WO1999057147A1 - Mutants de la thrombopoietine humaine - Google Patents

Mutants de la thrombopoietine humaine Download PDF

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Publication number
WO1999057147A1
WO1999057147A1 PCT/JP1999/002293 JP9902293W WO9957147A1 WO 1999057147 A1 WO1999057147 A1 WO 1999057147A1 JP 9902293 W JP9902293 W JP 9902293W WO 9957147 A1 WO9957147 A1 WO 9957147A1
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protein
tpo
residue
activity
human
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PCT/JP1999/002293
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English (en)
Japanese (ja)
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Ryota Kuroki
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Kirin Brewery Company, Limited
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Priority to AU35387/99A priority Critical patent/AU3538799A/en
Publication of WO1999057147A1 publication Critical patent/WO1999057147A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention refers to human thrombopoietin activity (an activity that promotes proliferation and differentiation of megakaryocyte progenitor cells or specifically stimulates or enhances the production of platelets in vivo.
  • Thrombopoietin is "TPO" TPo protein having the following formula:
  • the present invention relates to a TPO mutant having high stability, particularly high thermostability and / or high biological activity, DNA encoding the TPO mutant, a method for producing the mutant, or
  • the present invention relates to a pharmaceutical composition comprising the TPO variant, a platelet-increasing agent or a therapeutic agent for thrombocytopenia containing the TP ⁇ protein variant as an active ingredient.
  • the present invention also relates to a protein in which a water-soluble polymer such as polyethylenedaricol is bound to the TPo variant, a pharmaceutical composition comprising such a protein, a thrombocytosis agent, and a therapeutic agent for thrombocytopenia.
  • a water-soluble polymer such as polyethylenedaricol
  • Human thrombopoietin is a protein that has been cloned as a ligand for Mp1, a member of the receptor superfamily of cytokines (de Sauvage et al., Nature (London) 369, 533-565, (1994), Bartley, TD et al., Cell 77, 1117-1124, (1994)). All of these Mp1 ligands have been detected in the serum and plasma of thrombocytopenic animals (humans, mice, dogs), and their involvement in megakaryocyte formation and platelet formation has been confirmed.
  • the present applicant also used the activity of promoting megakaryocyte production from megakaryocyte progenitor cells highly purified from rat bone marrow as an index to determine thrombocytopenia. Purify rat TP ⁇ from rat plasma,
  • the present applicant has administered the human TPO to a thrombocytopenic mouse in which myelosuppression has occurred by administration of an anticancer drug or an immunosuppressant, or irradiation or bone marrow transplantation (BMT).
  • BMT bone marrow transplantation
  • the present applicant has prepared various derivatives in which a part of the amino acid sequence of the following Sequence Listing: SEQ ID NO: 1 has been deleted or substituted, or an amino acid has been inserted or added, and these derivatives have been used as human TPO. It has been confirmed that the activity is maintained (JP-A-8-228781).
  • other reports on the splicing variant of the TPO gene Snow Brand Milk Products: JP-A-8-168386, Design: International Patent Publication No. WO 96/03434, Yamanouchi Pharmaceutical Co., Ltd .: Japanese Patent Application Laid-Open No. 8-89252, Asahi Kasei Kogyo Co., Ltd., Japanese Patent Application Laid-Open No. 8-89253) and a report on deletion / substitution of TPO (GDSearle: International Publication No.
  • Proteins are generally known to be unstable. For example, in proteins, denaturation and inactivation during heating or vibration, inactivation by organic solvents and denaturing agents, and inactivation by proteolytic enzymes are observed. In addition, the metabolism of proteins in living organisms is triggered by digestion by proteases and certain chemical modifications, so that unstable proteins are more likely to be metabolized. No. Therefore, a protein having a stable three-dimensional structure, that is, a protein that is thermodynamically stable, can be expected to exhibit resistance to such causes of inactivation.
  • thermodynamically stable proteins Elucidation of the principles for designing thermodynamically stable proteins is still under study, but some knowledge has already been gathered. Recent studies on the relationship between the structure and stability of thermophilic bacterium proteins have revealed that the amino acid sequence of hyperthermophiles contains a large amount of phosphorus residues. Some useful stabilization methods have been derived from protein engineering studies. However, most of these require accurate information on the three-dimensional structure of the protein, making it difficult to take effective measures. One of the methods currently used to stabilize proteins is to randomly introduce amino acid substitutions and screen the stabilized proteins ( still obtaining stable proteins). At present, it is necessary to prepare a large number of mutants and evaluate them.
  • thermodynamic stability if the stability of the tertiary structure of TPO (especially thermodynamic stability) can be further enhanced, it will not only be less susceptible to degradation and inactivation by heat, but also to proteases. Therefore, there is a possibility that resistance may be improved, and at the same time, its biological activity may be expected to be improved.
  • the present inventors conducted research to further increase the stability of the tertiary structure of TPO (particularly, thermodynamic stability), and gradually increased the temperature of the protein solution by increasing the temperature of the protein solution. Changes upon exposure
  • the present invention relates to an amino acid sequence comprising at least one of the amino acid sequences of positions 1 to 163 of the amino acid sequence of SEQ ID NO: 1.
  • a protein having a mutation including substitution of a glutamine residue at position 5 with a lysine residue, an arginine residue or a tyrosine residue, and having human TPO activity.
  • the present invention relates to an amino acid sequence comprising at least the amino acid sequence from position 7 to position 151 of the amino acid sequence of SEQ ID NO: 1;
  • the present invention provides a protein having a mutation containing substitution of a glutamine residue at position 5 with a lysine residue, an arginine residue or a tyrosine residue, and having human TPO activity.
  • the protein comprises a protein having a methionine residue added at position 11.
  • the protein comprises a protein having a methionine residue at position 12 and a lysine residue at position 11.
  • the protein of the present invention may be bound to a water-soluble polymer, and examples of such a water-soluble polymer include polyethylene glycol.
  • the present invention provides a DNA comprising a nucleotide sequence encoding the above protein.
  • the present invention provides a recombinant vector comprising the above DNA.
  • the present invention also provides a prokaryotic cell transformed with the above DNA.
  • Prokaryotic cells are, for example, bacteria such as E. coli.
  • the present invention further provides a eukaryotic cell transformed with the above DNA.
  • Eukaryotic cells are, for example, mammalian cells.
  • the present invention provides a method for culturing the above-described prokaryotic or eukaryotic cells to separate and purify the produced protein having human TP ⁇ activity. And a method for producing a protein having human TP ⁇ activity.
  • prokaryotic cells are bacteria such as Escherichia coli, and examples of eukaryotic cells are mammalian cells.
  • the present invention provides, in yet another aspect, a TPII-containing pharmaceutical composition
  • a TPII-containing pharmaceutical composition comprising a protein as defined above and a pharmaceutically acceptable carrier.
  • the present invention also provides a highly stable TPo-containing pharmaceutical composition comprising a protein as defined above.
  • the present invention further provides a TPO-containing pharmaceutical composition having high heat stability, which comprises the protein as defined above.
  • the present invention also provides a platelet increasing agent comprising a protein as defined above as an active ingredient.
  • the present invention further provides a therapeutic agent for thrombocytopenia comprising a protein as defined above as an active ingredient.
  • a mutant human TPO protein having improved stability (particularly, thermostability) and / or improved biological activity is provided.
  • One such protein is a sequence comprising at least the amino acid sequence at positions 7 to 151 of SEQ ID NO: 1 (1 to 332), and at least the glutamate at position 115. It is a TP ⁇ mutant having a mutation including substitution of a residue with a lysine residue, an arginine residue or a tyrosine residue.
  • such proteins include a lysine residue and a arginine residue of at least a glutamine residue at position 115 in the amino acid sequence at positions 1 to 163 of SEQ ID NO: 1.
  • a TPO mutant having a mutation containing a substitution at a tyrosine residue is provided.
  • ⁇ a lysine residue of at least a glutamine residue at position 115
  • j means that the glutamine residue at position 115 is substituted with a lysine residue, an arginine residue, or a tyrosine residue
  • other mutations such as substitutions, deletions, insertions, and additions may be included in other parts of the sequence as long as they have TPO activity and maintain stability, particularly thermal stability. I do. Examples of such other mutations and methods for introducing mutations are disclosed, for example, in Japanese Patent Application Laid-Open No. 8-22881 of the present applicant, and the disclosure thereof is incorporated herein by reference.
  • the human TP protein of the present invention may have, for example, a methionine residue further added at position 11 in addition to the amino acid substitution described above, _ A methionine residue may be added at position 2, and a lysine residue may be added at position 11.
  • the serine residue at position 1 may be substituted with an alanine residue, and the alanine residue at position 3 may be substituted with a valinine residue.
  • the TPO mutant protein of the present invention is prepared by preparing a DNA encoding the same, incorporating it into an appropriate vector so that it can be expressed, introducing the DNA into an appropriate host, and culturing the host in an appropriate medium. It can be obtained by expressing DNA. "Expressably" means capable of transcription and translation into protein under the control of a promoter.
  • the DNA encoding the TP ⁇ variant can be obtained by chemical synthesis using conventional techniques such as the phosphoramidite method, preferably using a D ⁇ Synthesis machine, or the cTP of human TP ⁇ It can also be obtained by modifying DNA using a well-known site-directed mutagenesis method and by using a combination of PCR (polymerase chain reaction) techniques.
  • site-directed mutagenesis by PCR can be used.
  • TPO variants of the present invention such as site-directed mutagenesis, PCR, and vector cloning, transformation, etc.
  • the basic techniques for obtaining such proteins are described, for example, in J. Sambrook et al., Molecular Cloning A Laboratory Manual, 2hR, 1989, Cold Spring Harbor Laboratory Press, FM Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, 1995, John Wiley & Sons, Inc.
  • plasmid p HTF1 having a DNA containing a nucleotide sequence encoding amino acid sequence positions 1 to 33 (see SEQ ID NO: 3) is carried on Escherichia coli DH5, and By the Institute of Biotechnology and Industrial Technology, Institute of Biotechnology, Japan, on March 24, 1994 (1-3, Higashi, Tsukuba, Ibaraki, Japan) under the accession number FE RM BP—4 6 17 Has been deposited.
  • the TPO mutant of the present invention is preferably obtained by separating and purifying from a culture of a host cell transformed with a recombinant vector containing DNA encoding the TPO mutant.
  • prokaryotic cells for example, bacteria, preferably Escherichia coli
  • eukaryotic cells for example, yeast, insects, plant cells, and mammals
  • mammalian cells include COS cells, Chinese hamster ovary (CHO) cells, X63.6.5.3. Cells, C-127 cells, BHK (Baby Hamster Kidney) cells, and human-derived cells (eg, HeLa cells).
  • yeast include baker's yeast
  • insect cells include silkworm cultured cells (for example, Sf21 cells).
  • plant cells are dicot or monocot plant cells
  • the DNA is prepared by using a host-specific regulatory sequence (particularly, including a promoter.
  • a plasmid or viral vector or transgene may be constructed, and spontaneous uptake using calcium chloride calcium phosphate, electroporation , Micro-injection, lipovection, spheroplast,
  • the cells can be transferred into the cells using conventional techniques such as the protoplast method and the agrobacterium method.
  • the DNA may be capable of being expressed in the form of a plasmid capable of self-replication, or may be expressed by integration on a cell chromosome by homologous recombination.
  • the expression vector can further include an origin of replication, a ribosome binding site, a transcription termination signal, a selectable genetic marker, and the like.
  • the vector commercially available ones, those described in the literature, depositable deposits, and the like can be used, but if necessary, they may be used after modification.
  • pBluescript (Stratagene), pQ E series (Qiagen), pET series (Novagen), pUC19 (Gene, 33: 103, 1985), Yepl3 (ATCC 37115), pHS19, pHS15, pXTl ( Vectors such as Stratagene, pBPV, pSG, pSVL SV40 (Pharmacia), pcDNA1 / Amp, and pREP4 (Invitrogen) can be used, but are not limited thereto.
  • TPO variants of the present invention After culturing host cells and lysing cells as necessary, salting out, dialysis, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography Conventional techniques such as chromatography, electrophoresis, and isoelectric focusing can be used alone or in combination to purify the TPO variants of the present invention.
  • the TPO protein of the present invention includes not only the various TPo mutant proteins having the above-mentioned human TPO activity, but also a chemical modification in which the mutant protein is bound to at least one water-soluble polymer. Also included are TPo mutant proteins.
  • the TPO protein of the present invention includes a chemically modified TPO mutant protein obtained by reacting the aforementioned TP ⁇ mutant protein with a reactive poly (ethylene glycol) (PEG) molecule to add PEG to the TP ⁇ mutant protein. including. Such addition can be performed by a PEGylation reaction such as acylation or alkylation, as described below.
  • PEG is added to the ⁇ -amino group of a lysine residue, and PEG is added to the ⁇ -amino group at the ⁇ -terminal of the protein.
  • the yield and homogeneity of such monoPEGylation reactions are enhanced through reductive alkylation, which selectively modifies the single amino group at the N-terminal residue of the TP ⁇ protein portion.
  • This provides for the addition of a water-soluble polymer to the N-terminus of the protein.
  • a production example of a chemically modified TPO protein mutant to which PEG has been added is shown in Example 5 below.
  • Such chemically modified forms of proteins can effectively block proteolytic enzymes from physically contacting the protein structure itself to prevent degradation.
  • a further advantage is that, under certain circumstances, it increases the stability and circulation time of therapeutic proteins and reduces their immunogenicity. However, it should be noted that it is impossible to predict the effect of modifying a particular protein.
  • polyethylene glycol molecules can be attached to proteins via reactive groups found on the protein.
  • amino groups such as amino groups on lysine residues or on the N-terminus, or carboxyl groups on aspartic or glutamic acid residues or carboxyl groups on the C-terminus It is advantageous for such binding.
  • U.S. Pat. No. 4,002,531 describes reductive alkylation for attaching a poly (ethylene glycol) molecule to an enzyme.
  • EP-A-0 539 167 (Wri ght) states that peptides and organic compounds with free amino groups are modified with imidized derivatives of PEG or related water-soluble organic polymers.
  • U.S. Patent No. 4,904,584 discloses the modification of a lysine residue in a protein that binds to a poly (ethylene dalicol) molecule through a reactive amino group.
  • Other literatures and reviews describing protein modification and PEG modification include, for example, the following, and the technology disclosed therein may be used in the present invention.
  • Frans is, Focus on Growth Factors 3: 4-10 (May 1992) (From Medisc ript Mountview Court, Friern Barnet Lane, Shiondon N20, OLD, UK) ⁇ S ada,, J. Fermentation Bioengineer ing 71: 137-139 (1991), U.S. Pat. No. 4,179,337, U.S. Pat.
  • water-soluble polymer examples include, in addition to PEG, copolymers of ethylene glycolone Z-propylene glycolone, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and poly (ethylene glycol). 1,3-Dioxolane, other poly (1,3,6) -trioxane, ethylene // maleic anhydride copolymer, other water-soluble polymers such as polyamino acids (either homopolymers or random copolymers)
  • the functional polymer can be exemplified.
  • the present invention further provides a pharmaceutical composition containing the TPO protein of the present invention.
  • the pharmaceutical composition of the present invention can contain a stabilizer, a diluent, a solubilizer, a preservative, an antioxidant, an excipient, an isotonic agent, and the like, depending on the purpose of the preparation.
  • compositions containing the TPO protein of the present invention may be in the form of solutions, suspensions, tablets according to various administration routes including parenteral injections, pulmonary administration, nasal administration, and oral administration. Pills, capsules, granules, freeze-dried preparations and the like.
  • the pharmaceutical composition containing the TP ⁇ protein of the present invention usually contains 0.05 / z gZkg body weight to 1 mg Z kg body weight as an active ingredient per day depending on sex, administration route, etc. It can be administered once to several times.
  • the TP ⁇ protein of the present invention has improved stability, particularly heat stability, it is more suitable for use as a long-term storage preparation, a cold-flow preparation, or for long-term transport of the preparation.
  • a platelet-increasing agent for a large number of disease patients requiring an increase in platelets comprising the TPo protein of the present invention as an active ingredient.
  • a therapeutic agent for thrombocytopenia in patients undergoing chemotherapy or radiation therapy by administration of an anticancer drug or immunosuppressant, or bone marrow transplantation (BMT), PBSCT, or CBSCT is provided.
  • therapeutic agents are provided for a number of diseases characterized by thrombocytopenia, for example, thrombocytopenia due to impaired platelet production or shortened platelet life (enhanced platelet destruction or increased platelet consumption).
  • Examples include congenital Fanconi anemia, aplastic anemia associated with chemotherapy and radiation therapy, myelodysplastic syndrome, acute myeloid leukemia, or thrombocytopenia due to myelodysplasia such as bone marrow transplantation. It can be used to promote the recovery of platelet count in such patients.
  • thrombocytopenia due to abnormal TPO production examples include idiopathic thrombocytopenic purpura, acquired immunodeficiency syndrome (AIDS), disseminated intravascular coagulation, and thrombotic thrombocytopenia.
  • AIDS acquired immunodeficiency syndrome
  • thrombotic thrombocytopenia examples include idiopathic thrombocytopenic purpura, acquired immunodeficiency syndrome (AIDS), disseminated intravascular coagulation, and thrombotic thrombocytopenia.
  • AIDS acquired immunodeficiency syndrome
  • thrombotic thrombocytopenia examples include idiopathic thrombocytopenic purpura, acquired immunodeficiency syndrome (AIDS), disseminated intravascular coagulation, and thrombotic thrombocytopenia.
  • the TPO proteins of the present invention are also effective in treating platelet disorders caused by, for example, other chemicals or medicaments, or transient platelet loss or damage due to therapeutic measures.
  • the TPo protein of the present invention can be used to enhance the release of new "intact" platelets in such patients.
  • the liver is one of the major organs producing TP ⁇ , and it is also used in various liver diseases that cause thrombocytopenia, such as biliary atresia, liver transplantation, liver cirrhosis, and hepatitis.
  • thrombocytopenia such as biliary atresia, liver transplantation, liver cirrhosis, and hepatitis.
  • TPO protein administration is expected. It also stops stored platelets. It is also useful for recovering blood clot formation ability.
  • H6T (1-163) protein As the TPO protein from which the TP ⁇ mutant is designed, the applicant's earlier application (Japanese Patent Application No. 7-63298; Japanese Patent Application Laid-Open No. 8-228781) was published on September 10, 1996.
  • H6T (1-163) protein (see Sequence Listing: SEQ ID NO: 2 for amino acid sequence) described in (1).
  • the amino acid sequence of h6T (1-163) protein and the nucleotide sequence encoding it are shown in the sequence listing: SEQ ID NO: 2
  • Plasmid pBluescriptIISK + manufactured by Toyobo Co., Ltd.
  • Plasmid pCFM536-h6T l-163 (ligation of DNA sequence encoding h6T (1-163) shown in SEQ ID NO: 2 to expression vector pCFM536 was treated with restriction enzymes Xbal and Hindlll.
  • pBluescript IISK + was treated with alkaline phosphatase (Takara Shuzo), and a small fragment derived from plasmid DNA was recovered from the pCFM536-h6T digest by agarose gel electrophoresis. These were ligated with T4 DNA ligase (Takara Shuzo) to obtain plasmid pBluescript-h6T.
  • PCR was used to prepare a derivative Q115K in which the G1n residue at position 115 of human TPO was substituted with a Lys residue.
  • sequences of the primers used for PCR are as follows:
  • T3 5 '-ATTAACCCTCACTAAAGGG-3' (system IJ number 4)
  • the first stage PCR was performed using 140 ng of plasmid pBluescript-h6T as type I and 30 pmol of each synthesized primer (the combination of primers was [1] T3 and 115K (-), [ 2] ⁇ 13—21 and Q115K).
  • Pfu DNA Polymerase Toyobo Co., Ltd.
  • GeneArapPCR System9600 Perkin Elmer
  • PCR product was collected after electrophoresis on an agarose gel and dissolved in sterile water ( ⁇ ).
  • the second-stage PCR was performed using the collected PCR products.
  • ⁇ type quantity Uses [1] 8 ⁇ 1 [2] 2 ⁇ 1, and the ply is a combination of # 3 and M13-21.
  • the PCR product was treated with FUNOL / Kokuguchi form, precipitated with ethanol, and dissolved in 101 sterile water. Of these, 2 ⁇ l was designated as type I and subjected to the third stage PCR.
  • Pfu DNA Polymerase manufactured by Toyobo Co., Ltd.
  • incubate for 5 minutes at 95 ° C without adding a ply and then cycle for 20 minutes at 95 ° C for 30 seconds, 55 ° C for 1 minute, and 72 ° C for 1 minute, and The test was performed at 72 ° C for 10 minutes.
  • ply T30 and M13-21 were added and incubated at 95 ° C for 5 minutes, followed by incubation at 95 ° C for 30 seconds, 55 ° C for 1 minute, and 72 ° C for 1 minute for 20 cycles and 72 ° C for 10 minutes. .
  • This PCR product was collected by phenol phenol treatment and ethanol precipitation, and digested with restriction enzymes Xbal and Hindlll. The digested product was treated with phenol Z-cloth form and precipitated with ethanol, and then dissolved in 10 ⁇ l of ⁇ buffer. This fragment was similarly digested with a restriction enzyme, and subcloned into an expression vector pCFM536 which had been treated with allyl phosphatase (Takara Shuzo).
  • the host used was E. coli # 261 for PCFM536-h6T expression. Five clones were selected from the obtained transformants, and plasmid DNA was prepared. The method was performed essentially as described in Molecular Cloning [Sambrook et al., Cold Spring Harbor Laboratory Press (1989)]. The nucleotide sequence was confirmed using Applied Biosystems 373A DNA Sequencer using the purified plasmid DNA Taq Dye Deoxy TM Terminater Cycle Sequencing Kit (Applied Biosystems). .
  • the plasmid derived from the PCR product contained the DNA encoding the TPO-substituted derivative (Q115K) with the intended amino acid substitution [Glnll5 (CAG) ⁇ Lys (AAG)] was done.
  • a Q115R Q115Y Q115E mutant was prepared in the same manner as above.
  • Xba I-Hind III fragments were excised from all the pSKT PO mutants prepared in Example 2, inserted between the Xba I-Hind III cleavage sites of plasmid pCFM536, and transformed into E. coli 261. A transformant expressing the desired TP ⁇ mutant was obtained.
  • Example 4
  • the expression of the expression plasmid pCFM536 is controlled by the PL promoter itself, but is itself under the control of the cI857 repressor gene.
  • the transformant obtained in Example 3 was shake-cultured at 30 ° C in 60 ml of an LB medium containing 50 / xg / ml of ampicillin and 12.5 / ig / ml of tetracycline at 30 ° C. 25 ml of the solution was added to 1000 ml of LB medium containing 50 g / ml of ampicillin, and the OD was cultured at 30 ° C with shaking until the A600 reached 1.0 to 1.2. Then, about 330 ml LB medium at 65 ° C was added so that the final temperature became 42 ° C, and the cells were cultured with shaking at 42 ° C for 3 hours to induce the expression of the TPO mutant.
  • the column was equilibrated with about 300 mL of 10 raM phosphate buffer (pH 7.2) containing 15% glycerol.
  • the target sample was eluted at a flow rate of 1 ml / min using a linear concentration gradient from 10 mM phosphate buffer containing 15% glycerol to the same buffer containing 0.5 M salt.
  • the elution of the protein from the column was monitored by ultraviolet absorption at 280 nm, and the fraction containing the target protein was collected (about 30 to 40 ml). This fraction was concentrated to about 2 ml using a centrifugal concentrator (Centriprep 10, Amicon), and then purified using reverse phase HPLC (Waters).
  • a PEGylated TPII mutant (Q115K) was prepared as follows.
  • Was first concentrated protein sample was dialyzed in 20mM-phosphate buffer (P H 6.5), to about 1 mg / ml protein concentration using cents Li Gong (manufactured by amine co down).
  • the protein sample about 2 ml (about 2 mg), 5-fold molar excess of main butoxy - poly ethylene glycidyl Korua Rudehi de (MePEG; i.e. 0HC- (CH 2) 2 0- ( CH 2 - CH 2 0) "- CH 3 ;
  • n is the number of repetitions at which the molecular weight is about 12 KDa.;
  • Shearwater Polymers I got it.
  • sodium monocyanoborohydride NaCNBH.
  • PEG-modified TPO was produced by ion-exchange HPLC using ToyoSoda SP-5PW (Tosoichi).
  • the protein was eluted from the column using 2011 ⁇ Tris (Tris TM ) pH 6.8 phosphate buffer (20 mM Tris solution adjusted to pH 6.8 with phosphoric acid). Elution was performed with a linear concentration gradient in which the concentration gradient was changed from 0.1M to 0.5M in 32 minutes. The effluent of the elution solvent was 1. Oml / min. Elution of the protein from the column was monitored by ultraviolet absorption at 280 ⁇ m.
  • the molecular weight of the protein contained in each of the obtained fractions was analyzed by SDS-PAGE, and a TPII fraction having only one PEG molecule was pooled. This protein sample was sterilized through a 0.2 micron filtration membrane and used for in vivo activity evaluation.
  • PEGylated products of TPO mutants Q115K, Q115R and Q115Y
  • PEG-Q115K PEGylated products of TPO mutants
  • PEG-Q115R PEG-Q115R
  • PEG-Q115YJ PEG-Q115YJ
  • Thermodynamic characterization test of purified ⁇ ⁇ ⁇ mutant One measure of the thermal stability of a protein is the denaturation temperature of the protein.
  • the denaturation temperature is usually determined by changing the temperature of the protein solution over time using a spectroscopic method that can monitor the non-denaturing and denaturing state of the protein (a heating rate of 1 ° C per minute is most commonly used). ) Measure the temperature that leads to denaturation.
  • Thermostability of the TPO mutant was performed in 10 mM acetate buffer at pH 5.0.
  • CD circular dichroism
  • M-07 e-Atsusei human megakaryoblastic cell line
  • GM-CSF human megakaryoblastic cell line
  • IL-3 human megakaryoblastic cell line
  • SCF human megakaryoblastic cell line
  • the eluted mutants were collected, dialyzed against 2 L of 10 mM phosphate buffer for 2 days, and centrifuged (Centriprep 10, Amicon Was concentrated and concentrated, and the concentrate was supplied to an M-07 e-assay.
  • Q115R and Q115K both had a biological activity 1.5 times or more higher than that of the unsubstituted h6T (1-163) (see Table 2).
  • the relative activity of the test sample is determined by drawing a dose-response curve between the standard sample and the test sample.If the activity of the test sample is n times the activity of the standard sample, the relative activity of the sample is determined. Is n.
  • In vivo activity test of the purified TPO mutant The in vivo activity of the TPO mutant was determined using 8-week-old BALB / c male mice. Dilute the sample using PBS containing 0.5% (w / v) BSA as the diluent, and dilute the sample two-fold so that the dose-response curve includes the expected protein concentration range. A dosing solution was prepared. Divide the mice into groups of 4 or 5 each in the same number, and subcutaneously administer 0.2 ml of the sample solution to each mouse once a day for 4 consecutive days. did.
  • orbital blood was collected using EDTA-2K as an anticoagulant, and the blood sample was diluted 4-fold with a special diluent (Cell pack; Sy smex).
  • the platelet count was measured using (K-4500; Sysmex).
  • sample tube was transferred into a reaction vial, 6 N hydrochloric acid containing 1% phenol was added at 200 / z 1 to the bottom of the reaction vial, and gas phase hydrolysis was performed at 110 ° C under reduced pressure for 24 hours. After the completion of the hydrolysis, the sample tube was taken out, and 100 ml of 0.02N hydrochloric acid was added to obtain a sample solution. This sample solution was analyzed using an automatic 8500 amino acid analyzer manufactured by Hitachi, Ltd.
  • the protein concentration was calculated from the alanine peak area of the amino acid standard mixture solution analyzed at the same time, and the concentration of alanine in the sample solution was calculated.
  • the known number of alanine residues PEG 153: 12 residues, PEG 163: 14 residues, PEG 174: 14 residues).
  • SEQ ID NO: 4 shows a promoter sequence corresponding to the T3 promoter region of pBluescript II SK +.
  • SEQ ID NO: 5 Shows the promoter sequence corresponding to the downstream region of pBluescript II SK + downstream of manolytic crossing site.
  • SEQ ID NO: 6 shows a sense primer for mutation of Glnll5 ⁇ Lys in SEQ ID NO: 2.
  • SEQ ID NO: 7 Antisense primer for mutation of Glnll5 ⁇ Lys in SEQ ID NO: 2

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Abstract

L'invention concerne des protéines présentant des mutations et participant à la substitution d'au moins Glu en position 115 en Lys, Arg ou Tyr dans la séquence d'acides aminés contenant au moins les positions de 7- à 151- (par exemples, les positions de 1- à 163-) dans la séquence d'acides aminés de l'érythropoïétine humaine mûre (TPO) et possédant l'activité de la TPO humaine ; l'ADN codant pour ces protéines ; un procédé pour produire ces protéines ; et des compositions médicinales contenant ces protéines. Comparées à une protéine intacte, les protéines susmentionnées manifestent une stabilité élevée (notamment à la chaleur) et une activité biologique intense.
PCT/JP1999/002293 1998-04-30 1999-04-28 Mutants de la thrombopoietine humaine WO1999057147A1 (fr)

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AU35387/99A AU3538799A (en) 1998-04-30 1999-04-28 Human thrombopoietin mutants

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JP12057998 1998-04-30
JP10/120579 1998-04-30

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WO1999057147A1 true WO1999057147A1 (fr) 1999-11-11

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002015926A1 (fr) * 2000-08-24 2002-02-28 Kirin Beer Kabushiki Kaisha Compositions medicinales contenant des ligands c-mpl, destinees a l'augmentation des plaquettes et des erythrocytes
GB2382580A (en) * 2001-11-29 2003-06-04 Cheil Jedang Corp Erythropoietin- thrombopoietin fusion protein
JP2007524403A (ja) * 2003-07-26 2007-08-30 メデックスジェン カンパニー リミテッド 生物応答修飾タンパク質の効力を改善する方法及び典型的な突然変異タンパク質
JP2014519510A (ja) * 2011-06-10 2014-08-14 ノヴォ ノルディスク アー／エス ポリペプチド

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08277296A (ja) * 1994-02-14 1996-10-22 Kirin Brewery Co Ltd Ｔｐｏ活性を有するタンパク質

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08277296A (ja) * 1994-02-14 1996-10-22 Kirin Brewery Co Ltd Ｔｐｏ活性を有するタンパク質

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002015926A1 (fr) * 2000-08-24 2002-02-28 Kirin Beer Kabushiki Kaisha Compositions medicinales contenant des ligands c-mpl, destinees a l'augmentation des plaquettes et des erythrocytes
GB2382580A (en) * 2001-11-29 2003-06-04 Cheil Jedang Corp Erythropoietin- thrombopoietin fusion protein
GB2382580B (en) * 2001-11-29 2003-10-08 Cheil Jedang Corp Fusion protein having enhanced in vivo activity of erythropoietin
JP2007524403A (ja) * 2003-07-26 2007-08-30 メデックスジェン カンパニー リミテッド 生物応答修飾タンパク質の効力を改善する方法及び典型的な突然変異タンパク質
JP4747238B2 (ja) * 2003-07-26 2011-08-17 メデックスジェン カンパニー リミテッド 生物応答修飾タンパク質の効力を改善する方法及び典型的な突然変異タンパク質
JP2014519510A (ja) * 2011-06-10 2014-08-14 ノヴォ ノルディスク アー／エス ポリペプチド
JP2014519509A (ja) * 2011-06-10 2014-08-14 ノヴォ ノルディスク アー／エス ポリペプチド

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