MPL ligand analogs

专利摘要:
A mpl ligand analog with one or more altered glycosylation sites is described in comparison to the naturally occurring mpl ligand sequence of the corresponding amino acid number. The invention also relates to DNA sequences encoding such mpl ligand analogs, recombinant plasmids for expression of the analogs and host cells and therapeutic compositions comprising such analogs.
公开号:KR19980702206A
申请号:KR1019970705601
申请日:1996-02-14
公开日:1998-07-15
发明作者:스티븐 지. 엘리어트
申请人:스티븐 엠. 오드리；암겐 인코포레이티드；
IPC主号:

专利说明:

MPL ligand analogs
MGDF or megakaryocyte growth and differentiation factor is a recently cloned cytokine and has been shown to be a major regulator of circulating platelet levels. Bartley, T. D. Et al., Cell 77: 1117-1124 (1994); Locke, S. Group 3 Nature: 565-568 (1994); De Sovezi, F. second. Et al., Nature 369: 533-538 (1994); Miyazake, H. Exp. Hematol. 22: 838 (1994); And Cooter, D. second. See PNAS USA, 91: 11104-11108 (1998). Bartley, T. D. MGDF is called thrombopoietin (TPO), mpl ligand and megapoietin as described by Cell 77: 1117-1124 (1994). Here, the term mpl ligand is generally used to refer to any polypeptide that activates a mpl receptor, including TPO and MGDF. The mpl receptor is a cell surface protein that results in the production and / or differentiation of megakaryocytes and platelets upon activation. See WO 92/07074.
A mpl ligand analog is a polypeptide that differs from the native sequence in a way that affects the number, location, or type of hydrocarbon binding site. Such polypeptides are also an object of the present invention. The mature natural human mpl ligand is a protein with a total of 332 amino acids. The sequence of this protein (attached to the leader sequence 21 amino acids long) and the corresponding cDNA are shown in FIG. 1 (SEQ ID NOS: 1 and 2).
Recombinant mpl ligands produced in both Chinese Hamster Ovary (CHO) and Escherichia coli cells have been found to have biological activities that specifically stimulate or increase megakaryocytes and / or platelets in mice, rats and monkeys. For example hunt, blood. See, Blood 84 (10): 390A (1994). Human mpl ligand molecules (compared to the 332 amino acid proteins encoded by human cDNA) that have been cleaved starting at amino acid position 22 in FIG. 1 and continuing to at least 151 amino acids, have biological activity in vivo. FIG. 2 (SEQ ID NOS: 3 and 4) shows an example of a cleaved mpl ligand molecule having 174 amino acids as its mature form and having biological activity. In Figure 2, a 174 amino acid long protein is attached to the 21 amino acid N-terminal leader sequence. This molecule was used to create some mpl ligand analogs in the examples below. Other analogs are based on amino acids 1-199, 1-191 and 1-183 of FIG. 1. The first six amino acids at the N-terminus of the mature human mpl ligand protein sequence can be removed and biological activity can be maintained. Therefore, it can be seen that the biological activity is present in amino acids 7 to 151 (inclusive) of the mature amino acid sequence of the human mpl ligand shown in FIG. 1.
In general, secreted proteins produced from many cell surfaces and eukaryotic cells are modified by one or more oligopolysaccharide groups. Such modifications, also referred to as glycosylation, can have important effects on protein stability, secretion and intracellular location as they have a significant impact on the physical properties of the protein. Suitable glycosylation may also be necessary for biological activity. In fact, some genes from eukaryotes result in proteins with little or no activity due to the lack of glycosylation when expressed in bacteria that lack cellular processes for glycosylating proteins (eg, E. coli).
Glycosylation occurs at specific positions or sites of the polypeptide backbone and is usually of two types: O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides (chain) Is attached to an Asparagine (Asn) residue when they are part of an Asn-X-Ser / Thr, where X may be any amino acid except proline. X is preferably one of 19 naturally occurring amino acids except proline. The structures of N-linked oligosaccharides and O-linked oligosaccharides and the sugar residues bound in each type are different. One type of saccharide commonly found on both sides is N-acetylneuraminic acid (hereinafter referred to as sialic acid). Sialic acid is the terminal residue of N-linked and O-linked oligosaccharides and can impart acidic properties to the glycoprotein due to its negative charge.
As used herein, glycosylation sites are amino acid residues that can be structurally linked to glycosyl residues, but such sites may or may not be substantially bonded to glycosyl residues. As mentioned above, the O-linked site is a Ser or Thr residue while the N-linked site is Asn-X-Ser or Asn-X-Thr, where X is any amino acid except Pro as defined above. (Preferably one of the 19 amino acids of natural output). Whether a given position is glycosylated by the glycosyl chain is determined by the host cell in which the molecule expressed amino acids and other factors around the site.
As used herein, the number of chains attached to a given mpl ligand analog will be the average number of hydrocarbon (ie glycosyl) chains bound to a given mpl ligand molecule expressed by a particular host cell. The glycosylation sites for natural and corresponding recombinant mpl ligands are generally the same, but the number of chains depends on whether the particular cell host used for recombinant expression attaches the glycosyl chain to the same site compared to the natural source. Will be different. When comparing recombinant and natural mpl ligand analogs, the same number of amino acids are compared regardless of whether the natural source substantially produces mpl ligand molecules of that length. Thus, the term natural does not mean the length of a molecule substantially expressed in natural sources, but refers to sequences used in a particular species (such as humans).
Naturally occurring mpl ligands are glycosylated molecules. The glycosylation pattern of the native mpl ligand is linked to two major domains found in the mpl ligand. The first sequence corresponds to the active part of the mpl ligand of mature humans, which is about 151 amino acids and has a significant homology with erythropoietin, a cytokine capable of stimulating red blood cell production. Referred to as the domain. The remaining amino acids of the natural protein make up the so-called N-linked hydrocarbon domain because they include most if not all of the natural sites of N-linked glycosylation. In human mpl ligands, there are all six N-linked glycosylation sites contained in the N-linked glycosylation domain. Both domains contain O-linked glycosylation sites. Among the molecules are an estimated 12 to 14 O-linked glycosylation chains. Experiments with human mpl ligand DNA recombinantly expressed in CHO cells show that at least two O-linked sites in the EPO-like domain are glycosylated at positions 1 (Ser) and 37 (Thr).
Glycoproteins such as mpl ligands can be separated into differently charged forms using techniques such as isoelectric focusing (IEF). For example, several have reported IEF studies of crude erythropoietin preparations and partially purified erythropoietin preparations (Lukovsky gang, J. Biochem. 50: 909 (1972); Shelton gang, Biochem. Med) 12: 45 (1975); Fuhr et al., Biochem. Biophys. Res. Comm. 98: 930 (1981).
Despite the information on glycosylation of the mpl ligand molecules described above, there was still a need to obtain mpl ligand molecules with different glycosylation patterns and with improved biological activity.
It is therefore an object of the present invention to provide novel glycosylated mpl ligand molecules called mpl ligand analogs. Another object of the present invention is to provide a pharmaceutical composition containing the above-described molecule and a treatment method capable of treating mpl ligand using the mpl ligand analog of the present invention.
Summary of the Invention
In one embodiment, the invention provides an analog of a mpl ligand comprising an amino acid sequence having one or more added, one or more deleted and / or one or more added and deleted glycosylation sites compared to the corresponding native mpl ligand sequence It is about. The added or deleted glycosylation site (s) can result in a large or small number of hydrocarbon chains and a high or low sialic acid content relative to the corresponding natural mpl ligand sequence, in particular human mpl ligands. For example, one type of analog may include the deletion of one or more N- or O-linked sites and the addition of one or more N- or O-linked sites at the same or different positions.
As another example of the foregoing embodiments, the invention relates to mpl ligand analogs comprising amino acid sequences in which one or more N- or O-linked glycosylation sites are replaced with one or more non-naturally occurring sites. Thus N-linked sites can be replaced with different N-linked sites; N-linked sites can be replaced with O-linked sites; O-linked sites can be replaced with different O-linked sites; O-linked sites can also be replaced with N-linked sites.
Combinations of the above-described modifications are also included within the scope of the present invention.
The invention also includes DNA sequences encoding such mpl ligand analogs and recombinant plasmids and host cells for analog expression.
In all of the above cases, a change in the glycosylation site results in a change in the number, amount, position or type (N- versus O-) of the glycosyl chains in the resulting mpl ligand analogs and maintains the biological activity of the mpl ligand. That is, the analog may activate the mpl receptor. Activation of the mpl receptors means that megakaryocyte formation is enhanced, leading to an increase in platelets in vivo.
The present invention relates to mpl ligand analogs having one or more modified O- or N-linked glycosylation sites. The invention also relates to DNA sequences encoding these mpl ligand analogs, recombinant plasmids for expressing analogs and host cells.
1 depicts the DNA and amino acid sequences and mature amino acid sequences (1-332) of human native mpl ligands comprising signal peptides (amino acids -21 to -1).
FIG. 2 shows the DNA and amino acid sequences of mpl ligands corresponding to amino acids 1-174 of the mature mpl ligand sequence of a person attached to a signal peptide of 21 amino acids in length.
3 shows western blots performed with E. coli and CHO expressed mpl ligands. MK means Met-Lys added to the N-terminus of the mpl ligand for expression in E. coli and can be degraded using a dipeptidase such as cathepsin C. A molecule from which MK has been removed is called desMK. Glycosidase neuraminidase and O-glycanase treatments are also indicated.
4 shows the in vivo activity of E. coli and CHO expressing mpl ligands in normal mice by platelet count. This data indicates that the glycosylated mpl ligand (CHO material) has much better activity than the non-glycosylated (E. coli) material. This may be the result of increased half-life in glycosylated material. For example CHO 332 refers to human mpl ligand amino acids 1-332 (FIG. 1) expressed in CHO cells.
FIG. 5 shows Western blot analysis of COS cell supernatants of human prepared mpl ligands and analogs 4, 6, 7, 9, 10 and 11. FIG. The preparation of analogs is described in Example 4. Analogs 4, 7, 10 have one or more additional hydrocarbon chains as can be seen from the slow gel mobility. This analogue number is identical to the analogue number shown in Table 1 (eg 11 corresponds to analogue N11). The control is N1 in Table 1.
FIG. 6 depicts Western blot analysis of COS cell supernatants of human recombinant mpl ligands and analogs 4, 5, 13, 14 and 15. FIG. The preparation of analogs is described in Example 4. Analogs 4, 13, 14 and 15 have one or more additional hydrocarbon chains as can be seen from the slow gel mobility.
FIG. 7 shows Western blot analysis of COS cell supernatants of indicated mpl ligand analogs after treatment with human mpl ligand and N-glycanase. The results indicate that the analogs have different glycosylation patterns.
8 shows the results of a megakaryocyte growth biopsy in humans using mpl ligand analogs. Panels A and D are for the positive and negative controls, respectively. The wells shown in Panel A show substantial megakaryocyte growth as containing 37.5 pg of the wild type (ie native sequence) mpl ligand 1-174 COS-1 treated medium. Panel D contains 1.5 μl of COS-1 blank treated medium and shows no growth. Panels B and C are mpl ligand 1-174 analogs 7 and 10, respectively. Panel B contains 9.0 pg of mpl ligand COS-1 treated medium, while Panel C contains 27 pg and all show good megakaryocyte growth.
9 shows Western blot analysis of CHO mpl ligands 1-174 and analogs N4 and N15 (see Table 1). Slow gel shift indicates that analog N4 (4B) has one additional oligosaccharide, while analog N15 (15-8) has two additional oligosaccharides.
FIG. 10 shows Western blot of CHO cell producing mpl ligand analogs with or without N-glycanase treatment. Slow gel transfer after N-glycanase treatment indicates the presence of N-linked oligosaccharides.
11 shows platelet counts from mice treated with various forms of mpl ligand at various doses. This data indicates that an increase in the amount of N- and / or O-linked hydrocarbons results in an increase in activity in vivo.
12 shows western blot analysis of COS-producing mpl ligand 1-174 and analogs N10, N15, N33, N39, N31, N35, and N40. The number of added N-linked glycosyl moieties is also shown. This figure shows that as the number of N-linked sites increases, the mobility of the mpl ligand decreases due to the increase in the amount of N-linked hydrocarbons.
FIG. 13 depicts Western blot analysis of COS-producing mpl ligand 1-174 and analogs N15, N29, N30 and N38. The number of N-linked glycosyl chains is also shown.
The present invention provides mpl ligands having different glycosylation sites compared to native mpl ligands having corresponding sequences. Preferably, the resulting molecule is a molecule having additional glycosylation sites occupied by glycosyl chains when expressed in mammalian cells (such as COS, CHO and human cells).
In an embodiment of the invention, the invention is a mpl ligand comprising an amino acid sequence comprising one or more added, one or more deleted and / or one or more added and deleted glycosylation sites as compared to the corresponding native sequence mpl ligand Relates to analogues. Added or deleted glycosylation sites can result in some hydrocarbon chain and some sialic acid content compared to the mpl ligand of the corresponding native sequence, in particular human mpl ligand. Deletion of one site and addition of another site at one time will result in no change in the number of sites but a change in the location and / or type of site. Such combined modified analogs are included in the present invention.
In another embodiment of the invention, the invention relates to mpl ligand analogs comprising amino acid sequences, including replacing one or more N- or O-linked glycosylation sites with one or more non-naturally occurring sites. Thus N-linked sites can be replaced with different N-linked sites; N-linked sites can be replaced with O-linked sites; O-linked sites can be replaced with different O-linked sites; And / or the O-linked site may be replaced with an N-linked site. Substituting one site for another at substantially the same location may increase glycosylation efficiency or have other effects at that site. Evidence is provided that, for example, Thr residues in place of Ser residues can increase glycosylation efficiency at O-linked sites.
The term mpl ligand, as used herein, refers to macromolecules and / or platelets that have a two-fold amino acid sequence and glycosylation efficiency of not only the naturally occurring mpl ligand, the naturally occurring mpl ligand, but also the cleavage of the naturally occurring mpl ligand. Non-naturally occurring polypeptides having biological activity that specifically stimulate growth, differentiation and / or production. Preferred are mpl ligand analogs based on at least amino acids 7-151 to amino acids 1-332 of FIG. 1.
In a preferred embodiment of the invention, the mpl ligand is an expression product of an exogenous DNA sequence transfected with a eukaryotic host cell, ie the mpl ligand is a recombinant mpl ligand. Preferred eukaryotic hosts are mammals, particularly preferably CHO cells. Recombinant mpl ligands are advantageously prepared according to the methods described herein and the methods described in the literature cited herein with respect to cloning and expression of the mpl ligands.
Some additional preferred mpl ligand molecules have the following amino acid sequences based on FIG. 1:
mpl ligand 1-332 amino acids 1-332 of FIG.
mpl ligand 1-199 amino acids 1-199 of FIG.
mpl ligand 1-191 amino acids 1-191 of FIG. 1
mpl ligand 1-183 amino acids 1-183 of FIG.
mpl ligand 1-174 amino acids 1-174 of FIG.
mpl ligand 1-163 amino acids 1-163 of FIG. 1
mpl ligand 1-153 amino acids 1-153 of FIG.
mpl ligand 1-152 amino acids 1-152 of FIG.
mpl ligand 1-151 amino acids 1-151 of FIG.
mpl ligand 7-332 amino acids 7-332 of FIG.
mpl ligand 7-199 amino acids 7-199 of FIG.
mpl ligand 7-191 amino acids 7-191 of Figure 1
mpl ligand 7-183 amino acids 7-183 of FIG.
mpl ligand 7-174 amino acids 7-174 of FIG.
mpl ligand 7-163 amino acids 7-163 of FIG.
mpl ligand 7-153 amino acids 7-153 of FIG.
mpl ligand 7-152 amino acids 7-152 of FIG.
mpl ligand 7-151 amino acids 7-151 of FIG.
It should be appreciated, for example, 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 their shorter sequences. In this case, Met-Lys may be further included at its N-terminus.
In vivo inactivation referred to herein is a relative measure of inactivation ex vivo but not an absolute value of ex vivo inactivation. Inactivation is used in the present invention only to compare the relative activity of mpl ligand analogs analyzed using the same assay including the same internal criteria and using the same assay with the same assay data used to calculate the inactivation.
As used herein, an analog of a mpl ligand or an mpl ligand analog has one or more changes in the amino acid sequence of the mpl ligand such that the type of hydrocarbon attachment site (N- or O-bonds that may affect the amount of hydrocarbon attached). Refers to mpl ligands that can result in a change in number or position. In a preferred embodiment, the change in glycosylation sites results in a change in the number of glycosyl chains attached to the mpl ligand molecule. In a more preferred embodiment, the change in the glycosylation site adds one or more (generally 1-6, preferably 1-5, particularly preferably 2-4) glycosyl chains, most preferably this Chains are added via N-bonds. In a particularly preferred embodiment, the mpl ligand analog has a biological activity equal to or greater than that of the mpl ligand (eg human mpl ligand) of the native sequence and has substantially higher activity in vivo as measured by a biological activity assessment. Can be. Such assays include methods for detecting megakaryocytes or platelet production.
To prepare such mpl ligand analogs, they are preferably produced by site specific mutations that result in the addition, deletion or substitution of amino acid residues that add, remove or alter sites useful for glycosylation. By altered is meant that a site is deleted while another site is added at the same or a different location as the deleted site. However, as is known to those skilled in the art, such methods are included in the present invention as other methods can also induce a gene encoding the same amino acid sequence. The resulting analogs may have some (preferably more) attached hydrocarbon chains as compared to the natural human / recombinant mpl ligand.
Adding at least one hydrocarbon (ie glycosyl) chain to the mpl ligand is an important object of the present invention. Compared to those found in the corresponding naturally occurring amino acid sequences (such as 1-332 or 1-174, etc.), mpl ligand analogs with more hydrocarbon chains do not substantially affect the secondary or tertiary structure such that they do not substantially reduce biological activity. It is produced by adding a misfired site. As used herein, a naturally occurring mpl ligand refers to an amino acid sequence having a corresponding number of amino acids as in the relevant analog, even if a specific length of mpl ligand species is not substantially expressed in the native species. Advantageously, the mpl ligand analog has up to 6 additional sites for N-glycosylation or O-glycosylation, so 1 to 6 additional N-linked or O-linked hydrocarbon chains (or combinations thereof). Results in the addition of.
For example, at position 30 Pro is replaced with Asn and Val at position 32 is replaced with Thr to generate the sequence Asn-Glu-Thr, which provides a new N-glycosylation site (hereinafter analog N4; see Table 1).
Analogs with two or more additional N-linked chains can be prepared by combining mutations; For example, analogs N4 and N10 described in Table 1 can be combined to yield analogs with two additional hydrocarbon addition sites (ie analogue N15 in Table 1). Similarly, analogs with three or more additional chains can be prepared. As will be appreciated by those skilled in the art, the present invention includes many different analogues (in terms of number, type or location of sites) of mpl ligands having different glycosylation sites. The mpl ligand analogs of the present invention are in all cases based on mpl ligands having in particular human amino acid sequences (see FIGS. 1 and 2). However, analogs based on mpl ligand sequences from other species (eg dogs, pigs, monkeys, mice or mice) may also be included.
It also includes inserting amino acids to make glycosylation sites. For example, Glu at position 57 is replaced by Thr and inserts Asn immediately after Met at position 55:

This creates new glycosylation sites (amino acids 55 ', 56 and 57). See analog N23 below.
Analogs having one or more amino acids extending from the carboxy terminal end of the mpl ligand, where the carboxy terminal extension provides one or more additional hydrocarbon sites, are also included within the analogs of the present invention. The carboxy terminus of the mpl ligand will differ depending on the particular form of the mpl ligand used (eg mpl ligand 1-332 amino acids, or mpl ligand 1-163 amino acids). Additional hydrocarbon sites may be added to the carboxy terminus of the mpl ligand species by adding amino acids containing one or more N- or O-linked glycosylation sites to the carboxy terminus.
Tables 1 and 6 list some examples of mpl ligand analogs with additional sites for N-linked hydrocarbon chains. These analogs have N-linked sites with Asn-X-Ser or Asn-X-Thr sequences included at various positions in the human mpl ligand polypeptide chain based on human amino acid sequences. Tables 4 and 7 list analogs to which one or more additional N-linked hydrocarbon chains are added, as can be seen by the transport of glycoproteins on SDS gels (eg, Example 6 and Figures 3, 5, 6, 7, 9, 10, 12 and 13). These tables also include truncated species (ie, N1, N16, N17 and N31) that are not analogs defined herein. These are listed in the table to show how various cut papers can be produced.
Also included within the scope of the invention are DNA sequences encoding mpl ligand analogs described herein, preferably those having additional sites for N-linked chains. The methods used to introduce changes in the mpl ligand DNA sequence to create, delete and / or alter attachment sites for hydrocarbons are described in Examples 4 and 14.
These mpl ligand analogs can be produced by expression products of exogenous DNA sequences, ie recombinant DNA techniques, which can be chemically synthesized or prepared by combined methods. Exogenous DNA sequences include cDNA, genomic DNA or chemically synthesized DNA encoding mpl ligand analogs. Also provided are recombinant DNA plasmids and eukaryotic host cells useful for expression of such analogs. Expression vectors include vectors that are capable of expressing cloned DNA sequences in eukaryotic host cells, particularly those used in COS and CHO cells. Examples of such vectors include plasmids pDSRα and pDSRα2. Mol. Cell. Biol. 8: 466-472 (1988); WO 91/13160 (1991); And WO 90/14363 (1990). Cultivation of COS and CHO host cells expressing mpl ligand analogs was carried out using standard methods known to those skilled in the art.
Changing the number, type, position or amount of hydrocarbon chains attached to the mpl ligand confers beneficial properties such as increased solubility, greater resistance to proteolytic enzymes, reduced immunogenicity, increased serum half-life and increased or altered biological activity. You can do it.
Treatment medium from COS cells expressing mpl ligand analogs N2-N15 (N1 is human mpl ligand 1-® Figure 2) was analyzed for in vitro biological activity and the results are listed in Table 4 below.
Treatment media from COS cells expressing mpl ligand analogs / cut N15-N40 were analyzed for in vitro biological activity and the results are listed in Table 7 below.
In vivo biological activity for various forms is shown in FIG. 11 (see Example 13).
Other embodiments of the present invention provide for mpl ligands or analogs of mpl ligands having a specific number or more of sialic acid per molecule, such as mpl ligands 1-332, 1-199, 1-191 produced naturally or recombinantly in eukaryotic cells. Mammalian (eg, Chinese Hamster Ovary) host cells that specifically synthesize 1-183, 1-174, 1-163, 1-153, 1-152 or 1-151. The in vitro activity of analogs N4 and N15 along with the full length and various truncated species expressed in CHO cells are listed in Table 5.
Sialic acid content of the mpl ligand can affect biological activity in vivo. For example, tetra-branched N-linked oligosaccharides most often provide four possible sites for sialic acid attachment, while bivalent and tetravalent oligosaccharides that can substitute for quaternary forms at asparagine-linked sites are often high. Have three or three attached sialic acids. O-linked oligosaccharides provide two sites for sialic acid attachment. Thus, mpl ligand molecules with N-linked hydrocarbons substituted for O-linked hydrocarbons can accommodate two additional sialic acids per chain, with the exception that the N-linked oligosaccharides are four branched. Screening mammalian cell cultures for cells that specifically add a four branched chain to the recombinant mpl ligand maximizes the number of sites for sialic acid attachment.
CHO cells lacking dihydrofolate reductase (DHFR) are commonly used host cells for the production of recombinant glycoproteins, including recombinant mpl ligands.
Also included in the present invention are compositions comprising a therapeutically effective amount of a mpl ligand analog and suitable diluents, adjuvants and / or carriers useful for the treatment of mpl ligand. A therapeutically effective amount as used herein refers to an amount that provides a therapeutic effect for a given condition and range of administration.
The composition of the present invention can be administered parenterally to the system. Alternatively, the composition may be administered intravenously or subcutaneously. When administered systemically, the therapeutic composition for use in the present invention may be in the form of a parenterally acceptable aqueous solution free of pyrogenic substances. Formulations of such pharmaceutically acceptable protein solutions are within the scope of the present invention in terms of pH, isotonicity, stability and the like. The specific path depends on the conditions to be processed. Administration of the mpl ligand or mpl ligand analog is preferably carried out as part of a combination containing a suitable carrier such as human serum albumin, a suitable diluent such as buffered saline and / or a suitable adjuvant. The dosage required is sufficient to increase the amount of platelets in the patient and will vary depending on the severity of the condition to be treated, the method of administration and the like.
Conditions to be treated by the methods and compositions of the present invention generally include existing megakaryocytic / platelet deficiency or prospective megakaryocytic / platelet deficiency (eg, due to scheduled surgery). This condition will be the result of a lack of active mpl ligand (temporary or permanent) in vivo. The common name for thrombocytopenia is thrombocytopenia and the methods and compositions of the present invention are generally useful for treating thrombocytopenia.
Thrombocytopenia (platelet deficiency) can exist for a variety of reasons, including chemotherapy, bone marrow transplantation and various drug treatments, radiation therapy, surgery, blood loss from accidents, and certain other disease states. Examples of specific disease states that can be treated according to the present invention, including thrombocytopenia, include: aplastic anemia, idiopathic thrombocytopenia, metastatic tumors resulting in thrombocytopenia, systemic lupus erythematosus, gynecosis, facroni syndrome, Vitamin B12 deficiency, folic acid deficiency, may-hegrin abnormality, whisco-Aldrich syndrome and paroxysmal nocturnal hemoglobinuria. Certain treatments of AIDS also result in thrombocytopenia (such as AZT). Certain wound healing disorders can also benefit from increased platelet counts.
For example, with regard to platelet deficiency, which is expected due to future surgery or future thrombocytopenic induction therapy, the mpl ligand analogs of the present invention may be administered several days to several hours prior to the need for platelets. For acute situations, such as accidental mass loss of blood, mpl ligand analogs can be administered with blood or purified platelets.
Dosage regimens involved in the methods for treating the above-described conditions may include a variety of factors that alter the action of the drug, such as the age, condition, weight, sex and nutritional status of the patient, the severity of the infection, the timing of administration and other clinical factors. Will be determined by In general, the daily dose should be 0.01 to 1000 mg of mpl ligand analog, preferably 0.1 to 10 mg per kg body weight.
The methods, compositions and polypeptides of the present invention may be used alone or in other diseases as well as other cytokines, soluble mpl (ie mpl ligand) receptors, hematopoietic factors, interleukins, growth factors in the treatment of disease conditions characterized by platelet deficiency. Or in combination with an antibody. The mpl ligand analog molecules are expected to be useful for treating some forms of thrombocytopenia in combination with common hematopoietic stimulators such as IL-3 or MG-CSF. Other megakaryocyte stimulating factors such as meg-CSF, hepatocyte factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity can be used with the mpl ligand. Examples of additional cytokines or hematopoietic factors for such co-administration are IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulation Factor-1 (CSF-1), GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), IFN-beta, or IFN-gamma. It may be advantageous to simultaneously or sequentially administer an effective amount of soluble mammalian mpl receptor that appears to have the effect of causing megakaryocytes to fragment into platelets when the megakaryocytes reach maturation. Thus, adding mpl ligand analogs to increase the number of mature megakaryocytes and then administering soluble mpl receptors (to inactivate analogues and allow mature megakaryocytes to generate platelets) seem to be a particularly effective means to stimulate platelet production. . The dosage forms described above will be adjusted to compensate for the additional ingredients in the therapeutic composition. Progression of treated patients can be controlled by conventional methods.
Further modifications of the analogs of the invention can be made to increase activity, stability, half-life and the like. PEGylation (poly- or mono-) can be added to the mpl ligand analogs, for example, on proteins via amino groups or via hydrocarbon groups. Fatty acids or other polymers may also be attached to protein or hydrocarbon groups.
The following examples are presented to explain the present invention in more detail, but do not limit the scope of the invention. The mpl ligand standard used in the bioassay of the examples is a recombinant mpl ligand standard that is expressed in E. coli, regenerated and purified into active structures. Therefore only relative inactivity is measured.
Example 1
Preparation of mpl ligand 1-174
A person encoding amino acids 1-174 (starting with SPAPPA ...) of FIG. 2 by polymerase chain reaction (PCR) from a human fetal liver cDNA library (Battley et al., Cell 77: 1117-1124 (1994)). Mpl ligand gene was prepared. The 5 'PCR primers encoded the amino terminus, XbaI site, and optimized kozak sequence of human mpl ligands. The 3 'primer contained a stop codon and a SalI restriction site. The amplified DNA fragments were digested with XbaI and SalI and then ligated to pDSRα2 digested with XbaI and SalI. The resulting plasmid, pDSRα2 mpl ligand 1-174, was used for mammalian cell expression. The sequence of the gene produced (including signal peptide) is shown in FIG.
Plasmid DNA containing mpl ligand 1-174 was digested with XbaI and SalI restriction enzymes, the resulting DNA fragments were subjected to agarose gel electrophoresis and 605 nt mpl ligand 1-174 DNA fragments were prepared using GeneClean ^™ kit and manufacturer ( Isolated from the gel using a process provided by BIO 101, Inc.). Plasmid pDSRα2 as described in WO 90/14363 (1990) was digested with XbaI and SalI restriction enzymes and vector fragments were recovered. Two fragments were ligated to give pDSRα2 (mpl ligand 1-174).
Example 2
Expression and Purification of mpl Ligands in CHO Cells
Dihydrofolate reductase deficient (DHFR ⁻ ) CHO cells were transfected with pDSRα2-mpl ligand 1-174. CHO D ^- media (DMEM, 10% fetal calf serum, 1% penicillin / streptomycin / glutamine, 1% non-essential amino acids (Gibco) and 1% HT supplement (Gibco) in a 100 mm tissue culture dish the day before transfection ) Were inoculated with 1 × 10 ⁶ CHO DHFR ^- cells. Four transfections were performed. For each infection, plasmid DNA (50 μg) was linearized by digesting with PvuI and buffer H (Boehringer Mannheim). DNA precipitates were formed and added dropwise to the plates for the Mammalian Cell Transfection Kit (Speciality Media). After 24 hours in the tissue incubator, the medium was replaced with fresh CHO D-medium. Incubate 96 well tissues with cells with 100 μl of CHO selection medium (D-MEM, 5% dialyzed fetal bovine serum, 1% penicillin / streptomycin / glutamine, 1% non-essential amino acids (Gibco)) after 24 hours. The plates were distributed and transformants were selected. The medium was changed weekly until colonies appeared. Two weeks later, expression of the mpl ligand was screened using the 32D cell proliferation assay described below (see Example 9). Clones expressing excess 1 × 10 ⁵ units / ml were diluted and frozen at low temperature. One clone was diluted for roller bottle production and produced about 8 liters of treated medium.
Plasmid pDSRα2 containing mpl ligand 1-174 cDNA was transfected with DHFR-deficient CHO cells as described above. 2 liters of serum free CHO cell treatment medium (50% D-MEM, 50% HAMS-F12, 1% penicillin / streptomycin / glutamine, 1% from roller bottles inoculated with CHO cells expressing mpl ligand 1-174) Non-essential amino acids (Gibco)) were concentrated 15 fold using 2 L Amicon Model 2000 stirred cells and 10,000 Dalton molecular weight cutoff membrane (YM10, Amicon). 45 ml of concentrated treatment medium was directly hung on a 4 ml hu-MPL-X affinity column using Pharmacia FPLC at a flow rate of 0.4 ml / min. This affinity column was prepared by coupling 1.5 to 2.5 mg of Mpl-X (soluble extracellular domain of mpl receptor) per ml of Pharmacia CNBr activated Sepharose resin as recommended by the manufacturer. After loading, the column was washed with 16 ml phosphate buffered saline (PBS; 10 mM Na.PO ₄ pH 6.8 / 150 mM NaCl) followed by 24 ml of 10 mM Tris, pH 8.0 / 1M NaCl. Mpl ligand (1-174) was added to 40 ml of 20 mM CAPS (3- [cyclohexylamino] -1-propanesulfonic acid) pH 10.5 / 1M NaCl / 5 mM CHAPS (3- [3-colamidopropyl) dimethylammonio] -1-propanesulfonate) in 6 ml fractions. The second fraction showed a single band on 14% SDS gel. The material was concentrated and buffer exchanged with 0.9% NaCl saline solution and then biological activity was measured in vitro and in vivo. Other forms of CHO cells expressing mpl ligands were purified in a similar manner.
Example 3
In vivo biological activity of human recombinant mpl ligand
Platelet counts were determined from mice treated with various forms of mpl ligand. CHO-induced mpl ligands 1-332, 1-174, 1-163 and 1-153 were prepared and purified by Mpl receptor affinity chromatography. E. 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 prepared and subjected to conventional chromatography. Purification by
FIG. 4 shows platelet counts obtained from mice treated with recombinant mpl ligands of various types of CHO cell induced (dark line) or E. coli induced (dashed line) humans. Normally, female Balb / c mice were injected subcutaneously with a predetermined concentration of mpl ligand for 5 consecutive days. Twenty four hours after the last injection test blood was collected from the small flank cut in the tail vein. Blood cell analysis was performed with a Sysmex Electronic Blood Cell Analyzer (Bacter Diagnostics, Inc., Irvine, Calif.). Data are shown as mean, +/- standard deviation of measurements from four animals. Other blood cell variables such as total white blood cell count or red blood cell count were not affected by these treatments (data not shown).
The results indicate that CHO cells expressing various types of mpl ligand have higher in vivo activity than the same type of mpl ligand produced in E. coli. As shown in Example 6, CHO cells expressing various forms of mpl ligands contain both N and O-linked hydrocarbons, while E. coli expressing mpl ligand forms is not. This indicates that the hydrocarbons enhance the in vivo activity of the mpl ligands. The increased in vivo activity imparted by hydrocarbons may be the result of increased circulating half-life, increased stability, or both.
Example 4
Preparation of the Mpl Ligand Analog N2-N15
The method of generating additional glycosylation sites of the mpl ligand is as described below.
The following oligonucleotide primers were synthesized for use in ex vivo mutations to prepare analogs N2-N14 (see Table 1 for the structure of these analogs):
To prepare the m13mp18 mpl ligand 1-174, the gene of FIG. 2 was introduced into m13mp18 DNA digested with XbaI and SalI restriction enzymes. Kunkel group, Methods in Enzymol. 154: 367 (1987) and Messing, Methods in Enzymol. 101: 20 (1983) single chain DNA was recovered from the supernatant of E. coli strain RZ1032 infected with m13mp18 (mpl ligand 1-174). For ex vivo mutations, one of about 0.5 μg of single chain DNA and 0.125 pmole of sequential synthetic primers was added to 6 μl of buffer (250 mM Tris pH 7.8, 50 mM MgCl ₂ , 50 mM dithiothreitol and 1%). Mixed with bovine serum albumin (BSA-Pharmacia). This primer was activated with ATP and T4 polynucleotide kinase prior to the addition reaction. To anneal the primer to the template, the reaction volume was adjusted to 10 μl with water and the mixture was heated at 65 ° C. for 5 minutes and then cooled to room temperature. To prolong the reaction, 2.5 μl of each dTTP, dATP, dGTP and dCTP and 1 ml of ATP (10 μM) were added followed by 1 μl (1 unit) of E. coli DNA polymerase (cleno fragment) and 1 μl ( 1 unit) of T4 DNA ligase was added. The mixture was incubated overnight at 14 ° C. and used to transform Escherichia coli JM 109 (Yanish Peron et al., Gene 33, 103 (1985)) as described by meshing (homologous). To identify mutant clones by differential hybridization, nutrient agar-like plaques were transferred to a Gene Screen filter (New England Nuclear). DNA was crosslinked to the filter by irradiation with ultraviolet Stratalinker Model 1800 using autocrosslinking mode (produced by stratagene). They were incubated for 1 hour in 6 × SSC (0.9M NaCl / 0.09M Na. Citrate) containing 1% SDS at 60 ° C. To hybridize, the oligonucleotide primer (8 pmole) described above was end-labeled with T4 polynucleotide kinase and γ ³² P-labeled ATP and the filter was 6 x SSC, 0.5% SDS and 125 μg / ml of herring semen DNA Incubated overnight at. The hybridization temperature was chosen according to the estimated melting point of the oligonucleotides. In general, the hybridization temperature was about 10 ° C. below the melting point. The following day, the filter was washed twice with 6 x SSC / 1% SDS at hybridization temperature and then twice with 6 x SSC at hybridization temperature and treated by magnetic radiometry. If necessary, plaques with wild-type mpl ligand cDNA sequences were detected by washing the filter with 6 x SSC with increasing temperature until hybridization was no longer detected. Under these conditions, clones with positive hybridization signals were identified and retransfected with JM109 to obtain pure clones. Dideoxy chain termination sequence analysis indicates the presence of mutations.
Double-stranded m13 mpl ligand 1-174 DNA with the desired change was recovered from the JM109 transfected with the QIAGEN kit (manufactured by Chatsworth CA.) using the method recommended by the manufacturer. This DNA was digested with XbaI and SalI and the 605 bp mpl ligand DNA fragment was isolated. pDSRα2 was digested with XbaI and SalI. Vector fragments were isolated and ligated to the mpl ligand fragments described above. Recombinant plasmids were identified by restriction analysis. The resulting plasmid (denoted mpl ligand 1-174-NX, NX analogue number) contains DNA encoding a mpl ligand with altered amino acid residues at the indicated positions. The resulting plasmid was sequenced to confirm that the desired mutation was present.
Analog N15 with two additional N-linked glycosylation sites at positions 30 and 120 was created. PDSRα2 mpl ligand 174-N4 with Asn30 and Thr32 mutations was digested with XbaI and PstI restriction enzymes and about 385 nt DNA fragments were isolated. PDSRα2 mpl ligand 174-N10 containing Asn120 and Thr122 mutations was digested with PstI and SalI restriction enzymes to yield about 220 nt DNA fragments. pDSRα2 was digested with XbaI and SalI. Vector fragments were isolated and ligated to the mpl ligand fragments described above. This resulted in PDSRα2 mpl ligand 174-N15 containing Asn30, Thr32, Asn120 and Thr122 substitutions.
These general procedures were used to prepare the mpl ligand analogs shown in Table 1. The DNA sequence changes of each analogue are shown, with the remaining oligonucleotide primers used for mutations having sequences complementary to those of the human mpl ligand.
Note: Analogs N2-N15 are synonymously represented by analogs 2-15. As indicated herein, for example, the [Asn ²² ] mpl ligand may be defined as the substitution of an amino acid at position 22 with asparagine, given the sequence of a person having a particular mpl ligand species, preferably at least amino acids 7-151 of FIG. (Including the preferred human mpl ligand sequence as defined above). Thus, substitution of an asparagine residue in place of leucine residue at position 22 of mpl ligand 1-174 (human sequence) yields a mpl ligand analog that can be represented as [Asn ²² ] mpl ligand 1-174.
Plasmids represented by pDSRα 1-174-NX, where NX is an analogue number, were prepared by inserting mpl ligand DNA into pDSRα2. Expression vectors pDSRα2 are generally described in WO 90/14363 (1990). pDSRα2 mpl ligand 1-174-NX plasmid was prepared by digesting pDSRα2 with XbaI and SalI. Vector fragments were isolated and ligated to yield about 605 bp fragments containing the desired sequence.
Example 5
Expression of mpl ligand and mpl ligand N1-N15 in COS cells
The cDNA clones of human mpl ligands and mpl ligand analogs described in Table 1 were transformed into COS-1 cells (ATCC No. CRL-1650) by electroporation. Collect COS-1 cells from the semi-fusion plate and wash with medium (Dulbecco's denatured essential medium containing 10% fetal bovine serum and 1% L-glutamate / penicillin / streptomycin) (by Irvine Scientific). Resuspended at x 10 ⁶ cells / ml. Transfer 1/2 ml of cells to a 0.2 cm electroporation cuvette (manufactured by Biorad) and mpl ligand using a BTX electroporation system electrocell manipulator 600 at a low voltage setting of 650 kV and 130 volts. Electroporation was performed with 50 μg of plasmid DNA encoding the analog. Electroporated cells were plated on 100 mm tissue culture dishes in 10 ml medium. 12 to 24 hours after plating, the medium was replaced with 10 ml of fresh medium. Treated media were collected 3-5 days after electroporation.
Example 6
Characteristics of Mpl Ligand and Mpl Ligand N1-N15
A. Determination of Hydrocarbon Addition
A large amount of supernatant containing about 30-60 ng mpl ligand or mpl ligand analog from COS cells transfected with mpl ligand analog cDNA as described in Example 5 was overnight as rabbit anti-mpl ligand polyclonal antibody at room temperature. Immunoprecipitated. In some cases with low expression, a maximum volume of about 8-9 ml was used for immunoprecipitation. Antibodies to mpl ligand 1-163 expressed and purified from E. coli were prepared. 30 μl of 1: 1 protein A-Sepharose in phosphate buffered saline (PBS) containing 0.1% sodium azide was added to the immunoprecipitate and incubated for 1 hour at room temperature. Samples were centrifuged, washed with PBS and resuspended in SDS sample buffer (0.125 M Tris-HCl pH 6.8 / 4% SDS / 20% glycerol / 10% β-mercaptoethanol / 0.001% bromophenol blue). . Samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis, delivered to nitrocellulose and mouse anti-mpl ligands prepared for synthetic mpl ligand peptides (eg, corresponding to amino acid residues 47-62 of FIG. 1). Monoclonal antibodies were used for Western blot testing as described in Burnett, Anal. Biochem. 112: 195-203 (1981); Elliott, Gene 79: 167-180 (1989). A mpl ligand containing a band was visualized using an ECL kit (manufactured by Amersam).
FIG. 5 shows that COS cell supernatants obtained from cells transfected with analogs N4, N7 and N10 DNA have increased size compared to human sequence mpl ligand 174 (N1). FIG. 6 shows that COS cell supernatants obtained from cells transfected with analogs N13, N14 and N4 DNA have increased size compared to human sequence mpl ligands. This increase in size indicates the presence of additional N-linked hydrocarbon chains. N15 contains two additional N-linked glycosylation sites. FIG. 6 shows that the analog is much larger in size than the analog containing one additional N-linked glycosylation site. Protein size is estimated from their mobility on SDS-PAGE compared to protein standards of known molecular weight. The estimated magnitudes of the larger bands calculated from FIG. 6 are shown in Table 2. This result indicates that N15 contains two additional N-linked chains. Western blot analysis of other selected analogs is shown in FIG. 6.
N-linked carbohydrate estimates Mpl Ligand Analogs (1-174)Molecular Weight (Da)Molecular weight change (Da) (relative to natural sequence)Number of possible N-linked chains (@ 4 KDa / position) N1 (Native Sequence) N4N7N10N13N14N1523500287002720027200267002870033500052003700370032005200100000111112
Experiments were conducted to show that the increase in size of the Mpl ligand analog was due to N-linked carbohydrates. Media treated with COS cells and containing mpl ligand were immunoprecipitated and washed with PBS as above. Then 10 μl of 0.5% SDS was added to each test tube and each sample was heated for 3 minutes. The following components were then added: 10.8 μl 0.5 M NaPO ₄ (pH 8.6), 5 mL 7.5% nonidet P40 and 3 μl 250 units / ml N-glycanase (Genzyme). N-glycanase treatment removes N-linked carbohydrates. Samples were incubated at 37 ° C. for 6 hours. The reaction was stopped by addition of SDS-PAGE sample buffer followed by SDS-PAGE Western analysis (12% acrylamide) using the anti-mpl ligand monoclonal antibody and anti-mouse ECL Western Assay Kit (manufactured by Amersham). It was. Results of analysis of N-linked chains for human mpl ligand and mpl ligand analogs using this method are shown in FIG. 7. After treatment with N-glycanase the mobility on Western blot for N4, N7 and N10 decreased to the level of N1. As expected, treatment of N-glycanase on N1 had no effect on mobility because there was no N-linked glycosylation site on N1. The increase in size from these results is due to the addition of N-linked carbohydrates.
Analysis of O-linked Carbohydrates on B. mpl Ligands
In order to analyze the effect of O-linked carbohydrates on human mpl ligands, various types of proteins were purified from the medium treated with the CHO cells. Each protein was treated and untreated with O-glycanase (glycopeptide α-N-acetylgalactosaminiase, manufactured by Oxford Glycosystems). O-glycanase removed O-linked carbohydrates from glycoproteins. Each form of E. coli phenotype was used as a non-glycosylated control. In order to resolve the molecular weight difference caused by O-linked carbohydrates, it is necessary to first remove all N-linked carbohydrates. Since full-length mpl ligand 1-332 contains N-linked carbohydrates, full-length samples are expressed as well as CHO cells expressing the mpl ligand analog, except that N-glycanase treatment is overnight culture. CHO cells were treated with N-glycanase.
Prior to treatment with O-glycanase on full length (1-322) mpl ligands, the pH range of the samples was adjusted to pH 6.0-pH 7.0 using 100 mM acetic acid (pH 2.2) in 1/15 volume. 1 μg of protein was denatured by boiling in SDS for 3 minutes and 1 U / ml neuraminidase [sialidase, Artrobacter eure) in 1 mM calcium acetate (pH 6.8) and 20 mM sodium phosphate (pH 6.8). Extraction from Arthrobacter urefaciens , manufactured by Charinger Mannheim, Inc. was incubated for 60 minutes at 37 ℃.
5 mU of O-glycanase was then added in a final volume of 100 μl and incubated overnight at 37 ° C. Protein (0.2 μg / lane) was isolated by SDS-PAGE (15% acrylamide). After transferring to 0.2 μm nitrocellulose and overnight incubation with anti-mpl ligand monoclonal antibody, the mpl ligand protein was visualized using an anti-rabbit ECL western assay kit (manufactured by Amersham).
3 shows Western blots of four different forms of human mpl ligands. Full length mpl ligands 1-332 are lanes 1-3, mpl ligands 1-174 are lanes 4-6, mpl ligands 1-163 are lanes 7-9, and mpl ligands 1-153 are lanes 10-12. The neuraminidase and O-glycanase treatments shown in lanes 2, 5, 8 and 11 reduced the molecular weight to the non-glycosylated material, that is, to the molecular weights of lanes 3, 6, 9 and 12. In all cases, mobility increased to the level of unglycosylated protein expressed in E. coli. These results show that the large bands in lanes 1, 4, 7 and 10 are due to O-linked carbohydrates. The molecular weight of each band was estimated by comparing their mobility with a protein of known molecular weight.
As shown in Table 3 below, which shows the estimated molecular weights of the different proteins, there are 14 O-linked carbohydrate chains (about 950 Da / chain) for mpl ligand 1-332 from the apparent change in mobility and mpl ligand 1-174 It can be seen that there are 9 chains, 4 chains for mpl ligand 1-163, and 2 chains for mpl ligand 1-153. Sample in lane 2 is full length mpl ligand 1-332. This protein appears to be degraded because it was incubated in glycoenzyme for a long time at 37 ° C. Thus, unglycosylated ones expressed in Escherichia coli in lane 3 were used to calculate the estimated molecular weight of O-linked carbohydrates added to mpl ligand 1-332 expressed in CHO cells.
These results indicate the presence of carbohydrates on all forms of test mpl ligands expressed in CHO. The presence of O-linked carbohydrates was confirmed by direct analysis of monosaccharide compositions of mpl ligands 1-332, 1-174 and 1-163 expressed in CHO cells. Acid hydrolysis releases sialic acid, GalNAc and Gal from glycoproteins. Monosaccharides were detected by high pressure anion exchange chromatography and pulsed amperometric detection. All three sugars were detected in each type of mpl ligand. This result indicates the presence of sialic acid containing O-linked carbohydrates. This data is consistent with the in vivo experimental data of FIG. 4, which shows that all mpl ligands expressed in CHO cells are more active in in vivo experiments than the corresponding forms expressed in E. coli. Thus, the presence of carbohydrates enhances the in vivo activity of mpl ligands.
Calculation of O-linked Carbohydrates mpl ligand formO-glycanase treatment (+/-)Molecular Weight (Da)Molecular weight change (Da)Number of possible O-linked chains (@ 950 Da / chain) 1-3321-1741-1631-153Escherichia coli enzymes54200406002460016000184001450015200129001360086003900230014942
Example 7
Mpl Ligand ELISA Assay
Polyclonal Antibody Production: New Zealand white rabbits were overimmunized for 3 months with recombinant human mpl ligand 1-163 produced in Escherichia coli. Antisera were collected from six rabbits showing high antibody titers and certain anti-mpl ligand antibodies were purified by affinity.
Affinity Purification: Recombinant human mpl ligand 1-163 was covalently bound to Actigel-ALD (manufactured by Sterogen Bioseparation) according to the manufacturer's instructions. Fractions of rabbit antiserum were added to the mpl ligand affinity gel and the slurry was slowly stirred overnight on a stirrer at 4-8 ° C. Unbound serum proteins were washed from the gel using PBS, and specifically bound anti-mpl ligand antibodies were eluted using ImmunoPure Gentle antigen / antibody elution buffer (Pierce Chemical). The recovered antibody is dialyzed several times with PBS, the antibody solution is concentrated in an Amicon stirred cell ultrafiltration apparatus, and the resulting antibody concentrate is later used for specific anti-mpl ligands for well coating and preparation of urea conjugates. It becomes the material of an antibody.
ELISA Reagent: Immulon 4 Removawell Strips (manufactured by Dynatech Laboratories) were coated using a rabbit anti-mpl ligand antibody purified by affinity. Antibodies purified by affinity were diluted to a concentration of 2.5 μg / ml with 0.1 M sodium bicarbonate (pure prepared, about pH 8.2). Each well contains 100 μl of antibody and these culture plates were incubated for 24 hours in a sealed room temperature chamber. Then 200 μl of blocking solution of TEN (50 mM Tris 7.4 / 10 mM EDTA / 150 mM NaCl) containing 1% fetal bovine serum and 5% sucrose was added to each well and these culture plates were sealed. Further incubation for 24 hours in a room temperature room. The combined coating and blocking solution was removed from the wells. Additional overcoat / blocking steps were performed: 300 μl of SuperBlock blocking buffer in PBS (Pierce Chemicals Inc.) was added to each well. After standing at room temperature for about 5 minutes, the solution was removed and the wells were dried at room temperature for 24 hours. The coated wells were stored in sealed plastic bags at 4-8 ° C. until used for mpl ligand ELISA.
Anti-mpl ligand antibodies purified by affinity from rabbit antisera were covalently coupled with horseradish peroxidase (HRPO) for use as signal generating antibodies. Antibodies purified by affinity were derived using iminothiolane HCl (manufactured by Fluka Chemical Co., Ltd.). Separately, HRPO was induced using N-succinimidyl 6-maleimidocaproate (manufactured by Fluka Chemical Co., Ltd.). The two activated proteins were covalently coupled. The reaction mixture was then chromatographed through a FPLC Superrose 6 (Pharmacia) column to separate antibody: HRPO conjugates of the desired molecular weight (ie about 200 kD). Fractions containing the desired conjugate were collected, concentrated to Centricon 30 (Amicon Division, manufactured by W.R.Grace Co.) and stored at -20 ° C as a 50% glycerol solution. The anti-mpl ligand antibody: HRPO concentrate was diluted in 2% fetal bovine serum in PBS for use in ELISA. The final concentration of the conjugate used in the ELISA was 250-500 ng / ml.
Recombinant human mpl ligand 1-163 produced in E. coli was used for the preparation of standards. This mpl ligand was diluted with TEN buffer containing 0.05% thimerosal as preservative and 2% fetal bovine serum. The prepared standard contained 1.0, 0.5, 0.25, 0.125 and 0.062 ng / ml mpl ligand.
Assay: 100 [mu] l of mpl ligand standard or sample was added to the wells and incubated for 18-24 hours in a sealed room temperature wet room. The well contents and residual solution were then removed and the wells washed once with wash solution (0.05% Tween 20 in TEN buffer). Anti-mpl Ligand Antibody: HRPO conjugate solution (100 μl) was added to each well and incubated for 2 hours in a sealed room temperature room. The well contents were then removed and the wells washed four times with TEN buffer containing 0.05% Tween 20.
For color development, 100 μl of TMB / peroxidase substrate solution (1: 1 mixture of Kirkegaard and Perry solutions A and B) was added and incubated at room temperature for 20 minutes. The reaction was stopped by addition of 100 μl of termination solution (0.5 N sulfuric acid) and the absorbance was measured at 450 nm on a microtiter plate reader. The mpl ligand concentration in the sample was calculated from the green standard curve using the curve normalization program.
Example 8
Biological Activity of Mpl Ligand 1-174 Analogues in Short-term Liquid Culture Megakaryocyte Analysis
Analogs of Mpl ligand 1-174 were prepared and analyzed for growth stimulation of megakaryocytes in liquid culture. CD34 selective cells isolated from human leukocyte export units were cultured in 2 × 10 ⁵ / ml culture medium (IMDM / 1% Pen-Strep glutamine / 1% non-essential amino acids / 1% MEM Na-pyruvate / 1% MEM vitamin / 10 % Deionized BSA / 10% normal human AB plasma / 10 μM α-thiasylglycerol / 20 μg / ml L-asparagine). In addition, 1.5 μl COS-1 treatment medium containing mpl ligand (1-174) or mpl ligand 1-174 analog was added to each well. The final volume was 15 μl in a Terasaki type microtiter tissue culture plate (manufactured by Vanguard International). These cells were incubated for 8 days in a humidified vessel at 37 ° C., 5% CO ₂ , immobilized directly into culture wells with 1% glutaraldehyde, followed by anti-GPIb, anti-GPIIb (manufactured by Biodesign), and The cells were incubated with a monoclonal antibody cocktail consisting of anti-GPIb (Carpinterra, Calif.). The immune response was carried out with a streptavidin-β-galactosidase detection system (HistoMark, Kirkegaard and Perry). The megakaryocytes, which appear dark (blue in the real picture), are shown in FIG.
Panels A and D of FIG. 8 are positive and negative controls, respectively. The wells of panel A contain 37.5 pg of wild type mpl ligand 1-174 COS-1 treatment medium and show substantial megakaryocyte growth. Panel D included 1.5 μl COS-1 simulated treatment medium and showed no growth. Panels B and C of FIG. 8 are mpl ligand 1-174 analogs N7 and N10, respectively. Panel B contains 9.0 pg of mpl ligand COS-1 treatment medium, while Panel C contains 27 pg, both showing good megakaryocyte growth.
This experiment shows that the tested mpl ligand analogs can stimulate the growth of human megakaryocytes in vitro.
Example 9
Biological Activity of Mpl Ligand 1-174 Analogues in In Vitro Cell Proliferation Assays
Analogs of Mpl ligand 1-174 were prepared as above and analyzed for their ability to stimulate 32D-mpl cell proliferation. To produce 32D-mpl cells, full-length human mpl receptor sequences (I. Vigon et al., PNAS 89: 5640-5644 (1992)) were expressed in an expression vector containing a transcriptional promoter of the moroni murine sarcoma virus. Subcloned into. 6 μg of this construct and 6 μg of the packaging constructs of the trophic retroviruses (NRandau, DR Litman, Journal of Virology 66: 5110-5113 (1992)) were added to the CaPO ₄ Mammalian Transfection Kit (Stratagen). 3) was transfected to 3 × 10 ⁶ 293 cells. The same cells were retransfected after 2 and 4 days. On the day after the last transfection, 293 cells were transferred to an IL-3 dependent rat cell line [32D, clone 23; Greenberger group, PNAS 80: 2931-2936 (1983). After 24 hours, 32D cells were collected and banded by a BSA gradient (Path-o-cyte, manufactured by Miles). These cells were loosened in 1 ng / ml rat IL-3 and selected for incubation in 20% APK9 serum (Bartley, Cell 77: 1117-1124 (1994)). These cells were sorted according to receptors expressed at the cell surface by FACS using polyclonal rabbit antipeptide (MPL) serum. These cytokine dependent rat 32D-mpl cells respond to mpl ligands. 32D-MPL cells were incubated at a cell density of 1 × 10 ⁶ cells / ml in MEM medium containing 10% Fetal Clone II serum (manufactured by Cyclone Laboratories) and 1.0 ng / ml muIL3. These cells were collected by centrifugation (about 500 × G), washed twice in growth medium without muIL3 and resuspended at 1 × 10 ⁵ cells / ml.
Extended 12 point mpl ligand standard curve was prepared using mpl ligand 1-163 and ranged from 5000 to 1 pg / ml. Dilutions of 100 μl of standard mpl ligand or assay sample are added to the wells of a 96 well microtiter tissue culture plate containing 100 μl of resuspended cells (10,000 cells / well) and in a wet incubator at 37 ° C., 10% CO ₂ . Incubated. After 48 hours, 40 μl of MTS reagent (aqueous non-radioactive cell proliferation kit, manufactured by Promega) was added to each well and 14-18 hours later these plates were read on a 490 nM reader. In vitro activity of the samples was calculated from the dose response curves for each sample. One unit represents the amount of mpl ligand in each sample needed to achieve a 50% maximum stimulus. Inactivity was calculated by dividing biological activity (units / ml) by mpl ligand concentration (ng / ml) as determined by mpl ligand ELISA.
Specific biological activities of the mpl ligand analogs expressed by transfecting COS cells are shown in Table 4 below. The impact of amino acid substitutions on carbohydrate addition is also summarized. Purified human sequence mpl ligand exhibited 200-300 unit / ng of in vitro activity as measured by the above assay. The mpl ligand analogs containing additional N-linked carbohydrates from Table 4 are expressed similarly to the natural mpl ligand sequences even though they contain ancillary carbohydrate chains, N4 and N10 (as measured in section A of Example 6 above). This is clear. Both of these analogs showed complete in vitro biological activity. Thus, mpl ligand analogs containing N-linked carbohydrates can be normally expressed in mammalian cells and can exhibit normal or enhanced in vitro biological activity.
Mpl ligand form (amino acid length)orderNumber of N-linked chains (a)Elisa (ng / ml) (b)In vitro activity (units / ml) (c)Inactive (units / ng) (d) Simulation N1 (174) N1 (174) N1 (174) N2 (174) N3 (174) N4 (174) N4 (174) N5 (174) N6 (174) N7 (174) N7 (174) N9 (174) N10 (174) N10 (174) N11 (174) N11 (174) N13 (174) N14 (174) N15 (174)Natural Sequence Natural SequenceN22N25N30T32N30T32N38T40N86N82A83N82A83N92N120T122N120T122S36N38T40S36N38T40N53T55N58T60N30T32N120T1220NA000NA1 1000 to 10 to 10 11 NA00 to 10 to 10 to 20.082531.431.75NA1.8538241.20.4464.710.520.433.70.6251.36717.9261053758800NANA6368830NA10102660308019705943969010101800048506420125215280NANA344232NA822443655188291288168269271247
(a) The number of additional N-linked chains was estimated based on the mobility of analogue polypeptides in the SDS gel as described in Example 6.
(b) The amount of mpl ligand analog in CHO cell supernatant was determined by ELISA assay as described in the examples above.
(c) In vitro activity was determined by measuring uptake stimulation of thymidine in growth dependent 32D cells for mpl ligands.
(d) The ratio of in vitro activity of mpl ligand analogs measured by cell proliferation assay to the amount of mpl ligand analogs measured by Mpl ligand ELISA.
N.A .: Not measured.
Example 10
Expression in CHO Cells and Purification of Mpl Ligands 1-174, N4, and N15
PDSRα2 containing Mpl ligands 1-174, N4 and N15 cDNA were transfected into DHFR-deficient CHO cells using the experimental scheme of Example 2 modified as follows.
Each analog was transfected. Three weeks after transfection, mpl ligand expression was screened by mpl ligand ELISA. Three expression clones for each morphology were frozen in cold storage. The best expressing clones for each analog were mass produced in roller disease. For N4, 7.4 L of treated medium [50% D-MEM, 50% HAMS-F12, 1% penicillin / streptomycin / glutamine, 1% non-essential amino acid (manufactured by Gibco)] was prepared, and for N15 4.6 L of treated medium was prepared.
Medium treated with serum-free CHO cells from roller disease seeded with CHO cells expressing mpl ligands 1-174 (2.9 L), N4 (7.4 L) and N15 (4.4 L) was S1Y10 (molecular weight cutoff value 10,000 D). Amicon Spiral ultrafiltration cartridges were used to concentrate 12-, 19- and 12-fold, respectively. 150 ml of the treated concentrated medium was then loaded directly into a 3.3 ml hu-MPL-X (receptor) affinity column at a flow rate of 0.3 ml / min. Affinity columns were prepared by coupling 1.0-1.5 mg of Mpl-X (the soluble extracellular region of the Mpl receptor) per ml of Pharmacia CNBr-activated Sepharose resin as recommended by the manufacturer. After loading, the column was washed with 30 ml PBS (Phosphate Buffered Saline; 10 mM NaPO ₄ pH 6.8 / 150 mM NaCl) followed by 60 ml of 10 mM Tris pH 8.0 / 1 M NaCl / 1 mM CHAPS. . Mpl ligand 1-174 is 30 ml of 20 mM CAPS [3- (cyclohexylamino) -1 propanesulfonic acid] pH 10.5 / 1 M NaCl / 1 mM CHAPS {3-[(3-colamidopropyl) dimethylammonio] 1-propanesulfonate}.
To each eluted fraction, 0.6 ml of 1 M Tris (pH 7.0) was added to neutralize the fractions. SDS-PAGE analysis showed clear bleeding of 1-174 mpl ligands during washing with 10 mM Tris pH 8.0 / 1 M NaCl / 1 mM CHAPS. Elution fractions were analyzed by SDS-PAGE. These fractions containing Mpl ligand 1-174 were collected. The affinity purification was then repeated with the following modifications: loading and elution of 0.5 ml / min, and 10 mM Tris pH 8.0 / 1 M NaCl / 1 mM CHAPS wash omitted.
All fractions containing one mpl ligand band were concentrated using a YM10 (molecular weight cutoff value 10,000 D) membrane connected to a centricon device in 50 ml of agitated cells. The 0.5 ml concentrate was loaded directly at 0.25 ml / min onto a Pharmacia Superdex 200 HR 10/30 gel filtration column equilibrated with PBS and collected in 0.25 ml fractions. All elution fractions containing one mpl ligand band (based on SDS-PAGE analysis) were collected.
CHO cells of different forms (N4 and N5) expressing Mpl ligands were purified by a similar method (two affinity tablets were collected and loaded on one Superdex 200 gel filtration column).
Example 11
Carbohydrate Addition Test for N4 and N15 Expressed in CHO Cells
To determine whether N-linked carbohydrates were contained in the form of mpl ligands expressed in CHO cells, the treatment medium was analyzed by modifying the SDS-PAGE western blot as described in Example 6.
CHO D-treated medium from roller bottles was used. Samples were loaded into a Centricon-10 centrifuge concentrator (Beverley, Mass.) And spun at 6000 rpm for 1 hour in a Beckman J2-HS centrifuge using a fixed angle rotor (JA 20.1). Concentrated samples containing about 100 ng of mpl ligand analog were loaded on SDS-PAGE gel with SDS sample buffer (as described in Example 6). It also contained no carbohydrates and loaded mpl ligand MK 1-174 expressed in E. coli. 9 shows the difference in mobility consistent with the expected amount of carbohydrates. Following the fastest moving Met-Lys (1-174) Escherichia coli mpl ligand, 1-174 (CHO), N4 (CHO) and N15 (CHO) appear successively. See FIG. 9. The most plausible explanation for the increase in size relative to the non-glycosylated mpl ligand is that there are additional O-linked carbohydrates on mpl ligand 1-174 (CHO), and additional O-bonds on N4 (CHO). Carbohydrate and one additional N-linked oligosaccharide, and there is an additional O-linked carbohydrate and two additional N-linked oligosaccharides on N15 (CHO).
To confirm that the increase in molecular weight was due to the addition of N-linked carbohydrate chains, these samples were treated with N-glycanase to remove N-linked carbohydrates as described in Example 6. Each sample contained about 100 ng of mpl ligand analog purified from the treated medium.
After treatment with N-glycanase, the mobility of N4 (CHO) and N15 (CHO) decreased to the level of mpl ligand 1-174 (CHO). Treatment of Mpl ligand MK 1-174 (E. coli) or mpl ligand 1-174 (CHO) with N-glycanase did not affect mobility because they do not all contain N-linked carbohydrates. Compared with treatment with N-glycanase and no treatment, the size difference in N4 is consistent with the size of the N-linked carbohydrate chain, and the size difference in N15 is identical with the size of the two carbohydrate chains. . Thus addition of N-linked glycosylation sites to these two mpl ligand forms produced additional N-linked carbohydrates when these species were expressed in CHO cells. See FIG. 10.
Example 12
In Vitro Biological Activity of mpl Ligand Analogs Produced in CHO Cells
The mpl ligands and analogs expressed and purified in CHO cells or E. coli were assayed using the assays described in Example 9 except that factor dependent cell line 32D-MPL and activity were calculated using mpl ligand 1-332 produced in CHO cells as a standard. In vitro biological activity. Specific in vitro biological activities of the various forms are shown in Table 5 below. From this table it is clear that mpl ligand analogs containing additional carbohydrates, expressed in CHO cells, have biological activity in vitro.
In Vitro Activity of Mpl Ligands Mpl Ligand FormNumber of N-linked chainsIn vitro activity (U / mg × 10E6) MK174 (E. coli) 1-163 (CHO) 1-174 (CHO) N4 (CHO) N15 (CHO) 1-332 (CHO)000126138685609241
Example 13
In Vitro Biological Activity of Mpl Ligand Analogs
Platelet counts of mice treated with various forms of mpl ligand were measured and the results are shown in FIG. 11. CHO-induced mpl ligands 1-332, 1-174, N4 and N15 were produced and purified by mpl-receptor affinity chromatography. E. coli-induced Lys-mpl ligand 1-174 was produced and purified by conventional chromatography. Designated concentrations of each form were administered subcutaneously daily for 5 days to normal female Balb / c mice. Test blood was drawn from a small lateral incision in the tail vein 24 hours after the last dose. Blood cell analysis was performed using a Sysmex electronic blood cell analyzer (Baxter Diagnostics, Irvine, Calif.). Data show mean and +/- standard error of 4 measurements. Other blood cell variables such as white blood cell count or red blood cell count were not affected by these treatments (data not shown).
All forms increased platelet count. However, each activity varied. Relative in vitro activity was as follows: Mpl ligand MK 1-174 (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-linked carbohydrates increases in vivo activity. In addition, an increase in the amount of carbohydrates indicates a proportional increase in activity in vivo.
Example 14
Preparation of Mpl Ligand Analogs and Cleavage N16-N40 by Nested PCR
Analogs N16 to N40 (the structures of these analogues, see Table 6 below) were prepared by overlapping polymerase chain reaction (PCR) using the experimental scheme adopted from Cheng, PNAS 91, 5695 (1994). Usually 1 to 2 mutations were introduced into each construct.
The following oligonucleotide primers were synthesized and used to prepare analogs N16-N40:
F = forward
R = reverse
The construct introducing one new glycosylation site was made by two successive steps. In one step, two reactions were carried out using four different oligonucleotides. These oligos included 5 'forward primers, reverse mutagenic primers, forward mutagenic primers (usually complementary to reverse mutagenic primers) and reverse 3' primers. The reverse 3 ′ primer contained a sequence that introduced a stop codon after the SalI restriction site. Stop codons were introduced at the 175, 184, 192 and 200 sites. Thus, forms of lengths 1-174, 1-183 (N16), 1-191 (N17) and 1-199 (N31) could be made. PCR1 used template DNA (pDSRα2 containing mpl ligand 1-174 sequences or full length mpl ligand 1-332 sequences), 5 ′ forward primers and reverse mutagenic primers. PCR2 used template DNA, 3 ′ reverse primer and positive mutagenic primer. After performing the two PCR reactions, the amplified DNA fragments were separated by agarose gel electrophoresis. Small pieces of agarose containing DNA fragments of the correct size were cut from the gel.
DNA fragments obtained from PCR1 and PCR2 were joined together and a third PCR reaction was performed using 5 'forward and 3' reverse primers. This amplified the full-length DNA fragment containing the desired mutation inserted into the mpl ligand.
The amplified fragments were again separated by agarose gel electrophoresis, and DNA fragments of the correct size were purified according to the method provided by the manufacturer using a Geneclean ^™ apparatus (manufactured by Bio 101). Purified DNA was digested with XbaI and SalI and then purified again using the Geneclean ^™ device. The fragment was then bound to pDSRα2 cleaved with XbaI and SalI. Bound DNA was precipitated using 2 volumes of ethanol containing 0.3 M NaOAc (pH 5.2) in the presence of carrier tRNA and transformed into E. coli. Clones were examined by restriction analysis and agarose gel electrophoresis to identify clones containing the correct size DNA insert. Purified plasmid DNA was then prepared and sequenced for mpl ligand inserts to confirm the presence of the desired mutation and to ensure that there were no concomitant amino acid changes.
In some cases, two or more mutations have been combined at the same time, ie refer to N29, N33, N34, N35, N39 and N40. This can be done by introducing new substituents into DNA that already contains changes. For example, N33 was made by introducing N23 changes into N15. In this case the method was carried out using N23 mutagenic primers and N15 template DNA.
Alternatively, two changes could be introduced simultaneously into the template DNA. The template DNA may contain a sequence that encodes a mpl ligand form that contains a native sequence or that already contains a change. Step 1 involved 3 PCR reactions and 6 oligos. These oligos included 5 'forward primers, two pairs of forward and reverse mutagenic primers, and reverse 3' primers. Each pair of primers contained sequences complementary to each other and designed to introduce one new glycosylation site.
PCR1 included template DNA, 5 'forward primer and pair 1 reverse mutagenic primer. PCR2 included template DNA, pair 1 positive mutant primers and pair 2 reverse mutagenic primers, where pair 2 primers are 3 ′ → pair 1 primers. PCR3 included template DNA, pair 2 positive mutant primers and reverse 3 ′ primers.
DNA fragments obtained from each PCR reaction were separated by agarose gel electrophoresis as described above and then cleaved. Three DNA fragments were then joined together and again amplified by PCR using 5 'forward and 3' reverse primers.
The DNA fragments encoding the whole genes of interest together with the sequence containing the two new glycosylation sites were then purified as above, cleaved with XbaI and SalI, and bound to pDSRα2 cleaved with XbaI and SalI.
In addition, PCR reactions with templates that already contain mutations can be used to bind multiple mutations. For example, N39 was prepared by introducing N36 and N38 changes into the N15 template DNA. This was done using a different kind of primer [N36 (2)] than was used to prepare N36 [N36 (1)].
Longer mpl ligand forms could also be prepared. Thus, N40 was prepared in a similar manner to N39 except that the 3 ′ reverse primer (step 1) in PCR 3 and the PCR primer in step 2 were the primers used to prepare N31. The primer introduces a stop codon at site 200 following the SalI restriction site. In addition, the template DNA used in PCR 3 contained a sequence encoding a full length mpl ligand (1-332).
Conventional PCR reaction mixtures contained: 4 μl of each forward and reverse primer (5 pm / μl), 1 μl of template (50 ng), 10 μl of 5X LP buffer (100 mM Tricine pH 8.7 / 25 % Glycerol / 425 mM KOAc), 10 μl of dNTP mixture (1 mM each of dATP, dTTP, dCTP, dGTP), 0.8 μl rtTh polymerase (manufactured by Perkin Elmer; 2.5 U / μl), and 2 μl of Vent polymerase (Manufactured by Neb; 0.01 U / μl after 1: 100 dilution in 1 × LP buffer). Water was added to bring the final volume to 50 μl. PCR was started when all the ingredients were added together in the order indicated and 1 μl of 50 mM MgOAc was added and the temperature for the first cycle was above 60 ° C. The reaction conditions are as follows: 2 cycles of 94 ° C, 10 seconds / 45 ° C, 1 minute / 68 ° C, 5 minutes, followed by 25 cycles of 94 ° C, 10 seconds / 55 ° C, 1 minute / 68 ° C, 5 minutes.
These general methods were used to prepare the mpl ligand analogs and cleavages N16 to N40 specified in Table 6. Each type of DNA sequence change is indicated.

The symbol (i) in the above table means that the amino acid mentioned is inserted. For example, Glu ⁵⁷ → Asn ^{55 ′} (i), Thr ⁵⁷ (analogue N23 in Table 6) replaces Glu at 57 site with Thr, and also inserts Asn immediately after Met at 55 site, followed by amino acid Asn is set to 55 'to keep the number assigned to.
Embodiments, which include all changes from the previous embodiment, are indicated by the number of a particular analogue with a plus sign. See analogs N35, N39 and N40. The amino acid chain lengths of these analogues are indicated in parentheses. Thus, analog N35 contained a combination of all changes for N4, N23, N30 and N31. These changes indicated in N31 mean that analog N35 is 199 amino acids in length. The length of all analogs in Table 6 was 174 amino acids except for the different lengths indicated (or if the amino acids were inserted, the total length would increase by the number of amino acids inserted).
Example 15
Characterization of Mpl Ligand Analogs and Cleavage N16 to N40
A. Determination of expression level and in vitro biological activity of Mpl ligand analogs
Species N16 to N40 were transfected into COS cells using either the electroporation method (Example 5) or the CaPO ₄ method (mammal cell transfection kit; manufactured by Specialty Media). After 3-4 days, the cells-free treated medium was collected, fractionated and stored at -70 ° C. Expression levels were measured by ELISA assay as described in Example 7 above. Supernatants were also analyzed to determine biological activity as described in Example 9 above. These activities were calculated from standard curves using purified mpl ligands 1-332 expressed in CHO cells as standard.
The results are shown in Table 7 below. As shown in Table 7, most of the mpl ligand analogs were expressed and secreted. Some analogs have increased secretion. Biological analysis of these samples revealed that most of the inactivities were also similar to the unmodified form. Some analogs contained multiple N-linked carbohydrate chains (see below). This indicates that carbohydrate addition can increase the secretion of analogs and induce normal activity in vitro.
Mpl ligand analogs with chain sites of N-linked carbohydrates Mpl ligand form (amino acid length)orderNumber of N-linked chains (a)Elisa (ng / ml) (b)In vitro activity (units / ml) (c)Inactive (units / ng) (d) N1 (174) N15 (174) N16 (183) N17 (191) N18 (174) N19 (174) N20 (174) N21 (174) N22 (174) N23 (174) N24 (174) N25 (174) N26 ( 174) N27 (174) N29 (174) N30 (174) N31 (199) N33 (174) N34 (174) N35 (199) N36 (174) N37 (174) N38 (174) N39 (174) N40 (199)Natural sequence N30T32N120T1221-1831-191N23S25N37G38S39N39S41N54S56N52T54N55 '(i) T57N57S59N81T83N118S120N119S121N30S32N120S122T163N1641 -1994 + 10 + 234 + 23 + 3034 + 31N55N38N166N36N166N36N36T36N56N3800 to 20 NA0 NANA 0 to 10 to 110 NANA 00 to 20 to 11 or more 32 or more 4 or more 0 to 110 to 13 to 45 or more2845850.320.30.3302115.30.220.94.51512815678112172483225127134399170039276115NA104380856105945812396338162715592190001005713536131125808450439041766119735143156NANA2.5NANA14642896865591067510812212212912076121141156139147
(a) The number of additional N-linked chains was estimated according to the mobility of analogue polypeptides in the SDS gel.
(b) The amount of mpl ligand analog in CHO cell supernatant was determined by EIA.
(c) In vitro activity was determined by measuring proliferation of growth dependent 32D-MPL cells for mpl ligands.
(d) The ratio of in vitro activity of mpl ligand analogs measured by cell proliferation assay to the amount of mpl ligand analogs measured by Mpl ligand ELISA.
i: insert.
N.A .: Not measured.
B. Carbohydrate Addition Measurement
The analogs of Table 6 were examined to confirm the addition of N-linked carbohydrates using the method described in Example 6 above.
Some analogs (N21, N22, N30, N33 and N36) were also examined using a modified method. Monoclonal antibodies used to develop Western blots are generated for peptides comprising amino acid residues 47-62, and because some analogs of Table 6, such as N21, contain substituents that affect immune activity with the antibody, I need a way. Therefore, to analyze these analogs, supernatants were immunoprecipitated using monoclonal antibodies produced in mice against mpl ligands 1-163 expressed in E. coli.
Typically 15 μg of antibody was used to immunoprecipitate 50 ng of mpl ligand analog. Western blots using immunoprecipitated material incubated the immunoprecipitated bands by incubating these blots with rabbit anti-mpl ligand polyclonal antibodies (typically 1 μg / ml; produced for mpl ligands 1-163 expressed in Escherichia coli). Visualization was performed as described in Example 6 above except using the anti-rabbit ECL kit (manufactured by Amersham). The results of various experiments are shown in Table 7 above. Some analogs had increased sizes indicating the presence of N-linked carbohydrates (N21, N22, N23, N29, N30, N31, N33, N34, N35, N36, N38, N39 and N40). Some of these analogs, such as N29, N33, N34, N35, N39 and N40, had one or more N-linked chains. These analogs were secreted at normal or higher levels and had in vitro biological activity similar to mpl ligands 1-174. This indicates that multifunctional N-linked glycosylation sites can be introduced into the mpl ligand without deleterious effects on expression or biological activity.
To demonstrate that multiple oligosaccharide chains can be added to the mpl ligand, various analogs expressed in COS cells were analyzed by Western blot as described in Example 6 above. 12 shows that the mobility of the analogs decreases as the number of added N-linked carbohydrate sites increases. Analogs N39 and N40 with four new sites were prepared. Analogs with the most N-linked sites showed the slowest mobility. This result was observed for both mpl ligand 1-174 and 1-199 forms. This indicates that four or more analogs can be joined together to form new analogs with multiple N-linked carbohydrate chains.
Example 16
Comparison of Glycosylation Sites Containing Asn-X-Ser and Asn-X-Thr
N-linked glycosylation sites include Asn-X-Ser or Asn-X-Thr, where X can be one of 20 natural amino acids except Pro. We wanted to confirm whether Ser or Thr is preferred for the third site. Thus, to determine the effect on the% occupancy of N-linked glycosylation sites, two sets of analogs were examined, each with mpl ligand glycosylation analogs containing Ser or Thr at the third site of the sequence. It was. N15 contained two Asn-X-Thr sites while N29 contained two Asn-X-Ser sites in the exact same site. Similarly, N30 contained one Asn-X-Ser site while N38 contained one Asn-X-Thr site at the same site.
To compare these two sets of analogs, they were expressed in COS cells and the secreted mpl ligands were analyzed by Western blot as described in Example 6 above. 13 shows these results. N15 had glycosylated mpl ligands at a significantly increased rate compared to N29. In contrast, there was little difference in the ratio of glycosylated mpl ligands to unglycosylated mpl ligands when comparing N30 and N38. These results indicate that both Asn-X-Ser and Asn-X-Thr can be introduced into the mpl ligand, and both can act as sites for N-linked carbohydrate addition. Also, in some cases the Asn-X-Thr sequence may be preferred (ie, it may be glycosylated more efficiently).
Although the present invention has been described in the form of what is believed to be the preferred embodiment, it is not intended to be limited to the disclosed embodiment, but on the contrary, it is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims, the scope of which It should be consistent with extensive interpretation to include variants and equivalents.
Sequence table
(1) General Information:
(i) Applicants: Elliott, Steven G.
(ii) Name of the invention: Mpl ligand analog
(iii) number of sequences: 56
(iv) contact address:
(A) Recipient: Amgen Incorporated
(B) Street: Dehavilland Drive 1840
(C) City: Thousand Owls
(D) State: California
(E) Country: United States
(F) Zip code: 91320-1789
(v) computer readable mode:
(A) Medium form: floppy disk
(B) Computer: IBM PC Compatible
(C) working system: PC-DOS / MS-DOS
(D) Software: Patent In Release # 1.0, Version # 1.30
(vi) Current application data:
(A) Application number:
(B) filing date:
(C) Classification:
(2) Information about SEQ ID NO: 1
(i) Sequence Features:
(A) Length: 1342 base pair
(B) Type: nucleic acid
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(ix) characteristics:
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(ix) characteristics:
(A) Name / Key: sig_peptide
(B) Location: 1..98
(ix) characteristics:
(A) Name / Key: mat_peptide
(B) Location: 99..1094
(xi) sequence description:
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(ix) characteristics:
(A) Name / Key: mat_peptide
(B) location: 75..96
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(xi) sequence description:
(2) information for SEQ ID NO: 8:
(i) Sequence Features:
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(C) strands: single
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(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 9:
(i) Sequence Features:
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(xi) sequence description:
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(i) Sequence Features:
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(xi) sequence description:
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(i) Sequence Features:
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(xi) sequence description:
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(xi) sequence description:
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(C) strands: single
(D) topology: linear
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(xi) sequence description:
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(i) Sequence Features:
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(C) strands: single
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(A) Description: / desc = nucleic acid
(ix) characteristics:
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(i) Sequence Features:
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(xi) sequence description:
(2) information for SEQ ID NO: 22:
(i) Sequence Features:
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(xi) sequence description:
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(i) Sequence Features:
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(xi) sequence description:
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(i) Sequence Features:
(A) Length: 33 base pairs
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(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
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(i) Sequence Features:
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(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
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(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 27:
(i) Sequence Features:
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(C) strands: single
(D) topology: linear
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(A) Description: / desc = nucleic acid
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(i) Sequence Features:
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(C) strands: single
(D) topology: linear
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(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 29:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
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(A) Description: / desc = nucleic acid
(ix) characteristics:
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(B) Position: Complementary Strand (1..31)
(xi) sequence description:
(2) Information about SEQ ID NO: 30:
(i) Sequence Features:
(A) Length: 32 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 31:
(i) Sequence Features:
(A) Length: 32 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..32)
(xi) sequence description:
(2) information for SEQ ID NO: 32:
(i) Sequence Features:
(A) Length: 32 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 33:
(i) Sequence Features:
(A) Length: 32 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..32)
(xi) sequence description:
(2) information for SEQ ID NO: 34:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 35:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..31)
(xi) sequence description:
(2) information for SEQ ID NO: 36:
(i) Sequence Features:
(A) Length: 32 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 37:
(i) Sequence Features:
(A) Length: 32 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..32)
(xi) sequence description:
(2) information for SEQ ID NO: 38:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 39:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..31)
(xi) sequence description:
(2) information for SEQ ID NO: 40:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 41:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..31)
(xi) sequence description:
(2) information for SEQ ID NO: 42:
(i) Sequence Features:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 43:
(i) Sequence Features:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..24)
(xi) sequence description:
(2) information for SEQ ID NO: 44:
(i) Sequence Features:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 45:
(i) Sequence Features:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..24)
(xi) sequence description:
(2) information for SEQ ID NO: 46:
(i) Sequence Features:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 47:
(i) Sequence Features:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..27)
(xi) sequence description:
(2) information for SEQ ID NO: 48:
(i) Sequence Features:
(A) Length: 33 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..33)
(xi) sequence description:
(2) information for SEQ ID NO: 49:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 50:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..31)
(xi) sequence description:
(2) information for SEQ ID NO: 51:
(i) Sequence Features:
(A) Length: 26 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 52:
(i) Sequence Features:
(A) Length: 26 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..26)
(xi) sequence description:
(2) information for SEQ ID NO: 53:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 54:
(i) Sequence Features:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..31)
(xi) sequence description:
(2) information for SEQ ID NO: 55:
(i) Sequence Features:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(xi) sequence description:
(2) information for SEQ ID NO: 56:
(i) Sequence Features:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) strands: single
(D) topology: linear
(ii) Molecular type: other nucleic acid
(A) Description: / desc = nucleic acid
(ix) characteristics:
(A) Name / Key:-
(B) Position: Complementary Strand (1..27)
(xi) sequence description:

权利要求:
Claims (21)
[1" claim-type="Currently amended] An analog of a mpl ligand comprising an amino acid sequence comprising one or more added, one or more deleted or one or more altered glycosylation sites.
[2" claim-type="Currently amended] The analog of claim 1, wherein the glycosylation site is for an N-linked hydrocarbon chain.
[3" claim-type="Currently amended] The analog of claim 1, wherein the glycosylation moiety is for an O-linked hydrocarbon chain.
[4" claim-type="Currently amended] The analogue of claim 1 having one or more additional attached hydrocarbon chains when expressed in eukaryotes.
[5" claim-type="Currently amended] The analog of claim 1, wherein the hydrocarbon chain is an N-linked hydrocarbon chain.
[6" claim-type="Currently amended] The analog of claim 1, wherein the hydrocarbon chain is an O-linked hydrocarbon chain.
[7" claim-type="Currently amended] The analog of claim 1 which is the expression product of an exogenous DNA sequence in a eukaryotes.
[8" claim-type="Currently amended] The analog of claim 5 selected from the group consisting of:
[Asn ³⁰ , Thr ³² ] mpl ligands;
[Asn ⁸² , Ala ⁸³ ] mpl ligands;
[Asn ⁸⁷ , Thr ⁸⁹ ] mpl ligands;
[Asn ⁵³ , Thr ⁵⁵ ] mpl ligands;
[Asn ⁵⁸ , Thr ⁶⁰ ] mpl ligands;
[Asn ³⁰ , Thr ³² , Asn ¹²⁰ , Thr ¹²² ] mpl ligand;
[Asn ³⁷ , Gly ³⁸ , Ser ³⁹ ] mpl ligands;
[Asn ³⁹ , Ser ⁴¹ ] mpl ligand;
[Asn ⁵⁴ , Ser ⁵⁶ ] mpl ligands;
[Asn ⁵² , Thr ⁵⁴ ] mpl ligands;
[Asn ^{55 '(i)} , Thr ⁵⁷ ] mpl ligand;
[Asn ⁸¹ , Thr ⁸³ ] mpl ligand;
[Asn ¹¹⁸ , Ser ¹²⁰ ] mpl ligands;
^{[Asn 30, Ser 32, Asn} 120, Ser 122] mpl ligand;
[Thr ¹⁶³ , Asn ¹⁶⁴ ] mpl ligand;
[Asn ³⁰ , Thr ³² , Asn ¹²⁰ , Thr ¹²² , Asn ^{55 ′ (i)} , Thr ⁵⁷ ] mpl ligand;
[Asn ³⁰ , Thr ³² , Asn ^{55 '(i)} , Thr ⁵⁷ , Thr ¹⁶³ , Asn ¹⁶⁴ ] mpl ligand;
[Asn ⁵⁵ , Thr ⁵⁷ ] mpl ligands;
[Asn ⁵⁶ ] mpl ligand;
[Thr ¹⁶³ , Asn ¹⁶⁴ , Thr ¹⁶⁶ ] mpl ligand; And
[Asn ³⁰ , Thr ³² , Asn ¹²⁰ , Thr ¹²² , Asn ⁵⁵ , Thr ⁵⁷ , Thr ¹⁶³ , Asn ¹⁶⁴ , Thr ¹⁶⁶ ] mpl ligand.
[9" claim-type="Currently amended] The analog of claim 1, wherein the mpl ligand has an amino sequence selected from the group consisting of:
mpl ligand 1-332 amino acids 1-332 of FIG.
mpl ligand 1-199 amino acids 1-199 of FIG.
mpl ligand 1-191 amino acids 1-191 of FIG. 1
mpl ligand 1-183 amino acids 1-183 of FIG.
mpl ligand 1-174 amino acids 1-174 of FIG.
mpl ligand 1-163 amino acids 1-163 of FIG. 1
mpl ligand 1-153 amino acids 1-153 of FIG.
mpl ligand 1-152 amino acids 1-152 of FIG.
mpl ligand 1-151 amino acids 1-151 of FIG.
mpl ligand 7-332 amino acids 7-332 of FIG.
mpl ligand 7-199 amino acids 7-199 of FIG.
mpl ligand 7-191 amino acids 7-191 of Figure 1
mpl ligand 7-183 amino acids 7-183 of FIG.
mpl ligand 7-174 amino acids 7-174 of FIG.
mpl ligand 7-163 amino acids 7-163 of FIG.
mpl ligand 7-153 amino acids 7-153 of FIG.
mpl ligand 7-152 amino acids 7-152 of FIG.
mpl ligand 7-151 Amino acid 7-151 of FIG.
[10" claim-type="Currently amended] Analogs of human mpl ligands selected from the group consisting of:
[Asn ²² ] mpl ligand;
[Asn ²⁵ ] mpl ligand;
[Asn ³⁸ , Thr ⁴⁰ ] mpl ligands;
[Asn ⁸⁶ ] mpl ligand;
[Asn ⁹² ] mpl ligand;
[Asn ¹²⁰ , Thr ¹²² ] mpl ligand;
Ser ³⁶ , Asn ³⁸ , Thr ⁴⁰ ] mpl ligand;
[Asn ⁸⁸ , Thr ⁹⁰ ] mpl ligands;
[Asn ²³ , Ser ²⁵ ] mpl ligands;
[Asn ⁵⁷ , Ser ⁵⁹ ] mpl ligands; And
[Asn ¹¹⁹ , Ser ¹²¹ ] mpl ligand.
[11" claim-type="Currently amended] The analog of claim 10, wherein the mpl ligand is selected from the group consisting of:
mpl ligand 1-332 amino acids 1-332 of FIG.
mpl ligand 1-199 amino acids 1-199 of FIG.
mpl ligand 1-191 amino acids 1-191 of FIG. 1
mpl ligand 1-183 amino acids 1-183 of FIG.
mpl ligand 1-174 amino acids 1-174 of FIG.
mpl ligand 1-163 amino acids 1-163 of FIG. 1
mpl ligand 1-153 amino acids 1-153 of FIG.
mpl ligand 1-152 amino acids 1-152 of FIG.
mpl ligand 1-151 amino acids 1-151 of FIG.
mpl ligand 7-332 amino acids 7-332 of FIG.
mpl ligand 7-199 amino acids 7-199 of FIG.
mpl ligand 7-191 amino acids 7-191 of Figure 1
mpl ligand 7-183 amino acids 7-183 of FIG.
mpl ligand 7-174 amino acids 7-174 of FIG.
mpl ligand 7-163 amino acids 7-163 of FIG.
mpl ligand 7-153 amino acids 7-153 of FIG.
mpl ligand 7-152 amino acids 7-152 of FIG.
mpl ligand 7-151 Amino acid 7-151 of FIG.
[12" claim-type="Currently amended] The analog of any one of claims 8 to 11 which is an expression product of an exogenous DNA sequence in a eukaryotic cell.
[13" claim-type="Currently amended] 13. The analog of claim 12, wherein said eukaryotic cell is a mammalian cell.
[14" claim-type="Currently amended] The mplified product of [Asn ³⁰ , Thr ³² , Asn ¹²⁰ , Thr ¹²² , Asn ^{55 '(i)} , Thr ⁵⁷ , Thr ¹⁶³ , Asn ¹⁶⁴ , Thr ¹⁶⁶ ] mpl according to claim 1. Ligand 1-® Or an analog of [Asn ³⁰ , Thr ³² , Asn ¹²⁰ , Thr ¹²² , Asn ^{55 '(i)} , Thr ⁵⁷ , Thr ¹⁶³ , Asn ¹⁶⁴ , Thr ¹⁶⁶ ] mpl ligand 1-199.
[15" claim-type="Currently amended] The analog of claim 14, wherein said eukaryotic cell is a mammal.
[16" claim-type="Currently amended] The analog of claim 15, wherein said mammalian cell is CHO.
[17" claim-type="Currently amended] (a) the O-linked hydrocarbon moiety in the mpl ligand is substituted with an N-linked hydrocarbon moiety; (b) the N-linked hydrocarbon moiety in the mpl ligand is substituted with an O-linked hydrocarbon moiety; (c) the O-linked hydrocarbon moiety in the mpl ligand is substituted with a different O-linked hydrocarbon moiety; Or (d) an mpl ligand comprising an amino acid sequence in which the N-linked hydrocarbon moiety in the mpl ligand is substituted with a different N-linked hydrocarbon moiety.
[18" claim-type="Currently amended] A DNA sequence encoding an analog of a mpl ligand comprising one or more added, one or more deleted or one or more altered glycosylation sites.
[19" claim-type="Currently amended] A DNA sequence encoding an analog of the mpl ligand according to any one of claims 1-11 and 13-17.
[20" claim-type="Currently amended] Eukaryotic host cell transfected with a DNA sequence according to claim 19 such that the host cell expresses an analog of the mpl ligand.
[21" claim-type="Currently amended] A composition comprising a therapeutically effective amount of a mpl ligand analog according to any one of claims 1 to 11 and 13 to 17 and a pharmaceutically acceptable diluent, adjuvant or carrier.

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同族专利:

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公开号 | 公开日

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KR100492452B1|2005-12-02|

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US5696250A|1997-12-09|

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TW480272B|2002-03-21|

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US5756083A|1998-05-26|

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US7399833B1|2008-07-15|

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TW420688B|2001-02-01|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1995-02-15|Priority to US08/388,779

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1995-02-15|Priority to US08/388779

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1996-02-09|Priority to US08/591,070

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1996-02-09|Priority to US08/591,070

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1996-02-09|Priority to US8/591,070

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1996-02-14|Application filed by 스티븐 엠. 오드리, 암겐 인코포레이티드

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1998-07-15|Publication of KR19980702206A

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2005-12-02|Application granted

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2005-12-02|Publication of KR100492452B1

优先权:

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申请号 | 申请日 | 专利标题

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US08/388,779|US5696250A|1995-02-15|1995-02-15|DNA encoding megakaryocyte growth and development factor analogs|

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US08/388779|1995-02-15|

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US08/591,070|1996-02-09|

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US8/591,070|1996-02-09|

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US08/591,070|US5756083A|1995-02-15|1996-02-09|Mpl ligand analogs|