29 contains 2 Asn-X-Ser at the same position. Similarly, N30 contains an Asn-X-Ser, while N38 contains an Asn-X-Thr at the same position.

To compare the two sets of analogs, they were expressed in COS cells and the secreted mpl ligand was subjected to Western analysis as described in example 6. These results are presented in figure 13. The proportion of glycosylated mpl ligand in N15 was significantly increased compared to N29. In contrast, the ratio of glycosylated and unglycosylated mpl ligands differed very little when comparing N30 and N38. These results indicate that both Asn-X-Ser and Asn-X-Thr can introduce mpl ligands and that they can act as addition sites for N-type carbohydrates. In addition, Asn-X-Thr sequon is preferred in some cases (i.e., it can be glycosylated more efficiently).

While the invention has been described in terms of preferred embodiments, it is not intended to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims, which scope is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

Sequence listing

(1) General information:

(i) the applicant: elliott, Steven G.

(ii) The invention name is as follows: MPL ligand analogues

(iii) Sequence number: 56

(iv) Communication address:

(A) the contact person: AMGEN INC

(B) Street: 1840 Dehavolland Drive

(C) City: thousand Oaks

(D) State: CA

(E) The state is as follows: USA

(F) And (3) post code: 91320-1789

(v) A computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatibility

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patentin Release #1.0, version #1.30

(vi) The data of the application:

(A) application No.:

(B) application date:

(C) classification number:

(2) SEQ ID NO: 1 information

(i) Sequence characterization

(A) Length: 1342 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/key: CDS

(B) Position: 36..1094

(ix) Is characterized in that:

(A) name/key: signal peptide

(B) Position: 1..98

(ix) Is characterized in that:

(A) name/key: mature peptides

(B) Position: 99..1094

(xi) Sequence representation: SEQ ID NO: 1:

CAGGGAGCCA CGCCAGCCAA GACACCCCGG CCAGA ATG GAG CTG ACT GAA TTG 53

Met Glu Leu Thr Glu Leu

-21 -20

CTC CTC GTG GTC ATG CTT CTC CTA ACT GCA AGG CTA ACG CTG TCC AGC 101

Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu Ser Ser

15 -10 -5 1

CCG GCT CCT CCT GCT TGT GAC CTC CGA GTC CTC AGT AAA CTG CTT CGT 149

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

5 10 15

GAC TCC CAT GTC CTT CAC AGC AGA CTG AGC CAG TGC CCA GAG GTT CAC 197

Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His

20 25 30

CCT TTG CCT ACA CCT GTC CTG CTG CCT GCT GTG GAC TTT AGC TTG GGA 245

Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly

35 40 45

GAA TGG AAA ACC CAG ATG GAG GAG ACC AAG GCA CAG GAC ATT CTG GGA 293

Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly

50 55 60 65

GCA GTG ACC CTT CTG CTG GAG GGA GTG ATG GCA GCA CGG GGA CAA CTG 341

Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu

70 75 80

GGA CCC ACT TGC CTC TCA TCC CTC CTG GGG CAG CTT TCT GGA CAG GTC 389

Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val

85 90 95

CGT CTC CTC CTT GGG GCC CTG CAG AGC CTC CTT GGA ACC CAG CTT CCT 437

Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro

100 105 110

CCA CAG GGC AGG ACC ACA GCT CAC AAG GAT CCC AAT GCC ATC TTC CTG 485

Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu

115 120 125

AGC TTC CAA CAC CTG CTC CGA GGA AAG GTG CGT TTC CTG ATG CTT GTA 533

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

130 135 140 145

GGA GGG TCC ACC CTC TGC GTC AGG CGG GCC CCA CCC ACC ACA GCT GTC 581

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

150 155 160

CCC AGC AGA ACC TCT CTA GTC CTC ACA CTG AAC GAG CTC CCA AAC AGG 629

Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg

165 170 175

ACT TCT GGA TTG TTG GAG ACA AAC TTC ACT GCC TCA GCC AGA ACT ACT 677

Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr

180 185 190

GGC TCT GGG CTT CTG AAG TGG CAG CAG GGA TTC AGA GCC AAG ATT CCT 725

Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro

195 200 205

GGT CTG CTG AAC CAA ACC TCC AGG TCC CTG GAC CAA ATC CCC GGA TAC 773

Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr

210 215 220 225

CTG AAC AGG ATA CAC GAA CTC TTG AAT GGA ACT CGT GGA CTC TTT CCT 821

Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro

230 235 240

GGA CCC TCA CGC AGG ACC CTA GGA GCC CCG GAC ATT TCC TCA GGA ACA 869

Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr

245 250 255

TCA GAC ACA GGC TCC CTG CCA CCC AAC CTC CAG CCT GGA TAT TCT CCT 917

Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser Pro

260 265 270

TCC CCA ACC CAT CCT CCT ACT GGA CAG TAT ACG CTC TTC CCT CTT CCA 965

Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro

275 280 285

CCC ACC TTG CCC ACC CCT GTG GTC CAG CTC CAC CCC CTG CTT CCT GAC 1013

Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro Asp

290 295 300 305

CCT TCT GCT CCA ACG CCC ACC CCT ACC AGC CCT CTT CTA AAC ACA TCC 1061

Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser

310 315 320

TAC ACC CAC TCC CAG AAT CTG TCT CAG GAA GGG TAAGGTTCTC AGACACTGCC 1114

Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly

325 330

GACATCAGCA TTGTCTCGTG TACAGCTCCC TTCCCTGCAG GGCGCCCCTG GGAGACAACT 1174

GGACAAGATT TCCTACTTTC TCCTGAAACC CAAAGCCCTG GTAAAAGGGA TACACAGGAC 1234

TGAAAAGGGA ATCATTTTTC ACTGTACATT ATAAACCTTC AGAAGCTATT TTTTTAAGCT 1294

ATCAGCAATA CTCATCAGAG CAGCTAGCTC TTTGGTCTAT TTTCTGCA 1342

(2) SEQ ID NO: 2 information

(i) Sequence characterization

(A) Length: 353 amino acid

(B) Type (2): amino acids

(D) Topological shape: line shape

(ii) Molecular type: protein

(xi) Sequence representation: SEQ ID NO: 2:

Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala

-21 -20 -15 -10

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

5 1 5 10

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

15 20 25

Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55

Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

60 65 70 75

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

80 85 90

Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105

Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120

Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 130 135

Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala

140 145 150 155

Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu

160 165 170

Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr

175 180 185

Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly

190 195 200

Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu

205 210 215

Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly

220 225 230 235

Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro

240 245 250

Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu

255 260 265

Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr

270 275 280

Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu

285 290 295

His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser

300 305 310 315

Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu

320 325 330

Gly

(2) SEQ ID NO: 3 information

(i) Sequence characterization

(A) Length: 600 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/key: CDS

(B) Position: 12..596

(ix) Is characterized in that:

(A) name/key: signal peptide

(B) Position: 12..74

(ix) Is characterized in that:

(A) name/key: mature peptides

(B) Position: 75..96

(xi) Sequence representation: SEQ ID NO: 3:

TCTAGACCAC C ATG GAG CTG ACT GAA TTG CTC CTC GTG GTC ATG CTT CTC 50

Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu

-21 -20 -15 -10

CTA ACT GCAAGG CTA ACG CTG TCC AGC CCG GCT CCT CCT GCT TGT GAC 98

Leu Thr Ala Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp

5 1 5

CTC CGA GTC CTC AGT AAA CTG CTT CGT GAC TCC CAC GTC CTT CAC AGC 146

Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Vai Leu His Ser

10 15 20

AGA CTG AGC CAG TGC CCA GAG GTT CAC CCT TTG CCT ACA CCT GTC CTG 194

Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu

25 30 35 40

CTG CCT GCT GTG GAC TTT AGC TTG GGA GAA TGG AAA ACC CAG ATG GAG 242

Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu

45 50 55

GAG ACC AAG GCA CAG GAC ATT CTG GGA GCA GTG ACC CTT CTG CTG GAG 291

Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu

60 65 70

GGA GTG ATG GCA GCA CGG GGA CAA CTG GGA CCC ACT TGC CTC TCA TCC 331

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

75 80 85

CTC CTG GGG CAG CTT TCT GGA CAG GTC CGT CTC CTC CTT GGG GCC CTG 386

Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu

90 95 100

CAG AGC CTC CTT GGA ACC CAG CTT CCT CCA CAG GGC AGG ACC ACA GCT 434

Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala

105 110 115 120

CAC AAG GAT CCC AAT GCC ATC TTC CTG AGC TTC CAA CAC CTG CTC CGA 482

His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg

125 130 135

GGA AAG GTG CGT TTC CTG ATG CTT GTA GGA GGG TCC ACC CTC TGC GTC 530

Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val

140 145 150

AGG CGG GCC CCA CCC ACC ACA GCT GTC CCC AGC AGA ACC TCT CTA GTC 578

Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val

155 160 165

CTC ACA CTG AAC GAG CTC TAGG 600

Leu Thr Leu Asn Glu Leu

170

(2) SEQ ID NO: 4 information

(i) Sequence characterization

(A) Length: 195 amino acid

(B) Type (2): amino acids

(D) Topological shape: line shape

(ii) Molecular type: protein

(xi) Sequence representation: SEQ ID NO: 4:

Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala

-21 -20 -15 -10

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

5 1 5 10

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

15 20 25

Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala

30 35 40

Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys

45 50 55

Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met

60 65 70 75

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

80 85 90

Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu

95 100 105

Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp

110 115 120

Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val

125 130 135

Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala

140 145 150 155

Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu

160 165 170

Asn Glu Leu

(2) SEQ ID NO: 5 information

(i) Sequence characterization

(A) Length: 22 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 5:

CCCATGTCAA TCACAGCAGA CT 22

(2) SEQ ID NO: 6 information

(i) Sequence characterization

(A) Length: 22 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 6:

CTTCACAGCA ACCTGAGCCA GT 22

(2) SEQ ID NO: 7 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 7:

CAGTGCAACG AGACCCACCC TTTG 24

(2) SEQ ID NO: 8 information

(i) Sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 8:

GCCTACAAAT GTCACGCTGC CTGCT 25

(2) SEQ ID NO: 9 information

(i) Sequence characterization

(A) Length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 9:

CCCACTTGTA ACTCATCCCT C 21

(2) SEQ ID NO: 10 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 10:

CAACTGAACG CCACTTGTCT CTCA 24

(2) SEQ ID NO: 11 information

(i) Sequence characterization

(A) Length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 11:

ACTTGTCTCA ACTCCACCCT GGGGGA 26

(2) SEQ ID NO: 12 information

(i) Sequence characterization

(A) Length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: the term "nucleic acid"/desC ═ nucleic acid "

(xi) Sequence representation: SEQ ID NO: 12:

CTCCTGGGGA ACCTTTCTGG A 21

(2) SEQ ID NO: 13 information

(i) Sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 13:

GACCACAAAT CACACCGATC CCAAT 25

(2) SEQ ID NO: 14 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 14:

ACCCTTTGTC TACAAATGTC ACGCTGCCTG CT 32

(2) SEQ ID NO: 15 information

(i) Sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 15:

TCTCTCAAAC CTCACGGGGG AGCTT 25

(2) SEQ ID NO: 16 information

(i) Sequence characterization

(A) Length: 23 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 16:

TGGAAAAATC AGACGGAGGA GAC 23

(2) SEQ ID NO: 17 information

(i) Sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 17:

TGGAGGAGAA CAAGACACAG GACAT 25

(2) SEQ ID NO: 18 information

(i) Sequence characterization

(A) Length: 38 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 18:

CCCTCTAGAC CACCATGGAA CTGACTGAAT TGCTCCTC 38

(2) SEQ ID NO: 19 information

(i) Sequence characterization

(A) Length: 28 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..28)

(xi) Sequence representation: SEQ ID NO: 19:

GTGTGACTTG CTCGAGACTC AGCTGCCC 28

(2) SEQ ID NO: 20 information

(i) Sequence characterization

(A) Length: 29 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..29)

(xi) Sequence representation: SEQ ID NO: 20:

GAAGACCTAA CAACCTCACT CAGCTGCCC 29

(2) SEQ ID NO: 21 information

(i) Sequence characterization

(A) Length: 29 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..29)

(xi) Sequence representation: SEQ ID NO: 21:

AGTGACGGAG TCGGTCTATT CAGCTGCCC 29

(2) SEQ ID NO: 22 information

(i) Sequence characterization

(A) Length: 29 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 22:

CACGTCCTTA ACAGCAGCCT GAGCCAGTG 29

(2) SEQ ID NO: 23 information

(i) Sequence characterization

(A) Length: 29 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..29)

(xi) Sequence representation: SEQ ID NO: 23:

GTGCAGGAAT TGTCGTCGGA CTCGGTCAC 29

(2) SEQ ID NO: 24 information

(i) Sequence characterization

(A) Length: 33 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 24:

CCCTTTGCCT AACGGTTCCC TGCTGCCTGC TGT 33

(2) SEQ ID NO: 25 information

(i) Sequence characterization

(A) Length: 33 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..33)

(xi) Sequence representation: SEQ ID NO: 25:

GGGAAACGGA TTGCCAAGGG ACGACGGACG ACA 33

(2) SEQ ID NO: 26 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 26:

TGCCTACACC TAACCTGTCG CCTGCTGTGG A 31

(2) SEQ ID NO: 27 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 27:

ACGGATGTGG ATTGGACAGC GGACGACACC T 31

(2) SEQ ID NO: 28 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 28:

GGAAACCAA TATGTCGGAG ACCAAGGCAC A 31

(2) SEQ ID NO: 29 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 29:

CCTTTTGGTT ATACAGCCTC TGGTTCCGTG T 31

(2) SEQ ID NO: 30 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 30:

TGGGAGAATG GAACACCACG ATGGAGGAGA CC 32

(2) SEQ ID NO: 31 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..32)

(xi) Sequence representation: SEQ ID NO: 31:

ACCCTCTTAC CTTGTGGTGC TACCTCCTCT GG 32

(2) SEQ ID NO: 32 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 32:

AAAACCCAGA TGAACGAGAC GACCAAGGCA CA 32

4(2) SEQ ID NO: 33 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..32)

(xi) Sequence representation: SEQ ID NO: 33:

TTTTGGGTCT ACTTGCTCTG CTGGTTCCGT GT 32

(2) SEQ ID NO: 34 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 34:

CCCAGATGGA GAACACCTCG GCACAGGACA T 31

(2) SEQ ID NO: 35 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 35:

GGGTCTACCT CTTGTGGAGC CGTGTCCTGT A 31

(2) SEQ ID NO: 36 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 36:

CACGGGGACA AAACGGAACC ACTTGCCTCT CA 32

(2) SEQ ID NO: 37 information

(i) Sequence characterization

(A) Length: 32 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..32)

(xi) Sequence representation: SEQ ID NO: 37:

GTGCCCCTGT TTTGCCTTGG TGAACGGAGA GT 32

(2) SEQ ID NO: 38 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 38:

CAGGGCAGGA ACACATCTCA CAAGGATCCC A 31

(2) SEQ ID NO: 39 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 39:

GTCCCGTCCT TGTGTAGAGT GTTCCTAGGG T 31

(2) SEQ ID NO: 40 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 40:

GGGCAGGACC AACGCTAGCA AGGATCCCAA T 31

(2) SEQ ID NO: 41 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 41:

CCCGTCCTGG TTGCGATCGT TCCTAGGGTT A 31

(2) SEQ ID NO: 42 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 42:

CAGTGCAACG AGTCCCACCC TTGG 24

(2) SEQ ID NO: 43 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..24)

(xi) Sequence representation: SEQ ID NO: 43:

GTCACGTTGC TCAGGGTGGG AAAC 24

(2) SEQ ID NO: 44 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 44:

GACCACAAAT CACTCCGATC CCAA 24

(2) SEQ ID NO: 45 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..24)

(xi) Sequence representation: SEQ ID NO: 45:

CTGGTGTTTA GTGAGGCTAG GGTT 24

(2) SEQ ID NO: 46 information

(i) Sequence characterization

(A) Length: 27 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 46:

GTCCCCACCA ACACCTCTCT AGTCCTC 27

(2) SEQ ID NO: 47 information

(i) Sequence characterization

(A) Length: 27 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..27)

(xi) Sequence representation: SEQ ID NO: 47:

CAGGGGTGGT TGTGGAGAGA TCAGGAG 27

(2) SEQ ID NO: 48 information

(i) Sequence characterization

(A) Length: 33 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..33)

(xi) Sequence representation: SEQ ID NO: 48:

TGACCGAGAC CCGAAGACTT CACTCAGCTG CCC 33

(2) SEQ ID NO: 49 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 49:

GAAAACCCAG AACGAGACCA CCAAGGCACA G 31

(2) SEQ ID NO: 50 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 50:

CTTTTGGGTC TTGCTCTGGT GGTTCCGTGT C 31

(2) SEQ ID NO: 51 information

(i) Sequence characterization

(A) Length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 51:

CACCAAGGCA CAGGACATTC TGGGAG 26

(2) SEQ ID NO: 52 information

(i) Sequence characterization

(A) Length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..26)

(xi) Sequence representation: SEQ ID NO: 52:

GTGGTTCCGT GTCCTGTAAG ACCCTC. 26

(2) SEQ ID NO: 53 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 53:

GAAAACCCAG ATGAACGAGA CCAAGGCACA G 31

(2) SEQ ID NO: 54 information

(i) Sequence characterization

(A) Length: 31 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..31)

(xi) Sequence representation: SEQ ID NO: 54:

CTTTTGGGTC TACTTGCTCT GGTTCCGTGT C 31

(2) SEQ ID NO: 55 information

(i) Sequence characterization

(A) Length: 27 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(xi) Sequence representation: SEQ ID NO: 55:

GTCCCCACCA ACACCACTCT AGTCCTC 27

(2) SEQ ID NO: 56 information

(i) Sequence characterization

(A) Length: 27 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological shape: line shape

(ii) Molecular type: other nucleic acids

(A) The expression: nucleic acids "

(ix) Is characterized in that:

(A) name/key: -

(B) Position: complementation (1..27)

(xi) Sequence representation: SEQ ID NO: 56:

CAGGGGTGGT TGTGGTGAGA TCAGGAG 27

Claims

1. An mpl ligand analog, wherein

[Asn²⁵]an mpl ligand;

[Asn³⁰，Thr³²]an mpl ligand;

[Asn⁸²，Ala⁸³]an mpl ligand;

[Asn¹²⁰，Thr¹²²]an mpl ligand;

[Asn⁵³，Thr⁵⁵]an mpl ligand;

[Asn⁵⁸，Thr⁶⁰]an mpl ligand;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²]an mpl ligand;

[Asn⁵⁴，Ser⁵⁶]an mpl ligand;

[Asn⁵²，Thr⁵⁴]an mpl ligand;

[Asn⁸¹，Thr⁸³]an mpl ligand;

[Thr¹⁶³，Asn¹⁶⁴]an mpl ligand;

[Asn⁵⁵，Thr⁵⁷]an mpl ligand;

[Asn⁵⁶]an mpl ligand;

[Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]an mpl ligand; and

2. The analog of claim 1 which is

[Asn²⁵]mpl ligand 1-174;

[Asn³⁰，Thr³²]mpl ligand 1-174;

[Asn⁸²，Ala⁸³]mpl ligand 1-174;

[Asn¹²⁰，Thr¹²²]mpl ligand 1-174;

[Asn⁵³，Thr⁵⁵]mpl ligand 1-174;

[Asn⁵⁸，Thr⁶⁰]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²]mpl ligand 1-174;

[Asn⁵⁴，Ser⁵⁶]mpl ligand 1-174;

[Asn⁵²，Thr⁵⁴]mpl ligand 1-174;

[Asn⁸¹，Thr⁸³]mpl ligand 1-174;

[Thr¹⁶³，Asn¹⁶⁴]mpl ligand 1-174;

[Asn³⁰，Thr³²，Asn¹²⁰，Thr¹²²，Asn⁵⁵(i)，Thr⁵⁷]mpl ligand 1-174;

[Asn⁵⁵，Thr⁵⁷]mpl ligand 1-174;

[Asn⁵⁶]mpl ligand 1-174;

[Thr¹⁶³，Asn¹⁶⁴，Thr¹⁶⁶]mpl ligand 1-174;

3. A DNA sequence encoding the mpl ligand analog of claim 1.

4. A CHO or COS cell transfected with the DNA sequence of claim 3 in a manner such that the host cell expresses the mpl ligand analog.

5. A pharmaceutical composition comprising a therapeutically effective amount of an mpl ligand analog according to claim 1-in combination with a pharmaceutically acceptable diluent, adjuvant or carrier.

6. A DNA sequence encoding the mpl ligand analog of claim 2.

7. A CHO or COS cell transfected with the DNA sequence of claim 6 in a manner such that the host cell expresses an mpl ligand analog.

8. A pharmaceutical composition comprising a therapeutically effective amount of the mpl ligand analog of claim 2, and a pharmaceutically acceptable diluent, adjuvant or carrier.