WO2015199039A1 - 細胞の多核化を誘導するペプチドおよびその利用 - Google Patents
細胞の多核化を誘導するペプチドおよびその利用 Download PDFInfo
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Definitions
- the present invention relates to a synthetic peptide capable of polynucleating a target cell and use thereof. Specifically, the present invention relates to a multinucleation-inducing agent (pharmaceutical composition) containing the peptide and a method for inducing multinucleation using the peptide.
- a multinucleation-inducing agent pharmaceutical composition
- this application claims priority based on Japanese Patent Application No. 2014-128431 filed on June 23, 2014, the entire contents of which are incorporated herein by reference. ing.
- multinucleated cells are known. Examples of multinucleated cells that normally exist in a living body (cells that exhibit normal functions in the form of multinucleated cells) include megakaryocytes and osteoclasts. Osteoclasts are multinucleated cells differentiated from hematopoietic stem cells and are responsible for destroying old bone tissue (bone resorption) and are involved in bone remodeling (bone remodeling) and bone growth (non- Patent Document 1).
- Non-patent Document 2 Megakaryocytes are multinucleated cells differentiated from hematopoietic stem cells and are responsible for producing platelets.
- platelets are essential blood components (cell components in blood) for blood coagulation and hemostasis, and are in high demand in leukemia, bone marrow transplantation, surgical operations and the like.
- blood products by blood donation are used in many platelet transfusions for patients who require administration of platelets.
- the supply of platelets for blood transfusion tends to be unstable due to a short storage period (effective period) after blood collection and a decrease in donors.
- iPS cells induced pluripotent stem cells
- ES cells embryonic stem cells
- hematopoietic stem cells iPS cells, also called induced pluripotent stem cells
- ES cells embryonic stem cells
- hematopoietic stem cells iPS cells, also called embryonic stem cells
- hematopoietic stem cells iPS cells, also called embryonic stem cells
- hematopoietic stem cells hematopoietic stem cells
- Patent Document 1 describes a technique for establishing a megakaryocyte progenitor cell line immortalized by forcibly expressing a specific gene.
- Patent Document 2 discloses that a specific gene is forcibly expressed in order to improve the production amount of functional platelets (platelets retaining in vivo activity such as hemostasis) from megakaryocytes in vitro. A technique for promoting multinucleation of megakaryocytes is described.
- tumor cells are more active and cell growth rate is significantly higher than normal cells. Therefore, many anticancer drugs aimed at killing or inhibiting the growth of tumor cells use drugs that inhibit cell division of tumor cells (cancer cells). Examples of such drugs that inhibit cell division include alkylating agents, antimetabolites, and microtubule agents.
- An alkylating agent acts on DNA to crosslink double-stranded bases
- an antimetabolite such as 5-FU inhibits DNA synthesis
- a microtubule-acting agent is a microtubule polymerization or Inhibition of mitosis by inhibition of depolymerization inhibits cell division of tumor cells (cancer cells), respectively.
- anticancer agents anticancer compositions
- drugs that are more effective and have a lower risk of side effects, or drugs that are highly effective and inexpensive.
- a tumor cell can be inhibited by dividing the tumor cell artificially and actively into a multinuclear cell, it can be used as a new anticancer technique (anticancer agent).
- the present invention was created with the object of developing a technique capable of artificially multinucleating target cells. Specifically, it is a peptide having a relatively short chain length that can be artificially synthesized.
- An object of the present invention is to provide an artificial peptide that serves the purpose of inducing multinucleation of target cells.
- Another object is to provide a multinucleation inducer (pharmaceutical composition) containing such a peptide.
- Another object of the present invention is to provide a method for inducing multinucleation in a target cell using such a peptide.
- the present inventors diligently researched various peptides to contribute to the purpose of inducing multinucleation for the target cells. And surprisingly, by supplying a peptide synthesized to contain the amino acid sequence shown in SEQ ID NO: 1 or 2 to the target cell, it was found that the cell can be multinucleated or multinucleated can be promoted, The present invention has been completed. In addition, by inducing multinucleation of tumor cells, the present inventors can inhibit normal cell division, and can prevent or suppress the growth of target (target) tumor cells (cancer cells). It was confirmed.
- the cell in order to achieve the above object, can be multinucleated when supplied to a target cell (typically when added to a medium in which the cell is cultured), or the cell There is provided an artificially synthesized peptide characterized by having the ability to promote the multinucleation of the peptide (hereinafter also referred to as “multinucleation-inducing activity”).
- the synthetic peptide disclosed here includes the following amino acid sequences (A) and (B) in the peptide chain: (A) an amino acid sequence constituting a membrane-permeable peptide sequence; and (B) CPDGAKARC (SEQ ID NO: 1) constituting a multinucleation-inducing peptide sequence having an activity of inducing multinucleation in at least one eukaryotic cell.
- CPDGAKARC SEQ ID NO: 1
- CSRRSSKSKC SEQ ID NO: 2
- a modified amino acid sequence in which one or several amino acid residues in the amino acid sequence are substituted, deleted, and / or added.
- a modified amino acid sequence constituting a multinucleation-inducing peptide sequence having an activity of inducing multinucleation in at least one eukaryotic cell It is an artificially synthesized peptide having the activity of inducing multinucleation against at least one eukaryotic cell characterized by having
- a synthetic peptide (including a peptide comprising the above-mentioned modified amino acid sequence) containing a multinucleation-inducing peptide sequence and a membrane-permeable peptide sequence, that is, a synthetic peptide having multinucleation-inducing activity is referred to as “multinucleation-inducing property. Also called “synthetic peptide”.
- the multinucleation-inducing synthetic peptide disclosed herein has a multinucleation-inducing peptide sequence as shown in (B) above in the peptide chain.
- a target (target) cell typically in a medium in which the cell is cultured
- the target cell is induced to multinucleate or promote multinucleation. can do.
- such a multinucleation-inducing synthetic peptide has a membrane-permeable peptide sequence as shown in (A) above in the peptide chain, a cell (typically the cell) targeting the peptide (target) In the culture medium), the above-described multinucleation-inducing peptide sequence can be efficiently transferred from the outside of the eukaryotic cell (outside the cell membrane) into the cell.
- polynucleation can be induced in the target cells by supplying a synthetic peptide, there is no concern about insertion of a foreign gene into the genome due to gene transfer.
- the target by a simple processing method of supplying a multinucleation-inducing synthetic peptide to a target (target) cell (typically in a medium in which the cell is cultured). Induction of multinucleation of cells can be realized.
- the multinucleation-inducing synthetic peptide disclosed here can be easily and artificially produced by chemical synthesis (or biosynthesis). When such a multinucleation-inducing synthetic peptide is used, the target cell is multinucleated without using a large amount of expensive humoral factors such as cytokines (typically as an alternative to humoral factors). Therefore, multinucleation induction of target (target) cells can be realized at low cost.
- the multinucleation-inducing synthetic peptide disclosed here has a simple structure (typically a linear peptide chain), and has high structural stability, so that it is excellent in handleability.
- the amino acid sequence constituting the (A) membrane-permeable peptide sequence is any one selected from SEQ ID NOs: 3 to 11 Or a modified amino acid sequence in which one or several amino acid residues in the amino acid sequence are substituted, deleted, and / or added, and constitutes a membrane-permeable peptide sequence. It is characterized by being. All of the amino acid sequences shown in SEQ ID NOs: 3 to 11 are typical examples of amino acid sequences constituting a membrane-permeable peptide, and can be suitably employed as the membrane-permeable peptide sequence shown in (A) above.
- any amino acid sequence (typically SEQ ID NO: 3 to 6) which is a signal sequence for localizing a protein to a nucleolus in a nucleus and known as a nucleolus localization signal (NoLS). It is preferable to employ the amino acid sequence shown in FIG.
- the membrane-permeable peptide sequence composed of the amino acid sequence shown in SEQ ID NO: 3 is a typical example of the NoLS, and is particularly preferable from the viewpoint of the efficiency of transferring the multinucleation-inducing peptide sequence into cells.
- a preferred embodiment of the synthetic peptide (multinucleation-inducing synthetic peptide) disclosed herein is characterized in that the total amino acid sequence constituting the peptide chain is 30 or less.
- a peptide consisting of such a short peptide chain is easy to synthesize chemically, has a relatively low production cost, and is excellent in handleability. For this reason, for example, it can be preferably used as a component of a multinucleation inducer.
- the synthetic peptide multinucleation-inducing synthetic peptide
- the following amino acid sequence KKRTLRRKNDRKKRGCPDGAKARC (SEQ ID NO: 12); and KKRTLRKNDRKRKGCSRRSSKSK (SEQ ID NO: 13); It is characterized by having any of these.
- Such multinucleation-inducing synthetic peptides are particularly suitable for use in inducing multinucleation of cells derived from humans or non-human mammals.
- the present invention provides a multinucleation inducer used for inducing multinucleation of at least one eukaryotic cell, wherein the multinucleation induction according to any of the embodiments disclosed herein is performed.
- Multinucleation-inducing agent comprising a synthetic synthetic peptide and a pharmaceutically acceptable carrier (for example, at least one base material that contributes to improving the stability of the peptide, or a liquid medium such as physiological saline or various buffer solutions) (Pharmaceutical composition) is provided.
- the composition (the multinucleation-inducing synthetic peptide) is included in the composition (the multinucleation-inducing synthetic peptide) because the composition includes the above-described multinucleation-inducing peptide sequence (the multinucleation-inducing synthetic peptide).
- the multinucleation inducer disclosed here is preferably used for the purpose of multinucleating tumor cells derived from humans or mammals other than humans.
- target cells include malignant tumor cells (cancer cells), such as squamous cell carcinoma cells and adenocarcinoma cells.
- cancer cells malignant tumor cells
- the multinucleation inducer disclosed here By inducing multinucleation in tumor cells with the multinucleation inducer disclosed here, cell division of the multinucleated cells (multinucleated cells) is inhibited, and as a result, the growth of tumor cells is prevented or suppressed. can do.
- the multinucleation-inducing agent disclosed herein can be used as a composition for suppressing the growth of tumor cells derived from humans or non-human mammals. That is, since the multinucleation inducer disclosed here can exert an antitumor effect on the target tumor cells, it can be suitably used as an antitumor composition (anticancer agent).
- the present invention provides a method for multinucleating at least one eukaryotic cell in vivo or in vitro.
- One preferred embodiment is a method for inducing multinucleation on at least one eukaryotic cell in vitro, comprising: Preparing a cell culture containing the cells of interest in vitro, and in the cell culture, the multinucleation-inducing synthetic peptide of any of the embodiments disclosed herein (or a multinucleation-inducing agent containing the synthetic peptide) Is provided at least once, and culturing the cell culture supplied with the peptide.
- the target cell (and the cell) can be obtained by a simple method using a synthetic peptide (or a multinucleation inducing agent containing the synthetic peptide) having a simple structure as a multinucleation inducer as described above. It is possible to efficiently induce multinucleation (to promote multinucleation).
- the above-described method for inducing multinucleation disclosed herein can be suitably implemented for the purpose of inducing multinucleation of tumor cells derived from humans or mammals other than humans.
- the method for inducing multinucleation disclosed herein in particular, induction of multinucleation of malignant tumor cells (cancer cells), for example, cells derived from squamous cell carcinoma or adenocarcinoma can be easily realized. That is, the method for inducing multinucleation disclosed herein can inhibit cell division of tumor cells derived from humans or mammals other than humans, and can prevent or suppress the proliferation of such cells. Therefore, the multinucleation induction method disclosed here can be used as a method for treating a tumor (cancer).
- the multinucleation-inducing synthetic peptide (or multinucleation-inducing agent containing the peptide) disclosed herein is a multinucleated cell and a pre-multinucleated cell before multinucleation (for example, a meganuclear blast before multinucleation or osteoclast before multinucleation) It can be suitably used for the purpose of inducing multinucleation of progenitor cells and the like. Thereby, increase in the number of nuclei in the multinucleated cell (promotion of multinucleation) and production (manufacturing) of a multinucleated cell (for example, megakaryocyte, osteoclast) can be realized.
- a multinucleated cell for example, megakaryocyte, osteoclast
- a multinucleated cell can be produced (manufactured) by a simple method in which a synthetic peptide having a simple structure (that is, a multinucleation inducer containing the synthetic peptide) is used as a multinucleation inducer. This is preferable because it is possible.
- multinucleated cells eg, megakaryocytes, osteoclasts, etc.
- multinucleated cells eg, megakaryocytes, osteoclasts, etc.
- cells produced from the multinucleated cells for example, platelets
- biosynthetic materials for example, physiologically active substances such as secretory proteins and hormones
- megakaryocytes produced (manufactured) by the method disclosed herein that is, megakaryocytes that are sufficiently multinucleated
- produce cells for transplantation typically for bone marrow transplantation
- platelets in vitro can be used as a cultured cell line.
- a platelet preparation blood preparation
- a platelet preparation can be produced by using platelets produced from megakaryocytes produced (manufactured) by the method disclosed herein. Thereby, supply of platelets (platelet preparation) independent of blood donation can be realized.
- FIG. 1 is a fluorescence micrograph (image) obtained by examining the state of the HeLaS3 cell after adding the multinucleation-inducing synthetic peptide (sample 1) according to one example to the culture medium of HeLaS3 cell and culturing. It is an image obtained by superimposing (merging) a nuclear staining image by DAPI and a fluorescence image showing a result of examination by a cell immunostaining method using an anti-calreticulin antibody.
- FIG. 1 is a fluorescence micrograph (image) obtained by examining the state of the HeLaS3 cell after adding the multinucleation-inducing synthetic peptide (sample 1) according to the culture medium of HeLaS3 cell and culturing. It is an image obtained by superimposing (merging) a nuclear staining image by DAPI and a fluorescence image showing a result of examination by a cell immunostaining method using an anti-calreticulin antibody.
- FIG. 1 is a flu
- FIG. 2 is a fluorescence micrograph (image) of examining the state of the HeLaS3 cells after culturing by adding the multinucleation-inducing synthetic peptide (sample 2) according to one example to the culture solution of HeLaS3 cells, It is an image obtained by superimposing (merging) a nuclear staining image by DAPI and a fluorescence image showing a result of examination by a cell immunostaining method using an anti-calreticulin antibody.
- FIG. 3 shows the state of the HeLaS3 cells after culturing by adding a synthetic peptide (sample 3) constructed only with an amino acid sequence constituting a membrane-permeable peptide sequence as a comparative example to a culture solution of HeLaS3 cells.
- FIG. 4 is a fluorescence micrograph (image) of the state of the HeLaS3 cells after culturing HeLaS3 cells without the addition of a multinucleation-inducing synthetic peptide, a nuclear staining image by DAPI, and an anti-calreticulin antibody. It is the image which overlap
- amino acids are represented by one-letter code (in the sequence table, three-letter code) based on the nomenclature related to amino acids shown in the IUPAC-IUB guidelines.
- sequence table three-letter code
- synthetic peptide refers to an artificial chemical synthesis or biosynthesis (ie, production based on genetic engineering) that is not a peptide chain that exists independently and stably in nature. , Refers to a peptide fragment that can exist stably in a predetermined composition (for example, a multinucleation-inducing agent capable of inducing multinucleation in a subject cell).
- a predetermined composition for example, a multinucleation-inducing agent capable of inducing multinucleation in a subject cell.
- the “peptide” is a term indicating an amino acid polymer having a plurality of peptide bonds, and is not limited by the number of amino acid residues contained in the peptide chain, but typically the total number of amino acid residues.
- amino acid residue is a term encompassing the N-terminal amino acid and the C-terminal amino acid of a peptide chain, unless otherwise specified. In the amino acid sequences described herein, the left side is always the N-terminal side and the right side is the C-terminal side.
- a “modified amino acid sequence” with respect to a predetermined amino acid sequence refers to a function possessed by the predetermined amino acid sequence (for example, a cell possessed by the multinucleation-inducing activity or membrane-permeable peptide possessed by the multinucleation-inducing synthetic peptide) 1 or several (typically 1 to 5, for example 1, 2 or 3) amino acid residues are substituted, deleted and / or lost without impairing the ability to migrate from outside to inside the cell) Or an amino acid sequence formed by addition (insertion).
- sequences resulting from conservative ⁇ ⁇ ⁇ ⁇ amino ⁇ acid replacement where one or several (typically two or three) amino acid residues are conservatively substituted
- a sequence substituted with a basic amino acid residue for example, mutual substitution of a lysine residue and an arginine residue), or one or several (typically about 1 to 5, for example, 1) of a given amino acid sequence
- a sequence in which (2, 2 or 3) amino acid residues are added (inserted) or deleted is a typical example included in the modified amino acid sequence referred to in the present specification. Therefore, the multinucleation-inducing synthetic peptide disclosed herein includes one or several amino acids in the amino acid sequence shown in each SEQ ID NO.
- Synthetic peptides composed of amino acid sequences in which amino acid residues are substituted (for example, the above-mentioned conservative substitutions), deleted and / or added, and similarly include synthetic peptides exhibiting multinucleation-inducing activity.
- tumor is a term that is interpreted in a broad sense and refers to tumors in general (typically malignant tumors) including carcinomas and sarcomas or blood and hematopoietic tissue lesions (leukemia, lymphoma, etc.).
- the “tumor cell” refers to a cell that forms such a tumor. Typically, it refers to a cell (so-called cancerous cell) that has abnormally grown independently of the surrounding normal tissue. Therefore, unless otherwise specified, a cell that is classified as a tumor cell (cancer cell) rather than a normal cell is referred to as a tumor cell regardless of the origin and properties of the cell.
- Epithelial tumors (squamous cell carcinoma, adenocarcinoma, etc.), non-epithelial tumors (various sarcomas, osteosarcomas, etc.), various cell tumors (neuroblastoma, retinoblastoma, etc.), lymphomas, melanomas, etc.
- the constituent cells can be included in the tumor cells referred to herein.
- multinucleated cell is a term that means a cell that normally exists as a cell having a plurality of nuclei (typically 2 or more, for example, 4 or more) in vivo, that is, in vivo. Is a term that means a cell whose normal form is a form having multiple nuclei. Examples include megakaryocytes, osteoclasts, skeletal muscle cells, syncytial trophoblast cells present in placental villi, and foreign body giant cells that are macrophage-derived phagocytes.
- pre-multinuclear cell before multinucleation refers to a polynuclear cell that has not been polyploidized, and is typically a mononuclear cell (with two nuclei in the mitotic phase (M phase)). Cell).
- pre-multinucleated cells before multinucleation include megakaryocyte precursor cells (megakaryoblasts), osteoclast precursor cells (osteoclast precursor cells), and the like.
- inducing multinucleation or “promoting multinucleation” means relatively increasing the number of nuclei in a cell as compared with a cell before inducing (promoting) multinucleation. It means to make it. For example, obtaining a cell having 2 or more (typically 4 or more, for example, 8 or more) nuclei from a mononuclear cell, or increasing the number of nuclei in a multinucleated cell induces multinucleation. (Promotion). In addition, typically, the ploidy becomes higher as the number of nuclei in the cell increases, so that “inducing (promoting) multinucleation” is relatively more ploidy than the cell before multinucleation induction.
- a cell having a chromosome number of 2N obtaining a cell having a chromosome number of 4N or more (typically 8N or more, for example, 16N or more) is called induction (promotion) of multinucleation. it can.
- the multinucleation-inducing synthetic peptide disclosed herein is supplied to a cultured cell of a predetermined eukaryotic cell (typically, a cell derived from a human or non-human mammal, bird, or other animal) ( It is typically added to the medium in which the cells are cultured) and can induce multinucleation or promote multinucleation (ie, increase the number of nuclei in the cells).
- a synthetic peptide ie, a multinucleation-inducing synthetic peptide first discovered by the present inventors to have an action, ie, a multinucleation-inducing activity.
- the multinucleation inducer disclosed herein is a composition (pharmaceutical composition) capable of inducing (promoting) multinucleation to a target cell, and is at least one of the above-mentioned multinucleation-inducing synthetic peptides. Is contained as an active ingredient (that is, a substance involved in inducing multinucleation of a target cell).
- the multinucleation-inducing synthetic peptide disclosed herein includes, as a partial amino acid sequence (peptide motif), an amino acid sequence constituting the membrane-permeable peptide sequence defined in (A) above and (B) above. And the amino acid sequence constituting the multinucleation-inducing peptide sequence defined in (1).
- the (B) multinucleation-inducing peptide sequence of the multinucleation-inducing synthetic peptide is a sequence having multinucleation-inducing activity, and (A) a peptide having good multinucleation-inducing activity by combining with a membrane-permeable peptide sequence.
- the multinucleation-inducing peptide sequence has the following amino acid sequence: CPDGAKARC (SEQ ID NO: 1); and CSRRSKSKC (SEQ ID NO: 2); Or a modified amino acid sequence thereof.
- the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is the first amino acid sequence from the N-terminal side of the amino acid sequence information obtained by the inventor by independently translating the RNA sequence constituting the human-derived centrin 2 siRNA. 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th amino acid residues from the C-terminal side to the 1st, 2nd, respectively Total 9 amino acids obtained by reversing and rearranging to the 3rd, 4th, 5th, 6th, 7th, 8th, 9th amino acid residues An artificial amino acid sequence of residues.
- centrin is a centrosome-related protein that exists in the centrosome of eukaryotes and is involved in centriole duplication and microtubule cleavage as one of the centrosome constituent proteins. It is one of the proteins belonging to the Centrin family (typically Centrin 1, Centrin 2, Centrin 3, etc.) (Non-patent Document 3).
- the amino acid sequence constituting the (A) membrane-permeable peptide sequence of the multinucleation-inducing synthetic peptide disclosed herein is particularly an amino acid sequence that constitutes a membrane-permeable peptide that can pass through the cell membrane and / or the nuclear membrane.
- Many amino acid sequences that can function suitably as membrane-permeable peptides ie, membrane-permeable peptide sequences
- amino acid sequences related to NoLS (Nucleolar localization signal) (modified) (Including the amino acid sequence) is preferable as the amino acid sequence of the membrane-permeable peptide sequence of the multinucleation-inducing synthetic peptide.
- Preferred examples of the membrane-permeable peptide sequence related to the NoLS and other membrane-permeable peptide sequences (including modified amino acid sequences) are shown in SEQ ID NOs: 3 to 11. Specifically, it is as follows.
- amino acid sequence of SEQ ID NO: 3 is from the 491st amino acid residue to the 503rd amino acid residue of LIM kinase 2 (LIM Kinase 2) present in human endothelial cells, which is one type of protein kinase involved in intracellular signal transduction.
- LIM Kinase 2 LIM Kinase 2
- SEQ ID NO: 4 corresponds to NoLS consisting of a total of 14 amino acid residues derived from FGF2 (basic fibroblast growth factor).
- the amino acid sequence of SEQ ID NO: 5 corresponds to NoLS consisting of a total of 8 amino acid residues contained in the N protein (nucleocapsid protein) of IBV (avian infectious bronchitis virus).
- the amino acid sequence of SEQ ID NO: 6 corresponds to NoLS consisting of a total of 13 amino acid residues derived from adenovirus PTP (pre-terminal protein) 1 and PTP2.
- the amino acid sequence of SEQ ID NO: 7 corresponds to a membrane-permeable peptide sequence consisting of a total of 11 amino acid residues derived from a protein transduction domain contained in TAT of HIV (Human Immunodeficiency Virus).
- the amino acid sequence of SEQ ID NO: 8 corresponds to a membrane-permeable peptide sequence consisting of a total of 11 amino acid residues of the protein transduction domain (PTD4) modified with the TAT.
- the amino acid sequence of SEQ ID NO: 9 corresponds to a membrane-permeable peptide sequence consisting of a total of 16 amino acid sequences derived from Antennapedia ANT, a mutant of Drosophila.
- the amino acid sequence of SEQ ID NO: 10 corresponds to a membrane-permeable peptide sequence consisting of a total of 9 consecutive arginine residues as polyarginine.
- the amino acid sequence of SEQ ID NO: 11 corresponds to a membrane-permeable peptide sequence consisting of a total of 19 amino acid residues derived from a MyoD (myoblast determination) family inhibitory domain-containing protein.
- the above-mentioned membrane-permeable peptide sequences shown in the sequence listing are merely examples, and usable peptide sequences are not limited thereto.
- Various membrane-permeable peptide sequences that can be used in the practice of the present invention are described in numerous publications published at the time of filing this application. The amino acid sequences of these membrane-permeable peptide sequences can be easily known by general search means.
- the amino acid sequence shown in SEQ ID NO: 3 (including the modified amino acid sequence) described in Patent Document 3 is preferable as the membrane-permeable peptide sequence.
- the membrane-permeable peptide sequence shown in SEQ ID NO: 3 and the above-described multinucleation-inducing peptide sequence, a synthetic peptide having high multinucleation-inducing activity can be obtained.
- the peptide chain (amino acid sequence) of the multinucleation-inducing synthetic peptide disclosed here is constructed by appropriately combining (B) the multinucleation-inducing peptide sequence and (A) the membrane-permeable peptide sequence as described above. can do. Any of the multinucleation-inducing peptide sequence and the membrane-permeable peptide sequence may be arranged relatively on the C-terminal side (N-terminal side). In addition, the multinucleation-inducing peptide sequence and the membrane-permeable peptide sequence are preferably arranged adjacent to each other.
- the number of residues is about 1 to 3 preferable.
- one or several (typically one, two or three) amino acid residues (eg one or two) that function as a linker between a multinucleation-inducing peptide sequence and a membrane-permeable peptide sequence Several glycine (G) residues).
- the multinucleation-inducing synthetic peptide disclosed herein is preferably one in which at least one amino acid residue is amidated. By amidating the carboxyl group of an amino acid residue (typically the C-terminal amino acid residue of the peptide chain), the structural stability (eg, protease resistance) of the synthetic peptide can be improved.
- the multinucleation-inducing synthetic peptide disclosed herein is a sequence (amino acid residue) other than the amino acid sequence constituting the multinucleation-inducing peptide sequence and the membrane-permeable peptide sequence as long as the multinucleation-inducing activity is not lost. May include a portion.
- a partial amino acid sequence is preferably a sequence capable of maintaining the three-dimensional shape (typically a linear shape) of the multinucleation-inducing synthetic peptide sequence and the membrane-permeable peptide sequence portion.
- the total number of amino acid residues constituting the peptide chain is suitably 100 or less, desirably 60 or less, and preferably 50 or less.
- a synthetic peptide of 30 or less is particularly preferable.
- Such a peptide having a short chain length is easy to chemically synthesize, and can provide a multinucleation-inducing synthetic peptide at low cost.
- the peptide conformation (three-dimensional structure) is not limited as long as it exhibits multinucleation-inducing activity that induces multinucleation on the target eukaryotic cell in the environment used (in vitro or in vivo).
- the multinucleation-inducing synthetic peptide applied to the multinucleation-inducing agent is linear and has a relatively low molecular weight (typically 50 amino acid residues or less, particularly preferably 30 or less amino acid residues). Base number) is preferred.
- Proportion of the multinucleation-inducing peptide sequence and the membrane-permeable peptide sequence to the entire amino acid sequence (peptide chain) ie, the multinucleation-inducing peptide occupying the total number of amino acid residues constituting the peptide chain of the multinucleation-inducing synthetic peptide
- the number of amino acid residues constituting the sequence and the membrane-permeable peptide sequence is not particularly limited as long as it does not lose the multinucleation-inducing activity for inducing multinucleation in the target cell, but the ratio is approximately 60% or more. Is preferable, and 80% or more is preferable. 90% or more is particularly preferable.
- a peptide consisting of a multinucleation-inducing peptide sequence and a membrane-permeable peptide sequence that is, if these sequences are 100% of the total amino acid sequence or contain one to several linkers occupy other than the linker.
- the multinucleation-inducing synthetic peptide of the present invention is preferably one in which all amino acid residues are L-type amino acids, but does not lose the multinucleation-inducing activity for inducing multinucleation in the target eukaryotic cell. As long as a part or all of the amino acid residue is substituted with a D-type amino acid.
- a preferred embodiment of the multinucleation-inducing synthetic peptide disclosed herein is the following amino acid sequence: KKRTLRRKNDRKKRGCPDGAKARC (SEQ ID NO: 12); and KKRTLRKNDRKRKGCSRRSSKSK (SEQ ID NO: 13);
- the amino acid sequence selected from any of the above or a modified amino acid sequence of the selected amino acid sequence is particularly preferably contained.
- the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13 comprises a multinucleation-inducing peptide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and an amino acid sequence derived from NoLS of LIM kinase 2 shown in SEQ ID NO: 3. It is an amino acid sequence consisting of a total of 24 amino acid residues constructed by combining via a linker consisting of one glycine (G) residue.
- the multinucleation-inducing synthetic peptide disclosed here can be easily produced according to a general chemical synthesis method.
- any conventionally known solid phase synthesis method or liquid phase synthesis method may be employed.
- a solid phase synthesis method in which Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) is applied as an amino-protecting group is preferred.
- the multinucleation-inducing synthetic peptide disclosed here is obtained by a solid phase synthesis method using a commercially available peptide synthesizer (for example, available from Intavis AG, Protein Technologies, etc.), Peptide chains having modified (such as C-terminal amidation) moieties can be synthesized.
- a multinucleation-inducing synthetic peptide may be biosynthesized based on a genetic engineering technique. That is, a polynucleotide (typically DNA) having a nucleotide sequence (including the ATG start codon) encoding the amino acid sequence of a desired multinucleation-inducing synthetic peptide is synthesized. And it includes various regulatory elements (promoter, ribosome binding site, terminator, enhancer, and various cis elements that control the expression level) for expressing the synthesized polynucleotide (DNA) and the amino acid sequence in the host cell.
- a recombinant vector having a gene construct for expression consisting of) is constructed according to the host cell.
- This recombinant vector is introduced into a predetermined host cell (for example, yeast, insect cell, plant cell) by a general technique, and the host cell or a tissue or an individual containing the cell is cultured under a predetermined condition. Thereby, the target peptide can be expressed and produced in the cell. Then, the target multinucleation-inducing synthetic peptide can be obtained by isolating the peptide from the host cell (in the medium if secreted) and performing refolding, purification, etc. as necessary.
- a predetermined host cell for example, yeast, insect cell, plant cell
- a method for constructing a recombinant vector and a method for introducing the constructed recombinant vector into a host cell a method conventionally used in the field may be employed as it is, and such method itself particularly characterizes the present invention. Since it is not a thing, detailed description is abbreviate
- a fusion protein expression system can be used for efficient mass production in a host cell. That is, a gene (DNA) encoding an amino acid sequence of a target multinucleation-inducing synthetic peptide is chemically synthesized, and the synthetic gene is converted into an appropriate fusion protein expression vector (for example, pET series and Amersham Bio provided by Novagen). It is introduced into a suitable site of GST (Glutathione S-transferase) fusion protein expression vector such as pGEX series provided by Science. A host cell (typically E. coli) is transformed with the vector. The obtained transformant is cultured to prepare the desired fusion protein. The protein is then extracted and purified.
- a gene DNA
- encoding an amino acid sequence of a target multinucleation-inducing synthetic peptide is chemically synthesized, and the synthetic gene is converted into an appropriate fusion protein expression vector (for example, pET series and Amersham Bio provided by Novagen). It is introduced into a suitable site of GST
- the obtained purified fusion protein is cleaved with a predetermined enzyme (protease), and the released target peptide fragment (designed multinucleation-inducing synthetic peptide) is recovered by a method such as affinity chromatography. Further, refolding is performed by an appropriate method as necessary.
- a conventionally known fusion protein expression system for example, the GST / His system provided by Amersham Biosciences can be used
- the multinucleation-inducing synthetic peptide disclosed herein is produced. Can do.
- a template DNA for a cell-free protein synthesis system that is, a synthetic gene fragment containing a nucleotide sequence encoding the amino acid sequence of a multinucleation-inducing synthetic peptide
- various compounds ATP, RNA polymerase necessary for peptide synthesis
- the target polypeptide can be synthesized in vitro using a so-called cell-free protein synthesis system.
- cell-free protein synthesis systems for example, Shimizu et al. (Shimizu et al., Nature Biotechnology, 19, 751-755 (2001)), Madin et al. (Madin et al., Proc. Natl. Acad. Sci.
- a single-stranded or double-stranded polynucleotide comprising a nucleotide sequence encoding the multinucleation-inducing synthetic peptide disclosed herein and / or a nucleotide sequence complementary to the sequence can be easily produced by a conventionally known method ( Synthesis). That is, by selecting a codon corresponding to each amino acid residue constituting the designed amino acid sequence, a nucleotide sequence corresponding to the amino acid sequence of the multinucleation-inducing synthetic peptide is easily determined and provided.
- a polynucleotide (single strand) corresponding to the desired nucleotide sequence can be easily obtained using a DNA synthesizer or the like. Furthermore, using the obtained single-stranded DNA as a template, various enzymatic synthesis means (typically PCR) can be employed to obtain the desired double-stranded DNA.
- the polynucleotide may be in the form of DNA or RNA (mRNA or the like). DNA can be provided as double-stranded or single-stranded. When provided as a single strand, it may be a coding strand (sense strand) or a non-coding strand (antisense strand) having a sequence complementary thereto.
- the polynucleotide thus obtained is a material for constructing a recombinant gene (expression cassette) for producing a multinucleated inducible synthetic peptide in various host cells or in a cell-free protein synthesis system as described above. Can be used as
- the multinucleation-inducing synthetic peptide disclosed here may be in the form of a salt as long as the multinucleation-inducing activity is not impaired.
- a salt for example, an acid addition salt of the peptide that can be obtained by addition reaction of an inorganic acid or an organic acid usually used according to a conventional method can be used.
- another salt for example, metal salt
- the “peptide” described in the present specification and claims includes such a salt form.
- the multinucleation-inducing agent disclosed herein is pharmaceutically (pharmaceutical) acceptable depending on the form of use as long as the multinucleation-inducing synthetic peptide that is an active ingredient can be retained without losing its multinucleation-inducing activity.
- Various (at least one) carriers that can be made can be included. Carriers generally used in peptide medicine as diluents, excipients and the like are preferred. Typically, water, a physiological buffer, and various organic solvents can be mentioned, although it may vary depending on the use and form of the multinucleation inducer. It can be a non-drying oil such as an aqueous solution of alcohol (such as ethanol) of a suitable concentration, glycerol, olive oil.
- the secondary component that can be contained in the multinucleation inducer include various fillers, extenders, binders, moistening agents, surfactants, dyes, and fragrances.
- the form of the multinucleation inducer includes solutions, suspensions, emulsions, aerosols, foams, granules, powders, tablets, capsules, ointments, aqueous gels and the like.
- it can also be set as the freeze-dried material and granulated material for melt
- compositions using multinucleation-inducing synthetic peptides (main components) and various carriers (subcomponents) as materials may be in accordance with conventionally known methods. Since the formulation method itself does not characterize the present invention, a detailed description is omitted. As a detailed information source on prescription, for example, Comprehensive Medicinal Chemistry, supervised by Corwin Hansch, published by Pergamon Press (1990) can be mentioned. The entire contents of this book are incorporated herein by reference.
- the cells to which the multinucleation inducer (multinucleation-inducing synthetic peptide) disclosed herein is not particularly limited, and induces multinucleation in eukaryotic cells of various species (or induces multinucleation). Can be promoted).
- cells of humans or non-human animals typically vertebrates, particularly mammals
- tumor cells particularly malignant tumor cells (cancer cells), for example, squamous cell carcinoma cells and adenocarcinoma cells are suitable as target cells.
- multinucleated cells for example, megakaryocytes and osteoclasts
- pre-multinucleated cells before multinucleation for example, megakaryocytes or osteoclasts
- a cell or a precursor cell of a megakaryocyte before multinucleation for example, a megakaryocyte before multinucleation
- an osteoclast precursor cell before multinucleation osteoclast precursor cell
- the multinucleation-inducing agent (multinucleation-inducing synthetic peptide) disclosed herein can be used in a method or dosage depending on its form and purpose. For example, when inducing multinucleation on a target cell (eg, tumor cell, or multinucleated cell or pre-multinucleated cell before multinucleation) cultured (passaged) in vitro (in vitro), An appropriate amount of the disclosed multinucleation-inducing agent (ie, a multinucleation-inducing synthetic peptide) is applied to a cultured cell (cell culture) to be subjected to multinucleation at any stage (preferably for a predetermined period).
- a target cell eg, tumor cell, or multinucleated cell or pre-multinucleated cell before multinucleation
- An appropriate amount of the disclosed multinucleation-inducing agent ie, a multinucleation-inducing synthetic peptide
- the cultured cells include established cell lines and primary cultured cells, cell materials (cells, living tissues, cell masses, etc.) temporarily or permanently removed from living organisms, or stem cells (eg, iPS cells and ES).
- Cell material typically cells including tissues, cell masses, organs, etc. obtained by inducing differentiation from cells, hematopoietic stem cells, and the like.
- the supply amount and the supply frequency of the multinucleation inducer are the conditions such as the type of cultured cells, cell density (cell density at the start of culture), passage number, culture conditions, type of medium, etc.
- the concentration of the polynuclearization-inducing synthetic peptide in the medium is generally within the range of 0.1 ⁇ M to 100 ⁇ M, preferably Is supplied to a cultured cell (cell culture) one or more times so that it falls within a range of 0.5 ⁇ M to 80 ⁇ M (for example, 1 ⁇ M to 50 ⁇ M) It is preferable that additional supply is made according to the circumstances.
- the multinucleation inducer disclosed herein ie, An appropriate amount of the polynuclearization-inducing synthetic peptide
- a target cell for example, a tumor cell, a multinuclear cell or a multinucleated cell before multinucleation
- the multinucleation inducer disclosed herein ie, An appropriate amount of the polynuclearization-inducing synthetic peptide
- a solid form such as a tablet or a gel or aqueous jelly such as an ointment is directly applied to a target tissue (for example, an affected area of a tumor or a tissue in which multinucleation of target cells such as bone marrow or bone) occurs. It can be administered in the vicinity of the tissue. Alternatively, it may be administered orally or in the form of suppositories. Thereby, multinucleation can be induced with respect to cells in a living body, typically target cells existing in or around the target tissue.
- derivation agent namely, multinucleation induction synthetic peptide
- the multinucleation inducer (multinucleation inducing synthetic peptide) disclosed here is used in combination with a humoral factor (eg, cytokine) or a composition (eg, inhibitor) according to the type and purpose of the target cell. can do.
- a humoral factor eg, cytokine
- a composition eg, inhibitor
- anticancer agents include alkylating agents, antimetabolites, microtubule agonists, platinum preparations, anticancer antibiotics, hormone agents, molecular targeted therapeutic agents, and the like.
- multinucleation-inducing factors include, for example, thrombopoietin (TPO), various interleukins (for example, IL) in the case of inducing (promoting) multinucleation to megakaryocytes (or megakaryocytes before multinucleation).
- TPO thrombopoietin
- IL interleukins
- GM-CSF granulocyte macrophage colony-stimulating factor
- EPO Erythropoietin, erythropoietin
- SCF stem cell factor
- ROCK Ro-associated coiled-coil forming kinase inhibitors
- HDAC histone deacetylase
- TNF tumor necrosis factor
- RANKL receptor activator of NF- ⁇ B ligand
- Tumor necrosis factor a factor belonging to the superfamily
- M-CSF macrophage colony stimulating factor
- the multinucleation-inducing agent multinucleation-inducing synthetic peptide
- the multinucleation-inducing activity causes the tumor cells to become multinucleated.
- the cell division of the tumor cell can be suppressed (inhibited), and the cell proliferation of the tumor cell can be suppressed (inhibited).
- the multinucleation inducer disclosed here can be used as a pharmaceutical composition (antitumor composition, anticancer agent) that can be used for tumor treatment (cancer treatment).
- the multinucleation-inducing agent (multinucleation-inducing synthetic peptide) disclosed herein is administered so that multinucleated cells (for example, megakaryocytes, osteoclasts, etc.) in the body are supplied to the tissue that becomes multinucleated.
- multinucleated cells eg, megakaryocytes and osteoclasts
- pre-multinucleated cells prior to multinucleation eg, megakaryoblasts before multinucleation, osteoclast precursor cells before multinucleation
- an appropriate amount of a multinucleation-inducing agent (multinucleation-inducing synthetic peptide) is applied to a target cell (for example, a multinuclear cell or a pre-multinuclear cell before multinucleation), and the target cell is induced to be multinucleated.
- a target cell for example, a multinuclear cell or a pre-multinuclear cell before multinucleation
- the target cell is induced to be multinucleated.
- a cell material live tissue or cell mass, for example, cell culture
- a stem cell cultured in vitro (passaged) Predetermined cells (including tissues and organs composed of the cells) obtained by inducing differentiation from iPS cells (for example, iPS cells, ES cells, hematopoietic stem cells, etc.) can be used.
- iPS cells for example, iPS cells, ES cells, hematopoietic stem cells, etc.
- a cell ie, a multinucleated cell produced by the technology disclosed herein, or a multinucleated cell having an increased number of nuclei in the cell
- a tissue in which the cell functions ie, in a patient's body
- multinucleated cell material multinucleated cells, or a cell mass or tissue containing multinucleated cells
- cells and biosynthetic products for example, secreted proteins and Physiologically active substances such as hormones
- the technique disclosed here can be suitably used in a method for producing platelets from megakaryocytes.
- Sufficiently multinucleated megakaryocytes produce a larger number of platelets per cell (tens to hundreds of times) than megakaryocytes that are insufficiently multinucleated, thus producing platelets per megakaryocyte In order to increase the amount, it is important to promote multinucleation of megakaryocytes.
- a blood product (platelet product) can be manufactured using the platelets produced (manufactured) according to the present invention.
- Example 1 Peptide synthesis> A total of three types of peptides (samples 1 to 3) were produced using the peptide synthesizer described below. Table 1 lists information such as amino acid sequences of these synthetic peptides.
- the peptides according to Samples 1 and 2 have a multinucleation-inducing synthetic peptide sequence on the C-terminal side of the peptide chain, and two glycine (G) residues on the N-terminal side.
- the peptide of sample 2 (SEQ ID NO: 13) is a peptide having a total of 24 amino acid residues having the amino acid sequence shown in SEQ ID NO: 2 as a multinucleation-inducing synthetic peptide sequence.
- the peptide according to sample 3 is a peptide having a total of 13 amino acid residues consisting of only the amino acid sequence (SEQ ID NO: 3) derived from LIM kinase 2 which is a membrane-permeable peptide sequence.
- the synthetic peptides according to Samples 1 to 3 are linear peptides, and the synthetic peptide is a solid phase synthesis method (Fmoc method) according to the manual using a commercially available peptide synthesizer (product of Intavis AG). And synthesized. In addition, since the usage mode itself of the peptide synthesizer does not characterize the present invention, detailed description thereof is omitted.
- the synthesized peptides according to samples 1 to 3 were dissolved in PBS ( ⁇ ) or DMSO to prepare a peptide stock solution.
- Example 2 Test for evaluating the multinucleation-inducing activity of synthetic peptides against tumor cells-1> Regarding the multinucleation-inducing activity of the multinucleation-inducing synthetic peptides (sample 1 and sample 2) obtained in Example 1 above, cell immunostaining (fluorescence immunostaining) employing calreticulin as a cytoplasmic marker was performed. Evaluation was performed by performing nuclear staining with DAPI (4 ′, 6-diamidino-2-phenylindole). HeLaS3 cells (ATCC (registered trademark), CCL2.2), which is a cultured cell line derived from human cervical cancer, were used as test cells. Details of the evaluation test are as follows.
- HeLaS3 cells were seeded in each well of a 4-well (well) cell culture slide (also referred to as a chamber) so that the number of cells was about 1 ⁇ 10 3 per well, and 5% CO 2 at 37 ° C. The culture was performed overnight in an incubator under conditions.
- the medium is a general DMEM medium, that is, a DMEM (manufactured by Wako Pure Chemicals, Cat No. 043-30085) containing 10% FBS, 100 units / mL penicillin and 100 ⁇ g / mL streptomycin. Using.
- the medium in the culture vessel was added to the above-mentioned DMEM medium containing FBS, penicillin and streptomycin, and the peptide of any of Sample 1, Sample 2 and Sample 3 in an amount of 50 ⁇ M peptide concentration was contained.
- the medium was exchanged, and the culture was continued for 4 days under the same conditions.
- a peptide-free group in which only the same amount of PBS (-) as the peptide stock solution added to the sample addition group was added was provided.
- the multinucleated state in the cells of each test group was examined by the following cell immunostaining.
- PBS ( ⁇ ) containing 3% BSA PBS ( ⁇ ) containing 3% BSA
- blocking treatment was performed at room temperature for 1 hour.
- 3% BSA-containing PBS ( ⁇ ) was removed and washed 3 times with cold PBS ( ⁇ ).
- cell immunostaining for calreticulin was performed using an anti-calreticulin antibody and a secondary antibody that recognizes the antibody.
- an anti-calreticulin monoclonal [FMC75] antibody (derived from mouse, manufactured by Abcam, Cat No. ab22683) was diluted 400 times with 1% BSA / PBS ( ⁇ ) (PBS ( ⁇ ) containing 1% BSA).
- the diluted primary antibody was added to the above HeLaS3 cell culture vessel and allowed to stand at 4 ° C. overnight (about 16 to 18 hours). After a predetermined time, the primary antibody dilution was removed and washed 6 times with cold 0.1% BSA / PBS ( ⁇ ) (PBS ( ⁇ ) containing 0.1% BSA). Next, a secondary antibody obtained by diluting an anti-mouse IgG antibody (goat: Life Technologies, A11029) labeled with a fluorescent dye (Alexa (registered trademark) 488) as a secondary antibody 200-fold with 1% BSA / PBS ( ⁇ ) Diluent was added and allowed to stand at room temperature for 2 hours.
- a fluorescent dye Alexa (registered trademark) 488
- the secondary antibody diluted solution was removed and washed 6 times with cold 0.1% BSA / PBS ( ⁇ ). Then, the cells of each test section subjected to the above-described cell immunostaining were encapsulated using a DAPI-containing encapsulating solution, Slow Fade (Life Technologies, Cat. No. S36936) and a cover glass, and a confocal laser microscope was used. Fluorescence observation was performed.
- FIGS. These drawings are fluorescent micrographs showing the state of multinucleation of cells in each test group. Fluorescence images showing the results of examining the expression state of calreticulin by the above immunofluorescent antibody method, nuclear staining images by DAPI, and Is an image that is superimposed (merged). FIGS. 1 to 3 show the results of the samples 1 to 3 added group corresponding to the numbers of the figures, respectively, and FIG. 4 shows the results of the peptide-free group.
- Example 3 Evaluation test 2 of multinucleation-inducing activity of synthetic peptide against tumor cells> The multinucleation induction ratio of the multinucleation-inducing synthetic peptides (sample 1 and sample 2) obtained in Example 1 was examined. As a test cell, the same HeLaS3 cell as used in Example 2 was used. Details of the evaluation test are as follows.
- HeLaS3 cells were seeded in each well of an 8-well cell culture slide (also referred to as a chamber) so that the number of cells was about 0.5 ⁇ 10 3 per well, and 5% CO 2 , 37 The culture was performed overnight in an incubator under the condition of ° C.
- the medium is a general DMEM medium, that is, a DMEM (manufactured by Wako Pure Chemicals, Cat No. 043-30085) containing 10% FBS, 100 units / mL penicillin and 100 ⁇ g / mL streptomycin. Using.
- the medium in the culture vessel was further mixed with DMEM medium containing 3% FBS, 100 units / mL penicillin and 100 ⁇ g / mL streptomycin in the above-mentioned DMEM in an amount of a sample having a peptide concentration of 50 ⁇ M. Or it replaced
- a peptide-free group in which only the same amount of PBS (-) as the peptide stock solution added to the sample addition group was added was provided.
- cytoplasmic marker calreticulin
- cell immunostaining and nuclear staining of cytoplasmic marker were performed on the cells in each test group in the same manner as in Example 2. Then, the cells in each test section after cell immunostaining and nuclear staining are observed with fluorescence using a confocal laser microscope, and the total number of cells and two or more nuclei are observed for the cells observed in one field of view. And the number of cells (number of multinucleated cells) that were observed.
- multinucleation induction rate ratio of the number of cells having two or more nuclei to the total number of cells observed in one visual field of microscopic observation:%
- multinucleation induction rate (%) (Number of cells having two or more nuclei) / (total number of cells) ⁇ 100;
- the test on the conditions similar to the evaluation test of the said multinucleation induction rate was repeated 3 times mutually independently, and the average multinucleation induction rate was computed by averaging the multinucleation induction rate obtained by each test. The results are shown in Table 2.
- the synthetic peptide (multinucleation-inducing synthetic peptide) according to sample 1 or sample 2 is at least 30% or more of the test tumor cells (human cervical cancer cells: HeLaS3 cells) within 4 days after supplying the peptides. It was confirmed that multinucleation can be induced on cells (typically 35% or more).
- the efficiency (multinucleation induction rate, multinucleation induction frequency) of the synthetic peptide (multinucleation-inducing synthetic peptide) according to sample 1 or sample 2 to induce multinucleation on the test tumor cells (HeLaS3 cells) is , At least 30% or more (typically 35% or more).
- the average multinucleation induction efficiency of the synthetic peptide according to sample 1 is 40% or more, and can induce multinucleation more efficiently than the synthetic peptide according to sample 2 (that is, has high multinucleation induction activity). It was confirmed.
- the multinucleation-inducing synthetic peptide disclosed herein (that is, the multinucleation-inducing agent containing the peptide) has high multinucleation-inducing activity against eukaryotic cells (for example, human-derived tumor cells). It is shown that.
- Example 4 Cell proliferation inhibitory activity evaluation test of synthetic peptide against tumor cells> Regarding the cell growth inhibitory activity of the polynuclearization-inducing synthetic peptides (sample 1 and sample 2) obtained in Example 1 above, a cell proliferation test (WST-8) by an absorbance method using WST-8 (water-soluble tetrazolium salt). Assay). As a test cell, the same HeLaS3 cell as used in Example 2 was used. Details of the evaluation test are as follows.
- HeLaS3 cells were seeded into each well of a 96-well (well) plates such that the number of cells is approximately 5 ⁇ 10 3 cells per well, for 6 hours of culture in a 5% CO 2, 37 incubator under conditions of °C It was.
- the medium is a general DMEM medium, that is, DMEM containing 2 mM L-glutamine (Gibco, product number 11965-092), 10% FBS (Hyclone Laboratories, KSD28662), 100 units / mg penicillin and Using a medium containing 100 ⁇ g / mL streptomycin (hereinafter, this medium is also referred to as “basic medium for proliferation assay”), the amount of medium per well was set to 100 ⁇ L.
- the medium in the culture vessel was replaced with a medium in which the peptide according to Sample 1 or 2 having a peptide concentration of 50 ⁇ M was further added to the basic medium for proliferation assay.
- a peptide-free group in which only the same amount of PBS (-) as the peptide stock solution added to the sample addition group was added was provided.
- a 96-well (well) plate was placed in a CO 2 incubator and statically cultured at 37 ° C. under 5% CO 2 conditions. Then, when the peptide was added (at the start of treatment, Day 0) and at one day (24 hours) after the start of the culture in the presence of the peptide (Day 1), the survival state (number of viable cells) of the test cells was measured by a commercially available color measurement. Measurement was performed using a kit (Cell counting kit-8, manufactured by Dojindo Laboratories).
- the amount of water-soluble formazan in the medium is measured by the absorbance method (measurement wavelength: 450 nm, reference wavelength: 650 nm).
- the number of viable cells was measured by In addition, it performed according to the manual attached to the said measurement kit except the operation explained in full detail below. Specifically, 10 ⁇ L / well of a reagent containing “water-soluble tetrazolium salt (WST-8)” as a coloring substrate is added to the cell culture well after the predetermined culture time has elapsed, and 5% CO 2 , 37 Incubated for 1 hour under the condition of ° C.
- WST-8 water-soluble tetrazolium salt
- the absorbance at a wavelength of 450 nm (A 450 ) and the absorbance at a wavelength of 650 nm (A 650 ) were measured using a spectrophotometer (microplate reader) for the cell culture medium to which the coloring reagent was added, and A 450 was measured as A 650.
- the value A 450-650 corrected by the above was calculated.
- cell survival rate (%) ⁇ (A 450 of the sample addition group Day1 -650) / (a 450-650 sample addition group Day0) ⁇ ⁇ ⁇ (a 450-650 of a 450-650) / (no peptide addition group Day 0 peptide untreated silage Day1) ⁇ ⁇ 100; calculated by .
- the test tumor cells human cervical cancer cells: HeLaS3 cells
- the test tumor cells markedly suppressed cell proliferation as compared with the cells in the group without addition of peptide.
- any of the synthetic peptides (multinucleation-inducing synthetic peptides) according to Sample 1 or Sample 2 induces multinucleation on the test tumor cells (HeLaS3 cells), so that the multinucleated cells (multinuclear cells) Cell division), and as a result, it was confirmed that the growth of tumor cells could be prevented or suppressed.
- the cell growth inhibition rate of the synthetic peptides (multinucleation-inducing synthetic peptides) according to Sample 1 and Sample 2 with respect to the test tumor cells (HeLaS3 cells) was at least 20% or more.
- the cell growth inhibition rate of the synthetic peptide according to Sample 1 was 30% or more, and it was confirmed that the synthetic peptide according to Sample 2 has a higher cell growth inhibitory activity than the synthetic peptide according to Sample 2.
- Example 5 Preparation of granules> After mixing 50 mg of the synthetic peptide (multinucleation-inducing synthetic peptide) according to Sample 1 and Sample 2, 50 mg of crystallized cellulose and 400 mg of lactose, 1 mL of a mixed solution of ethanol and water was added and kneaded. This kneaded product was granulated according to a conventional method to obtain a granule (granular composition) mainly composed of the multinucleation-inducing synthetic peptide disclosed herein.
- the target eukaryotic cell has a multinucleation-inducing activity that multinucleates the target eukaryotic cell
- the target eukaryotic cell (particularly derived from a human). It can be suitably used for the purpose of inducing multinucleation of cells). For example, it can be used for the purpose of suppressing the growth of tumor cells by inducing multinucleation of tumor cells. Alternatively, it can be used for the purpose of promoting megakaryocyte maturation by inducing multinucleation of megakaryocytes before multinucleation (that is, megakaryocyte progenitor cells), thereby promoting the production of platelets. Therefore, the multinucleation inducer disclosed here can be suitably used as, for example, a composition for regenerative medicine or a composition for cancer treatment.
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Abstract
Description
なお、本出願は2014年6月23日に出願された日本国特許出願2014-128431号に基づく優先権を主張しており、当該日本国出願の全内容は本明細書中に参照として援用されている。
生体内に通常存在する多核細胞(多核細胞の形態で正常な機能を発揮する細胞)の例として、巨核球や破骨細胞が挙げられる。破骨細胞は造血幹細胞から分化した多核細胞であって、古い骨組織を破壊(骨吸収)する役割を担う細胞であり、骨の再構築(骨リモデリング)や骨の成長に関与する(非特許文献1)。また、上記骨吸収により骨中のカルシウムを血液中に供給することで、血中カルシウム濃度の調整(恒常性の維持)にも関与する(非特許文献2)。巨核球は、造血幹細胞から分化した多核細胞であり、血小板を産生する役割を担う細胞である。
即ち、ここで開示される合成ペプチドは、そのペプチド鎖中に、以下の(A)および(B)のアミノ酸配列:
(A)膜透過性ペプチド配列を構成するアミノ酸配列;および
(B)少なくとも一種の真核細胞に対して多核化を誘導する活性を有する多核化誘導性ペプチド配列を構成するCPDGAKARC(配列番号1)またはCSRRSKSKC(配列番号2)のいずれかで表されるアミノ酸配列、若しくは、該アミノ酸配列のうちの1個又は数個のアミノ酸残基が置換、欠失及び/又は付加された改変アミノ酸配列であって、少なくとも一種の真核細胞に対して多核化を誘導する活性を有する多核化誘導性ペプチド配列を構成する改変アミノ酸配列;
を有することを特徴とする少なくとも一種の真核細胞に対して多核化を誘導する活性を有する人為的に合成されたペプチドである。
なお、本明細書において、多核化誘導性ペプチド配列および膜透過性ペプチド配列を含む合成ペプチド(上記改変アミノ酸配列からなるペプチドを含む)、即ち多核化誘導活性を有する合成ペプチドを「多核化誘導性合成ペプチド」とも呼称する。
また、ここで開示される多核化誘導性合成ペプチドは、化学合成(若しくは生合成)によって容易に人為的に製造することができる。このような多核化誘導性合成ペプチドを用いると、サイトカインに代表される高価な液性因子等を大量に使用することなく(典型的には液性因子等の代替物として)対象細胞を多核化させることができるため、対象(標的)細胞の多核化誘導を低コストで実現することができる。さらに、ここで開示される多核化誘導性合成ペプチドは、単純な構造(典型的には直鎖状のペプチド鎖)であり、構造安定性が高いため、取扱い性に優れる。
配列番号3~11に示すアミノ酸配列はいずれも膜透過性ペプチドを構成するアミノ酸配列の典型例であり、上記(A)に示す膜透過性ペプチド配列として好適に採用することができる。なかでも、タンパク質を核内の核小体へ局在させるシグナル配列であり核小体局在シグナル(NoLS:Nucleolar localization signal)として知られるいずれかのアミノ酸配列(典型的には配列番号3~6に示すアミノ酸配列)を採用することが好ましい。特に、配列番号3に示すアミノ酸配列から構成される膜透過性ペプチド配列は、上記NoLSの典型例であり、多核化誘導性ペプチド配列を細胞内へ移送する効率の観点から特に好ましい。
KKRTLRKNDRKKRGGCPDGAKARC(配列番号12);および
KKRTLRKNDRKKRGGCSRRSKSKC(配列番号13);
のうちのいずれかを有することを特徴とする。
このような多核化誘導性合成ペプチドは、特にヒト若しくはヒト以外の哺乳動物由来の細胞に対して多核化を誘導する用途に好適である。
かかる構成の組成物によると、上記多核化誘導性ペプチド配列を有する合成ペプチド(多核化誘導性合成ペプチド)を含むため、該組成物(該多核化誘導性合成ペプチド)を対象(標的)とする細胞(典型的には当該細胞を培養する培地中)に供給することにより、該対象細胞の多核化を誘導すること(多核化を促進すること)ができる。
ここで開示される多核化誘導剤により腫瘍細胞に多核化を誘導することで、該多核化された細胞(多核化細胞)の細胞分裂が阻害され、その結果、腫瘍細胞の増殖を阻止若しくは抑制することができる。換言すると、ここで開示される多核化誘導剤は、ヒト若しくはヒト以外の哺乳動物由来の腫瘍細胞の増殖を抑制するための組成物として使用することができる。即ち、ここで開示される多核化誘導剤は、対象の腫瘍細胞に対して抗腫瘍効果を発揮し得るため、抗腫瘍組成物(抗がん剤)として好適に使用することができる。
好ましい一態様は、インビトロにおいて、少なくとも一種の真核細胞に対して多核化を誘導する方法であって、
インビトロにおいて対象の細胞を含む細胞培養物を準備すること、当該細胞培養物中に、ここで開示されるいずれかの態様の多核化誘導性合成ペプチド(あるいは該合成ペプチドを含む多核化誘導剤)を少なくとも1回供給すること、および、該ペプチドを供給した上記細胞培養物を培養すること、を含む多核化誘導方法である。
かかる多核化誘導方法によると、上述のとおり単純な構成の合成ペプチド(あるいは該合成ペプチドを含む多核化誘導剤)を多核化誘導因子として使用するという簡易な方法によって、目的の細胞(および該細胞からなる組織体)に対して効率的に多核化を誘導すること(多核化を促進すること)ができる。
換言すれば、多核細胞および多核化前のプレ多核細胞を対象にここで開示される多核化誘導性合成ペプチドを供給することを特徴とする多核化誘導方法を実施することにより、十分に多核化が誘導(促進)された多核細胞(例えば巨核球や破骨細胞等)を生産(製造)することができる。かかる方法によると、上述のとおり単純な構成の合成ペプチド(即ち、該合成ペプチドを含む多核化誘導剤)を多核化誘導因子として使用するという簡易な方法によって多核細胞を生産(製造)することができるため好ましい。
また、本明細書中で引用されている全ての文献の全ての内容は本明細書中に参照として組み入れられている。
また、本明細書において「ペプチド」とは、複数のペプチド結合を有するアミノ酸ポリマーを指す用語であり、ペプチド鎖に含まれるアミノ酸残基の数によって限定されないが、典型的には全アミノ酸残基数が概ね100以下(好ましくは50以下、より好ましくは30以下)のような比較的分子量の小さいものをいう。
また、本明細書において「アミノ酸残基」とは、特に言及する場合を除いて、ペプチド鎖のN末端アミノ酸及びC末端アミノ酸を包含する用語である。
なお、本明細書中に記載されるアミノ酸配列は、常に左側がN末端側であり右側がC末端側である。
また、本明細書において「多核化前のプレ多核細胞」とは、多倍体化していない多核細胞をいい、典型的には単核の細胞(有糸分裂期(M期)に2核となる細胞を含む)をいう。多核化前のプレ多核細胞の具体例として、巨核球の前駆細胞(巨核芽球)、破骨細胞の前駆細胞(破骨前駆細胞)、等が例示される。
CPDGAKARC(配列番号1);および
CSRRSKSKC(配列番号2);
のうちのいずれか若しくはその改変アミノ酸配列からなる。配列番号1又は配列番号2に示されるアミノ酸配列は、本発明者がヒト由来のセントリン2のsiRNAを構成するRNA配列を独自に翻訳して得たアミノ酸配列情報について、N末端側から1個目、2個目、3個目、4個目、5個目、6個目、7個目、8個目、9個目のアミノ酸残基が、それぞれC末端側から1個目、2個目、3個目、4個目、5個目、6個目、7個目、8個目、9個目のアミノ酸残基となるように逆転して再配列することで得られた合計9アミノ酸残基の人為的なアミノ酸配列である。
ここで、セントリンとは、真核生物の中心体に存在し、中心子の構成タンパク質の一つとして中心子の複製や微小管の切断に関与する中心体関連タンパク質であり、セントリン2とは、セントリンファミリー(典型的には、セントリン1、セントリン2、セントリン3等)に属するタンパク質のうちの一つである(非特許文献3)。
配列番号4のアミノ酸配列は、FGF2(塩基性線維芽細胞増殖因子)由来の合計14アミノ酸残基から成るNoLSに対応する。
配列番号5のアミノ酸配列は、IBV(トリ伝染性気管支炎ウイルス:avian infectious bronchitis virus)のNタンパク質(nucleocapsid protein)に含まれる合計8アミノ酸残基から成るNoLSに対応する。
配列番号6のアミノ酸配列は、アデノウイルスのPTP(新生末端タンパク質:pre-terminal protein)1及びPTP2由来の合計13アミノ酸残基からなるNoLSに対応する。
配列番号7のアミノ酸配列は、HIV(ヒト免疫不全ウイルス:Human Immunodeficiency Virus)のTATに含まれるタンパク質導入ドメイン由来の合計11アミノ酸残基から成る膜透過性ペプチド配列に対応する。
配列番号8のアミノ酸配列は、上記TATを改変したタンパク質導入ドメイン(PTD4)の合計11アミノ酸残基から成る膜透過性ペプチド配列に対応する。
配列番号9のアミノ酸配列は、ショウジョウバエ(Drosophila)の変異体であるAntennapediaのANT由来の合計16アミノ酸配列から成る膜透過性ペプチド配列に対応する。
配列番号10のアミノ酸配列は、ポリアルギニンとして、連続した合計9個のアルギニン残基から成る膜透過性ペプチド配列に対応する。
配列番号11のアミノ酸配列は、MyoD(筋芽細胞決定因子:myoblast determination)ファミリー阻害ドメイン含有タンパク質由来の合計19アミノ酸残基から成る膜透過性ペプチド配列に対応する。
なお、配列表に示した上述の膜透過性ペプチド配列はあくまでも例示であり、使用可能なペプチド配列はこれに限定されない。本発明の実施に使用可能な様々な膜透過性ペプチド配列が本願出願当時に出版されている数々の文献に記載されている。それら膜透過性ペプチド配列のアミノ酸配列は一般的な検索手段によって容易に知ることができる。
ここで開示される多核化誘導性合成ペプチドは、少なくとも一つのアミノ酸残基がアミド化されているものが好ましい。アミノ酸残基(典型的にはペプチド鎖のC末端アミノ酸残基)のカルボキシル基のアミド化により、合成ペプチドの構造安定性(例えばプロテアーゼ耐性)を向上させることができる。
このような鎖長の短いペプチドは、化学合成が容易であり、安価に多核化誘導性合成ペプチドを提供することができる。なお、ペプチドのコンホメーション(立体構造)については、使用する環境下(生体外若しくは生体内)で対象の真核細胞に対して多核化を誘導する多核化誘導活性を発揮する限りにおいて、特に限定されるものではないが、免疫原(抗原)になり難いという観点から直鎖状又はヘリックス状のものが好ましい。このような形状のペプチドはエピトープを構成し難い。かかる観点から、多核化誘導剤に適用する多核化誘導性合成ペプチドとしては、直鎖状であり比較的低分子量(典型的には50以下のアミノ酸残基数、特に好ましくは30以下のアミノ酸残基数)のものが好適である。
なお、本発明の多核化誘導性合成ペプチドとしては、全てのアミノ酸残基がL型アミノ酸であるものが好ましいが、対象の真核細胞に対して多核化を誘導する多核化誘導活性を失わない限りにおいて、アミノ酸残基の一部又は全部がD型アミノ酸に置換されているものであってもよい。
KKRTLRKNDRKKRGGCPDGAKARC(配列番号12);および
KKRTLRKNDRKKRGGCSRRSKSKC(配列番号13);
のうちから選択されるいずれかのアミノ酸配列または該選択されたアミノ酸配列の改変アミノ酸配列を特に好ましく含有する。配列番号12又は配列番号13に示すアミノ酸配列は、上記配列番号1又は配列番号2に示す多核化誘導性ペプチド配列と、上記配列番号3に示すLIMキナーゼ2のNoLS由来のアミノ酸配列とを、2個のグリシン(G)残基から成るリンカーを介して組み合わせることにより構築された合計24アミノ酸残基からなるアミノ酸配列である。
ここで開示される多核化誘導性合成ペプチドは、市販のペプチド合成機(例えば、Intavis AG社、Protein Technologies社等から入手可能である。)を用いた固相合成法により、所望するアミノ酸配列、修飾(C末端アミド化等)部分を有するペプチド鎖を合成することができる。
一般的な技法によって、この組換えベクターを所定の宿主細胞(例えばイースト、昆虫細胞、植物細胞)に導入し、所定の条件で当該宿主細胞又は該細胞を含む組織や個体を培養する。このことにより、目的とするペプチドを細胞内で発現、生産させることができる。そして、宿主細胞(分泌された場合は培地中)からペプチドを単離し、必要に応じてリフォールディング、精製等を行うことによって、目的の多核化誘導性合成ペプチドを得ることができる。
なお、組換えベクターの構築方法及び構築した組換えベクターの宿主細胞への導入方法等は、当該分野で従来から行われている方法をそのまま採用すればよく、かかる方法自体は特に本発明を特徴付けるものではないため、詳細な説明は省略する。
或いは、無細胞タンパク質合成システム用の鋳型DNA(即ち多核化誘導性合成ペプチドのアミノ酸配列をコードするヌクレオチド配列を含む合成遺伝子断片)を構築し、ペプチド合成に必要な種々の化合物(ATP、RNAポリメラーゼ、アミノ酸類等)を使用し、いわゆる無細胞タンパク質合成システムを採用して目的のポリペプチドをインビトロ合成することができる。無細胞タンパク質合成システムについては、例えばShimizuらの論文(Shimizu et al., Nature Biotechnology, 19, 751-755(2001))、Madinらの論文(Madin et al., Proc. Natl. Acad. Sci. USA, 97(2), 559-564(2000))が参考になる。これら論文に記載された技術に基づいて、本願出願時点において既に多くの企業がポリペプチドの受託生産を行っており、また、無細胞タンパク質合成用キット(例えば、日本の(株)セルフリーサイエンスから入手可能なPROTEIOS(商標)Wheat germ cell-free protein synthesis kit)が市販されている。
こうして得られるポリヌクレオチドは、上述のように、種々の宿主細胞中で又は無細胞タンパク質合成システムにて、多核化誘導性合成ペプチド生産のための組換え遺伝子(発現カセット)を構築するための材料として使用することができる。
多核化誘導剤の形態に関して特に限定はない。例えば、典型的な形態として、液剤、懸濁剤、乳剤、エアロゾル、泡沫剤、顆粒剤、粉末剤、錠剤、カプセル、軟膏、水性ジェル剤等が挙げられる。また、使用直前に生理食塩水又は適当な緩衝液(例えばPBS)等に溶解して薬液を調製するための凍結乾燥物、造粒物とすることもできる。
なお、多核化誘導性合成ペプチド(主成分)及び種々の担体(副成分)を材料にして種々の形態の薬剤(組成物)を調製するプロセス自体は従来公知の方法に準じればよく、かかる製剤方法自体は本発明を特徴付けるものでもないため詳細な説明は省略する。処方に関する詳細な情報源として、例えばComprehensive Medicinal Chemistry, Corwin Hansch監修,Pergamon Press刊(1990)が挙げられる。この書籍の全内容は本明細書中に参照として援用されている。
例えば、生体外(インビトロ)で培養(継代)している対象細胞(例えば、腫瘍細胞、又は多核細胞若しくは多核化前のプレ多核細胞等)に対して多核化を誘導する場合は、ここで開示される多核化誘導剤(即ち多核化誘導性合成ペプチド)の適当量を、多核化を行う対象の培養細胞(細胞培養物)に対し、培養過程のいずれかの段階(好ましくは所定期間の培養(増殖)や継代を行った後)で培地に少なくとも1回供給するとよい。上記培養細胞の例としては、樹立細胞株および初代培養細胞、あるいは生体から一時的に又は永久的に摘出した細胞材料(細胞、あるいは生組織や細胞塊等)、あるいは幹細胞(例えばiPS細胞やES細胞、造血幹細胞等)から分化誘導して得られる細胞材料(組織、細胞塊、器官等を含む、典型的には細胞)等が挙げられる。
多核化誘導剤(即ち多核化誘導性合成ペプチド)の供給量及び供給回数は、培養細胞の種類、細胞密度(培養開始時の細胞密度)、継代数、培養条件、培地の種類、等の条件によって異なり得るため特に限定されない。例えば、ヒト又はヒト以外の動物(典型的には脊椎動物、特に哺乳動物)由来の細胞を培養する場合、培地中の多核化誘導性合成ペプチド濃度が概ね0.1μM~100μMの範囲内、好ましくは0.5μM~80μM(例えば1μM~50μM)の範囲内となるように、培養細胞(細胞培養物)に対して1~複数回供給する(例えば培養開始時ならびに細胞の継代時や培地交換時に合わせて追加供給する)ことが好ましい。
例えば、腫瘍細胞に対して多核化を誘導する場合であれば、他の抗腫瘍効果を有する組成物(抗がん剤)と併用することができる。かかる抗がん剤としては、例えば、アルキル化剤、代謝拮抗剤、微小管作用薬、白金製剤、抗がん性抗生物質、ホルモン剤、分子標的治療薬等が挙げられる。
あるいは、多核細胞または多核化前のプレ多核細胞に対して多核化を誘導する場合であれば、他の多核化誘導因子および分化誘導因子等や、多核化誘導効果を有する組成物と併用することができる。かかる多核化誘導因子としては、巨核球(若しくは多核化前の巨核球)に対して多核化を誘導(促進)する場合であれば、例えば、トロンボポエチン(Thrombopoietin、TPO)、各種インターロイキン(例えばIL-1、IL-3、IL-4、IL-7、IL-11等)、GM-CSF(granulocyte macrophage colony-stimulating factor、顆粒球単球コロニー刺激因子)、EPO(Erythropoietin、エリスロポエチン)、SCF(stem cell factor、幹細胞因子)、その他のサイトカインファミリーに属する因子、ROCK(Rho-associated coiled-coil forming kinase、Rho結合キナーゼ)阻害剤、HDAC(histone deacetylase、ヒストン脱アセチル化酵素)阻害剤等が挙げられる。また、破骨細胞(若しくは多核化前の破骨細胞)に対して多核化を誘導(促進)する場合であれば、例えば、RANKL(receptor activator of NF-κB ligand)等のTNF(tumor necrosis factor、腫瘍壊死因子)スーパーファミリーに属する因子、M-CSF(macrophage colony stimulating factor)、TGF-β1等のTGF-βスーパーファミリーに属する因子、その他のサイトカインファミリーに属する因子、等が挙げられる。
また、ここで開示される多核化誘導方法(インビトロでの多核化誘導方法)や多核化誘導性合成ペプチドを採用することによって生体外(インビトロ)で効率よく多核化を誘導(促進)された目的細胞(即ち、ここで開示される技術によって生産された多核細胞、又は細胞内の核数を増大された多核細胞)を該細胞が機能する組織(即ち患者の生体内)に導入することにより、該細胞の多核化に要する時間を短縮することができる。
合計3種類のペプチド(サンプル1~3)を後述するペプチド合成機を用いて製造した。表1には、これら合成ペプチドのアミノ酸配列等の情報を列挙している。
即ち、サンプル1のペプチド(配列番号12)は、多核化誘導性合成ペプチド配列として配列番号1に示すアミノ酸配列を有する合計24アミノ酸残基のペプチドである。
また、サンプル2のペプチド(配列番号13)は、多核化誘導性合成ペプチド配列として配列番号2に示すアミノ酸配列を有する合計24アミノ酸残基のペプチドである。
また、サンプル3に係るペプチドは、表1に示すように、膜透過性ペプチド配列であるLIMキナーゼ2由来のアミノ酸配列(配列番号3)のみから成る合計13アミノ酸残基のペプチドである。
合成したサンプル1~3に係るペプチドは、PBS(-)又はDMSOに溶かし、ペプチドストック液を調製した。
上記実施例1で得られた多核化誘導性合成ペプチド(サンプル1およびサンプル2)の多核化誘導活性について、細胞質のマーカーとしてカルレティキュリンを採用した細胞免疫染色(蛍光免疫染色)を行うとともに、DAPI(4',6-diamidino-2-phenylindole)による核染色を行うことで評価した。供試細胞としてヒト子宮頸癌由来の培養細胞株であるHeLaS3細胞(ATCC(登録商標)、CCL2.2)を用いた。評価試験の詳細は以下のとおりである。
まず、各試験区の細胞を固定し、その後ブロッキング処理を行った。具体的には、まず、各試験区の培養容器中の培地を除去し、冷PBS(-)で2回洗浄した。次いで、メタノール1容量とアセトン1容量とを混和した溶液(メタノール/アセトン=1:1溶液)を添加し、氷上に15分静置してHeLaS3細胞を固定した。その後、メタノール/アセトン=1:1溶液を除去し、冷PBS(-)で3回洗浄した。そして、3%のBSA含有PBS(-)(3%のBSAを含むPBS(-))を添加して室温で1時間のブロッキング処理を行った。所定時間経過後、3%のBSA含有PBS(-)を除去し、冷PBS(-)で3回洗浄した。
次に、抗カルレティキュリン抗体と該抗体を認識する二次抗体とを用いて、カルレティキュリンに対する細胞免疫染色を行った。まず、抗カルレティキュリンモノクローナル[FMC75]抗体(マウス由来、Abcam社製、Cat No. ab22683)を1%BSA/PBS(-)(1%のBSAを含むPBS(-))で400倍希釈した一次抗体希釈液を上記HeLaS3細胞の培養容器中に添加し、4℃で一晩(約16~18時間)静置した。そして、所定時間経過後、上記一次抗体希釈液を除去し、冷0.1%BSA/PBS(-)(0.1%のBSAを含むPBS(-))で6回洗浄した。次いで、二次抗体として蛍光色素(Alexa(登録商標)488)で標識した抗マウスIgG抗体(ヤギ:Life Technologies社製品、A11029)を1%BSA/PBS(-)で200倍希釈した二次抗体希釈液を添加し、室温で2時間静置した。上記所定時間経過後、二次抗体希釈液を除去し、冷0.1%BSA/PBS(-)で6回洗浄した。
そして、上記の細胞免疫染色を行った各試験区の細胞をDAPI含有封入液であるSlow Fade(Life Technologies社製、Cat. No. S36936)とカバーガラスを用いて封入し、共焦点レーザー顕微鏡による蛍光観察を行った。
上記実施例1で得られた多核化誘導性合成ペプチド(サンプル1およびサンプル2)について、多核化誘導率を調べた。供試細胞として、実施例2と同様のHeLaS3細胞を用いた。評価試験の詳細は以下のとおりである。
これらの結果は、ここで開示される多核化誘導性合成ペプチド(即ち、該ペプチドを含む多核化誘導剤)が真核細胞(例えばヒト由来の腫瘍細胞)に対して高い多核化誘導活性を有することを示している。
上記実施例1で得られた多核化誘導性合成ペプチド(サンプル1およびサンプル2)の細胞増殖阻害活性について、WST-8(水溶性テトラゾリウム塩)を用いた吸光度法による細胞増殖試験(WST-8アッセイ)を行うことで評価した。供試細胞として、実施例2と同様のHeLaS3細胞を用いた。評価試験の詳細は以下のとおりである。
上記所定時間の培養後、培養容器中の培地を、上記増殖アッセイ用基本培地に更にペプチド濃度50μMとなる量のサンプル1または2に係るペプチドを含有させた培地に交換した。なお、コントロール区として、サンプル添加区に添加したペプチドストック液と同容量のPBS(-)のみを添加したペプチド無添加区を設けた。
具体的には、上記所定の培養時間が経過した細胞培養ウェル中に、発色基材として「水溶性テトラゾリウム塩(WST-8)」含む試薬を1ウェルあたり10μL添加し、5%CO2、37℃の条件下で1時間インキュベートした。その後、該発色試薬を添加した細胞培養液について、波長450nmの吸光度(A450)および波長650nmの吸光度(A650)を分光光度計(マイクロプレートリーダ)を用いて測定し、A450をA650で補正した値A450-650を算出した。そして、ペプチド無添加区における細胞生存率を100%としたときの各サンプル添加区の細胞生存率(%)を、以下の式:細胞生存率(%)={(サンプル添加区Day1のA450-650)/(サンプル添加区Day0のA450-650)}÷{(ペプチド無添加区Day1のA450-650)/(ペプチド無添加区Day0のA450-650)}×100;により算出した。そして、ペプチド無添加区の細胞生存率(%)を基準として、各サンプル添加区の細胞生存率の減少率を細胞増殖阻害率(%)として算出した。即ち、以下の式:細胞増殖阻害率(%)=100-(サンプル添加区の細胞生存率);より細胞増殖阻害率を算出した。結果を表3に示す。
また、サンプル1およびサンプル2に係る合成ペプチド(多核化誘導性合成ペプチド)の供試腫瘍細胞(HeLaS3細胞)に対する細胞増殖阻害率は少なくとも20%以上であることを確認した。特に、サンプル1に係る合成ペプチドの細胞増殖阻害率は30%以上であり、サンプル2に係る合成ペプチドよりも高い細胞増殖阻害活性を有することを確認した。
これらの結果は、ここで開示される多核化誘導性合成ペプチド(即ち、該ペプチドを含む多核化誘導剤)が腫瘍細胞(例えばヒト由来の腫瘍細胞)に対して高い抗腫瘍活性(腫瘍細胞増殖阻害活性)を有することを示している。
上記サンプル1およびサンプル2に係る合成ペプチド(多核化誘導性合成ペプチド)50mgと結晶化セルロース50mg及び乳糖400mgとを混合した後、エタノールと水の混合液1mLを加え混練した。この混練物を常法に従って造粒し、ここで開示される多核化誘導性合成ペプチドを主成分とする顆粒剤(顆粒状組成物)を得た。
Claims (9)
- ペプチド鎖中に、以下の(A)および(B)のアミノ酸配列:
(A)膜透過性ペプチド配列を構成するアミノ酸配列;および
(B)少なくとも一種の真核細胞に対して多核化を誘導する活性を有する多核化誘導性ペプチド配列を構成するCPDGAKARC(配列番号1)またはCSRRSKSKC(配列番号2)のいずれかで表されるアミノ酸配列、若しくは、該アミノ酸配列のうちの1個又は数個のアミノ酸残基が置換、欠失及び/又は付加された改変アミノ酸配列であって、少なくとも一種の真核細胞に対して多核化を誘導する活性を有する多核化誘導性ペプチド配列を構成する改変アミノ酸配列;
を有する、少なくとも一種の真核細胞に対して多核化を誘導する活性を有する人為的に合成されたペプチド。 - 前記(A)膜透過性ペプチド配列を構成するアミノ酸配列は、配列番号3~11のうちから選択されるいずれかのアミノ酸配列、若しくは、該アミノ酸配列のうちの1個又は数個のアミノ酸残基が置換、欠失及び/又は付加された改変アミノ酸配列であって膜透過性ペプチド配列を構成する改変アミノ酸配列である、請求項1に記載の合成ペプチド。
- 前記(A)膜透過性ペプチド配列を構成するアミノ酸配列が、以下のアミノ酸配列:
KKRTLRKNDRKKR(配列番号3)
である、請求項1または2に記載の合成ペプチド。 - ペプチド鎖を構成する全アミノ酸残基数が30以下である、請求項1~3のいずれか一項に記載の合成ペプチド。
- 以下のアミノ酸配列:
KKRTLRKNDRKKRGGCPDGAKARC(配列番号12);および
KKRTLRKNDRKKRGGCSRRSKSKC(配列番号13);
のうちのいずれかを有する、請求項4に記載の合成ペプチド。 - 少なくとも一種の真核細胞に対して多核化を誘導するために用いられる多核化誘導剤であって、
請求項1~5のいずれか一項に記載の合成ペプチドと、
薬学的に許容可能な担体と、
を含む、多核化誘導剤。 - 前記真核細胞がヒト若しくはヒト以外の哺乳動物由来の腫瘍細胞である、請求項6に記載の多核化誘導剤。
- インビトロにおいて、少なくとも一種の真核細胞に対して多核化を誘導する方法であって、
インビトロにおいて対象の細胞を含む細胞培養物を準備すること、
前記細胞培養物中に、請求項1~5の何れか一項に記載の合成ペプチドのいずれかを少なくとも1回供給すること、および
該ペプチドを供給した前記細胞培養物を培養すること、
を含む、多核化誘導方法。 - 前記真核細胞がヒト若しくはヒト以外の哺乳動物由来の腫瘍細胞である、請求項8に記載の多核化誘導方法。
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JP7096990B1 (ja) | 2022-04-20 | 2022-07-07 | 東亞合成株式会社 | 外来物質導入用構築物およびその利用 |
WO2023204138A1 (ja) * | 2022-04-20 | 2023-10-26 | 東亞合成株式会社 | 外来物質導入用構築物およびその利用 |
JP2023159685A (ja) * | 2022-04-20 | 2023-11-01 | 東亞合成株式会社 | 外来物質導入用構築物およびその利用 |
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