WO2020262617A1 - Cell fusion-accelerating agent - Google Patents

Cell fusion-accelerating agent Download PDF

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Publication number
WO2020262617A1
WO2020262617A1 PCT/JP2020/025250 JP2020025250W WO2020262617A1 WO 2020262617 A1 WO2020262617 A1 WO 2020262617A1 JP 2020025250 W JP2020025250 W JP 2020025250W WO 2020262617 A1 WO2020262617 A1 WO 2020262617A1
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cell
peg
peptide
cells
cell fusion
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PCT/JP2020/025250
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French (fr)
Japanese (ja)
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裕治 寺村
彬文 吉原
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国立大学法人東京大学
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Priority to JP2021527776A priority Critical patent/JP7335000B2/en
Publication of WO2020262617A1 publication Critical patent/WO2020262617A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention relates to an agent that promotes cell fusion and a cell fusion method using the cell fusion promoter.
  • Cell fusion is a phenomenon in which two or more cells are fused, and is a phenomenon that occurs not only between allogeneic cells but also between heterologous cells.
  • cell fusion between heterologous cells has long been used as a method for producing cells having new traits different from those of cells before fusion.
  • hybridomas in which antibody-producing B cells and myeloma cells are fused to prepare monoclonal antibodies have long been used as cells that are replicable and produce the desired antibody.
  • a method of transplanting fusion cells of mesenchymal cells and pancreatic islet cells has been proposed (Non-Patent Document 1).
  • the fusion cells of mesenchymal cells and islet cells maintained the islet function even after culturing for about 20 days, and when this was transplanted into rats, a decrease in blood glucose was observed for about 3 months.
  • a Sendai virus method As described above, cell fusion is widely used in the research field and the medical field.
  • a Sendai virus method As a method for fusing cells with each other, a Sendai virus method, a PEG (polyethylene glycol) method and an electrical fusion method are mainly known.
  • Sendai virus method it is considered that Sendai virus acts as a bridge between cell membranes and promotes fusion.
  • This method makes it difficult to completely avoid contamination of the fused cell fraction with the virus and is not well suited for medical use of the fused cells.
  • the PEG method does not require a dedicated device, the reagent is inexpensive, and has been mainly used in the past.
  • this method has low cell fusion efficiency and reproducibility, and the cell fusion efficiency varies greatly depending on the cell type.
  • an AC voltage is applied to bring cells that exist between electrodes into contact with each other, and then a DC pulse voltage is applied to open a perforation in the cell membrane, causing fusion between the cells in contact with each other.
  • the method can fuse cells more efficiently than the above two methods.
  • this method has problems such as the need for a special device and the decrease in cell viability due to the application of voltage.
  • the selectivity of cells is poor, and it is difficult to fuse cells of different species.
  • Non-patent document 3 a method of binding a single-stranded polyDNA to a PEG-lipid complex, introducing it into a cell to be adhered to, and adhering the cells to each other using a complementary sequence of the single-stranded DNA.
  • This method is an effective method for adhering cells to each other, but in this method, since the molecule does not exist other than the adhering site (see Fig. 2 of Non-Patent Document 3), one cell is used. It is considered difficult to fuse multiple cells (3 or more) with the cells of. Under the above circumstances, it is desired to establish an efficient cell fusion method that overcomes the problems of the conventional method.
  • the present invention can also be used in the medical field such as cell transplantation, and a method capable of cell fusion more efficiently than the conventional method, and a cell fusion promoter used in such a method Development is an issue.
  • the cell fusion-promoting compound comprises a lipid moiety that interacts with the cell membrane, a peptide moiety that holds one of the peptide pairs that form a dimer, and a moiety that connects the lipid moiety and the peptide moiety (eg, PEG). ..
  • the peptides that bind to each other are A 1 and A 2 (A 1 and A 2 bind)
  • a cell fusion-promoting compound having A 1 on the cell surface of cell 1 and a cell fusion-promoting compound having A 2 on the cell surface of cell 2 are introduced.
  • the interaction between A 1 and A 2 causes cell 1 and cell 2 to approach each other and promote mutual fusion.
  • the cell fusion promoting compound according to the present invention is one without accumulating at the adhesion site between cells. Fusion of a plurality of cells can also be induced with respect to the cells of the above (for example, the lower right figure of FIG. 1).
  • X is an atomic group containing a peptide that forms a dimer with another peptide
  • Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen
  • Z is a polymer electrolyte or Water-soluble polymer
  • (2) The compound according to (1) above, a salt thereof, or a solvent thereof, wherein the peptide is a peptide containing repeating amino acids of 7 residues represented by the following formula (2). Japanese products or their hydrates.
  • a and d are hydrophobic amino acids, e, f and g are charged amino acids, b is any of serine, asparagine, glutamine and threonine, and c is alanine]
  • the Y is a chain hydrocarbon group having 10 to 50 carbon atoms which may contain oxygen, sulfur, phosphorus or nitrogen.
  • W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond]
  • n is an integer of 50 or more and 1250 or less]
  • a cell fusion promoter comprising the compound according to any one of (1) to (9) above or a salt thereof, a solvate thereof or a hydrate thereof.
  • a cell fusion promoting kit containing at least two types of cell fusion promoting agents according to (10) above.
  • a method for fusing cell A and cell B which comprises the following (a) and (b).
  • step (B) The step of treating cell B with the cell fusion promoter according to (10) above, which binds to the cell fusion promoter used in step (a), and the step (c) (c).
  • cell fusion can be induced more efficiently than the conventional method.
  • a plurality of cells can be fused to one cell, and cells of not only allogeneic cells but also heterologous cells can be fused.
  • FIG. 1 An example of the cell fusion promoting compound according to the present invention (upper figure), a diagram schematically showing the modification of the cell surface by the cell fusion promoting compound (lower left figure), and a cell fused by the cell fusion promoting method according to the present invention. (Lower right figure) is shown. Fluorescence micrographs (A) of cells surface-modified with FITC-fuP1-PEG-lipid over time and results (B) of measuring the abundance of FITC-fuP1-PEG-lipid on the cell surface by flow cytometry are shown. Shown. Control is when FITC-fuP1-PEG-lipid is not used to modify the cell surface. The value is the average of 3 experiments.
  • the results of evaluating the interaction of fuP2 with fuP1-PEG-lipid are shown.
  • the results of observing the state of cell adhesion using fuP1-PEG-lipid and fuP2-PEG-lipid are shown.
  • the results of measuring the cell adhesion frequency (A) and the cell adhesion area (B) using fuP1-PEG-lipid and fuP2-PEG-lipid are shown. The value is the average of 3 experiments.
  • Cell surface modification with peptide-PEG-lipid (hereinafter also referred to as "cell modification method") and PEG treatment (PEG method) are performed together to induce cell fusion (cell fusion between cells of the same type) with a fluorescence microscope.
  • the observed results are shown.
  • the upper figure shows the results immediately after cell fusion, and the lower figure shows the results 24 hours after cell fusion.
  • the results of flow cytometry measurement of the cell fusion rate of the cells 24 hours after the cell fusion shown in FIG. 6 are shown.
  • A is the result of quantifying the flow cytometry histogram
  • B is the result of quantifying the cell fusion rate.
  • the number regarded as fused cells indicates the number of cells not surrounded by gate P1 and gate P2, and the fusion rate is the total minus the ratio of P1 and P2.
  • the value is the average of 3 experiments.
  • the results of measuring the cell viability 24 hours after the cell fusion shown in FIG. 6 by trypan blue staining are shown.
  • Control is when the cell surface is not modified with peptide-PEG-lipid.
  • the value is the average of 3 experiments.
  • the results of observing cells that have been induced to undergo cell fusion (cell fusion between different types of cells) by combining cell surface modification with peptide-PEG-lipid and PEG treatment (PEG method) with a confocal laser scanning microscope are shown.
  • the upper figure shows the results immediately after cell fusion, and the lower figure shows the results 24 hours after cell fusion.
  • the results of flow cytometry measurement of the cell fusion rate of the cells 24 hours after the cell fusion shown in FIG. 9 are shown. The value is the average of 3 experiments.
  • the first embodiment of the present invention is a compound represented by the following formula (1) or a salt thereof, a solvate thereof or a hydrate thereof (hereinafter, "cell fusion promoting compound of the present invention, etc.”” Also described).
  • X is an atomic group containing a peptide that forms a dimer with another peptide
  • Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen
  • Z is a polymer electrolyte or Water-soluble polymer
  • the "fusion" of cells means not only the adhesion between cell surfaces (cell membranes) but also the fusion between cell membranes and the mixing of cytoplasm.
  • the "peptide forming another peptide dimer" contained in X is, for example, peptide A or peptide B when peptide A and peptide B are combined to form a dimer. That is.
  • a peptide that forms a dimer with another peptide (hereinafter, also referred to as “dimer-forming peptide”) will be described by taking the above peptides A and B as an example.
  • the length thereof is not particularly limited as long as it is easy to use, and for example, the amino acid is 5 to 100 residues, preferably 10 to 50 residues, and more preferably 20 to 50 residues.
  • Examples of the peptide forming a dimer include the peptide of the following formula (2).
  • a and d are the same or different hydrophobic amino acids, for example, amino acids selected from the group consisting of alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan and tyrosine.
  • e, f and g are the same or different charged amino acids, eg, amino acids selected from the group consisting of arginine, aspartic acid, glutamic acid, lysine and histidine. It is desirable that e and f have different charges.
  • b is not particularly limited, but is a highly polar uncharged amino acid, and for example, an amino acid selected from the group consisting of serine, asparagine, glutamine and threonine is preferable.
  • c is not particularly limited, but alanine and the like can be mentioned as an example.
  • Examples of the peptide of the above formula (2) include peptides containing the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 described in Examples. Furthermore, as a typical example of the peptide of the above formula (2), a peptide having a double coil (coiled-coil) structure can be mentioned without limitation.
  • peptides having a double coil structure many peptides are known in the art, such as the peptides described in Table 1 of Hodges et al., J. Peptide Res., 58: 477-492 2001. Needless to say, it is not limited to these.
  • X in the formula (1) may contain a linker (or a part thereof) for binding Z and the dimer-forming peptide.
  • a linker containing a maleimide group or a part thereof may be contained in X, and in addition, a thiol group, an amino group, and a carboxyl group may be contained.
  • the X may contain a linker containing a group, a hydroxyl group, an azide group, or a part thereof.
  • Y in the formula (1) is a "hydrocarbon group which may contain oxygen, sulfur, phosphorus or nitrogen".
  • the "hydrocarbon” of Y is a chain hydrocarbon (which may be linear or branched), a cyclic hydrocarbon, an aromatic hydrocarbon, and the like, and the structure thereof is not particularly limited and is saturated. It may be a hydrocarbon or an unsaturated hydrocarbon, and a hydrocarbon having high hydrophobicity or lipophilicity is particularly preferable.
  • the "hydrocarbon group which may contain oxygen, sulfur, phosphorus or nitrogen” of Y in the present embodiment is not limited, but is, for example, a hydrocarbon having 10 to 50 carbon atoms, and in some cases, oxygen.
  • Substituents containing sulfur, phosphorus or nitrogen in particular, hydrocarbons having 10 to 50 carbon atoms and in some cases consisting of chain hydrocarbons containing oxygen, sulfur, phosphorus or nitrogen, etc. may be mentioned. it can. More specifically, as the "hydrocarbon group which may contain oxygen, sulfur, phosphorus or nitrogen" of Y in this embodiment, a substituent composed of a lipid (containing a lipid) can be mentioned.
  • examples of the lipid include simple lipids such as acylglycerol and ceramide, complex lipids such as phospholipids, glycolipids, lipoproteins and sulfolipids, fatty acids, terpenoids, steroids (sterols, cholesterol and the like), carotenoids and the like. be able to. Among them, phospholipids, ceramides, steroids and the like are preferable, and phospholipids are particularly preferable.
  • the phospholipid of the formula (3) can be mentioned.
  • W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond
  • DPPE 1,2-Dipalmitoyl- sn -Glycero-3-Phosphoethanolamine
  • DSPE 1,2-Distearoyl- sn -Glycero-3-Phosphoethanolamine
  • DSPE 1,2-Distearoyl- sn -Glycero-3-Phosphoethanolamine
  • DMPE 1,2-Dimyristoyl- sn -Glycero-3-Phosphoethanolamine
  • DOPE 1,2-Dioleoyl- sn -Glycero-3-Phosphoethanolamine
  • Y in formula (1) may contain a linker (or a part thereof) for binding Y and Z in addition to "hydrocarbons which may contain oxygen, sulfur, phosphorus or nitrogen".
  • Y may contain a linker containing a maleimide group, a thiol group, an amino group, a carboxyl group, a hydroxyl group, an azide group, or a part thereof.
  • Z is a polymer electrolyte or a water-soluble polymer. More specifically, it is a linear or branched water-soluble type having 100 to 4000 carbon atoms, preferably 150 to 3500 carbon atoms, and more preferably 200 to 2500 carbon atoms, which may contain oxygen, sulfur or nitrogen. It is a hydrocarbon chain.
  • the "water-soluble hydrocarbon chain that may contain oxygen, sulfur, or nitrogen” that can be used as Z is not particularly limited, but in addition to a water-soluble polymer such as a sugar chain, for example, the following formula ( Polyethylene glycol (PEG) represented by 4) is preferable.
  • the molecular weight thereof is preferably, for example, preferably about 2 kDa to 50 kDa, more preferably about 4 kDa to 45 kDa, and in this case, n in the formula (2) is an integer and is preferable. Is about 50 ⁇ n ⁇ 1250, more preferably about 100 ⁇ n ⁇ 1125.
  • the compound of the general formula (1) can be expressed as the following general formula (1').
  • X is an atomic group containing a peptide that forms a dimer with another peptide
  • Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen
  • n is 50 or more and 1250. The following integers]
  • the hydrocarbon chain of Z of the formula (1) When the hydrocarbon chain of Z of the formula (1) is bound to the dimer-forming peptide contained in X, it may be bound via an appropriate linker or directly.
  • the dimer-forming peptide and Z can be easily linked by using a well-known technique in the art. The method of binding the dimer-forming peptide and the hydrocarbon chain of Z will be described below with an example of the case where Z is PEG. 1.
  • a method using the reaction of a maleimide group and a thiol group This is a method of reacting a maleimide group-introduced PEG with a dimer-forming peptide having a thiol group. According to this method, the maleimide group functions as a linker.
  • the maleimide group introduced into PEG by the Michael addition reaction reacts with the thiol group of the dimer-forming peptide, and PEG and the dimer-forming peptide bind to each other.
  • Any method may be used to introduce the thiol group into the dimer-forming peptide.
  • the thiol group can be introduced into the dimer-forming peptide by adding cysteine to the dimer-forming peptide. it can.
  • the amino group of the dimer-forming peptide (the N-terminal amino group or the lysine residue added to the dimer-forming peptide) Introduce a maleimide group into the amino group, etc.).
  • EMCS N- (6-Maleimidocaproyloxy) succinimide
  • the maleimide-introduced dimer-forming peptide with the thiol-introduced PEG, the PEG and the dimer-forming peptide can be bound to each other.
  • the maleimide group-introduced PEG and the thiol group-introduced PEG can be prepared by using well-known techniques in the art, and commercially available products (NOF Corporation, etc.) may be used.
  • the coupling reaction between PEG having COOH and the amino group or OH group of the dimer-forming peptide can be used to bind PEG to the dimer-forming peptide (amide). Form a bond or ester bond).
  • a coupling reagent such as N, N'-dichlorohexyl carbodiimide (DCC) is used to activate the COOH group of PEG and the amino group of the dimer-forming peptide (such as the amino group of Lys or the N-terminal amino group).
  • DCC N, N'-dichlorohexyl carbodiimide
  • it can be bonded to an OH group (such as an OH group of Ser or Thr).
  • the coupling reaction between NH 2 groups of PEG and COOH group of dimer-forming peptide can be used to bind PEG and dimer-forming peptide (amide bond). Formation).
  • a coupling reagent such as N, N'-dichlorohexylcarbodiimide (DCC) can be used to activate the COOH group of the dimer-forming peptide and bind it to the transamination of PEG.
  • DCC N, N'-dichlorohexylcarbodiimide
  • the PEG and the dimer-forming peptide can be bound to each other by utilizing the coupling reaction between the OH group of PEG and the COOH group of the dimer-forming peptide (forming an ester bond). ..
  • a coupling reagent such as N, N'-dichlorohexyl carbodiimide (DCC) can be used to activate the COOH group of the dimer-forming peptide and bind it to the OH group of PEG.
  • DCC N, N'-dichlorohexyl carbodiimide
  • Z and Y of the formula (1) When Z and Y of the formula (1) are bonded, they may be bonded via an appropriate linker or may be directly bonded.
  • Y has a COOH group and Z has an amino group or a hydroxyl group, or when Y has an amino group or a hydroxyl group and Z has a COOH group, a coupling reaction using COOH is carried out to obtain Y.
  • Z can be combined.
  • a coupling reagent such as N, N'-dichlorohexylcarbodiimide (DCC) and then bonded to the amino or hydroxyl group of Z. Can be made to.
  • DCC N, N'-dichlorohexylcarbodiimide
  • Y has an N-hydroxyl succinimide group (NHS) which is an active ester group and Z has an amino group or a hydroxyl group
  • NHS can be utilized to bond Y and Z by utilizing the reaction with an amino group or a hydroxyl group.
  • Y can be bonded to Z having an N-hydroxyl succinimide group (NHS) which is an active ester group.
  • Z in the formula (1) includes, in addition to the “polymer electrolyte or water-soluble polymer”, a linker (or a part thereof) for binding Z and X and / or Z and Y. May be.
  • Y may contain a linker containing a maleimide group, a thiol group, an amino group, a carboxyl group, a hydroxyl group, an azide group, or a part thereof.
  • the second embodiment of the present invention is a cell fusion promoter containing the cell fusion promoter compound of the present invention (hereinafter, "cell fusion promoter of the present invention").
  • the present embodiment is represented by the formula (1). It is based on the fact that the cell fusion promoting compound has an effect of promoting fusion between cell membranes and fusion between cells, and can be used as a reagent or the like for promoting fusion when performing fusion between desired cells.
  • the cell fusion-promoting agent of the present embodiment may contain only the cell fusion-promoting compound or the like, or may be dissolved in an appropriate solvent.
  • two types of cell fusion promoters which separately contain two types of cell fusion promoter compounds that bind to each other, are added to each cell to be fused. use. Therefore, when the cell fusion promoter of the present invention promotes cell fusion, two types of cell fusion promoters (including cell fusion promoter compounds that bind to each other) are used as a set.
  • a third embodiment of the present invention is a cell fusion promoting kit containing at least two kinds of cell fusion promoting agents of the present invention.
  • the cell fusion promoting kit according to the present invention is characterized by containing at least two types of cell fusion promoting agents that separately contain cell fusion promoting compounds and the like that bind to each other.
  • reagents and the like for example, PBS, PEG, medium, etc.
  • the cell fusion promoting kit according to the present invention may include an instruction manual, or may include an information document or the like containing information such as a website containing the usage instructions. ..
  • the instruction manual may be recorded and attached to a recording medium such as a CD or DVD.
  • two types of cells to be fused are separately contained, such as a cell fusion promoting compound which is a cell fusion promoting compound of the present invention and binds to each other (via binding between peptides).
  • a cell fusion promoting compound which is a cell fusion promoting compound of the present invention and binds to each other (via binding between peptides).
  • This is a method in which the two cells are mixed and co-cultured after each treatment with the cell fusion promoter of the above, and the two cells are fused.
  • the present embodiment is a cell fusion method including the following (a) and (b) when the cells to be fused are cells A and B.
  • a step of treating cell A with the cell fusion promoter of the present invention (B) A step of treating cell B with a cell fusion promoter containing a cell fusion promoter that binds to a cell fusion promoter contained in the cell fusion promoter used in step (a), and a step (c) (c).
  • the "cell” may be not only the established cell but also the primary cultured cell (primary cell) collected from the tissue of an animal (human and non-human animal), and the tissue from which the cell is derived is also limited. Not done. Further, the cells to be fused may be allogeneic cells or heterologous cells.
  • each of the two cells to be fused is treated with a cell fusion promoter. Specifically, cells and a cell fusion promoter are mixed and incubated at a temperature that does not adversely affect the cells (for example, about 4 ° C to 37 ° C) for an appropriate time (for example, about 15 minutes to 1 hour).
  • the Y portion of the formula (1) is introduced into the cell membrane, and the X portion is presented on the cell surface.
  • Confirmation that the cell fusion promoting compound or the like has been introduced into the cell membrane may be, for example, indexed by the localization of the cell fusion promoting compound or the like labeled with a fluorescent label on the cell surface.
  • Mixing ratio of cell to cell fusion promoting agent is not particularly limited, for example, 10 4 with respect to 10 6 about cells, the final concentration of such cell fusion promoter compound 0.05 mg / mL or more, preferably 0.1 mg It may be added and mixed so as to be at least / mL.
  • the cells to be fused Prior to seeding the cells to be fused, they may be washed with a suitable solution (eg, PBS) to remove excess cell fusion promoters.
  • a suitable solution eg, PBS
  • the cell density at the time of seeding the cells can be freely selected as appropriate according to the purpose of use of the cells and the properties of the cells.
  • the mixture of cells to be fused may be treated with PEG and then co-cultured.
  • the cell mixture is collected by centrifugation or the like, the PEG solution having an appropriate concentration (for example, about 10 wt% to 50 wt%) and the cell mixture are mixed, and the cells are diffused into the PEG solution for a short time (for example, about 10 wt% to 50 wt%).
  • PEG may be removed by centrifugation or the like, and co-culture may be performed.
  • fuP1 EIAALEKEIAALEKEIAALEKGGC (SEQ ID NO: 1)
  • fuP2 KIAALKEKIAALKEKIAALKEGGGC (SEQ ID NO: 2)
  • FITC-fuP1 FITC-EIAALEKEIAALEKEIAALEKGGGC Diethyl ether, dichloromethane, dimethyl sulfoxide (DMSO) and triethylamine were purchased from Kanto Chemical Co., Inc.
  • FIG. 1 shows the structure of a peptide-bonded PEG lipid (upper figure), a schematic view of a cell surface modified by a peptide-bonded PEG lipid (lower left figure), and a representative micrograph of cells adhered via the peptide-bonded lipid. (Lower right figure) is shown.
  • the fluorescence intensity of the solution of the hemolytic cells was measured with a fluorometer, and the number of molecules of FITC-fuP1-PEG-DPPE was calculated from the calibration curve obtained by the measurement of FITC-fuP1.
  • the stability of peptide-bonded PEG lipids modified on the cell surface was evaluated.
  • CCRF-CEM cells were modified with 1 mg / mL FITC-fuP1-PEG-DPPE solution, suspended in medium and then incubated at 37 ° C. Cells were analyzed with a flow cytometer (Fig. 2B) 0 hours, 3 hours, and 24 hours after the start of incubation, and observed with a confocal microscope (Fig. 2A).
  • the present invention provides a technique for efficiently fusing cells with each other. Therefore, the present invention is expected to be used in fields related to cell biology and the like and in the medical field.

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Abstract

The present invention addresses the problem of providing an efficient cell fusion method, and a cell fusion-accelerating agent used in said method. More specifically, the present invention is a compound represented by general formula (1) or a salt thereof, or a solvate thereof or a hydrate thereof. (1): X-Z-Y [in formula (1), X represents an atomic group including a peptide that forms a dimer together with another peptide, Y represents a hydrocarbon group optionally containing oxygen, sulfur, phosphorus, or nitrogen, and Z represents a polymer electrolyte or a water-soluble polymer]. Furthermore, the present invention is a cell fusion-accelerating agent containing a compound represented by general formula (1) or a salt thereof, or a solvate thereof or a hydrate thereof.

Description

細胞融合促進剤Cell fusion promoter
　本発明は、細胞融合を促進する剤、および当該細胞融合促進剤を用いた細胞融合法に関する。 The present invention relates to an agent that promotes cell fusion and a cell fusion method using the cell fusion promoter.
　細胞融合は、2つ以上の細胞が融合する現象のことであり、同種細胞間のみならず、異種細胞間においても生じる現象である。特に、異種細胞同士の細胞融合は、融合前の細胞の形質とは異なる新たな形質を持つ細胞を生み出す方法として、以前から利用されてきた。
　例えば、モノクローナル抗体を調製するために、抗体産生B細胞とミエローマ細胞を融合させたハイブリドーマは、複製可能であり、かつ、所望の抗体を産生する細胞として、古くから使用されてきた。また、近年では、糖尿病の新たな治療方法として、間葉系細胞と膵島細胞の融合細胞を移植する方法が提唱されている（非特許文献１）。間葉系細胞と膵島細胞の融合細胞は、20日程度の培養後においても膵島機能が維持されており、これをラットに移植すると、3ヶ月程度にわたり血糖低下が認められた。 Cell fusion is a phenomenon in which two or more cells are fused, and is a phenomenon that occurs not only between allogeneic cells but also between heterologous cells. In particular, cell fusion between heterologous cells has long been used as a method for producing cells having new traits different from those of cells before fusion.
For example, hybridomas in which antibody-producing B cells and myeloma cells are fused to prepare monoclonal antibodies have long been used as cells that are replicable and produce the desired antibody. Further, in recent years, as a new therapeutic method for diabetes, a method of transplanting fusion cells of mesenchymal cells and pancreatic islet cells has been proposed (Non-Patent Document 1). The fusion cells of mesenchymal cells and islet cells maintained the islet function even after culturing for about 20 days, and when this was transplanted into rats, a decrease in blood glucose was observed for about 3 months.
　以上のように、細胞融合は、研究分野および医療分野において広く利用されている。細胞同士を融合する方法としては、主として、センダイウイルス法、PEG（ポリエチレングリコール）法および電気的融合法が知られている。
　センダイウイルス法は、センダイウイルスが細胞膜同士の橋渡しとなり融合を促進すると考えられている。この方法は、融合細胞の画分へのウイルスの混入を完全に回避することが難しく、融合した細胞の医療への使用にはあまり適していない。
　PEG法は、専用機器を必要とせず、試薬も安価であり、従来から主に用いられてきた方法である。しかしながら、この方法は、細胞融合効率および再現性が低く、また細胞の種類によっても細胞融合効率が大きく異なる。
　電気的融合法は、交流電圧を印加することで、電極間に存在する細胞を接触させ、次いで直流パルス電圧を印加することで、細胞膜に穿孔が開け、接触している細胞同士の融合を引き起こす方法である。この方法は、前記2つの方法よりも効率良く細胞を融合することができる。しかし、この方法は、特別な装置が必要であること、電圧の印加により細胞の生存率が低下するなどの問題点を有している。また、細胞の選択性に乏しく、異種同士の細胞を融合させることは困難である。 As described above, cell fusion is widely used in the research field and the medical field. As a method for fusing cells with each other, a Sendai virus method, a PEG (polyethylene glycol) method and an electrical fusion method are mainly known.
In the Sendai virus method, it is considered that Sendai virus acts as a bridge between cell membranes and promotes fusion. This method makes it difficult to completely avoid contamination of the fused cell fraction with the virus and is not well suited for medical use of the fused cells.
The PEG method does not require a dedicated device, the reagent is inexpensive, and has been mainly used in the past. However, this method has low cell fusion efficiency and reproducibility, and the cell fusion efficiency varies greatly depending on the cell type.
In the electrical fusion method, an AC voltage is applied to bring cells that exist between electrodes into contact with each other, and then a DC pulse voltage is applied to open a perforation in the cell membrane, causing fusion between the cells in contact with each other. The method. This method can fuse cells more efficiently than the above two methods. However, this method has problems such as the need for a special device and the decrease in cell viability due to the application of voltage. In addition, the selectivity of cells is poor, and it is difficult to fuse cells of different species.
　また、近年、一本鎖ポリDNAをPEG-脂質複合体に結合させ、これを接着させたい細胞に導入し、一本鎖DNAの相補配列を利用して細胞同士を接着させる方法（非特許文献２および非特許文献３）が報告されている。この方法は、細胞同士を接着させる上では有効な方法であるが、この方法では、当該分子が接着箇所以外には存在しないため（非特許文献３のFig.2など参照のこと）、1個の細胞に対し複数（3以上）の細胞を融合させることは難しいと考えられる。
　以上のような状況において、従来法の問題点を克服した効率のよい細胞融合法の確立が望まれている。 Further, in recent years, a method of binding a single-stranded polyDNA to a PEG-lipid complex, introducing it into a cell to be adhered to, and adhering the cells to each other using a complementary sequence of the single-stranded DNA (non-patent document). 2 and Non-Patent Document 3) have been reported. This method is an effective method for adhering cells to each other, but in this method, since the molecule does not exist other than the adhering site (see Fig. 2 of Non-Patent Document 3), one cell is used. It is considered difficult to fuse multiple cells (3 or more) with the cells of.
Under the above circumstances, it is desired to establish an efficient cell fusion method that overcomes the problems of the conventional method.
　上記事情に鑑み、本発明は、細胞移植などの医療分野においても使用可能であり、従来法よりも効率良く細胞融合が可能になる方法、およびそのような方法に使用される細胞融合促進剤の開発を課題とする。 In view of the above circumstances, the present invention can also be used in the medical field such as cell transplantation, and a method capable of cell fusion more efficiently than the conventional method, and a cell fusion promoter used in such a method Development is an issue.
　発明者らは、２量体を形成するペプチド、ポリエチレングリコール（PEG）および脂質からなる化合物（以下、「細胞融合促進化合物」とする）を細胞と混合したところ、従来のPEG法よりも高効率で細胞融合を誘導することができることを見いだし、本発明を完成させた。
　本発明に係る細胞融合促進化合物は、細胞膜と相互作用する脂質部分と２量体を形成するペプチドペアの1つを保持するペプチド部分および脂質部分とペプチド部分をつなげる部分（例えば、PEG）からなる。例えば、互いに結合するペプチドをA1およびA2とした場合（A1とA2は結合する）、A1を有する細胞融合促進化合物で細胞1を処理し、A2を有する細胞融合促進化合物で細胞2を処理すると、細胞1の細胞表面上にA1を有する細胞融合促進化合物が、細胞2の細胞表面上にA2を有する細胞融合促進化合物が、各々、導入される。その後、細胞1と細胞2を混合するとA1とA2の相互作用により、細胞1と細胞2が接近し、互いの融合が促進される。しかも、一本鎖DNAを使用する従来の分子（非特許文献２および非特許文献３）とは異なり、本発明にかかる細胞融合促進化合物は、細胞同士の接着箇所に集積することなく、1個の細胞に対し複数の細胞の融合も誘導することができる（例えば、図１の右下図）。 When the inventors mixed a compound consisting of a dimer-forming peptide, polyethylene glycol (PEG) and a lipid (hereinafter referred to as "cell fusion promoting compound") with cells, the efficiency was higher than that of the conventional PEG method. The present invention was completed by finding that cell fusion can be induced in the cell.
The cell fusion-promoting compound according to the present invention comprises a lipid moiety that interacts with the cell membrane, a peptide moiety that holds one of the peptide pairs that form a dimer, and a moiety that connects the lipid moiety and the peptide moiety (eg, PEG). .. For example, if the peptides that bind to each other are A 1 and A 2 (A 1 and A 2 bind), treat cell 1 with a cell fusion-promoting compound having A 1 and use a cell fusion-promoting compound with A 2. When cell 2 is treated, a cell fusion-promoting compound having A 1 on the cell surface of cell 1 and a cell fusion-promoting compound having A 2 on the cell surface of cell 2 are introduced. After that, when cell 1 and cell 2 are mixed, the interaction between A 1 and A 2 causes cell 1 and cell 2 to approach each other and promote mutual fusion. Moreover, unlike conventional molecules that use single-stranded DNA (Non-Patent Documents 2 and 3), the cell fusion promoting compound according to the present invention is one without accumulating at the adhesion site between cells. Fusion of a plurality of cells can also be induced with respect to the cells of the above (for example, the lower right figure of FIG. 1).
　すなわち、本発明の以下の（１）～（１３）である
（１）下記式（１）で表される化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
Figure JPOXMLDOC01-appb-C000007
 ［式（１）中、Ｘは他のペプチドと２量体を形成するペプチドを含む原子団、Ｙは酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基、Ｚは高分子電解質または水溶性高分子］
（２）前記ペプチドが、以下の式（２）で表される７残基のアミノ酸の繰り返しを含むペプチドであることを特徴とする上記（１）に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
Figure JPOXMLDOC01-appb-C000008
 ［式（２）中、ａおよびｄは疎水性アミノ酸であり、ｅ、ｆおよびｇは荷電アミノ酸であり、ｂはセリン、アスパラギン、グルタミンおよびトレオニンのいずれかであり、ｃは、アラニンである］
（３）前記Ｙが酸素、硫黄、リンまたは窒素を含んでいてもよい炭素数１０～５０の鎖状炭化水素基であることを特徴とする上記（１）または（２）のいずれかに記載の化合物もしくはその塩、またはそれらの溶媒和物もしくは水和物。
（４）前記Ｙが酸素、硫黄、リンまたは窒素を含んでいてもよい脂質からなる炭化水素基であることを特徴とする上記（３）に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくは水和物。
（５）前記脂質が、リン脂質であることを特徴とする上記（４）に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
（６）前記リン脂質が下記の式（３）で表されるものであることを特徴とする上記（５）に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
Figure JPOXMLDOC01-appb-C000009
 ［式（３）中、Ｗは、二重結合を含んでもよい炭素数１０～２０の炭化水素鎖］
（７）前記Ｚが酸素、硫黄または窒素を含んでいてもよい炭素数１５０～４０００の水溶性の炭化水素鎖であることを特徴とする上記（１）ないし（６）のいずれかに記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
（８）前記Ｚが下記の式（４）であることを特徴とする上記（７）に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
Figure JPOXMLDOC01-appb-C000010
 ［式（４）中、ｎは５０以上１２５０以下の整数］
（９）前記Ｙが下記式（３）を含む置換基であり、前記Ｚが下記式（４）で表される酸素を含む炭化水素鎖であることを特徴とする上記（１）ないし（８）のいずれかに記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
Figure JPOXMLDOC01-appb-C000011
 ［式（３）中、Ｗは、二重結合を含んでもよい炭素数１０～２０の炭化水素鎖］
Figure JPOXMLDOC01-appb-C000012
 ［式（４）中、ｎは５０以上１２５０以下の整数］
（１０）上記（１）ないし（９）のいずれかに記載される化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物を含む、細胞融合促進剤。
（１１）上記（１０）に記載される少なくとも２種類の細胞融合促進剤を含む細胞融合促進用キット。
（１２）以下の（ａ）および（ｂ）を含む、細胞Ａと細胞Ｂを融合させる方法。
（ａ）上記（１０）に記載の細胞融合促進剤で細胞Ａを処理する工程、
（ｂ）上記（１０）に記載の細胞融合促進剤であって、工程（ａ）で用いた細胞融合促進剤と結合する細胞融合促進剤で細胞Ｂを処理する工程、および
（ｃ）工程（ａ）および工程（ｂ）の処理後の細胞Ａと細胞Ｂを混合し、培養する工程
（１３）前記工程（Ｃ）において、細胞Ａと細胞Ｂの混合物をPEGで処理することを含む、上記（１２）に記載の方法。 That is, (1) the compound represented by the following formula (1) or a salt thereof, or a solvate thereof or a hydrate thereof, which are the following (1) to (13) of the present invention.
Figure JPOXMLDOC01-appb-C000007
 [In formula (1), X is an atomic group containing a peptide that forms a dimer with another peptide, Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen, and Z is a polymer electrolyte or Water-soluble polymer]
(2) The compound according to (1) above, a salt thereof, or a solvent thereof, wherein the peptide is a peptide containing repeating amino acids of 7 residues represented by the following formula (2). Japanese products or their hydrates.
Figure JPOXMLDOC01-appb-C000008
 [In formula (2), a and d are hydrophobic amino acids, e, f and g are charged amino acids, b is any of serine, asparagine, glutamine and threonine, and c is alanine]
(3) The above-mentioned (1) or (2), wherein the Y is a chain hydrocarbon group having 10 to 50 carbon atoms which may contain oxygen, sulfur, phosphorus or nitrogen. Compounds or salts thereof, or solvates or hydrates thereof.
(4) The compound according to (3) above, a salt thereof, or a solvate thereof, wherein Y is a hydrocarbon group composed of a lipid which may contain oxygen, sulfur, phosphorus or nitrogen. Or hydrate.
(5) The compound or salt thereof according to (4) above, which is characterized in that the lipid is a phospholipid, or a solvate thereof or a hydrate thereof.
(6) The compound or salt thereof according to (5) above, or a solvate thereof or a hydrate thereof, wherein the phospholipid is represented by the following formula (3).
Figure JPOXMLDOC01-appb-C000009
 [In formula (3), W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond]
(7) The above-mentioned (1) to (6), wherein the Z is a water-soluble hydrocarbon chain having 150 to 4000 carbon atoms which may contain oxygen, sulfur or nitrogen. Compounds or salts thereof, or solvates thereof or hydrates thereof.
(8) The compound or salt thereof according to (7) above, or a solvate thereof or a hydrate thereof, wherein Z is of the following formula (4).
Figure JPOXMLDOC01-appb-C000010
 [In equation (4), n is an integer of 50 or more and 1250 or less]
(9) The above (1) to (8), wherein Y is a substituent containing the following formula (3), and Z is a hydrocarbon chain containing oxygen represented by the following formula (4). ), A salt thereof, a solvate thereof, or a hydrate thereof.
Figure JPOXMLDOC01-appb-C000011
 [In formula (3), W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond]
Figure JPOXMLDOC01-appb-C000012
 [In equation (4), n is an integer of 50 or more and 1250 or less]
(10) A cell fusion promoter comprising the compound according to any one of (1) to (9) above or a salt thereof, a solvate thereof or a hydrate thereof.
(11) A cell fusion promoting kit containing at least two types of cell fusion promoting agents according to (10) above.
(12) A method for fusing cell A and cell B, which comprises the following (a) and (b).
(A) A step of treating cell A with the cell fusion promoter according to (10) above.
(B) The step of treating cell B with the cell fusion promoter according to (10) above, which binds to the cell fusion promoter used in step (a), and the step (c) (c). The step (13) of mixing and culturing cells A and B after the treatments of a) and step (b), which comprises treating the mixture of cells A and B with PEG in the step (C). The method according to (12).
　本発明によると、従来法よりも効率のよい細胞融合を誘導することができる。 According to the present invention, cell fusion can be induced more efficiently than the conventional method.
　また、本発明によると、1個の細胞に対して複数の細胞を融合させることができ、かつ、同種細胞同士のみならず異種細胞同士の細胞を融合させることができる。 Further, according to the present invention, a plurality of cells can be fused to one cell, and cells of not only allogeneic cells but also heterologous cells can be fused.
本発明にかかる細胞融合促進化合物の１例（上図）、細胞融合促進化合物による細胞表面の修飾を模式的に示した図（左下図）および本発明にかかる細胞融合促進方法により細胞融合した細胞の１例（右下図）を示す。An example of the cell fusion promoting compound according to the present invention (upper figure), a diagram schematically showing the modification of the cell surface by the cell fusion promoting compound (lower left figure), and a cell fused by the cell fusion promoting method according to the present invention. (Lower right figure) is shown. FITC-fuP1-PEG-lipidで細胞表面修飾した細胞の経時的な蛍光顕微鏡写真（Ａ）およびFITC-fuP1-PEG-lipidの細胞表面上における存在量をフローサイトメトリーにより測定した結果（Ｂ）を示す。Control（コントロール）はFITC-fuP1-PEG-lipidで細胞表面修飾を行わなかった場合である。値は3回の実験の平均である。Fluorescence micrographs (A) of cells surface-modified with FITC-fuP1-PEG-lipid over time and results (B) of measuring the abundance of FITC-fuP1-PEG-lipid on the cell surface by flow cytometry are shown. Shown. Control is when FITC-fuP1-PEG-lipid is not used to modify the cell surface. The value is the average of 3 experiments. fuP1-PEG-lipidに対するfuP2の相互作用を評価した結果を示す。水晶振動子マイクロバランス装置（QCM-D）にfuP1-PEG-lipid、BSA、fuP2 peptideの順に流した結果（Ａ）とBSA、fuP2 peptideの順に流した結果（Ｂ）である。The results of evaluating the interaction of fuP2 with fuP1-PEG-lipid are shown. The results (A) of flowing fuP1-PEG-lipid, BSA, and fuP2 peptide in this order through the quartz crystal microbalance device (QCM-D) and the results (B) of flowing BSA and fuP2 peptide in this order. fuP1-PEG-lipidとfuP2-PEG-lipidを用いた細胞接着の様子を観察した結果を示す。The results of observing the state of cell adhesion using fuP1-PEG-lipid and fuP2-PEG-lipid are shown. fuP1-PEG-lipidとfuP2-PEG-lipidを用いた細胞接着頻度（Ａ）および細胞接着面積（Ｂ）を測定した結果を示す。値は3回の実験の平均である。The results of measuring the cell adhesion frequency (A) and the cell adhesion area (B) using fuP1-PEG-lipid and fuP2-PEG-lipid are shown. The value is the average of 3 experiments. ペプチド-PEG-lipidによる細胞表面修飾（以下「細胞修飾法」とも記載する）とPEG処理（PEG法）を併せて行い細胞融合（同種の細胞同士の細胞融合）を誘導した細胞を蛍光顕微鏡で観察した結果を示す。上図は細胞融合直後、下図は細胞融合から24時間後の結果である。Cell surface modification with peptide-PEG-lipid (hereinafter also referred to as "cell modification method") and PEG treatment (PEG method) are performed together to induce cell fusion (cell fusion between cells of the same type) with a fluorescence microscope. The observed results are shown. The upper figure shows the results immediately after cell fusion, and the lower figure shows the results 24 hours after cell fusion. 図６に示す細胞融合から24時間後の細胞について、細胞融合率をフローサイトメトリーで測定した結果を示す。Ａはフローサイトメトリーのヒストグラムを、Ｂは細胞融合率を数値化した結果である。ここで、融合した細胞とみなした数は、ゲートP1とゲートP2に囲まれていない細胞数を示し、融合率は、全体からP1とP2の割合を差し引いたものである。値は3回の実験の平均である。The results of flow cytometry measurement of the cell fusion rate of the cells 24 hours after the cell fusion shown in FIG. 6 are shown. A is the result of quantifying the flow cytometry histogram, and B is the result of quantifying the cell fusion rate. Here, the number regarded as fused cells indicates the number of cells not surrounded by gate P1 and gate P2, and the fusion rate is the total minus the ratio of P1 and P2. The value is the average of 3 experiments. 図６に示す細胞融合から24時間後の細胞の生存率をトリパンブルー染色で測定した結果を示す。Control（コントロール）はペプチド-PEG-lipidで細胞表面修飾を行わなかった場合である。値は3回の実験の平均である。The results of measuring the cell viability 24 hours after the cell fusion shown in FIG. 6 by trypan blue staining are shown. Control is when the cell surface is not modified with peptide-PEG-lipid. The value is the average of 3 experiments. ペプチド-PEG-lipidによる細胞表面修飾とPEG処理（PEG法）を併せて行い細胞融合（異種の細胞同士の細胞融合）を誘導した細胞を共焦点レーザー顕微鏡で観察した結果を示す。上図は細胞融合直後、下図は細胞融合から24時間後の結果である。The results of observing cells that have been induced to undergo cell fusion (cell fusion between different types of cells) by combining cell surface modification with peptide-PEG-lipid and PEG treatment (PEG method) with a confocal laser scanning microscope are shown. The upper figure shows the results immediately after cell fusion, and the lower figure shows the results 24 hours after cell fusion. 図９に示す細胞融合から24時間後の細胞について、細胞融合率をフローサイトメトリーで測定した結果を示す。値は3回の実験の平均である。The results of flow cytometry measurement of the cell fusion rate of the cells 24 hours after the cell fusion shown in FIG. 9 are shown. The value is the average of 3 experiments.
　本発明の第１の実施形態は、下記式（１）で表される化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物である（以下「本発明の細胞融合促進化合物等」とも記載する）。
Figure JPOXMLDOC01-appb-C000013
 ［式（１）中、Ｘは他のペプチドと２量体を形成するペプチドを含む原子団、Ｙは酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基、Ｚは高分子電解質または水溶性高分子］
　本明細書中、細胞の「融合」とは、単に、細胞表面（細胞膜）同士の接着に止まらず、細胞膜同士の融合ならびに細胞質の混合が生じている状態を意味する。
　一般式（１）において、Ｘに含まれる「他のペプチド２量体を形成するペプチド」とは、例えば、ペプチドＡとペプチドＢが結合して２量体を形成する場合、ペプチドＡまたはペプチドＢのことである。
　ここで、「他のペプチドと２量体を形成するペプチド」（以下「２量体形成ペプチド」とも記載する）について、上記ペプチドＡとペプチドＢを例にして説明を行う。また、その長さも使用し易い長さであれば特に限定されず、例えば、アミノ酸が5～100残基、好ましくは10～50残基、より好ましくは20～50残基である。
　２量体を形成するペプチドとしては、例えば、以下の式（２）ペプチドを挙げることができる。なお、「ａ」がN末端側である。
Figure JPOXMLDOC01-appb-C000014
 　式（２）中、ａおよびｄは、同一または相異なる疎水性アミノ酸で、例えば、アラニン、バリン、グリシン、イソロイシン、ロイシン、フェニルアラニン、プロリン、トリプトファンおよびチロシンからなるグループから選択されるアミノ酸である。ｅ、ｆおよびｇは、同一または相異なる荷電アミノ酸で、例えば、アルギニン、アスパラギン酸、グルタミン酸、リジンおよびヒスチジンからなるグループから選択されるアミノ酸である。ｅおよびｆは異なる荷電であることが望ましい。また、ｂは特に限定はされないが、極性の大きい非荷電のアミノ酸で、例えば、セリン、アスパラギン、グルタミンおよびトレオニンからなるグループから選択されるアミノ酸などが好ましい。さらに、ｃは、特に限定されないが、アラニンなどを例として挙げることができる。
　上記式（２）のペプチドとして、例えば、実施例に記載される配列番号１および配列番号２で表されるアミノ酸配列を含むペプチドを挙げることができる。さらに、上記式（２）のペプチドの典型的な例として、限定はしないが、２重コイル（coiled-coil）構造を有するペプチドを挙げることができる。２重コイル構造を有するペプチドとして、例えば、Hodgesら, J. Peptide Res., 58:477-492 2001のTable1などに記載されているペプチドなど、当該技術分野において多数のペプチドが知られており、これらに限定されるものではないことは言うまでもない。 The first embodiment of the present invention is a compound represented by the following formula (1) or a salt thereof, a solvate thereof or a hydrate thereof (hereinafter, "cell fusion promoting compound of the present invention, etc."" Also described).
Figure JPOXMLDOC01-appb-C000013
 [In formula (1), X is an atomic group containing a peptide that forms a dimer with another peptide, Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen, and Z is a polymer electrolyte or Water-soluble polymer]
In the present specification, the "fusion" of cells means not only the adhesion between cell surfaces (cell membranes) but also the fusion between cell membranes and the mixing of cytoplasm.
In the general formula (1), the "peptide forming another peptide dimer" contained in X is, for example, peptide A or peptide B when peptide A and peptide B are combined to form a dimer. That is.
Here, "a peptide that forms a dimer with another peptide" (hereinafter, also referred to as "dimer-forming peptide") will be described by taking the above peptides A and B as an example. The length thereof is not particularly limited as long as it is easy to use, and for example, the amino acid is 5 to 100 residues, preferably 10 to 50 residues, and more preferably 20 to 50 residues.
Examples of the peptide forming a dimer include the peptide of the following formula (2). In addition, "a" is the N-terminal side.
Figure JPOXMLDOC01-appb-C000014
 In formula (2), a and d are the same or different hydrophobic amino acids, for example, amino acids selected from the group consisting of alanine, valine, glycine, isoleucine, leucine, phenylalanine, proline, tryptophan and tyrosine. e, f and g are the same or different charged amino acids, eg, amino acids selected from the group consisting of arginine, aspartic acid, glutamic acid, lysine and histidine. It is desirable that e and f have different charges. Further, b is not particularly limited, but is a highly polar uncharged amino acid, and for example, an amino acid selected from the group consisting of serine, asparagine, glutamine and threonine is preferable. Further, c is not particularly limited, but alanine and the like can be mentioned as an example.
Examples of the peptide of the above formula (2) include peptides containing the amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 described in Examples. Furthermore, as a typical example of the peptide of the above formula (2), a peptide having a double coil (coiled-coil) structure can be mentioned without limitation. As peptides having a double coil structure, many peptides are known in the art, such as the peptides described in Table 1 of Hodges et al., J. Peptide Res., 58: 477-492 2001. Needless to say, it is not limited to these.
　式（１）のＸには、２量体形成ペプチドの他、Ｚと２量体形成ペプチドを結合するためのリンカー（またはその一部）が含まれていてもよい。例えば、マレイミド基をリンカーとして２量体形成ペプチドとＺを結合させた場合には、マレイミド基を含むリンカーまたはその一部がＸに含まれていてもよく、その他、チオール基、アミノ基、カルボキシル基、ヒドロキシル基、アジド基などを含むリンカーまたはその一部がＸに含まれていてもよい。 In addition to the dimer-forming peptide, X in the formula (1) may contain a linker (or a part thereof) for binding Z and the dimer-forming peptide. For example, when Z is bonded to a dimer-forming peptide using a maleimide group as a linker, a linker containing a maleimide group or a part thereof may be contained in X, and in addition, a thiol group, an amino group, and a carboxyl group may be contained. The X may contain a linker containing a group, a hydroxyl group, an azide group, or a part thereof.
　式（１）のＹは「酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基」である。Ｙの「炭化水素」とは、鎖状炭化水素（直鎖型または分岐型のいずれであってもよい）、環状炭化水素、芳香族炭化水素など、特にその構造は限定されず、また、飽和炭化水素であっても不飽和炭化水素であってもよく、特に、疎水性または親油性が高い炭化水素が好ましい。
　本実施形態におけるＹの「酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基」として、限定はしないが、例えば、炭素数10～50の炭化水素であって、場合によっては酸素、硫黄、リンまたは窒素が含まれる置換基、特に、炭素数10～50の炭化水素であって、場合によっては酸素、硫黄、リンまたは窒素を含む鎖状炭化水素からなる置換基などを挙げることができる。
　より具体的には、本実施形態におけるＹの「酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基」として、脂質からなる（脂質を含んでなる）置換基を挙げることができる。さらに、前記脂質としては、例えば、アシルグリセロール、セラミドなどの単純脂質、リン脂質、糖脂質、リポタンパク質、スルホ脂質などの複合脂質、脂肪酸、テルペノイド、ステロイド（ステロール、コレステロールなど）、カロテノイドなどを挙げることができる。中でも、好ましくは、リン脂質、セラミド、ステロイドなどで、特に好ましくはリン脂質である。 Y in the formula (1) is a "hydrocarbon group which may contain oxygen, sulfur, phosphorus or nitrogen". The "hydrocarbon" of Y is a chain hydrocarbon (which may be linear or branched), a cyclic hydrocarbon, an aromatic hydrocarbon, and the like, and the structure thereof is not particularly limited and is saturated. It may be a hydrocarbon or an unsaturated hydrocarbon, and a hydrocarbon having high hydrophobicity or lipophilicity is particularly preferable.
The "hydrocarbon group which may contain oxygen, sulfur, phosphorus or nitrogen" of Y in the present embodiment is not limited, but is, for example, a hydrocarbon having 10 to 50 carbon atoms, and in some cases, oxygen. Substituents containing sulfur, phosphorus or nitrogen, in particular, hydrocarbons having 10 to 50 carbon atoms and in some cases consisting of chain hydrocarbons containing oxygen, sulfur, phosphorus or nitrogen, etc. may be mentioned. it can.
More specifically, as the "hydrocarbon group which may contain oxygen, sulfur, phosphorus or nitrogen" of Y in this embodiment, a substituent composed of a lipid (containing a lipid) can be mentioned. Further, examples of the lipid include simple lipids such as acylglycerol and ceramide, complex lipids such as phospholipids, glycolipids, lipoproteins and sulfolipids, fatty acids, terpenoids, steroids (sterols, cholesterol and the like), carotenoids and the like. be able to. Among them, phospholipids, ceramides, steroids and the like are preferable, and phospholipids are particularly preferable.
　上記リン脂質のより具体的な例として、式（３）のリン脂質を挙げることができる。
Figure JPOXMLDOC01-appb-C000015
 ［式（３）中、Ｗは、二重結合を含んでもよい炭素数10～20の炭化水素鎖］
 　より具体的には、特に限定はしないが、例えば、
１，２－ジパルミトイル－ｓｎ－グリセロ－３－ホスホエタノールアミン
（1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine(DPPE)）、
１，２－ジステアロイル－ｓｎ－グリセロ－３－ホスホエタノールアミン
（1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine(DSPE)）、
１，２－ジミリストイル－ｓｎ－グリセロ－３－ホスホエタノールアミン
（1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine(DMPE)）、
１，２－ジオレオイル－ｓｎ－グリセロ－３－ホスホエタノールアミン
（1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine(DOPE)）などを挙げることができる。 As a more specific example of the above phospholipid, the phospholipid of the formula (3) can be mentioned.
Figure JPOXMLDOC01-appb-C000015
 [In formula (3), W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond]
More specifically, although not particularly limited, for example,
1,2-Dipalmitoyl- sn -Glycero-3-Phosphoethanolamine (DPPE),
1,2-Distearoyl- sn -Glycero-3-Phosphoethanolamine (DSPE), 1,2-Distearoyl- sn -Glycero-3-Phosphoethanolamine (DSPE),
1,2-Dimyristoyl- sn -Glycero-3-Phosphoethanolamine (DMPE), 1,2-Dimyristoyl- sn -Glycero-3-Phosphoethanolamine (DMPE),
1,2-Dioleoyl- sn -Glycero-3-Phosphoethanolamine (DOPE) and the like can be mentioned.
　式（１）のＹには、「酸素、硫黄、リンまたは窒素を含んでもよい炭化水素」以外に、ＹとＺを結合するためのリンカー（またはその一部）が含まれていてもよい。例えば、マレイミド基、チオール基、アミノ基、カルボキシル基、ヒドロキシル基、アジド基などを含むリンカーまたはその一部がＹに含まれていてもよい。 Y in formula (1) may contain a linker (or a part thereof) for binding Y and Z in addition to "hydrocarbons which may contain oxygen, sulfur, phosphorus or nitrogen". For example, Y may contain a linker containing a maleimide group, a thiol group, an amino group, a carboxyl group, a hydroxyl group, an azide group, or a part thereof.
　式（１）中、Ｚは高分子電解質または水溶性高分子である。より具体的には、酸素、硫黄または窒素を含んでいてもよい炭素数100～4000、好ましくは炭素数150～3500、より好ましくは炭素数200～2500の直鎖型または分岐型の水溶性の炭化水素鎖である。
　Ｚとして使用可能な「酸素、硫黄または窒素を含んでいてもよい水溶性の炭化水素鎖」としては、特に限定はしないが、糖鎖などの水溶性高分子の他、例えば、下記の式（４）で示されるポリエチレングリコール（polyethylene glycol：PEG）などが好ましい。
Figure JPOXMLDOC01-appb-C000016
 　式（１）のＺがPEGの場合、その分子量は、例えば、好ましくは2kDa～50kDa、より好ましくは4kDa～45kDa程度が好ましく、この場合、式（２）中のｎは整数であって、好ましくは50≦ｎ≦1250、より好ましくは100≦ｎ≦1125程度である。 In formula (1), Z is a polymer electrolyte or a water-soluble polymer. More specifically, it is a linear or branched water-soluble type having 100 to 4000 carbon atoms, preferably 150 to 3500 carbon atoms, and more preferably 200 to 2500 carbon atoms, which may contain oxygen, sulfur or nitrogen. It is a hydrocarbon chain.
The "water-soluble hydrocarbon chain that may contain oxygen, sulfur, or nitrogen" that can be used as Z is not particularly limited, but in addition to a water-soluble polymer such as a sugar chain, for example, the following formula ( Polyethylene glycol (PEG) represented by 4) is preferable.
Figure JPOXMLDOC01-appb-C000016
 When Z in the formula (1) is PEG, the molecular weight thereof is preferably, for example, preferably about 2 kDa to 50 kDa, more preferably about 4 kDa to 45 kDa, and in this case, n in the formula (2) is an integer and is preferable. Is about 50 ≦ n ≦ 1250, more preferably about 100 ≦ n ≦ 1125.
　式（１）のＺがPEGの場合に、一般式（１）の化合物は、下記の一般式（１’）として表すことができる。
Figure JPOXMLDOC01-appb-C000017
 ［式（１’）中、Ｘは他のペプチドと２量体を形成するペプチドを含む原子団、Ｙは酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基、ｎは50以上1250以下の整数］ When Z of the formula (1) is PEG, the compound of the general formula (1) can be expressed as the following general formula (1').
Figure JPOXMLDOC01-appb-C000017
 [In formula (1'), X is an atomic group containing a peptide that forms a dimer with another peptide, Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen, and n is 50 or more and 1250. The following integers]
　式（１）のＺの炭化水素鎖と、Ｘに含まれる２量体形成ペプチドを結合させる場合、適当なリンカーを介して結合させても、直接結合させてもよい。２量体形成ペプチドとＺは、当該技術分野における周知技術を用いることで、容易に結合させることができる。２量体形成ペプチドとＺの炭化水素鎖を結合させる方法について、ＺがPEGの場合について、以下に例を挙げて説明する。
１．マレイミド基とチオール基の反応を利用する方法
　マレイミド基を導入したPEGとチオール基を有する２量体形成ペプチドを反応させる方法である。この方法によると、マレイミド基がリンカーとして機能する。すなわち、マイケル付加反応によりPEGに導入されたマレイミド基と２量体形成ペプチドのチオール基が反応して、PEGと２量体形成ペプチドが結合する。２量体形成ペプチドへのチオール基の導入は、如何なる方法を用いてもよいが、例えば、システインを２量体形成ペプチドに付加することで、チオール基を２量体形成ペプチドに導入することができる。
　あるいは、架橋剤であるN-(6-Maleimidocaproyloxy)succinimide(EMCS)などを用いて、２量体形成ペプチドのアミノ基（Ｎ末端のアミノ基または、２量体形成ペプチドに付加したリシン残基のアミノ基など）にマレイミド基を導入する。このマレイミドを導入した２量体形成ペプチドとチオール基を導入したPEGを反応させることで、PEGと２量体形成ペプチドを結合させることができる。
　マレイミド基を導入したPEG、チオール基を導入したPEGは、当該技術分野における周知技術を用いて調製可能であり、また、市販のもの（日油株式会社など）を使用してもよい。 When the hydrocarbon chain of Z of the formula (1) is bound to the dimer-forming peptide contained in X, it may be bound via an appropriate linker or directly. The dimer-forming peptide and Z can be easily linked by using a well-known technique in the art. The method of binding the dimer-forming peptide and the hydrocarbon chain of Z will be described below with an example of the case where Z is PEG.
1. 1. A method using the reaction of a maleimide group and a thiol group This is a method of reacting a maleimide group-introduced PEG with a dimer-forming peptide having a thiol group. According to this method, the maleimide group functions as a linker. That is, the maleimide group introduced into PEG by the Michael addition reaction reacts with the thiol group of the dimer-forming peptide, and PEG and the dimer-forming peptide bind to each other. Any method may be used to introduce the thiol group into the dimer-forming peptide. For example, the thiol group can be introduced into the dimer-forming peptide by adding cysteine to the dimer-forming peptide. it can.
Alternatively, using a cross-linking agent such as N- (6-Maleimidocaproyloxy) succinimide (EMCS), the amino group of the dimer-forming peptide (the N-terminal amino group or the lysine residue added to the dimer-forming peptide) Introduce a maleimide group into the amino group, etc.). By reacting the maleimide-introduced dimer-forming peptide with the thiol-introduced PEG, the PEG and the dimer-forming peptide can be bound to each other.
The maleimide group-introduced PEG and the thiol group-introduced PEG can be prepared by using well-known techniques in the art, and commercially available products (NOF Corporation, etc.) may be used.
２．PEGのCOOH基を利用する方法
　COOHを有するPEGと、２量体形成ペプチドのアミノ基またはOH基とのカップリング反応を利用して、PEGと２量体形成ペプチドを結合させることができる（アミド結合またはエステル結合を形成）。例えば、N,N’-ジクロロヘキシルカルボジイミド(DCC)などのカップリング試薬を用いて、PEGのCOOH基を活性化し、２量体形成ペプチドのアミノ基（Lysのアミノ基やN末端アミノ基など）またはOH基（SerやThrのOH基など）と結合させることができる。 2. 2. Method of Utilizing COOH Group of PEG The coupling reaction between PEG having COOH and the amino group or OH group of the dimer-forming peptide can be used to bind PEG to the dimer-forming peptide (amide). Form a bond or ester bond). For example, a coupling reagent such as N, N'-dichlorohexyl carbodiimide (DCC) is used to activate the COOH group of PEG and the amino group of the dimer-forming peptide (such as the amino group of Lys or the N-terminal amino group). Alternatively, it can be bonded to an OH group (such as an OH group of Ser or Thr).
３．PEGのNH2基を利用する方法
　PEGのNH2基と２量体形成ペプチドのCOOH基とのカップリング反応を利用して、PEGと２量体形成ペプチドを結合させることができる（アミド結合を形成）。例えば、N,N’-ジクロロヘキシルカルボジイミド(DCC)などのカップリング試薬を用いて、２量体形成ペプチドのCOOH基を活性化し、PEGのアミノ基と結合させることができる。 3. 3. Method using NH 2 groups of PEG The coupling reaction between NH 2 groups of PEG and COOH group of dimer-forming peptide can be used to bind PEG and dimer-forming peptide (amide bond). Formation). For example, a coupling reagent such as N, N'-dichlorohexylcarbodiimide (DCC) can be used to activate the COOH group of the dimer-forming peptide and bind it to the transamination of PEG.
４．PEGのOH基を利用する方法
　PEGのOH基と２量体形成ペプチドのCOOH基とのカップリング反応を利用して、PEGと２量体形成ペプチドを結合させることができる（エステル結合を形成）。例えば、N,N’-ジクロロヘキシルカルボジイミド(DCC)などのカップリング試薬を用いて、２量体形成ペプチドのCOOH基を活性化し、PEGのOH基と結合させることができる。 4. Method of utilizing OH group of PEG The PEG and the dimer-forming peptide can be bound to each other by utilizing the coupling reaction between the OH group of PEG and the COOH group of the dimer-forming peptide (forming an ester bond). .. For example, a coupling reagent such as N, N'-dichlorohexyl carbodiimide (DCC) can be used to activate the COOH group of the dimer-forming peptide and bind it to the OH group of PEG.
５．PEGの-N3とプロパルギル基との反応を利用する方法
　クリック反応により、PEGのアジド基（N3）とプロパルギル基を銅触媒存在下で結合させる。プロパルギル基を有するアミノ酸を含有する２量体形成ペプチドを調製し、アジド基を末端に有するPEGと反応させ、PEGと２量体形成ペプチドを結合させることができる。 5. Method of utilizing the reaction between -N 3 of PEG and propargyl group By click reaction, the azide group (N 3 ) of PEG and propargyl group are bonded in the presence of a copper catalyst. A dimer-forming peptide containing an amino acid having a propargyl group can be prepared and reacted with a PEG having an azide group at the end to bind the PEG to the dimer-forming peptide.
　式（１）のＺとＹを結合させる場合、適当なリンカーを介して結合させても、直接結合させてもよい。
　ＹがCOOH基を有しＺがアミノ基もしくはヒドロキシル基を有する場合、または、Ｙがアミノ基もしくはヒドロキシル基を有しＺがCOOH基を有する場合には、COOHを利用したカップリング反応によりＹとＺを結合させることができる。例えば、Ｙがカルボン酸を有するアルキル鎖からなる場合、ＹのCOOH基をN,N’-ジクロロヘキシルカルボジイミド(DCC)などのカップリング試薬で活性化した後、Ｚのアミノ基またはヒドロキシル基と結合させることができる。
　あるいは、Ｙが活性エステル基であるN-ヒドロキシルスクシンイミド基（NHS）を有しＺがアミノ基もしくはヒドロキシル基を有する場合、または、Ｙがアミノ基もしくはヒドロキシル基を有しＺが活性エステル基であるN-ヒドロキシルスクシンイミド基（NHS）を有する場合、NHSを利用して、アミノ基もしくはヒドロキシル基との反応を利用して、ＹとＺを結合させることができる。例えば、実施例で示すように、Ｙが、アミノ基を有するDPPEやDSPEなどの場合には、活性エステル基であるN-ヒドロキシルスクシンイミド基（NHS）を持つＺと結合させることができる。
　なお、ＹまたはＺに対する、アミノ基、ヒドロキシル基、カルボキシル基またはN-ヒドロキシルスクシンイミド基の導入は、当該技術分野における周知の方法によって容易に実施することができる。
　また、式（１）のＺには、「高分子電解質または水溶性高分子」以外に、ＺとＸ、および／または、ＺとＹを結合するためのリンカー（またはその一部）が含まれていてもよい。例えば、マレイミド基、チオール基、アミノ基、カルボキシル基、ヒドロキシル基、アジド基などを含むリンカーまたはその一部がＹに含まれていてもよい。 When Z and Y of the formula (1) are bonded, they may be bonded via an appropriate linker or may be directly bonded.
When Y has a COOH group and Z has an amino group or a hydroxyl group, or when Y has an amino group or a hydroxyl group and Z has a COOH group, a coupling reaction using COOH is carried out to obtain Y. Z can be combined. For example, if Y consists of an alkyl chain with a carboxylic acid, the COOH group of Y is activated with a coupling reagent such as N, N'-dichlorohexylcarbodiimide (DCC) and then bonded to the amino or hydroxyl group of Z. Can be made to.
Alternatively, when Y has an N-hydroxyl succinimide group (NHS) which is an active ester group and Z has an amino group or a hydroxyl group, or when Y has an amino group or a hydroxyl group and Z is an active ester group. If it has an N-hydroxyl succinimide group (NHS), NHS can be utilized to bond Y and Z by utilizing the reaction with an amino group or a hydroxyl group. For example, as shown in Examples, in the case of DPPE or DSPE having an amino group, Y can be bonded to Z having an N-hydroxyl succinimide group (NHS) which is an active ester group.
The introduction of an amino group, a hydroxyl group, a carboxyl group or an N-hydroxyl succinimide group into Y or Z can be easily carried out by a method well known in the art.
Further, Z in the formula (1) includes, in addition to the “polymer electrolyte or water-soluble polymer”, a linker (or a part thereof) for binding Z and X and / or Z and Y. May be. For example, Y may contain a linker containing a maleimide group, a thiol group, an amino group, a carboxyl group, a hydroxyl group, an azide group, or a part thereof.
　本発明の第２の実施形態は、本発明の細胞融合促進化合物等を含む細胞融合促進剤（以下「本発明の細胞融合促進剤）である。本実施形態は、式（１）で示される細胞融合促進化合物が細胞膜同士の融合ならびに細胞同士の融合を促進する効果を有することに基づいており、所望の細胞同士の融合を行う際に、融合を促進するための試薬等として使用することができる。本実施形態の細胞融合促進剤は、細胞融合促進化合物等のみを含むものであっても、適当な溶媒に溶解したものであってもよい。
　本発明の細胞融合促進剤を用いて細胞融合を行う場合、互いに結合する２種類の細胞融合促進化合物等を、別々に含む２種類の細胞融合促進剤を、各々、融合させる各細胞に対して使用する。従って、本発明の細胞融合促進剤で細胞融合を促進する場合には、２種類の細胞融合促進剤（互いに結合する細胞融合促進化合物等を含む）を１組にして使用する。 The second embodiment of the present invention is a cell fusion promoter containing the cell fusion promoter compound of the present invention (hereinafter, "cell fusion promoter of the present invention"). The present embodiment is represented by the formula (1). It is based on the fact that the cell fusion promoting compound has an effect of promoting fusion between cell membranes and fusion between cells, and can be used as a reagent or the like for promoting fusion when performing fusion between desired cells. The cell fusion-promoting agent of the present embodiment may contain only the cell fusion-promoting compound or the like, or may be dissolved in an appropriate solvent.
When cell fusion is performed using the cell fusion promoter of the present invention, two types of cell fusion promoters, which separately contain two types of cell fusion promoter compounds that bind to each other, are added to each cell to be fused. use. Therefore, when the cell fusion promoter of the present invention promotes cell fusion, two types of cell fusion promoters (including cell fusion promoter compounds that bind to each other) are used as a set.
　本発明の第３の実施形態は、少なくとも２種類の本発明の細胞融合促進剤を含む細胞融合促進用キットである。
　本発明にかかる細胞融合促進用キットは、互いに結合する細胞融合促進化合物等を、別々に含む２種の細胞融合促進剤を少なくとも含むことを特徴とする。また、細胞融合を実施する上で必要な試薬等（例えば、PBS、PEGおよび培地など）が含まれていてもよい。
　本発明にかかる細胞融合促進用キットには、使用説明書が含まれていてもよく、または、当該使用方法を掲載したウェッブサイトなどの情報が記載された情報書面等が含まれていてもよい。使用説明書は、CDやDVDなどの記録媒体に記録されて添付されてもよい。 A third embodiment of the present invention is a cell fusion promoting kit containing at least two kinds of cell fusion promoting agents of the present invention.
The cell fusion promoting kit according to the present invention is characterized by containing at least two types of cell fusion promoting agents that separately contain cell fusion promoting compounds and the like that bind to each other. In addition, reagents and the like (for example, PBS, PEG, medium, etc.) necessary for carrying out cell fusion may be contained.
The cell fusion promoting kit according to the present invention may include an instruction manual, or may include an information document or the like containing information such as a website containing the usage instructions. .. The instruction manual may be recorded and attached to a recording medium such as a CD or DVD.
　本発明の第４の実施形態は、融合させる２細胞を、本発明の細胞融合促進化合剤であって互いに結合する細胞融合促進化合物等（ペプチド同士の結合を介して）を別々に含む２種類の細胞融合促進剤で、各々、処理した後、当該２細胞を混合して共培養し、当該２細胞を融合させる方法である。
　本実施形態は、融合させたい細胞を細胞Ａおよび細胞Ｂとした場合、以下の（ａ）および（ｂ）を含む、細胞融合方法である。
（ａ）本発明の細胞融合促進剤で細胞Ａを処理する工程、
（ｂ）工程（ａ）で用いた細胞融合促進剤に含まれる細胞融合促進化合物等と結合する細胞融合促進化合物等を含む細胞融合促進剤で細胞Ｂを処理する工程、および
（ｃ）工程（ａ）および工程（ｂ）の処理後の細胞Ａと細胞Ｂを混合し、培養する工程 In the fourth embodiment of the present invention, two types of cells to be fused are separately contained, such as a cell fusion promoting compound which is a cell fusion promoting compound of the present invention and binds to each other (via binding between peptides). This is a method in which the two cells are mixed and co-cultured after each treatment with the cell fusion promoter of the above, and the two cells are fused.
The present embodiment is a cell fusion method including the following (a) and (b) when the cells to be fused are cells A and B.
(A) A step of treating cell A with the cell fusion promoter of the present invention,
(B) A step of treating cell B with a cell fusion promoter containing a cell fusion promoter that binds to a cell fusion promoter contained in the cell fusion promoter used in step (a), and a step (c) (c). A step of mixing and culturing cells A and B after the treatments of a) and step (b).
　ここで「細胞」とは、株化された細胞のみならず、動物（ヒトおよび非ヒト動物）の組織から採取した初代培養細胞（プライマリー細胞）であってもよく、細胞が由来する組織も限定されない。また、融合させる細胞は、同種細胞同士であっても、異種細胞同士であってもよい。
　第４の実施形態にかかる細胞融合方法では、まず、融合させる２つの細胞を、各々、細胞融合促進剤で処理を行う。具体的には、細胞と細胞融合促進剤を混合し、当該細胞に悪影響を及ぼさない温度（例えば、4℃～37℃程度）にて、適当な時間（例えば、15分間～1時間程度）インキュベートし、式（１）のＹの部分を細胞膜に導入させ、Ｘの部分を細胞表面上に提示させる。細胞融合促進化合物等が細胞膜へ導入されたことの確認は、例えば、蛍光標識等を施した細胞融合促進化合物等が細胞表面に局在することを指標にしてもよい。
　細胞と細胞融合促進剤との混合比率は、特に限定されないが、例えば、104～106 程度の細胞に対して、細胞融合促進化合物等の終濃度が0.05 mg/mL以上、好ましくは0.1 mg/mL以上となるように加えて混合してもよい。融合させる細胞同士を播種する前に、余分な細胞融合促進剤を除去するために、適当な溶液（例えば、PBSなど）で洗浄してもよい。細胞を播種する際の細胞密度は、適宜、当該細胞の使用目的および当該細胞の性質に応じて、自由に選択することができる。 Here, the "cell" may be not only the established cell but also the primary cultured cell (primary cell) collected from the tissue of an animal (human and non-human animal), and the tissue from which the cell is derived is also limited. Not done. Further, the cells to be fused may be allogeneic cells or heterologous cells.
In the cell fusion method according to the fourth embodiment, first, each of the two cells to be fused is treated with a cell fusion promoter. Specifically, cells and a cell fusion promoter are mixed and incubated at a temperature that does not adversely affect the cells (for example, about 4 ° C to 37 ° C) for an appropriate time (for example, about 15 minutes to 1 hour). Then, the Y portion of the formula (1) is introduced into the cell membrane, and the X portion is presented on the cell surface. Confirmation that the cell fusion promoting compound or the like has been introduced into the cell membrane may be, for example, indexed by the localization of the cell fusion promoting compound or the like labeled with a fluorescent label on the cell surface.
Mixing ratio of cell to cell fusion promoting agent is not particularly limited, for example, 10 4 with respect to 10 6 about cells, the final concentration of such cell fusion promoter compound 0.05 mg / mL or more, preferably 0.1 mg It may be added and mixed so as to be at least / mL. Prior to seeding the cells to be fused, they may be washed with a suitable solution (eg, PBS) to remove excess cell fusion promoters. The cell density at the time of seeding the cells can be freely selected as appropriate according to the purpose of use of the cells and the properties of the cells.
　第４の実施形態において、融合させる細胞の混合物をPEGで処理した後、共培養してもよい。具体的には、細胞の混合物を遠心等で回収し、適当な濃度のPEG溶液（例えば、10wt%～50wt%程度）と細胞混合物を混合し、細胞をPEG溶液中に拡散させ、短時間（例えば、数分間）インキュベートしたのち、遠心等でPEGを除去し、共培養を行ってもよい。 In the fourth embodiment, the mixture of cells to be fused may be treated with PEG and then co-cultured. Specifically, the cell mixture is collected by centrifugation or the like, the PEG solution having an appropriate concentration (for example, about 10 wt% to 50 wt%) and the cell mixture are mixed, and the cells are diffused into the PEG solution for a short time (for example, about 10 wt% to 50 wt%). For example, after incubating for several minutes, PEG may be removed by centrifugation or the like, and co-culture may be performed.
　本明細書が英語に翻訳されて、単数形の「a」、「an」、および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数も含むものとする。
　以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 If this specification is translated into English and contains the singular words "a", "an", and "the", not only the singular, unless the context clearly indicates otherwise. It shall include more than one.
Hereinafter, the present invention will be described with reference to examples, but the present examples are merely examples of embodiments of the present invention, and do not limit the scope of the present invention.
１．材料
　１，２－ジパルミトイル－ｓｎ－グリセロ－３－ホスホエタノールアミン（1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine(DPPE)）、１，２－ジミリストイル－ｓｎ－グリセロ－３－ホスホエタノールアミン（1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine(DMPE)）、およびα－Ｎ－ヒドロキシスクシンイミジル－ω－マレイミジル　ポリ（エチレングリコール）（α-N-hydroxysuccinimidyl-ω-maleimidyl poly(ethylene glycol)）（NHS-PEG-Mal,　Mw: 5000)は、 日油株式会社から購入した。
　以下の合成ペプチドは、GenScript（Piscataway, NJ, USA）にその合成を委託した。
fuP1：EIAALEKEIAALEKEIAALEKGGGC（配列番号１）
fuP2：KIAALKEKIAALKEKIAALKEGGGC（配列番号２）
FITC-fuP1：FITC-EIAALEKEIAALEKEIAALEKGGGC
　ジエチルエーテル、ジクロロメタン、ジメチルスルホキシド（DMSO）およびトリエチルアミンは、関東化学株式会社から購入した。 1. 1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (1,2-Dipalmitoyl- sn -Glycero-3-Phosphoethanolamine (DPPE)), 1,2-dipalmitoyl-sn-glycero-3-phospho Ethanolamine (1,2-Dimyristoyl- sn -Glycero-3-Phosphoethanolamine (DMPE)), and α-N-hydroxysuccinimidyl-ω-maleimidyl poly (ethylene glycol) (α-N-hydroxysuccinimidyl-ω-maleimidyl) poly (ethylene glycol)) (NHS-PEG-Mal, Mw: 5000) was purchased from Nichiyu Co., Ltd.
The following synthetic peptides were outsourced to GenScript (Piscataway, NJ, USA).
fuP1: EIAALEKEIAALEKEIAALEKGGC (SEQ ID NO: 1)
fuP2: KIAALKEKIAALKEKIAALKEGGGC (SEQ ID NO: 2)
FITC-fuP1: FITC-EIAALEKEIAALEKEIAALEKGGGC
Diethyl ether, dichloromethane, dimethyl sulfoxide (DMSO) and triethylamine were purchased from Kanto Chemical Co., Inc.
２．実験方法と実験結果
１－２．ペプチド結合PEG脂質の合成
　Mal-PEG-DPPEを合成し、細胞の表面修飾に使用した。ジクロロメタン中でNHS-PEG-Mal（180 mg）、トリエチルアミン（50 mg）、DPPE（20 mg）を混合し、室温で36時間反応させた。ジエチルエーテルを用いた再沈殿により、Mal-PEG-DPPEが白色粉末として得られた。また、Mal-PEG-DMPEを合成し、SP2/O細胞（メラノーマ）の細胞表面修飾に使用した。ジクロロメタン中でNHS-PEG-Mal（180 mg）、トリエチルアミン（50 mg）、DMPE（19 mg）を混合し、室温で36時間反応させた。ジエチルエーテルを用いた再沈殿により、Mal-PEG-DMPEが白色粉末として得られた。
　3種類のペプチド（fuP1、fuP2およびFITC-fuP1）を、それぞれ、Mal-PEG-DPPEに結合させた。チオール-マレイミド反応を介してペプチドをMal-PEG-DPPEに結合させるため、ペプチドのC末端にシステインを加えた。1 mgのMal-PEG-DPPEをfuP1、fuP2、FITC-fuP1ペプチド（それぞれ0.51 mg、0.51 mg、0.61 mg）と1 mL DMSO中で混合した。室温で24時間インキュベートし、スピンカラムで精製した。これらの材料を細胞の表面修飾に使用した。また、SP2/O細胞（メラノーマ）には、fuP1-PEG-DMPEを使用した。1 mgのMal-PEG-DMPEをfuP1ペプチド（それぞれ0.51 mg）と1 mL DMSO中で混合した。室温で24時間インキュベートし、スピンカラムで精製した。
　図１にペプチド結合PEG脂質の構造（上図）、ペプチド結合PEG脂質によって修飾される細胞表面の模式図（下左図）およびペプチド結合脂質を介して接着した細胞の代表的な顕微鏡写真を示す（右下図）を示す。 2. 2. Experimental method and experimental results 1-2. Synthesis of Peptide Bonded PEG Lipid Mal-PEG-DPPE was synthesized and used for surface modification of cells. NHS-PEG-Mal (180 mg), triethylamine (50 mg) and DPPE (20 mg) were mixed in dichloromethane and reacted at room temperature for 36 hours. Reprecipitation with diethyl ether gave Mal-PEG-DPPE as a white powder. In addition, Mal-PEG-DMPE was synthesized and used for cell surface modification of SP2 / O cells (melanoma). NHS-PEG-Mal (180 mg), triethylamine (50 mg) and DMPE (19 mg) were mixed in dichloromethane and reacted at room temperature for 36 hours. Reprecipitation with diethyl ether gave Mal-PEG-DMPE as a white powder.
Three peptides (fuP1, fuP2 and FITC-fuP1) were each bound to Mal-PEG-DPPE. Cysteine was added to the C-terminus of the peptide to attach the peptide to Mal-PEG-DPPE via a thiol-maleimide reaction. 1 mg of Mal-PEG-DPPE was mixed with fuP1, fuP2 and FITC-fuP1 peptides (0.51 mg, 0.51 mg and 0.61 mg, respectively) in 1 mL DMSO. It was incubated at room temperature for 24 hours and purified on a spin column. These materials were used to modify the surface of cells. In addition, fuP1-PEG-DMPE was used for SP2 / O cells (melanoma). 1 mg of Mal-PEG-DMPE was mixed with fuP1 peptide (0.51 mg each) in 1 mL DMSO. It was incubated at room temperature for 24 hours and purified on a spin column.
FIG. 1 shows the structure of a peptide-bonded PEG lipid (upper figure), a schematic view of a cell surface modified by a peptide-bonded PEG lipid (lower left figure), and a representative micrograph of cells adhered via the peptide-bonded lipid. (Lower right figure) is shown.
１－３．細胞表面上のプチド結合PEG脂質の定量分析
　CCRF-CEM細胞の細胞膜に修飾されたペプチド結合PEG脂質の分子数を評価した。FITC-fuP1-PEG-DPPE溶液（50μL, 1, 0.1, 0.01, 0.001 mg/mL in PBS）をCCRF-CEM細胞のペレット（1.0 × 106 cells）に加え、室温で30分インキュベートした。遠心によってPBSでリンスしたのち、細胞を溶血させるため細胞ペレットに0.2 mLの純水を加えた。溶血した細胞の溶液の蛍光強度を蛍光光度計にて測定し、FITC-fuP1の測定によって得られた検量線からFITC-fuP1-PEG-DPPEの分子数を計算した。
　細胞表面に修飾されたペプチド結合PEG脂質の安定性を評価した。上記で述べた通りに、CCRF-CEM細胞を1 mg/mL FITC-fuP1-PEG-DPPE溶液で修飾し、培地で懸濁したのち、37℃でインキュベートした。インキュベート開始から0時間後、3時間後、24時間後にそれぞれ細胞をフローサイトメーターで解析し（図２Ｂ）、共焦点顕微鏡で観察した（図２Ａ）。
　ペプチド結合PEG脂質で細胞を処理すると、処理した時点で細胞表面上に蛍光が観察され、ペプチド結合脂質で細胞表面が修飾されたことが確認できた（図２ＡおよびＢ、0h）。細胞表面上のペプチド結合脂質は、時間経過に伴って脱離することが確認された（図２ＡおよびＢ、3h、24h） 1-3. Quantitative analysis of petit-bonded PEG lipids on the cell surface The number of molecules of peptide-bonded PEG lipids modified on the cell membrane of CCRF-CEM cells was evaluated. FITC-fuP1-PEG-DPPE solution (50μL, 1, 0.1, 0.01 , 0.001 mg / mL in PBS) was added to the pellet (1.0 × 10 6 cells) of CCRF-CEM cells were incubated for 30 minutes at room temperature. After rinsing with PBS by centrifugation, 0.2 mL of pure water was added to the cell pellet to hemolyze the cells. The fluorescence intensity of the solution of the hemolytic cells was measured with a fluorometer, and the number of molecules of FITC-fuP1-PEG-DPPE was calculated from the calibration curve obtained by the measurement of FITC-fuP1.
The stability of peptide-bonded PEG lipids modified on the cell surface was evaluated. As mentioned above, CCRF-CEM cells were modified with 1 mg / mL FITC-fuP1-PEG-DPPE solution, suspended in medium and then incubated at 37 ° C. Cells were analyzed with a flow cytometer (Fig. 2B) 0 hours, 3 hours, and 24 hours after the start of incubation, and observed with a confocal microscope (Fig. 2A).
When the cells were treated with the peptide-bonded PEG lipid, fluorescence was observed on the cell surface at the time of treatment, confirming that the cell surface was modified with the peptide-bonded lipid (FIGS. 2A and B, 0h). It was confirmed that the peptide-binding lipids on the cell surface were eliminated over time (FIGS. 2A and B, 3h, 24h).
１－４．QCM-D法によるfuP2およびfuP1-PEG-脂質間の相互作用の解析
　fuP1ペプチドとfuP2ペプチドの結合性を調べるため、水晶振動子マイクロバランス装置（QCM-D）による測定を行った。測定には、１－ドデカンチオール（1-dodecanethiol）で修飾しCH3-SAMが作製された金センサーチップを用いた。最初に、PBSでシグナルの安定化を行った。続いて、fuP1-PEG-DPPE溶液 (1 mg/mL in PBS)、BSA溶液 (1 mg/mL in PBS)、fuP2 ペプチド溶液 (5, 10, 100μg/mL in PBS)を順にセンサーチップに流した。この際、各ステップでPBSによるリンスを行った。振動数変化から、導入されたfuP1-PEG-DPPE量およびfuP2ペプチドの吸着量を評価した。fuP1-PEG-DPPE、BSA、fuP2ペプチドの順に流した場合には振動数変化は検出されたが（図３Ａ）、fuP1-PEG-DPPEを流さなかった場合には検出されなかった（図３Ｂ）。この結果から、fuP1-PEG-DPPEとfuP2ペプチドが特異的に結合することが確認できた。 1-4. Analysis of interaction between fuP2 and fuP1-PEG-lipid by QCM-D method In order to investigate the binding property between fuP1 peptide and fuP2 peptide, measurement was performed by a quartz crystal microbalance device (QCM-D). For the measurement, a gold sensor chip modified with 1-dodecanethiol to prepare CH 3 -SAM was used. First, the signal was stabilized with PBS. Subsequently, fuP1-PEG-DPPE solution (1 mg / mL in PBS), BSA solution (1 mg / mL in PBS), and fuP2 peptide solution (5, 10, 100 μg / mL in PBS) were flowed through the sensor chip in this order. .. At this time, a PBS rinse was performed at each step. The amount of fuP1-PEG-DPPE introduced and the amount of fuP2 peptide adsorbed were evaluated from the frequency change. The frequency change was detected when fuP1-PEG-DPPE, BSA, and fuP2 peptide were flowed in this order (Fig. 3A), but not when fuP1-PEG-DPPE was not flowed (Fig. 3B). .. From this result, it was confirmed that fuP1-PEG-DPPE and fuP2 peptide specifically bind to each other.
１－５．細胞間接着の確認
　CCRF-CEM細胞をCelltracker Green（緑色）またはCelltracker Orange（赤色）によって染色した。遠心によってPBSでリンスしたのち、50μLのfuP1-PEG-DPPE溶液（1, 0.1, 0.01, 0.001 mg/mL in PBS）およびfuP2-PEG-DPPE溶液（1, 0.1, 0.01, 0.001 mg/mL in PBS）を、それぞれCelltracker Greenラベル化細胞、Celltracker Orangeラベル化細胞に加え、室温で30分インキュベートした。PBSでリンスしたのち、培地で懸濁した。以上のようにして、ペプチド結合PEG脂質修飾細胞を用意した。
　1.0 × 106個のfuP1-PEG-DPPE修飾CCRF-CEM細胞と、同数のfuP2-PEG-DPPE修飾CCRF-CEM細胞を2 mL培地中で混合し、ガラスボトムディッシュ中で5分間インキュベートした。その後、細胞を共焦点顕微鏡で観察し（図４）、細胞接着頻度（図５Ａ）および細胞接着面積を評価した（図５Ｂ）。細胞接着面積は、接着部の長さをImage J測定し、測定した長さを直径とした面積で近似した。
　fuP1-PEG-DPPE修飾CCRF-CEM細胞とfuP2-PEG-DPPE修飾CCRF-CEM細胞が選択的に接着することが確認され、ペプチドPEG脂質の濃度（または、ペプチドPEG脂質の導入分子数）が高くなるにつれて、細胞接着頻度が高くなった（図４および図５Ａ）。また、細胞接着面積は、ペプチドPEG脂質の導入分子数に依存することが分かった（図５Ｂ）。 1-5. Confirmation of cell-cell adhesion CCRF-CEM cells were stained with Celltracker Green (green) or Celltracker Orange (red). After rinsing with PBS by centrifugation, 50 μL of fuP1-PEG-DPPE solution (1, 0.1, 0.01, 0.001 mg / mL in PBS) and fuP2-PEG-DPPE solution (1, 0.1, 0.01, 0.001 mg / mL in PBS) ) Was added to Celltracker Green labeled cells and Celltracker Orange labeled cells, respectively, and incubated at room temperature for 30 minutes. After rinsing with PBS, it was suspended in medium. As described above, peptide-bonded PEG lipid-modified cells were prepared.
1.0 × 10 6 fuP1-PEG-DPPE-modified CCRF-CEM cells and the same number of fuP2-PEG-DPPE-modified CCRF-CEM cells were mixed in 2 mL medium and incubated in a glass bottom dish for 5 minutes. The cells were then observed under a confocal microscope (FIG. 4) to evaluate cell adhesion frequency (FIG. 5A) and cell adhesion area (FIG. 5B). The cell adhesion area was approximated by measuring the length of the adhesion portion with Image J and using the measured length as the diameter.
It was confirmed that fuP1-PEG-DPPE-modified CCRF-CEM cells and fuP2-PEG-DPPE-modified CCRF-CEM cells adhered selectively, and the concentration of peptide PEG lipid (or the number of molecules into which peptide PEG lipid was introduced) was high. As the cell adhered frequently, the frequency of cell adhesion increased (FIGS. 4 and 5A). It was also found that the cell adhesion area depends on the number of introduced molecules of the peptide PEG lipid (Fig. 5B).
１－６．同種細胞同士の融合
　同種細胞の融合実験を行った。CCRF-CEM細胞をDiOまたはDiIによって染色した。上記の通りに1.0 × 106個のDiOラベル化fuP1-PEG-DPPE修飾CCRF-CEM細胞と、同数のDiIラベル化fuP2-PEG-DPPE修飾CCRF-CEM細胞を用意してガラスボトムディッシュ中で接着させたのち、懸濁液を15 mLファルコンチューブに穏やかに移し、瞬間的に遠心した。上清をほとんど取り除き、先端の口を切って広げたピペットチップで細胞を吸い、1mLのPEG溶液（PEG分子量：6kDa、50 wt% in PBS）中に加えた。1分間穏やかに攪拌したのち、10 mLの培地を加え、遠心によってPEGを取り除き、2 mLの培地で再懸濁してガラスボトムディッシュに移した。直後および24時間後に共焦点顕微鏡にて細胞を観察し（図６）、24時間後の細胞をフローサイトメトリーにて評価した（図７）。また、24時間後の細胞の生存率をトリパンブルー染色にて測定した（図８）。
　ペプチド結合PEG脂質で細胞表面を修飾し（細胞表面修飾法）、さらに、PEG溶液で処理すると（PEG法）、PEG単独処理と比較して、細胞融合効率が明らかに上昇した（図７Ｂ）。また、5kDaのPEGを含むペプチド結合PEG脂質で処理した場合には、PEG法を併用しなくても、PEG単独処理の場合よりも、細胞融合効率は優れていた（図７Ｂ）。ペプチド結合PEG脂質とPEG法を併用すると、細胞の生存率が若干減少するが、大きな影響ではないと言える（図８）。 1-6. Fusion of allogeneic cells A fusion experiment of allogeneic cells was performed. CCRF-CEM cells were stained with DiO or DiI. Prepare 1.0 x 10 6 DiO-labeled fuP1-PEG-DPPE-modified CCRF-CEM cells and the same number of DiI-labeled fuP2-PEG-DPPE-modified CCRF-CEM cells as described above and adhere them in a glass bottom dish. After that, the suspension was gently transferred to a 15 mL falcon tube and centrifuged momentarily. Most of the supernatant was removed, and the cells were sucked with a pipette tip opened by cutting the tip of the mouth and added to 1 mL of PEG solution (PEG molecular weight: 6 kDa, 50 wt% in PBS). After gentle stirring for 1 minute, 10 mL of medium was added, PEG was removed by centrifugation, resuspended in 2 mL of medium and transferred to a glass bottom dish. Immediately after and 24 hours later, the cells were observed with a confocal microscope (Fig. 6), and the cells after 24 hours were evaluated by flow cytometry (Fig. 7). In addition, the cell viability after 24 hours was measured by trypan blue staining (Fig. 8).
When the cell surface was modified with a peptide-bonded PEG lipid (cell surface modification method) and further treated with a PEG solution (PEG method), the cell fusion efficiency was clearly increased as compared with the PEG alone treatment (FIG. 7B). In addition, when treated with a peptide-bonded PEG lipid containing 5 kDa PEG, the cell fusion efficiency was superior to that in the case of PEG alone treatment, even without the combined use of the PEG method (FIG. 7B). The combined use of peptide-bonded PEG lipids and the PEG method slightly reduces cell viability, but it can be said that this is not a significant effect (Fig. 8).
１－７．異種細胞同士の融合
　異種細胞の融合実験を行った。SP2/O細胞（メラノーマ）をDiOで、ラット脾臓細胞をDiIで染色した。CCRF-CEM細胞と同様の方法で、1.0 × 106個のDiOラベル化fuP1-PEG-DMPE修飾SP2/O細胞と、1.0 × 107個のDiIラベル化fuP2-PEG-DPPE修飾ラット脾臓細胞を用意してガラスボトムディッシュ中で接着させたのち、懸濁液を15 mLファルコンチューブに穏やかに移し、瞬間的に遠心した。上清をほとんど取り除き、先端の口を切って広げたピペットチップで細胞を吸い、1 mLの50 wt% PEG溶液 (in PBS) 中に加えた。1分間穏やかに攪拌したのち、10 mLの培地を加え、遠心によってPEGを取り除き、2 mLの培地で再懸濁してガラスボトムディッシュに移した。直後および24時間後に共焦点顕微鏡にて細胞を観察し（図９）、24時間後の細胞をフローサイトメトリーにて評価した（図１０）。
　その結果、PEG単独処理と比較して、ペプチド結合PEG脂質単独処理およびペプチド結合PEG脂質とPEG併用処理を行った細胞の融合効率の方が、細胞融合効率が優れていた（図１０）。 1-7. Fusion of heterologous cells We conducted a fusion experiment of heterologous cells. SP2 / O cells (melanoma) were stained with DiO and rat spleen cells were stained with DiI. 1.0 x 10 6 DiO-labeled fuP1-PEG-DMPE-modified SP2 / O cells and 1.0 x 10 7 DiI-labeled fuP2-PEG-DPPE-modified rat spleen cells in the same manner as CCRF-CEM cells. After being prepared and adhered in a glass bottom dish, the suspension was gently transferred to a 15 mL falcon tube and centrifuged momentarily. Most of the supernatant was removed and the cells were aspirated with a pipette tip that had been cut open at the tip and added to 1 mL of 50 wt% PEG solution (in PBS). After gentle stirring for 1 minute, 10 mL of medium was added, PEG was removed by centrifugation, resuspended in 2 mL of medium and transferred to a glass bottom dish. Immediately after and 24 hours later, the cells were observed with a confocal microscope (Fig. 9), and the cells after 24 hours were evaluated by flow cytometry (Fig. 10).
As a result, the cell fusion efficiency of the cells subjected to the peptide-bonded PEG lipid alone treatment and the peptide-bonded PEG lipid and PEG combined treatment was superior to that of the PEG alone treatment (FIG. 10).
　本発明は、細胞同士を効率的に融合させる技術を提供する。従って、本発明は、細胞生物学等に関連する分野および医学分野における利用が期待される。 The present invention provides a technique for efficiently fusing cells with each other. Therefore, the present invention is expected to be used in fields related to cell biology and the like and in the medical field.

Claims (13)

  1. 　下記式（１）で表される化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
    Figure JPOXMLDOC01-appb-C000001
     ［式（１）中、Ｘは他のペプチドと２量体を形成するペプチドを含む原子団、Ｙは酸素、硫黄、リンまたは窒素を含んでいてもよい炭化水素基、Ｚは高分子電解質または水溶性高分子］     A compound represented by the following formula (1) or a salt thereof, a solvate thereof or a hydrate thereof.
    Figure JPOXMLDOC01-appb-C000001
     [In formula (1), X is an atomic group containing a peptide that forms a dimer with another peptide, Y is a hydrocarbon group that may contain oxygen, sulfur, phosphorus or nitrogen, and Z is a polymer electrolyte or Water-soluble polymer]
  2. 　前記ペプチドが、以下の式（２）で表される７残基のアミノ酸の繰り返しを含むペプチドであることを特徴とする請求項１に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
    Figure JPOXMLDOC01-appb-C000002
     ［式（２）中、ａおよびｄは疎水性アミノ酸であり、ｅ、ｆおよびｇは荷電アミノ酸であり、ｂはセリン、アスパラギン、グルタミンおよびトレオニンのいずれかであり、ｃは、アラニンである］     The compound according to claim 1, a salt thereof, a solvate thereof, or a peptide thereof, wherein the peptide is a peptide containing repeating amino acids of 7 residues represented by the following formula (2). Hydrate.
    Figure JPOXMLDOC01-appb-C000002
     [In formula (2), a and d are hydrophobic amino acids, e, f and g are charged amino acids, b is any of serine, asparagine, glutamine and threonine, and c is alanine]
  3. 　前記Ｙが酸素、硫黄、リンまたは窒素を含んでいてもよい炭素数１０～５０の鎖状炭化水素基であることを特徴とする請求項１または２のいずれかに記載の化合物もしくはその塩、またはそれらの溶媒和物もしくは水和物。 The compound or a salt thereof according to claim 1 or 2, wherein Y is a chain hydrocarbon group having 10 to 50 carbon atoms which may contain oxygen, sulfur, phosphorus or nitrogen. Or their solvates or hydrates.
  4. 　前記Ｙが酸素、硫黄、リンまたは窒素を含んでいてもよい脂質からなる炭化水素基であることを特徴とする請求項３に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくは水和物。 The compound according to claim 3, a salt thereof, or a solvate or hydrate thereof, wherein Y is a hydrocarbon group composed of a lipid which may contain oxygen, sulfur, phosphorus or nitrogen. ..
  5. 　前記脂質が、リン脂質であることを特徴とする請求項４に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。 The compound or salt thereof according to claim 4, wherein the lipid is a phospholipid, a solvate thereof, or a hydrate thereof.
  6. 　前記リン脂質が下記の式（３）で表されるものであることを特徴とする請求項５に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
    Figure JPOXMLDOC01-appb-C000003
     ［式（３）中、Ｗは、二重結合を含んでもよい炭素数１０～２０の炭化水素鎖］     The compound according to claim 5, a salt thereof, a solvate thereof, or a hydrate thereof, wherein the phospholipid is represented by the following formula (3).
    Figure JPOXMLDOC01-appb-C000003
     [In formula (3), W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond]
  7. 　前記Ｚが酸素、硫黄または窒素を含んでいてもよい炭素数１５０～４０００の水溶性の炭化水素鎖であることを特徴とする請求項１ないし６のいずれかに記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。 The compound according to any one of claims 1 to 6, or a salt thereof, wherein Z is a water-soluble hydrocarbon chain having 150 to 4000 carbon atoms which may contain oxygen, sulfur or nitrogen. Their solvates or their hydrates.
  8. 　前記Ｚが下記の式（４）であることを特徴とする請求項７に記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
    Figure JPOXMLDOC01-appb-C000004
     ［式（４）中、ｎは５０以上１２５０以下の整数］     The compound or salt thereof according to claim 7, wherein Z is of the following formula (4), or a solvate thereof or a hydrate thereof.
    Figure JPOXMLDOC01-appb-C000004
     [In equation (4), n is an integer of 50 or more and 1250 or less]
  9. 　前記Ｙが下記式（３）を含む置換基であり、前記Ｚが下記式（４）で表される酸素を含む炭化水素鎖であることを特徴とする請求項１ないし８のいずれかに記載の化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物。
    Figure JPOXMLDOC01-appb-C000005
     ［式（３）中、Ｗは、二重結合を含んでもよい炭素数１０～２０の炭化水素鎖］
    Figure JPOXMLDOC01-appb-C000006
     ［式（４）中、ｎは５０以上１２５０以下の整数］     The invention according to any one of claims 1 to 8, wherein Y is a substituent containing the following formula (3), and Z is a hydrocarbon chain containing oxygen represented by the following formula (4). Compounds or salts thereof, or solvates thereof or hydrates thereof.
    Figure JPOXMLDOC01-appb-C000005
     [In formula (3), W is a hydrocarbon chain having 10 to 20 carbon atoms which may contain a double bond]
    Figure JPOXMLDOC01-appb-C000006
     [In equation (4), n is an integer of 50 or more and 1250 or less]
  10. 　請求項１ないし９のいずれかに記載される化合物もしくはその塩、またはそれらの溶媒和物もしくはそれらの水和物を含む、細胞融合促進剤。 A cell fusion promoter comprising the compound according to any one of claims 1 to 9 or a salt thereof, a solvate thereof or a hydrate thereof.
  11. 　請求項１０に記載される少なくとも２種類の細胞融合促進剤を含む細胞融合促進用キット。 A cell fusion promoting kit containing at least two types of cell fusion promoting agents according to claim 10.
  12. 　以下の（ａ）および（ｂ）を含む、細胞Ａと細胞Ｂを融合させる方法。
    （ａ）請求項１０に記載の細胞融合促進剤で細胞Ａを処理する工程、
    （ｂ）請求項１０に記載の細胞融合促進剤であって、工程（ａ）で用いた細胞融合促進剤と結合する細胞融合促進剤で細胞Ｂを処理する工程、および
    （ｃ）工程（ａ）および工程（ｂ）の処理後の細胞Ａと細胞Ｂを混合し、培養する工程     A method of fusing cell A and cell B, comprising: (a) and (b) below.
    (A) A step of treating cell A with the cell fusion promoter according to claim 10.
    (B) The step of treating cell B with the cell fusion promoter according to claim 10, which binds to the cell fusion promoter used in step (a), and (c) step (a). ) And the step of mixing and culturing the cells A and B after the treatment of the step (b).
  13. 　前記工程（Ｃ）において、細胞Ａと細胞Ｂの混合物をPEGで処理することを含む、請求項１２に記載の方法。

          The method of claim 12, wherein in step (C), the mixture of cells A and B is treated with PEG.
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