JP2003024076A - Tpo variant protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a protein having a highly stable human thrombopoietin(TPO) activity. SOLUTION: This protein having the human thrombopoietin activity includes a variation including a simultaneous substitution of cysteine residues for at least a proline residue on 38th and an alanine residue on 120th in an amino acid sequence at least including amino acid residues from 7th to 151st of a specific amino acid sequence, and a DNA encoding the protein, a vector and a host cell including the DNA, a method for producing the protein, and a medicinal composition including the protein are provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a TPO mutant protein having human thrombopoietin activity. Thrombopoietin is hereinafter referred to as "TPO". Specifically, the present invention relates to a TPO variant having high stability such as heat stability and storage stability and / or high biological activity,
DNA encoding a PO mutant, a method for producing the mutant,
Alternatively, it relates to a pharmaceutical composition containing the TPO variant, a thrombocytosis agent or a thrombocytopenia therapeutic agent containing the TPO variant as an active ingredient. The present invention also relates to a protein in which a water-soluble polymer such as polyethylene glycol is bound to the TPO variant, a pharmaceutical composition containing such protein as an active ingredient, a thrombocytopenic agent or a thrombocytopenic therapeutic agent. .

[0002]

2. Description of the Related Art Human TPO is a protein cloned as a ligand for Mpl, which is one of the receptor superfamily of cytokines (de Sauva
ge et al., Nature (London) 369, 533-565, (1994), Ba.
rtley, TD et al., Cell 77, 1117-1124, (199
Four)). All of these Mpl ligands were detected in the serum or plasma of thrombocytopenic animals (human, pig, rat, mouse, dog, etc.), and their involvement in megakaryocyte formation and platelet formation was confirmed.

With the development of a therapeutic agent for thrombocytopenia in mind, the present applicant also used the activity of promoting megakaryocyte production from megakaryocyte progenitor cells highly purified from rat bone marrow as an index, and the rat with thrombocytopenia Rat TPO was purified from plasma, and rat TPOc was purified based on its partial amino acid sequence.
DNA and even human TPO cDNA were cloned, and a large amount of uniform human TPO was obtained by gene recombination technology.
(H. Miyazaki et al., Exp. He
matol. 22, p. 838, (1994)). The human TPO successfully obtained by the present applicant has been found to have the same amino acid sequence as the factor obtained as a ligand for human Mpl described above (shown as SEQ ID NO: 1 in the sequence listing described below). ing.).

The applicant of the present invention applied the human TPO with an anticancer drug or an immunosuppressive drug, or irradiation or bone marrow transplantation (BM).
When administered to mice with thrombocytopenia in which myelosuppression was caused by T), a thrombocytopenia inhibitory effect, a platelet increase promoting effect, and an increase in hematopoietic function were observed.
Have been found to be effective in these thrombocytopenia.

Further, the present applicant has prepared various derivatives in which a part of the amino acid sequence of SEQ ID NO: 1 is deleted or substituted, or amino acids are inserted or added, which are active in human TPO. It has been confirmed that the above is maintained (Japanese Patent Laid-Open No. 8-228781, Japanese Patent No. 2991630, Japanese Patent No. 2991640, etc.). Regarding the derivative of human TPO,
Report on splicing variant of TPO gene (Snow Brand Milk Products: JP-A-8-168386, Eisai: International Publication No. 96/03434 pamphlet, Yamanouchi Pharmaceutical: JP-A-8-89
No. 252, Asahi Kasei Kogyo: JP 8-89253, and TP
There is a report on a deletion / substitution of O (GDSearle: WO 96/23888 pamphlet), but it is more stable and higher than the natural human TPO having the amino acid sequence of SEQ ID NO: 1. Those that are active are 115 of human TPO.
It is not known except for the amino acid substitutions at positions (Q115R, Q115K, Q115Y) (WO 99/57147 pamphlet).

[0006]

[Problems to be Solved by the Invention] Proteins are generally known to be unstable. For example, proteins are denatured and deactivated when heated or vibrated, deactivated by organic solvents and denaturing agents, and deactivated by proteolytic enzymes. Further, in vivo protein metabolism is triggered by digestion by a protease or some kind of chemical modification, and thus unstable proteins are more likely to be metabolized. 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 a cause of inactivation.

[0007] Although the elucidation of the principle for designing a thermodynamically stable protein is still under study, some knowledge has already been gathered. Recent correlation studies on the structure and stability of thermophilic bacteria have revealed that the amino acid sequence of thermophilic bacteria contains many proline residues. Also, some useful stabilization methods have been derived from protein engineering studies. However, most of these require highly accurate three-dimensional structural information of proteins, and thus it is difficult to take effective measures. One of the means currently used to stabilize proteins is to screen for stabilized proteins by randomly introducing amino acid substitutions here and there. At present, many mutants must be prepared and evaluated in order to obtain a stable protein.

Under such circumstances, if the stability of the three-dimensional structure of TPO (particularly, thermodynamic stability) can be further enhanced, not only will it be less susceptible to thermal decomposition and inactivation, but also protease It is considered that there is a possibility that the resistance to the bacterium may be improved, and at the same time, the biological activity thereof can be expected to be improved.

[0009]

[Means for Solving the Problems] The inventors of the present invention conducted studies to further enhance the stability of the three-dimensional structure of TPO (particularly, thermodynamic stability), and As a result of evaluation by measuring spectroscopic changes (ultraviolet absorption and molecular ellipticity) accompanied by gradually increasing the temperature of the protein solution, the proline (Pro) residue at position 38 and the alanine at position 120 of TPO ( Ala) residue and cysteine (Cy
It was found that the mutants substituted for the s) residue have improved stability, especially improved thermal stability. Also these TP
When the biological activity of the O mutant was measured, it was revealed that the activity was improved, and the present invention was completed.

That is, the present invention is, in one aspect,
At least 7 to 1 in the amino acid sequence of SEQ ID NO: 1
An amino acid sequence comprising the amino acid residue at position 51, having a mutation comprising the simultaneous substitution of at least the proline residue at position 38 and the alanine residue at position 120 with a cysteine residue,
A protein having human TPO activity is provided.

The invention, in another aspect, is SEQ ID NO: 1.
In the amino acid sequence from the 1st position to the 3rd position of the human T, which has a mutation including the simultaneous substitution of at least the proline residue at position 38 and the alanine residue at position 120 with a cysteine residue.
A protein having PO activity is provided. In another aspect, the present invention has the simultaneous substitution of the proline residue at position 38 and the alanine residue at position 120 with a cysteine residue in the amino acid sequence from position 1 to position 332 of SEQ ID NO: 1.
A protein having human TPO activity is provided.

The invention, in another aspect, is SEQ ID NO: 1.
In the amino acid sequence from position 1 to position 163 of T., which has a mutation involving the simultaneous substitution of at least the proline residue at position 38 and the alanine residue at position 120 with a cysteine residue.
A protein having PO activity is provided. In another aspect, the invention has the simultaneous substitution of the proline residue at position 38 and the alanine residue at position 120 with a cysteine residue in the amino acid sequence from position 1 to position 163 of SEQ ID NO: 1.
A protein having human TPO activity is provided.

In the protein of the above aspect of the present invention,
The protein may have a methionine residue added at the -1 position, or the protein may have a methionine residue added at the -2 position and a lysine residue at the -1 position. The protein of the present invention may be bound to a water-soluble polymer, and such a water-soluble polymer is exemplified by polyethylene glycol.

[0014] The present invention, in yet another aspect, provides a DNA containing a nucleotide sequence encoding the above protein. The present invention provides, in another aspect, a recombinant vector containing the above DNA. The present invention also provides a prokaryotic cell transformed with the above recombinant vector. Prokaryotic cells are, for example, bacteria such as Escherichia coli, Bacillus subtilis, Bacillus brevis, and Pseudomonas.

The present invention further provides a eukaryotic cell transformed with the above recombinant vector. Eukaryotic cells are, for example, fungi (fungi, basidiomycetes, etc.), yeast, plant cells, or insect cells or mammalian cells (Chinese hamster ovary (CH
O) animal cells such as cells).

In another embodiment, the present invention comprises culturing the above-described prokaryotic cell or eukaryotic cell and isolating and purifying the produced protein having human TPO activity.
Provided is a method for producing the above protein having TPO activity. Examples of prokaryotic cells and eukaryotic cells are as described above, in particular, prokaryotic cells are bacteria such as Escherichia coli, and eukaryotic cells are mammalian cells. In addition, the method of the present invention can further include a step of binding a water-soluble polymer to the obtained protein. A preferred example of the water-soluble polymer is polyethylene glycol.

The present invention provides, in a further aspect, a pharmaceutical composition comprising a protein as defined above and a pharmaceutically acceptable carrier. The present invention also provides a TPO variant-containing pharmaceutical composition, characterized in that the TPO variant is a protein as defined above, which has improved stability such as heat stability and storage stability. To do. The present invention also provides a platelet-increasing agent containing the above-defined protein as an active ingredient.

The present invention further provides a therapeutic agent for thrombocytopenia containing the above-defined protein as an active ingredient. The term “human TPO activity” as used herein refers to an activity that promotes proliferation and differentiation of megakaryocyte progenitor cells in human, or specifically stimulates or enhances production of platelets in a human body. The term "stability" as used herein means stability such as heat stability and storage stability.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, stability (particularly heat stability or storage stability) is improved (or increased) or biological activity is improved as compared with an unmutated protein,
Human TPO variant proteins are provided.

That is, the protein according to the present invention has at least a proline residue at position 38 in the amino acid sequence containing at least amino acid residues 7 to 151 in the amino acid sequence of SEQ ID NO: 1 (amino acid numbers 1 to 332). 12
It is a TPO variant having human TPO activity, which has a mutation involving the simultaneous substitution of the 0-position alanine residue with a cysteine residue.

As used herein, "7 to 151
"Amino acid residue at position" indicates an amino acid residue of a region required to retain its biological activity in the amino acid sequence of human TPO SEQ ID NO: 1 (amino acid Nos. 1 to 332). This means that the N-terminus and C of the human TPO protein of SEQ ID NO: 1
Various human TPOs with one or more amino acids deleted from the end
It was clarified by producing a deletion mutant and measuring its biological activity (Japanese Patent Publication No. 8-510921, particularly Examples 27 to 29). Therefore, in the amino acid sequence of SEQ ID NO: 1, for example, (1-7) to 244, (1-7) to 2
31, (1 to 7) to 232, (1 to 7) to 211, (1
~ 7) to 191, (1 to 7) to 177, (1 to 7) to 1
74, (1 to 7) to 171, (1 to 7) to 170, (1
~ 7) ~ 169, (1 ~ 7) ~ 163, (1 ~ 7) ~ 1
58, (1-7) -157, (1-7) -156, (1-
7) -155, (1-7) -154, (1-7) -15
3, (1-7) -151, (1-7) -163, (1-
7) to 232, (1 to 7) to 332 [where (1 to 7) to 163 are, for example, 1 to 163, 2 to 163, 3 to 163, and 4 to
163, 5-163, 6-163 and 7-163. ], Which has an amino acid sequence containing amino acid residues 7 to 151, has human TPO activity. In the present invention, the human TPO deletion mutant as described above also includes a mutant having a mutation including the simultaneous substitution of at least the proline residue at position 38 and the alanine residue at position 120 with a cysteine residue. .

In the present specification, "having a mutation including the simultaneous substitution of at least the 38-position proline residue and the 120-position alanine residue with the cysteine residue" means that the 38-position proline residue and the 120-position Alanine residue of is replaced with cysteine residue at the same time, as long as it has TPO activity and retains the above-mentioned stability, one of substitutions, deletions, insertions, additions, etc. in other parts of the sequence It also means that it may contain a plurality of mutations, preferably one or several mutations. Here, several means 2 to less than 10. Such other mutations and mutagenesis methods are described in, for example, the applicant's Japanese Patent
It is disclosed in Japanese Patent Publication No. 8-228781, Japanese Patent Publication No. 8-510921, etc., and the disclosure thereof is incorporated in the present specification as a reference. Here, various TPO derivatives obtained by substituting or deleting a part of the amino acid sequence of SEQ ID NO: 1 or inserting or adding an amino acid sequence different from the amino acid sequence are human TPO.
Examples of retaining activity are disclosed.

The following are examples of the protein of the present invention. In the amino acid sequence of positions 1 to 332 of SEQ ID NO: 1, at least the proline residue at position 38 and 1
A TPO mutant protein having human TPO activity, which has a mutation comprising the simultaneous substitution of the alanine residue at position 20 with a cysteine residue. In the amino acid sequence from the 1st position to the 332nd position of SEQ ID NO: 1, a TPO mutant protein having human TPO activity, which has a simultaneous substitution of a proline residue at position 38 and an alanine residue at position 120 with a cysteine residue [Cys ³⁸ , Cys ¹²⁰ ]
TPO (1-332).

In the amino acid sequence of positions 1 to 163 of SEQ ID NO: 1, at least the proline residue at position 38 and 120
A TPO mutant protein having human TPO activity, which has a mutation comprising the simultaneous substitution of the alanine residue at the position with a cysteine residue. In the amino acid sequence from positions 1 to 163 of SEQ ID NO: 1, a TPO mutant protein having human TPO activity, having simultaneous substitution of a proline residue at position 38 and an alanine residue at position 120 with a cysteine residue [Cys ³⁸ , Cys ¹²⁰ ] TPO (1-1
63).

[Thr³³, Cys³⁸, Cys¹²⁰, T
hr³³³, Ser³³⁴, Ile³³⁵, Gly³³⁶, Tyr
³³⁷, Pro³³⁸, Tyr³³⁹, Asp³⁴⁰, Val³⁴¹，
Pro^{34 2}, Asp³⁴³, Tyr³⁴⁴, Ala³⁴⁵, Gly
³⁴⁶, Val³⁴⁷, His³⁴⁸, His³⁴⁹, His³⁵⁰，
His³⁵¹, His³⁵², His³⁵³] TPO (1-33
2). [Asn^{twenty five}, Cys³⁸, Cys¹²⁰, Ly
s²³¹, Thr³³³, Ser³³⁴, Ile³³⁵, Gl
y³³⁶, Tyr³³⁷, Pro³³⁸, Tyr³³⁹, As
p³⁴⁰, Val^{34 1}, Pro³⁴², Asp³⁴³, Ty
r³⁴⁴, Ala³⁴⁵, Gly³⁴⁶, Val³⁴⁷, Hi
s³⁴⁸, His³⁴⁹, His³⁵⁰, His³⁵¹, Hi
s³⁵², His³⁵³] TPO (1-332). [As
n^{twenty five}, Cys³⁸, Cys¹²⁰] TPO (1-33
2). [Thr³³, Cys³⁸, Cys¹²⁰] TPO
(1-332). [ΔHis³³, Cys³⁸, Cys
¹²⁰] TPO (1-163). [Cys³⁸, ΔAr
g¹¹⁷, Cys¹²⁰] TPO (1-163). [Cys
³⁸, ΔGly¹¹⁶, Cys¹²⁰] TPO (1-16
3). [His³³, Thr³³, Pro³⁴, Cys³⁸，
Cys¹²⁰] TPO (1-163). [His³³, Al
a³³, Pro³⁴, Cys³⁸, Cys¹²⁰] TPO (1
-163). [Cys³⁸, Gly¹¹⁶, Asn¹¹⁶, A
rg¹¹⁷, Cys¹²⁰] TPO (1-163). [Cy
s³⁸, Gly¹¹⁶, Ala¹¹⁶, Arg¹¹⁷, Cys
¹²⁰] TPO (1-163). [Cys³⁸, Gl
y¹¹⁶, Gly¹¹⁶, Arg¹¹⁷, Cys¹²⁰] TPO
(1-163). [Ala¹, Val³, Cys³⁸, C
ys¹²⁰, Arg¹²⁹] TPO (1-163). [Al
a¹, Val³, Cys³⁸, Cys¹²⁰, Arg¹³³]
TPO (1-163). [Ala¹, Val³, Cy
s³⁸, Cys¹²⁰, Arg¹⁴³] TPO (1-16
3). [Ala¹, Val³, Cys³⁸, Leu⁸²，
Cys¹²⁰] TPO (1-163). [Ala¹, Val
³, Cys³⁸, Cys¹²⁰, Leu¹⁴⁶] TPO (1
-163). [Ala¹, Val³, Cys³⁸, Cys
¹²⁰, Pro¹⁴⁸] TPO (1-163). [Al
a¹, Val³, Cys³⁸, Arg⁵⁹, Cys¹²⁰]
TPO (1-163). [Ala¹, Val³, Cy
s³⁸, Arg¹¹⁵, Cys¹²⁰] TPO (1-16
3).

In the above example, for example, [Asn ²⁵ , Cys ³⁸ ,
Cys ¹²⁰ ] TPO (1-332) is amino acid sequence 1 to SEQ ID NO: 1
The amino acid at position 25 of 332 becomes asparagine (Asn),
It is shown that the amino acids at positions 38 and 120 are TPO mutant proteins in which cysteine (Cys) is substituted, respectively. Further, for example, ΔHis ³³ indicates that histidine (His) at the 33rd position is deleted. Furthermore, for example, T
hr ^{33 ′} indicates that threonine (Thr) is inserted (or added) between the amino acids at positions 33 and 34.

The TPO mutant protein may further contain the amino acid sequence [Met ^-2 -Lys ^-1 ], [Met ^-1 ] or [Gly ^-1 ]. Especially Met-Lys
Can be removed by treatment with an enzyme such as dipeptidase (eg, cathepsin C) after the target protein is expressed in bacterial cells and purified.

Alternatively, other specific mutations in the amino acid sequence of the TPO variant protein may also include modification of the site of glycosylation (such as serine, threonine or asparagine). Glycosylation recognition sites containing asparagine residues are composed of tripeptide sequences that are specifically recognized by the appropriate cellular glycosylation enzyme. This tripeptide sequence is Asn-Xaa-Thr or
Asn-Xaa-Ser (where Xaa is any amino acid except proline), which does not significantly change the three-dimensional structure (or conformation) of the TPO mutant protein and has substantially no biological activity. To the extent that it can be maintained, a new glycosylation recognition site can be introduced into the amino acid sequence of the TPO mutant protein. This allows the TPO variant protein resulting from glycosylation to have unnatural carbohydrate chains as well as the same carbohydrate chains as native (especially human).

The TPO mutant protein of the present invention is prepared by preparing a DNA encoding the TPO mutant protein, incorporating the DNA into an appropriate vector so that the protein can be expressed, introducing the DNA into an appropriate host, and then culturing the host in an appropriate medium, It can be obtained by expressing the DNA. As used herein, “expressibly” means capable of transcription and translation into a protein under the control of a promoter. DNA encoding the TPO variant is chemically synthesized using conventional techniques such as the phosphoramidite method,
Preferably it can be obtained using a DNA synthesizer, or by modifying the cDNA of human TPO using the well-known site-directed mutagenesis method, and by using a combination of PCR (polymerase chain reaction) techniques. You can also get it. For example, site-directed mutagenesis by PCR (si
te-directed mutagenesis by PCR) can be used. Such site-directed mutagenesis, PCR, cloning into a vector, transformation, etc.
The basic technique for obtaining TPO variants is, for example, J. Sa
mbrook et al., Molecular Cloning A Laboratory Mannual,
2nd Edition, 1989, Cold Spring Harbor Laboratory Press,
FM Ausubel et al., Short Protocols in Molecular Biol
Ogy, 3rd edition, 1995, John Wiley & Sons, Inc .. In addition, a plasmid pHTF1 having a DNA containing a nucleotide sequence encoding the amino acid sequences 1 to 332 (see Sequence Listing: SEQ ID NO: 2) is carried in Escherichia coli DH5,
Deposited with the deposit number FERM BP-46 at the Institute of Biotechnology, Institute of Biotechnology (new name: National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center) on March 24, 1994.
Deposited as 17

The TPO mutant of the present invention is obtained by separating and purifying from a cultured cell or a culture medium of a host cell transformed with a recombinant vector containing a DNA encoding the TPO mutant. Is preferred. Examples of the host include prokaryotic cells (for example, bacteria such as Escherichia coli, Bacillus subtilis, Bacillus brevis, and Pseudomonas), eukaryotes (for example, fungi such as fungi and basidiomycetes, yeasts, plants, animals such as insects and mammals). Can be used. Examples of mammalian cells include
COS cells, Chinese hamster ovary (CHO) cells, X63.6.5.3. Cells (official name: X63-Arg8.653; JD
Sato et al., In Vitro Cellular & Developmental Biology
(1988), 24 (12): 1223-1228), C-127 cells, BHK (Ba
by Hamster Kidney) cells, human-derived cells (eg HeL
a cell) and the like. Examples of yeast include baker's yeast (Saccharomyces cerevisiae) and methanol-assimilating yeast (Pichia pastoris). Examples of insect cells include silkworm cultured cells (eg, Sf21 cells) and the like. Examples of plant cells are dicotyledonous or monocotyledonous plant cells (eg tobacco, rice, corn, etc.). The culture can vary depending on the type of host, and general techniques and conditions can be used.

When the DNA encoding the TPO mutant of the present invention is introduced into prokaryotic or eukaryotic cells as described above,
Construction of a plasmid or viral vector or transgene containing DNA together with host-specific regulatory sequences (in particular including a promoter and optionally further enhancers), and spontaneous uptake using calcium chloride or calcium phosphate Method, electroporation method, microinjection method, lipofection, spheroplast method, protoplast method, Agrobacterium method and the like can be used to transfer into the cells. The DNA may be expressible in the form of a plasmid capable of self-replication, or may be expressed by being integrated into the cell chromosome by homologous recombination. The expression vector further comprises
Origin of replication, ribosome binding site, transcription termination signal,
Selectable genetic markers and the like may also be included. Commercially available vectors, those described in literatures, depositable deposits, and the like can be used as the vector, but if necessary, they may be modified and used. For example, pBluescript (Stratagene), p
QE series (manufactured by Qiagen), pET series (manufactured by Novagen), pUC19 (Gene, 33: 103, 1985), Yep13 (ATCC 37115),
pHS19, pHS15, pXT1 (Stratagene), pBPV, pMS
G, pSVL SV40 (Pharmacia), pcDNA1 / Amp, pREP4
Vectors such as (manufactured by Invitrogen) can be used, but are not limited thereto.

After culturing the host cells and, if necessary, lysing the cells, salting out, dialysis, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, electrophoresis, isoelectricity. Conventional techniques such as point electrophoresis can be used alone or in combination to purify the TPO variants of the invention.

The TPO mutant protein of the present invention includes
Not only the above-mentioned various TPO mutant proteins having human TPO activity, but also chemical modification in which the mutant proteins are bound to at least one water-soluble polymer (or polymer)
Also included are TPO variant proteins. Particularly, as the TPO mutant protein of the present invention, the above-mentioned TPO mutant protein is reacted with a reactive polyethylene glycol (PEG) molecule to induce P
Includes chemically modified TPO mutant proteins with EG added to the TPO molecule. Such additions can be carried out by PEGylation reactions such as acylation or alkylation, as described below.

Acylation or alkylation with PEG is carried out under conditions such that the major product exhibits mono- or poly-PEGylation. In polyPEG addition, generally, PEG is added to the ε-amino group of a lysine residue, and further PEG is added to the α-amino group at the N-terminal of the protein.
The mono-PEGylation reaction is performed by using the α
-Enhanced via reductive alkylation to selectively modify the amino group, which provides for the addition of a water-soluble polymer at the N-terminus of the protein. An example of producing a chemically modified TPO protein variant to which PEG is added is shown in Example 5 below.

Chemically modified forms of such proteins are
It may effectively block the physical contact of proteolytic enzymes with the protein structure itself to prevent degradation.
A further advantage is the increased stability and circulation time of therapeutic proteins and their reduced immunogenicity under certain circumstances. However, it should be noted that it is impossible to predict the effect of modifying a particular protein.

Various means known in the art can be used to attach the PEG molecule to the protein. Generally, polyethylene glycol molecules are capable of binding to proteins via reactive groups found on proteins. In particular, amino groups such as those on lysine residues or on the N-terminus, or carboxyl groups on aspartic acid or glutamic acid residues or on the C-terminus are convenient for such attachment. For example, US Pat. No. 4,002,531 describes reductive alkylation to attach a polyethylene glycol molecule to an enzyme. European Patent Application Publication No. 0539167 discloses that a peptide and an organic compound having a free amino group are PEG.
Of imidate derivatives or related water-soluble organic polymers. Further US patent
4,904,584 discloses the modification of lysine residues in proteins that bind polyethylene glycol molecules via a reactive amino group. Protein modification, PEG
Other documents and reviews describing modifications are as follows, for example, and the techniques disclosed therein can be used in the present invention. Fransis, Focus o
n Groth Factors 3: 4-10 (May 1992) (Published by Mediscript, Mountview Court, Friern Barnet Lane, London N2
0, OLD, UK), Sada et al., J. Fermentation Bioengineering
71: 137-139 (1991), U.S. Pat.No. 4,179,337, U.S. Pat.No. 4,002,531, Abuchowski et al.
S. Holcerberg and J. Roberts, eds., Pp367-383 (198
1)), European Patent 0 401 384, European Patent 0 473 268
No., European Patent No. 0 335 423, European Patent No. 0 442 724,
European Patent 0 154 316 etc.

As the water-soluble polymer usable in the present invention, in addition to PEG, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly- Other water soluble polymers such as 1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers) can be exemplified.

The present invention further provides a pharmaceutical composition containing the TPO mutant protein of the present invention. The pharmaceutical composition of the present invention comprises, in addition to a pharmaceutically acceptable carrier (diluent, excipient, etc.), a stabilizer, a solubilizer, an antiseptic, an antioxidant depending on the purpose of formulation thereof. Alternatively, additives such as a tonicity agent can be contained. For carriers and additives, see, for example, Remington, The Science and Practice of Pharmac
Those described in y, Nine edition (1995), Mack Publishing Company can be used.

Regarding the pharmaceutical composition containing the TPO variant protein of the present invention, a solution formulation as a formulation according to various administration routes including parenteral such as injection, pulmonary, nasal, and oral, Examples thereof include suspensions, tablets, pills, capsules, granules and freeze-dried preparations. It is also possible to prepare a sustained-release preparation. Sustained-release preparations combine enteric-coated substances and / or hydrophobic polymeric substances such as eudrazide with hydrophilic substances and simply disperse the drug in them to give a multiparticulate or multi-layered form, optionally with hydrophobic It can be obtained by coating with a functional coating agent. If necessary, additives such as antiseptics, surfactants, solubilizers, antioxidants and isotonic agents may be added. Further, the pharmaceutical composition of the present invention may be in a freeze-dried or dried dosage form, and may be dissolved in a pharmaceutically acceptable diluent before use and administered to a subject patient. The freeze-dried preparation may contain a saccharide or a surfactant as a stabilizer. Examples of diluents include buffers of various pH and ionic strength (eg Tris-HCl, acetate, phosphate), saline and the like.

Examples of surfactants are Tween 20, Tween 80, Pl
uronic F68, bile salts, etc. Examples of solubilizers are glycerol, polyethylene glycol and the like. Examples of antioxidants are ascorbic acid, sodium metabisulfite and the like. Examples of preservatives are thimerosal, benzyl alcohol, parabens and the like. Examples of tonicity agents are lactose, mannitol and the like. As other additives,
Albumin to prevent adsorption to the surface of the container,
Examples thereof include proteins such as gelatin, protease inhibitors, and gastrointestinal absorption enhancers.

The pharmaceutical composition of the present invention may also be prepared by complexing an active ingredient with a metal ion, or in a granular preparation of a polymerized compound such as polylactic acid, polyglycolic acid, hydrogel or the like, or in a liposome. , Microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or incorporation of the active ingredient into spheroplasts. The dose of the pharmaceutical composition of the present invention is usually (as a TPO variant protein) about 0.05 μg / kg body weight to about 1 mg / kg body weight, preferably 0.0.
5 μg / kg body weight to 300 μg / kg body weight, more preferably the dose is 1 μg / kg body weight to 100 μg / kg body weight. The dose may vary depending on age, medical condition, weight, sex, patient's diet, administration route, etc., and may be administered once or several times a day for about 1 to 2 weeks or more. can do. The TPO variant protein is not limited to the above dose range, and may be contained in the composition in a dose that does not cause side effects and in a dose that can achieve the desired therapeutic effect.

In addition to the above TPO mutant protein, the pharmaceutical composition of the present invention comprises erythropoietin (EPO),
Granulocyte colony stimulating factor (G-CSF), Granulocyte macrophage colony stimulating factor (GM-CSF), Macrophage colony stimulating factor (M-CSF), Colony stimulating factor (CSF) -1,
Interleukin (IL) -1, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-1
3, leukocyte migration inhibitory factor (LIF), stem cell factor (SCF),
One or more of factors such as interferon α, β, γ can be included as additional hematopoietic factors.

Since the TPO mutant protein of the present invention has improved stability, especially heat stability or storage stability, it is more suitable for long-term storage preparation, use as normal temperature distribution preparation, or long-distance transportation of the preparation. ing. According to the present invention, the TPO mutant protein of the present invention is used as an active ingredient,
Platelet increasing agents are provided for a number of diseased patients who require an increase in platelets. Furthermore, a therapeutic agent for thrombocytopenia in a patient who has undergone chemotherapy or radiation therapy by administration of an anticancer agent or an immunosuppressive agent, or bone marrow transplant (BMT), PBSCT or CBSCT is provided.

Further, there is provided a therapeutic agent for a number of diseases characterized by thrombocytopenia, for example, thrombocytopenia due to impaired platelet production or shortened platelet life (increased platelet destruction or accelerated platelet consumption). Examples include congenital Fanconi anemia, aplastic anemia associated with chemotherapy or radiation therapy, myelodysplastic syndrome, acute myelogenous leukemia, or thrombocytopenia due to myelodysplasia such as bone marrow transplantation. It can be used to promote platelet recovery in healthy patients.

It is also useful for thrombocytopenia due to abnormal TPO production. Examples of thrombocytopenia due to shortened lifespan of platelets and megakaryocytes include idiopathic thrombocytopenic purpura,
Acquired immunodeficiency syndrome (AIDS), disseminated intravascular coagulation syndrome, thrombotic thrombocytopenia, etc. are also useful in promoting platelet recovery in such patients. In addition, before surgery
It is also useful as a so-called autologous platelet transfusion, in which the PO variant is administered to increase one's own platelets and the platelets are used as blood transfusion platelets during one's own surgery.

Further, the TPO mutant protein of the present invention comprises
It is also effective in treating platelet disorders caused by, for example, transient deficiency or damage of platelets by other chemicals or pharmaceuticals or therapeutic measures. TPO of the present invention
The mutant protein can be used to promote the release of new "intact" platelets in such patients.
Furthermore, since it has been revealed that the liver is one of the major TPO producing organs, the TPO of the present invention can be applied to various liver diseases that cause thrombocytopenia, such as biliary atresia, liver transplantation, liver cirrhosis, and hepatitis. Clinical application of mutant protein administration is expected. It is also useful as an application for restoring the ability of stored platelets to form hemostatic thrombi.

[0047]

The present invention will be described below with reference to examples.
These are for illustrative purposes only and do not limit the invention. Hereinafter, production examples of the heat-stabilized TPO variant of the present invention and the results of its characteristic test will be shown.

<Example 1> Preparation of TPO mutant As a TPO protein which is the basis of the design of the TPO mutant, Japanese Patent Application Laid-Open No. 8-228781 filed by the present applicant (1996, 9)
The human MKTPO (1-163) protein (SEQ ID NO: 3) described in Jpn. The amino acid sequence of human MKTPO (1-163) protein and the nucleotide sequence encoding it are shown in SEQ ID NO: 3 (corresponding to SEQ ID NO: 11 in JP-A-8-228781). In other words, in the amino acid sequence of positions 1 to 163 of the human TPO amino acid sequence (SEQ ID NO: 1), an amino acid in which a methionine residue (M) is added at the -2 position and a lysine residue (K) is added at the -1 position. Have an array. In an attempt to improve the biological activity and stability of the TPO protein, TPO in which the proline residue at position 38 and the alanine residue at position 120 of SEQ ID NO: 3 were simultaneously replaced with cysteine residues
A mutant (hereinafter referred to as [Cys ³⁸ , Cys ¹²⁰ ] TPO (1-163)) was designed.

Example 2 TPO by in vitro mutation
Acquisition of DNA encoding mutant [Cys ³⁸ , Cys ¹²⁰ ] TPO (1-163) First, plasmid pKF18K'-, which serves as a template for mutant preparation
hMKT (1-163) was prepared as follows.

The plasmid pKF18K (manufactured by Takara Shuzo) and the plasmid pAMG11-hMKT (1-163) (the same as pAMG11-r-HuMGDF described in Example 14 of Japanese Patent Publication No. 10-510980) are restricted. It was treated with the enzymes XbaI and HindIII. Then pKF18
K was ethanol precipitated. The digested product of pAMG11-hMKT (1-163) was subjected to agarose gel electrophoresis followed by hMKT (1
The fragment on the low molecular weight side containing -163) was recovered. Combine these with DNA Ligation Kit Ver.2 (Takara Shuzo),
Escherichia coli JM109 (Takara Shuzo) was transformed to obtain plasmid pKF18K-hMKT (1-163).

Pro residue at position 38 of human TPO and A
Derivatives in which the la residue is replaced with a Cys residue [Cys ³⁸ , Cys ¹²⁰ ]
Mutan-Super Express Km (manufactured by Takara Shuzo Co., Ltd.) was used for the preparation of TPO (1-163), and the PCR primers shown below were used. In this kit, the selection primer (se
mutagenesis is performed using a mutation primer and a mutation primer for introducing a mutation. Since the pKF18K vector has a double amber mutation in the kanamycin resistance gene, it cannot show kanamycin resistance when introduced into a suppressor-free host such as Escherichia coli MV1184. On the other hand, the plasmid introduced with the mutation by PCR shows the kanamycin resistance even when introduced into Escherichia coli MV1184 by introducing the mutation into the kanamycin resistance gene by the selection primer. Utilizing this principle, only transformants having a mutated plasmid are selected.

Hereinafter, the plasmid which has been modified and acquired kanamycin resistance is referred to as pKF18K (+). The primers used in the following operations are as follows. P38C: 5 '
-CACCCGCTGCCGACCTGCGTTCTGCTTCCGGCTG-3 '(SEQ ID NO: 4)
A120C: 5'-AGGGCCGTACCACTTGCCACAAGGATCCGAAC-3 '(SEQ ID NO: 5) M13 RV-N: 5'-TGTGGAATTGTGAGCGG-3' (SEQ ID NO: 6) M13 M4: 5'-GTTTTCCAGTCACGAC-3 '(SEQ ID NO: 7)

The operation has gone through two steps, the first step is pKF18
K (+)-hMKT (1-163) P38C was prepared, and 2
PKF18K (+)-hMKT (1-163) P38C / A1 after the mutation operation of the step
You get 20C. The first step of the mutation operation is the template plasmid pKF.
10 ng of 18K-hMKT (1-163) and 5 pmol each of the selection primer and the P38C mutation primer were used.
TAKARA LA Taq (Takara Shuzo) and GeneAmp PCR Syste
m 9600 (manufactured by Perkin Elmer) at 95 ° C for 5 minutes,
Continuously 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 4 minutes 3
PCR reaction of 0 cycles was performed. The sample after the reaction was subjected to ethanol precipitation treatment, and this was used for E. coli MV11
84 competent cells (Takara Shuzo) were transformed.
Five clones were selected from the resulting transformants to prepare plasmid DNA. For the purified plasmid DNA, M1 was used using Big Dye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems Japan).
Using 3RV-N and M13 M4 as primers, 96
A PCR reaction was carried out at 25 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes. The nucleotide sequence of the PCR reaction product was confirmed by a 377 DNA sequencer manufactured by Applied Biosystems. The pKF18K (+)-hMKT (1-163) P38C and p
KF18K-to-mutation plasmid pKF18K-hMKT (1-163) P38
Got C. Subsequent operations are from pKF18K-hMKT (1-163) to pKF18K
(+)-hMKT (1-163) P38C according to the operation obtained. In the second step, a selection primer and an A120C mutation primer were used. PKF18K whose nucleotide sequence was confirmed in the same manner as above
(+)-hMKT (1-163) P38C / A120C was obtained.

<Example 3> Incorporation into expression vector Xba was obtained from pKF18K (+)-hMKT (1-163) P38C / A120C prepared above.
I-HindIII fragment was extracted and XbaI-Hind of plasmid pAMG11
After inserting between II cleavage sites, Escherichia coli JM109 was transformed to obtain the desired plasmid pAMG11-hMKT (1-163) P38C / A120C. With this plasmid, Escherichia coli 2008 (A
(manufactured by mgen) was transformed to obtain a transformant expressing the desired TPO mutant.

Example 4 Expression and Purification Example of TPO Variant in Escherichia coli The transformant obtained in Example 3 was shake-cultured overnight at 37 ° C. in 150 ml of LB medium containing 25 μg / ml of kanamycin, The whole amount of this culture solution was added to 1100 ml of LB medium containing 25 μg / ml of kanamycin, and the mixture was shake-cultured at 37 ° C. until the OD ₆₀₀ reached 0.7-1.0. Then isopropyl-β-
D-Thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further incubated for 3 hours at 37 ° C. with shaking to induce the expression of the TPO mutant.

About 30 mL of 20 mM Tris buffer (pH 8.5) containing 10 mM EDTA, 10 mM DTT, and 1 mM PMSF was added to the frozen cells (about 3 g) of the TPO mutant-producing recombinant thus obtained, and the suspension was suspended. Then, sonication (using an ultrasonic crusher) for 1 minute was performed 5 times with an interval of 1 minute under ice cooling. The disrupted cells were transferred to a centrifuge tube and centrifuged at 4 ° C. and 15000 rpm for 10 minutes to collect the pellet.

To this pellet was added 8M guanidine hydrochloride, 5
10 mM Tris buffer containing 1 mM PMSF and 1 mM PMSF (pH 8.
7) About 30 mL was added and suspended, about 50 mg DTT was further added, and the mixture was stirred at room temperature for 1 to 2 hours to reduce the protein sample. After the reduction, the pH of the sample was adjusted to 5 with a 2M hydrochloric acid solution and left at 4 ° C.

The reduced protein sample was diluted with 10 mM CAPS buffer (pH 10.5, about 1.5 L) containing 30% glycerol, 3 M urea, 3.33 mM cystamine and 0.67 mM L-cysteine. Dilutions were performed at room temperature overnight. The diluted sample solution was stirred at room temperature for 2 days to sufficiently oxidize the sample protein and then centrifuged at 8000 rpm for 45 minutes to remove the insoluble matter. The supernatant after centrifugation was adjusted to pH 6.8 with 6M phosphoric acid, diluted 2 times with ion-exchanged water, and the insoluble matter was filtered using two pieces of filter paper (Toyo Roshi Kaisha, # 2, diameter 90 mm). did. The filtrate is ion-exchange resin (CM-Sepharose f
ast flow, Pharmacia column (2.6 x 10 cm) and then washed with about 400 mL of 10 mM phosphate buffer (pH 6.8) containing 15% glycerol and 1 M urea, and then 15% glycerol. The column was equilibrated with about 300 mL of 10 mM phosphate buffer (pH 7.2) containing the column. For elution of the target sample, 0.5M sodium chloride,
A 10 mM phosphate buffer containing 15% glycerol was used, and the flow rate was 1 mL / min. The elution of protein from the column was followed by UV absorption at 280 nm and the fractions containing the protein of interest were collected (approximately 30-40 mL). This fraction was purified using reverse phase HPLC. At this time, μBondasphere C4 (3.9x150 mm) manufactured by Waters was used for the column, and 0.05% trifluoroacetic acid (TFA) was used for protein elution.
Using eluent A, which is a solution, and eluent B containing 70% 2-propanol, 30% acetonitrile, and 0.02% TFA, change the concentration of B eluent from 5% to 95% in 50 minutes to change the target protein. It was eluted. The TPO variant was eluted at approximately 40 minutes under these conditions. The elution fraction was dialyzed for 1 day against 20 mM Tris ( ^TM ) / phosphate buffer solution (20 mM Tris solution adjusted to pH 7.2 with phosphoric acid) having a pH of 7.2, followed by ion exchange HPLC (SHIMAZU). Product). ToyoSoda SP-5PW (manufactured by Tosoh Corporation) was used as the column, and 20 mM Tris ( ^TM ) -phosphate buffer solution (pH 7.
Using eluent A which is 2) and eluent B containing 20 mM Tris ^™ phosphate buffer (pH 7.2) and 0.5 M sodium chloride,
The target protein was eluted by changing the concentration of the eluent from 10% to 99% in 30 minutes. TPO mutant [Cys ³⁸ , Cys
¹²⁰ ] TPO (1-163) was eluted at about 20 minutes under these conditions.

Example 5 Production Example of PEGylated TPO Variant A PEGylated TPO variant was prepared as follows. First, the TPO mutant [Cys ³⁸ , Cys ¹²⁰ ] TPO (1-16 from Example 4
3) was dialyzed against 20 mM Tris ( ^TM ) / phosphate buffer solution (pH 7.2) for 1 day and night, and 0.1 mg of each of cathepsin C and cysteamine was added per 1 mg of this sample and left at room temperature for 90 minutes to give -2. The methionine residue at position 1 and the lysine residue at position -1 were removed. The pH of the sample solution was adjusted to 4.8 by adding 1M acetic acid to the protein sample solution, and then about 2 mL (about 2 mg) of the protein sample was added with a 5-fold molar excess of methoxy-polyethylene glycol aldehyde (MePEG; ie OH).
_{C- (CH 2) 2 O- (} CH 2 -CH 2 O) n-CH 3; wherein n has a molecular weight of about 20,
It is the number of repetitions of 000. ) (Shearwater Polymers
Manufactured) was added. After stirring for about 10 minutes, sodium - was added cyanoborohydride (NaCNBH ₄₎ to a final concentration of 15 mM. The reaction was stirred at 4 ° C for about 16 hours.

After diluting this reaction solution with purified water by 2 times,
PEG-modified TPO was generated by ion exchange HPLC using Toyo Soda SP-5PW (manufactured by Tosoh). To elute the protein from the column, Tris ^™ phosphate buffer of pH 7.2 was used, and the salt concentration gradient was eluted with a linear concentration gradient changing from 0.1 M to 0.5 M in 30 minutes. The flow rate of the eluting solvent was 1.0 mL / min. Elution of protein from the column was monitored by UV absorption at 280 nm. The molecular weight of the protein contained in each of the obtained fractions was analyzed by SDS-PAGE, TPO fractions having only one PEG molecule were pooled, sterilized through a 0.22 micron filtration membrane, and used for bioactivity evaluation. Using the above method, the TPO mutant [Cys ³⁸ , Cys
PEGylated product of ¹²⁰ ] TPO (1-163) PEG- [Cys ³⁸ , Cys
¹²⁰ ] TPO (1-163) was obtained.

Example 6 Thermodynamic Characterization Test of Purified TPO Variant One measure of the thermostability of a protein is the denaturation temperature of the protein. The denaturation temperature is usually a spectroscopic method capable of monitoring the non-denaturation state and denaturation state of the protein, and the temperature of the protein solution is changed with time (a heating rate of 1 ° C. per minute is most commonly used). ), And measure the temperature at which denaturation occurs.

The thermostability of the TPO mutant was 10 mM at pH 4.5.
Performed in acetate buffer. Protein (OD280nm = about 0.05)
The temperature of the solution containing C was increased by 1 ° C./min from about 20 ° C. to 90 ° C., and the intensity change of circular dichroism (CD) signal at 222 nm was measured. In each TPO mutant, the CD signal intensity decreased with increasing temperature, and thermal destruction of the three-dimensional structure in the protein was observed. The temperature change curve of the obtained CD signal intensity was calculated using the program EXAM (National Institute
of Standard and Technology, USA).
For the analysis, it was assumed that TPO was denatured in two states, undenatured and denatured. Two-state denaturation is observed in many small globular proteins. The analysis result of the thermal denaturation curve of TPO
This was in good agreement with this two-state modification. From the analysis results, each TP
The denaturation temperature of the O variant was obtained. The results are summarized in Table 1.

[0063]

[Table 1]

From Table 1, the TPO mutants of the present invention [Cys ³⁸ , Cy
s ¹²⁰ ] TPO (1-163) is a control TPO protein TPO (1-16
It can be seen that the denaturation temperature is much higher than that in 3). This indicates that TPO mutants having Cys at least at positions 38 and 120 have high thermostability.

<Example 7> Human TPO activity confirmation experiment of purified TPO mutant-assay system using human Mpl forced expression cell line (FDCP-hMpl5 assay) Human Mpl forced expression cell line FDCP-hMpl5 cells Is T in addition to mouse IL-3
Since it also responds to PO, it can be used as an alternative assay method for the rat CFU-MK assay system. The assay was performed as follows.

FDCP-hMp subcultured in the presence of mouse IL-3
l5 cells are collected, washed thoroughly, and then resuspended in IMDM culture medium containing 10% fetal bovine serum. Put FDCP-hMpl5 cells as number of cells per well into tissue culture 96-well flat bottom plate is 10 ^3, the addition of more standard or test sample, the final volume to 200 [mu] l / well . Place the plate in a 5% CO 2 incubator and incubate at 37 ℃ for 3 days.
Four hours before the end of the culture on the 3rd day, add 1 μCi (37 KBq) / well of ³ H-thymidine, and after the completion of the culture, collect the cells on a glass fiber filter with a cell harvester and collect the ³ H radioactivity in the liquid. It is measured with a scintillation counter (for example, beta plate; manufactured by Pharmacia).

To measure the activity of the purified TPO mutant, the eluted mutants were collected, dialyzed against 2 L of 10 mM phosphate buffer (pH 7.2) for 2 days, and then centrifuged ( Centriprep 10, Amicon) was used for concentration and FDCP
-Subjected to hMpl5 assay. Prepared TPO mutant [Cys
³⁸ , Cys ¹²⁰ ] TPO (1-163) was the TPO (1-163) used as a control.
The biological activity was about 7 times higher than that of

<Example 8> In vivo activity test of purified TPO mutant For in vivo activity of the TPO mutant, BALB / c male mice aged 10 weeks were used. The sample was diluted with 10 mM sodium acetate buffer containing 5% sorbitol as a dilution medium, and a 5-fold serial dilution sample administration solution was prepared so as to include the protein concentration range where the dose-response curve is expected to be linear. . The mice were divided into groups of 5 mice each, and each group of mice was given a sample administration solution at a concentration of 1
A single intravenous dose was given at a dose of 100 μL / g. Orbital blood was collected using EDTA-2K as an anticoagulant on days 2, 4, 6, 8, 10, and 12 after administration, and the platelet count of the blood sample was measured using an automatic hemocytometer (F-800; Sysmex). Measured. At this time, the dilution medium was used as a control.

TPO mutant [Cys ³⁸ , Cys ¹²⁰ ] TPO (1-163) and TPO
An increase in the platelet count of mice administered with (1-163) was observed,
Decreased after peaking on day 6. 100 and 500 μg / kg
TPO mutants [Cys ³⁸ , Cys
^{When 120} ] TPO (1-163) and TPO (1-163) were compared, almost no difference was found in the platelet counts of both (Table 2).

To quantify the protein, 30 mL of the sample and 500 nm
40 mL of l / mL norleucine was weighed in the same sample tube and dried under reduced pressure. The sample tube was transferred into a reaction vial, 200 μL of 6N hydrochloric acid containing 1% phenol was added to the bottom of the reaction vial, and gas phase hydrolysis was performed under reduced pressure at 110 ° C. for 24 hours. After completion of hydrolysis, the sample tube was taken out, and 100 mL of 0.02N hydrochloric acid was added to prepare a sample solution. This sample solution was analyzed using a L-8500 type amino acid automatic analyzer manufactured by Hitachi. The protein concentration was calculated from the peak area of alanine in the amino acid standard mixture that was analyzed at the same time, and the concentration of alanine in the sample solution was calculated. The known number of alanine residues (TPO (1-163): 14 residues) Calculated by dividing by.
Specific activity was determined using the results of in vivo assay and protein quantification (Table 2).

[0071]

[Table 2]

[0072]

INDUSTRIAL APPLICABILITY As demonstrated by the above examples, the present invention provides a TPO mutation having excellent stability and, particularly, thermostability and / or high biological activity as compared with a control unmutated protein. The body is provided, which can be expected to contribute significantly to the solution of stability problems during storage and distribution when TPO is used as a medicine.

[0073]

[Sequence list]                           SEQUENCE LISTING <110> Kirin Brewery Co., Ltd. <120> TPO Mutant Proteins <130> P01-0209 <140> <141> <160> 7 <170> Windows (registered trademark) 95 <210> 1 <211> 332 <212> PRT <213> Homo sapiens <400> 1 Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu   1 5 10 15 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val              20 25 30 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu          35 40 45 Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu      50 55 60 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln  65 70 75 80 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln                  85 90 95 Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu             100 105 110 Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe         115 120 125 Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu     130 135 140 Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala 145 150 155 160 Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn                 165 170 175 Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr             180 185 190 Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile         195 200 205 Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly     210 215 220 Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe 225 230 235 240 Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly                 245 250 255 Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser             260 265 270 Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu         275 280 285 Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro     290 295 300 Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr 305 310 315 320 Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly                 325 330 <210> 2 <211> 1721 <212> DNA <213> Homo sapiens <400> 2 gcggcacgag gggggtgtct ggctggcgtg gctccctgtt tggggcctct cccctgaatc 60 cttcctgggg ccatggaggc cagacagaca ccccggccag a 101 atg gag ctg act gaa ttg ctc ctc gtg gtc atg ctt ctc cta act gca 149 Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala     -20 -15 -10 agg cta acg ctg tcc agc ccg gct cct cct gct tgt gac ctc cga gtc 193 Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val  -5 1 5 10 ctc agt aaa ctg ctt cgt gac tcc cat gtc ctt cac agc aga ctg agc 245 Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser              15 20 25 cag tgc cca gag gtt cac cct ttg cct aca cct gtc ctg ctg cct gct 293 Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala          30 35 40 gtg gac ttt agc ttg gga gaa tgg aaa acc cag atg gag gag acc aag 341 Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys      45 50 55 gca cag gac att ctg gga gca gtg acc ctt ctg ctg gag gga gtg atg 389 Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met  60 65 70 75 gca gca cgg gga caa ctg gga ccc act tgc ctc tca tcc ctc ctg ggg 437 Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly                  80 85 90 cag ctt tct gga cag gtc cgt ctc ctc ctt ggg gcc ctg cag agc ctc 485 Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu              95 100 105 ctt gga acc cag ctt cct cca cag ggc agg acc aca gct cac aag gat 533 Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp         110 115 120 ccc aat gcc atc ttc ctg agc ttc caa cac ctg ctc cga gga aag gtg 581 Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val     125 130 135 cgt ttc ctg atg ctt gta gga ggg tcc acc ctc tgc gtc agg cgg gcc 629 Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala 140 145 150 155 cca ccc acc aca gct gtc ccc agc aga acc tct cta gtc ctc aca ctg 677 Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu                 160 165 170 aac gag ctc cca aac agg act tct gga ttg ttg gag aca aac ttc act 725 Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr             175 180 185 gcc tca gcc aga aca act ggc tct ggg ctt ctg aag tgg cag cag gga 773 Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly         190 195 200 ttc aga gcc aag att cct ggt ctg ctg aac caa acc tcc agg tcc ctg 821 Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu     205 210 215 gac caa atc ccc gga tac ctg aac agg ata cac gaa ctc ttg aat gga 869 Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn Gly 220 225 230 235 act cgt gga ctc ttt cct gga ccc tca cgc agg acc cta gga gcc ccg 917 Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro                 240 245 250 gac att tcc tca gga aca tca gac aca ggc tcc ctg cca ccc aac ctc 965 Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu             255 260 265 cag cct gga tat tct cct tcc cca acc cat cct cct act gga cag tat 1013 Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro Gly Gln Tyr         270 275 280 acg ctc ttc cct ctt cca ccc acc ttg ccc acc cct gtg gtc cag ctc 1061 Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu     285 290 295 cac ccc ctg ctt cct gac cct tct gct cca acg ccc acc cct acc agc 1109 His Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser 300 305 310 315 cct ctt cta aac aca tcc tac acc cac tcc cag aat ctg tct cag gaa 1157 Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu                 320 325 330 ggg taaggttctc agacactgcc gacatcagca ttgtctcgtg tacagctccc 1210 Gly ttccctgcag ggcgcccctg ggagacaact ggacaagatt tcctactttc tcctgaaacc1270 caaagccctg gtaaaaggga tacacaggac tgaaaaggga atcatttttc actgtacatt1330 ataaaccttc agaagctatt tttttaagct atcagcaata ctcatcagag cagctagctc1390 tttggtctat tttctgcaga aatttgcaac tcactgattc tctacatgct ctttttctgt1450 gataactctg caaaggcctg ggctggcctg gcagttgaac agagggagag actaaccttg1510 agtcagaaaa cagagaaagg gtaatttcct ttgcttcaaa ttcaaggcct tccaacgccc1570 ccatcccctt tactatcatt ctcagtggga ctctgatccc atattcttaa cagatcttta1630 ctcttgagaa atgaataagc tttctctcag aaatgctgtc cctatacact agacaaaact1690 gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1721 <210> 3 <211> 535 <212> DNA <213> Homo sapiens <400> 3 ctagaaaaaa ccaaggaggt aataaata 28 atg aaa agt cct gca cca cct gca tgt gat tta cgg gtc ctg tct aaa 76 Met Lys Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys          +1 5 10 ctg ctg cgc gac tct cac gtg ctg cac tct cgt ctg tcc cag tgc ccg 124 Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro  15 20 25 30 gaa gtt cac ccg ctg ccg acc ccg gtt ctg ctt ccg gct gtc gac ttc 172 Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe                  35 40 45 tcc ctg ggt gaa tgg aaa acc cag atg gaa gag acc aaa gct cag gac 220 Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp              50 55 60 atc ctg ggt gca gta act ctg ctt ctg gaa ggc gtt atg gct gca cgt 268 Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg          65 70 75 ggc cag ctt ggc ccg acc tgc ctg tct tcc ctg ctt ggc cag ctg tct 316 Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser      80 85 90 ggc cag gtt cgt ctg ctg ctc ggc gct ctg cag tct ctg ctt ggc acc 364 Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr  95 100 105 110 cag ctg ccg cca cag ggc cgt acc act gct cac aag gat ccg aac gct 412 Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala                 115 120 125 atc ttc ctg tct ttc cag cac ctg ctg cgt ggc aaa gtt cgt ttc ctg 460 Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu             130 135 140 atg ctg gtt ggc ggt tct acc ctg tgc gtt cgt cgg gcg ccg cca acc 508 Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr         145 150 155 act gct gtt ccg tct taatgaaagc tt 535 Thr Ala Val Pro Ser     160 <210> 4 <211> 34 <212> DNA <213> Artificial Seuqnce <220> <223> Description of Artificial Sequence: a mutation primer <400> 4 cacccgctgc cgacctgcgt tctgcttccg gctg 34 <210> 5 <211> 32 <212> DNA <213> Artificial Seuqnce <220> <223> Description of Artificial Sequence: a mutation primer <400> 5 agggccgtac cacttgccac aaggatccga ac 32 <210> 6 <211> 17 <212> DNA <213> Artificial Seuqnce <220> <223> Description of Artificial Sequence: a primer <400> 6 tgtggaattg tgagcgg 17 <210> 7 <211> 16 <212> DNA <213> Artificial Seuqnce <220> <223> Description of Artificial Sequence: a primer <400> 7 gttttccagt cacgac 16

[0074]

[Sequence Listing Free Text] SEQ ID NO: 4-Explanation of artificial sequence: Mutation primer SEQ ID NO: 5-Explanation of artificial sequence: Mutation primer SEQ ID NO: 6-Explanation of artificial sequence: Primer SEQ ID NO: 7-Explanation of artificial sequence :Primer

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12N 5/00 B C12P 21/02 A61K 37/02

Claims (23)

[Claims] 1. In the amino acid sequence containing at least amino acid residues 7 to 151 of the amino acid sequence of SEQ ID NO: 1, at least a proline residue at the 38th position and an alanine residue at the 120th position are simultaneously converted to cysteine residues. A protein having human TPO activity, which has a mutation comprising the substitution of 2. In the amino acid sequence of positions 1 to 332 of SEQ ID NO: 1, at least the proline residue at position 38 and 12
A protein having human TPO activity, having a mutation comprising the simultaneous substitution of the 0-position alanine residue with a cysteine residue. 3. A protein having human TPO activity, which comprises the simultaneous substitution of a proline residue at position 38 and an alanine residue at position 120 with a cysteine residue in the amino acid sequence from positions 1 to 332 of SEQ ID NO: 1. . 4. In the amino acid sequence of positions 1 to 163 of SEQ ID NO: 1, at least the proline residue at position 38 and 12
A protein having human TPO activity, having a mutation comprising the simultaneous substitution of the 0-position alanine residue with a cysteine residue. 5. A protein having human TPO activity, which comprises the simultaneous substitution of a proline residue at position 38 and an alanine residue at position 120 with a cysteine residue in the amino acid sequence from positions 1 to 163 of SEQ ID NO: 1. . 6. The protein according to claim 1, wherein a methionine residue is added at the -1 position. 7. A methionine residue is added to the -2 position and a lysine residue is added to the -1 position.
The protein according to any one of 1. 8. The protein according to claim 1, which is bound with a water-soluble polymer. 9. The protein according to claim 8, wherein the water-soluble polymer is polyethylene glycol. 10. A DNA containing a nucleotide sequence encoding the protein according to any one of claims 1 to 7. 11. A recombinant vector containing the DNA according to claim 10. 12. A prokaryotic cell transformed with the recombinant vector according to claim 11. 13. A bacterium transformed with the recombinant vector according to claim 11. 14. Escherichia coli transformed with the recombinant vector according to claim 11. 15. A eukaryotic cell transformed with the recombinant vector according to claim 11. 16. A mammalian cell transformed with the recombinant vector according to claim 11. 17. A method comprising culturing the cell according to any one of claims 12 to 16 and isolating and purifying the produced protein having human TPO activity.
The method for producing a protein having human TPO activity according to any one of 1. 18. The method of claim 17, further comprising attaching a water soluble polymer to the resulting protein. 19. The method of claim 18, wherein the water soluble polymer is polyethylene glycol. 20. A pharmaceutical composition comprising the protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. 21. The TPO variant-containing pharmaceutical composition, wherein the TPO variant has improved stability such as heat stability and storage stability as compared with the unmutated protein. A pharmaceutical composition, which is the protein according to item 1. 22. A platelet increasing agent comprising the protein according to claim 1 as an active ingredient. 23. A therapeutic agent for thrombocytopenia containing the protein according to any one of claims 1 to 9 as an active ingredient.