Peptide, and cell fusion agent and pharmaceutical composition for cancer therapy containing said peptide

Abstract

A purpose of the present invention is to provide a method for efficient cell fusion. Also, a purpose of the present invention is to provide a method for killing cancer cells or a method for fusing a viral envelope by membrane fusion.　The foregoing can be achieved by: (1) a polypeptide, which includes an amino acid sequence having a group represented by formula (I): -Z-Xm-Y (I) (in the formula: Z is a hydrophilic linker; X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine and cysteine; Y is a carboxyl group or an amino group; m is an integer 1-5; and, when m is 2-5, the hydrophilic amino acid residues may be the same or may be the combination or different amino acid residues) at the N-terminal of an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1-8; or (2) a polypeptide, which includes an amino acid sequence having a group represented by the formula (I) at the N-terminal of an amino acid sequence in which 1-4 amino acids have been deleted, substituted, inserted and / or added in an amino acid sequence represented by SEQ ID NOs: 1-8 and which has cell fusion activity or viral envelope fusion activity.

Description

Polypeptide, cell fusion agent containing same, and pharmaceutical composition for cancer treatment

[0001] The present invention relates to a polypeptide, a cell fusion agent containing the polypeptide, and a pharmaceutical composition for cancer treatment. According to the present invention, cells can be fused efficiently.

[0002] Cell fusion is a phenomenon discovered due to the ability of Sendai virus to fuse cells (Non-Patent Documents 1 and 2). Currently, cell fusion is used for breeding or monoclonal antibody production. In addition to using viruses, it is also known that cell fusion can occur using the protoplast-PEG method or electrical stimulation.

[0003] Cell fusion, 1970, Harvard University Press, Mass.Cell Fusion and some subcellular Properties of heterokaryons and hybrids, Journal of Cell Biology, VOLUME 67, 1975, pages 257-280

[0004] The present inventors wondered whether there was a method for efficient cell fusion. They also wondered whether cancer cells could be killed by cell fusion. Therefore, an object of the present invention is to provide an efficient method for cell fusion. It is also an object of the present invention to provide a method for killing cancer cells by cell fusion or a method for fusing viral envelopes by membrane fusion.

[0005] As a result of extensive research into efficient cell fusion methods, the present inventors have surprisingly found that cells can be efficiently fused by a novel hydrophilic polypeptide having a specific amino acid sequence. Accordingly, the present invention provides: [1] (1) a polypeptide having the following formula (I): -Z-X at the N-terminus of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 8; ｍ-Y (I) (wherein Z is a hydrophilic linker, X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine, Y is a carboxyl group or an amino group, m is an integer of 1 to 5, and when m is 2 to 5, the hydrophilic amino acid residues may be the same amino acid residue or a combination of different amino acid residues), or (2) a polypeptide comprising an amino acid sequence having a group represented by formula (I) at the N-terminus, in which 1 to 4 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NOs: 1 to 8, and having cell fusion activity; [2] the polypeptide according to [1], in which the amino acid sequence represented by SEQ ID NOs: 1 to 8 has a methyl group at the N-terminus; [3] the polypeptide according to [1], in which Z is -NH-(CH ２ CH ２ O) ｎ-CO-, where n is an integer of 1 to 4; [4] an antibody or antigen-binding fragment thereof that binds to the polypeptide of any one of [1] to [3]; [5] a cell fusion agent comprising, as an active ingredient, the polypeptide of any one of [1] to [3]; [6] a pharmaceutical composition comprising, as an active ingredient, the polypeptide of any one of [1] to [3]; [7] the pharmaceutical composition of [6], which is used for treating cancer; [8] An antiviral agent for an enveloped virus, comprising as an active ingredient: (A) the polypeptide according to any one of [1] to [3]; or (B) (b1) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8; (b2) a polypeptide comprising an amino acid sequence in which 1 to 4 amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NOs: 1 to 8, and having cell fusion activity; (b3) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8, and having a methyl group at the N-terminus; or (b4) a polypeptide comprising an amino acid sequence in which 1 to 4 amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NOs: 1 to 8, and having cell fusion activity, and having a methyl group at the N-terminus; [9] A method for treating cancer, comprising the step of administering an effective amount of the polypeptide according to any one of [1] to [3] to a subject in need of treatment;

[10] The polypeptide according to any one of [1] to [3], which is for treating cancer; and

[11] Use of the polypeptide according to any one of [1] to [3] in the production of a pharmaceutical composition for cancer treatment.

[0006] The hydrophilic polypeptide of the present invention can efficiently fuse cells. The hydrophilic polypeptide of the present invention can fuse viral envelopes. Furthermore, the hydrophilic polypeptide of the present invention can be used as an active ingredient in a pharmaceutical composition for cancer treatment.

[0007] 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 11. 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 12. 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 13. 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 14. 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 15. 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 16. 1 shows a micrograph (x100) of RFL cells (A) and RM4 cells (B) treated with peptide 17. 1 shows micrographs (100x) of RFL cells (A) and RM4 cells (B) treated with peptide 18. 1 shows electron micrographs of RFL cells treated with peptide 16. 1 shows a graph measuring the induction of apoptosis, measured by activation of caspase-3 / 7, when RFL cells were treated with peptide 16. 1 shows a graph measuring the induction of apoptosis, measured by activation of caspase-3 / 7, when RM4 cells were treated with peptide 16. 1 shows electron micrographs of HVJ virus (Sendai virus) treated with peptide 16. 100x (A) and 400x (B) photographs of tumor masses excised from mice administered with peptide 16, and 100x (C) and 400x (D) photographs of tumor masses excised from control mice.

[0008] [1] Polypeptide The polypeptide of the present invention is (1) a polypeptide having the following formula (I) at the N-terminus of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 8: ｍ-Y (I) (wherein Z is a hydrophilic linker, X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine, Y is a carboxyl group or an amino group, and m is an integer of 1 to 5, and when m is 2 to 5, the hydrophilic amino acid residues may be the same amino acid residue or a combination of different amino acid residues). That is, the polypeptide of the present invention has a hydrophilic amino acid linked via a hydrophilic linker. The linkage of the hydrophilic amino acid improves the solubility of the polypeptide in aqueous solution, allowing for efficient cell fusion. The amino acid sequences represented by SEQ ID NOs: 1 to 8 are as follows: Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (SEQ ID NO: 1) Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (SEQ ID NO: 2) Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (SEQ ID NO: 3) Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (SEQ ID NO: 4) Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (SEQ ID NO: 5) Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (SEQ ID NO: 6) Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (SEQ ID NO: 7) Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (SEQ ID NO: 8)

[0009] The C-terminal alanine (Ala) of the amino acid sequences represented by SEQ ID NOs: 1 to 8 is bonded to the group represented by formula (I) via a peptide bond (-CO-NH-). In this specification, the amino acid residue refers to the amino acid residue excluding the OH of the carboxyl group (COOH) and the amino group (NH ２) without H. Z in the group of formula (I) is a hydrophilic linker that connects the amino acid sequences represented by SEQ ID NOS: 1 to 8 with hydrophilic amino acid residues. By having the hydrophilic linker, the polypeptides of the amino acid sequences represented by SEQ ID NOS: 1 to 8 can exhibit the effects of the present invention, such as cell fusion activity, without being affected by the hydrophilic amino acid residues. The hydrophilic linker Z is not particularly limited, and examples thereof include hydrocarbon groups containing heteroatoms, such as "-NH-(CH ２ CH ２ O) ｎ -CO-" can be used. The n is, for example, 1 to 4, preferably 2 to 3, and more preferably 2. The hydrophilic amino acid residue is selected from serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine, and is preferably arginine, histidine, or lysine. The number of amino acid residues, m, is 1 to 5, and preferably 2 to 4. The hydrophilic amino acid residue may be an L-amino acid residue or a D-amino acid residue, but D-amino acids are preferred from the viewpoint of suppressing degradation in vivo. The amino acid residues may be the same amino acid residue or a combination of two or more types of amino acid residues. Specific amino acid residue sequences include D-Lys-D-Lys-D-Lys, D-Arg-D-Arg-D-Arg, and D-His-D-His-D-His.

[0010] The polypeptide of the present invention also comprises (2) an amino acid sequence in which 1 to 4 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NOs: 1 to 8, and which has a group represented by formula (I) at the N-terminus thereof, and has cell fusion activity.

[0011] The (1) polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8 and having a group represented by formula (I) at its N-terminus includes (1) a polypeptide consisting of an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8 and having a group represented by formula (I) at its N-terminus. Furthermore, (2) a polypeptide comprising an amino acid sequence formed by deletion, substitution, insertion, and / or addition of 1 to 4 amino acids in an amino acid sequence represented by SEQ ID NOs: 1 to 8 and having a group represented by formula (I) at its N-terminus, and having cell fusion activity (hereinafter, this may be referred to as a "functionally equivalent variant") includes (2) a polypeptide consisting of an amino acid sequence formed by deletion, substitution, insertion, and / or addition of 1 to 4 amino acids in an amino acid sequence represented by SEQ ID NOs: 1 to 8 and having a group represented by formula (I) at its N-terminus, and having cell fusion activity. The polypeptide of the present invention can exhibit cell fusion activity when it is a polypeptide consisting of the 10 amino acid sequence, such as the amino acid sequence represented by SEQ ID NOs: 1 to 8, and has a specific structure in the 10 amino acid sequence that exhibits cell fusion activity. Therefore, as long as the specific structure exhibiting cell fusion activity in the 10 amino acid sequence is not destroyed, the polypeptide of the present invention can exhibit cell fusion activity even if the group of formula (I), other amino acids, polypeptides, or proteins are bound to the polypeptide of the present invention.

[0012] <Functionally Equivalent Variants> Functionally equivalent variants are not particularly limited, as long as they are polypeptides comprising an amino acid sequence having a group represented by formula (I) at the N-terminus of the amino acid sequence obtained by deleting, substituting, inserting, and / or adding 1 to 4, preferably 1 to 3, more preferably 1 to 2, and most preferably 1 amino acid at one or more positions in the amino acid sequences represented by SEQ ID NOs: 1 to 8. For example, relative to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5 is a functionally equivalent variant in which one amino acid has been substituted, a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6 is a functionally equivalent variant in which two amino acids have been substituted, a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7 is a functionally equivalent variant in which three amino acids have been substituted, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 8 is a functionally equivalent variant in which four amino acids have been substituted.

[0013] The "deletion, substitution, insertion, and / or addition of 1 to 4 amino acids" in functionally equivalent variants is a conservative substitution that maintains the function of the polypeptide of the present invention. "Conservative substitution" is not limited to, but can be performed by, for example, replacing an amino acid residue with another chemically similar amino acid residue. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Nonpolar (hydrophobic) amino acids include, for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include, for example, glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include, for example, arginine, histidine, and lysine. Furthermore, examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, etc. Although not limited thereto, such conservative substitutions can result in functionally equivalent variants that have cell fusion activity.

[0014] The polypeptide of the present invention is preferably, but not limited to, methylated at the N-terminal proline. That is, the N-terminal proline is preferably methylated proline. Although not limited to, methylation of the N-terminal proline can result in even better cell fusion activity.

[0015] The polypeptides of the present invention are preferably produced by chemical synthesis.

[0016] <<Cell fusion activity>> The polypeptide of the present invention has cell fusion activity. Cell fusion mediated by the polypeptide of the present invention results in the fusion of several cells to form a fused cell having multiple nuclei. Furthermore, apoptosis is induced in the fused cells after cell fusion, resulting in cell death.

[0017] Antibodies (e.g., polyclonal or monoclonal antibodies) reactive with the polypeptide of the present invention can be obtained by directly administering the polypeptide of the present invention or a fragment thereof to various animals. Alternatively, antibodies can be obtained by the DNA vaccination method (Raz, E. et al., Proc. Natl. Acad. Sci. USA, 91, 9519-9523, 1994; or Donnelly, JJ et al., J. Infect. Dis., 173, 314-320, 1996) using a plasmid carrying a polynucleotide encoding the polypeptide of the present invention.

[0018] Polyclonal antibodies can be produced, for example, from the serum or eggs of animals (e.g., rabbits, rats, goats, chickens, etc.) that have been sensitized by intraperitoneal, subcutaneous, or intravenous immunization with an emulsion of the polypeptide of the present invention or a fragment thereof in a suitable adjuvant (e.g., Freund's complete adjuvant, etc.). Polyclonal antibodies can be separated and purified from the serum or eggs thus produced by conventional protein isolation and purification methods. Examples of such separation and purification methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, protein A agarose, etc.

[0019] Monoclonal antibodies can be easily produced by those skilled in the art, for example, by the cell fusion method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975). Specifically, a mouse is immunized by repeatedly injecting an emulsion of the polypeptide of the present invention or a fragment thereof into an appropriate adjuvant (e.g., Freund's complete adjuvant) intraperitoneally, subcutaneously, or intravenously several times every few weeks. After the final immunization, spleen cells are removed and fused with myeloma cells to produce hybridomas.

[0020] Examples of myeloma cells used to obtain hybridomas include myeloma cells (e.g., mouse myeloma cell line P3X63Ag8.U1) with markers such as hypoxanthine-guanine-phosphoribosyltransferase deficiency or thymidine kinase deficiency. Examples of fusogenic agents include polyethylene glycol. Furthermore, commonly used media such as Eagle's minimum essential medium, Dulbecco's modified minimum essential medium, or RPMI-1640 can be used with 10-30% fetal bovine serum added as appropriate. Fusogenic strains can be selected by the HAT selection method. Hybridomas can be screened using culture supernatants by well-known methods such as ELISA or immunohistochemical staining, and hybridoma clones secreting the desired antibody can be selected. Furthermore, the monoclonality of hybridomas can be ensured by repeated subcloning using limiting dilution. The hybridomas thus obtained can produce antibodies in a purifiable amount by culturing them in a medium for 2 to 4 days or in the abdominal cavity of a BALB / c mouse pretreated with pristane for 10 to 20 days.

[0021] The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites fluid by conventional polypeptide isolation and purification methods. Examples of such separation and purification methods include centrifugation, dialysis, salting out with ammonium sulfate, and chromatography using DEAE-cellulose, hydroxyapatite, or protein A agarose. Alternatively, a monoclonal antibody or an antibody fragment containing a portion thereof can be produced by incorporating all or part of the gene encoding the monoclonal antibody into an expression vector and introducing the vector into a suitable host cell (e.g., Escherichia coli, yeast, or animal cell).

[0022] The antibodies (including polyclonal and monoclonal antibodies) separated and purified as described above are digested with a polypeptide-degrading enzyme (e.g., pepsin or papain) in a conventional manner, and then separated and purified by a conventional polypeptide isolation and purification method to obtain antigen-binding fragments containing active antibody portions, such as F(ab) fragments. ２ , Fab, Fab, or Fv can be obtained.

[0023] Furthermore, antibodies reactive with the polypeptides of the present invention can be obtained as single-chain Fv or ab by the method of Clackson et al. or the method of Zebedee et al. (Clackson, T. et al., Nature, 352, 624-628, 1991; or Zebedee, S. et al., Proc. Natl. Acad. Sci. USA, 89, 3175-3179, 1992). Human antibodies can also be obtained by immunizing transgenic mice in which mouse antibody genes have been replaced with human antibody genes (Lonberg, N. et al., Nature, 368, 856-859, 1994).

[0024] [2] Cell Fusion Agent The cell fusion agent of the present invention contains the polypeptide of the present invention as an active ingredient. The cell fusion agent of the present invention may contain one type of polypeptide alone, or may contain two or more types of polypeptides in combination. The content of the polypeptide in the cell fusion agent is not particularly limited, but is, for example, 0.1 to 100% by weight, preferably 10 to 100% by weight, and more preferably 30 to 90% by weight. The cell fusion agent of the present invention may contain, as components other than the polypeptide, a carrier (e.g., water or a buffer solution), an excipient, a diluent, a preservative, a stabilizer, an antiseptic, or an antioxidant.

[0025] The cell fusion agent of the present invention can be used for plant breeding or monoclonal antibody production, etc. The cell fusion agent of the present invention can efficiently fuse cells. Cells that can be fused by the cell fusion agent of the present invention are not particularly limited, and examples include microbial cells, plant cells, and animal cells. Examples of animal cells include nucleated cells (e.g., blood cells, lymphatic cells, and cells that constitute internal organs) of vertebrates (e.g., mammals) such as mice, rats, rabbits, guinea pigs, goats, sheep, horses, and cows, and mammal-derived cancer cells.

[0026] The temperature for cell fusion is not particularly limited as long as cell fusion occurs, but is, for example, 0 to 40° C., preferably 10 to 38° C. The treatment time is not particularly limited, but is preferably 1 minute to 2 hours.

[0027] [3] Pharmaceutical Composition The pharmaceutical composition of the present invention contains the polypeptide of the present invention as an active ingredient. Diseases that can be prevented or treated by the pharmaceutical composition of the present invention are not particularly limited, but for example, the pharmaceutical composition can fuse cancer cells, kill the cancer cells, and treat cancer. Specifically, the peptide of the present invention can induce apoptosis in cells through cell fusion. Caspase-3 / 7 or Annexin V is activated in the fused cells, inducing apoptosis. Induction of apoptosis in the fused cells can kill the cancer cells. Cancers that can be treated by the pharmaceutical composition of the present invention include tongue cancer, gingival cancer, malignant lymphoma, malignant melanoma, maxillary cancer, nasal cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer, glioma, meningioma, glioma, neuroblastoma, papillary thyroid adenocarcinoma, thyroid follicular carcinoma, medullary thyroid carcinoma, primary lung cancer, squamous cell carcinoma, adenocarcinoma, alveolar cell carcinoma, large cell undifferentiated carcinoma, small cell undifferentiated carcinoma, carcinoid, testicular tumor, prostate cancer, Breast cancer, Paget's disease of the breast, breast sarcoma, bone tumor, thyroid cancer, stomach cancer, liver cancer, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute lymphoblastic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma, multiple myeloma, primary macroglobulinemia, childhood leukemia, esophageal cancer, Gastric cancer, gastric and colon leiomyosarcoma, gastric and intestinal malignant lymphoma, pancreatic and gallbladder cancer, duodenal cancer, colon cancer, primary liver cancer, hepatoblastoma, uterine intraepithelial carcinoma, cervical squamous cell carcinoma, uterine adenocarcinoma, uterine adenosquamous cell carcinoma, uterine corpus adenoid carcinoma, uterine sarcoma, uterine carcinosarcoma, uterine destructive mole, uterine malignant chorioepithelioma, uterine malignant melanoma, ovarian cancer, mesodermal mixed tumor, kidney cancer, renal pelvis transitional cell carcinoma, ureteral transitional cell carcinoma, bladder These include papillary carcinoma of the bladder, transitional cell carcinoma of the bladder, squamous cell carcinoma of the urethra, adenocarcinoma of the urethra, Wilms' tumor, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, synovial sarcoma, myxosarcoma, liposarcoma, Ewing's sarcoma, cutaneous squamous cell carcinoma, basal cell carcinoma of the skin, Bowen's disease of the skin, Paget's disease of the skin, cutaneous malignant melanoma, malignant mesothelial carcinoma, metastatic adenocarcinoma, metastatic squamous cell carcinoma, metastatic sarcoma, and mesothelioma.

[0028] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, and parenteral preparations such as injections, topical liquids, ointments, suppositories, topical creams, or eye drops. Oral preparations can be prepared according to conventional methods using excipients such as gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, silicic acid anhydride, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, flavorings, stabilizers, moisturizers, antiseptics, or antioxidants. Parenteral preparations include, for example, injections. In preparing an injection, in addition to the active ingredient, any of the following may be used: a water-soluble solvent such as physiological saline or Ringer's solution; a water-insoluble solvent such as vegetable oil or fatty acid ester; an isotonic agent such as glucose or sodium chloride; a solubilizing agent; a stabilizer; a preservative; a suspending agent; or an emulsifier.

[0029] When using a pharmaceutical composition, the dosage can be determined appropriately depending on, for example, the type of active ingredient used, the type of disease, the patient's age, sex, weight, and level of health, or the administration method, and can be administered orally or parenterally. For example, when the pharmaceutical composition of the present invention is orally ingested, the intake amount is preferably 0.01 to 100 mg / kg of polypeptide per day for an adult. Note that the above administration method is only one example, and other administration methods may also be used. The administration method, dosage, administration period, administration interval, etc. of the pharmaceutical composition to humans are desirably determined through controlled clinical trials.

[0030] Furthermore, the administration form is not limited to pharmaceuticals, and various forms, such as functional foods, health foods (including beverages), or feed, can be used. The pharmaceutical composition containing the polypeptide can be produced using known pharmaceutical production methods, except that the composition contains a polypeptide as an active ingredient.

[0031] The pharmaceutical composition of the present invention may contain other ingredients. Examples of the other ingredients include food ingredients and food additives such as edible fats and oils, water, emulsifiers such as edible oils and fats, water, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerin organic acid fatty acid esters, polyglycerin fatty acid esters, calcium stearoyl lactylate, sodium stearoyl lactylate, and polyoxyethylene sorbitan fatty acid esters; thickening and stabilizing agents such as locust bean gum, carrageenan, alginic acids, pectin, xanthan gum, crystalline cellulose, carboxymethylcellulose, methylcellulose, agar, glucomannan, gelatin, starch, and modified starch; salting agents such as salt and potassium chloride; acidulants such as acetic acid, lactic acid, and gluconic acid; sugars or sugar alcohols, sweeteners such as stevia and aspartame; coloring agents such as beta-carotene, caramel, and red koji pigment; antioxidants such as tocopherol and tea extract; flavorings; pH adjusters; food preservatives; and shelf-life enhancers. It is also possible to add functional materials such as various vitamins, coenzyme Q, plant sterols, or milk fat globule membranes. The total content of these other ingredients in the pharmaceutical composition of the present invention is preferably 8% by mass or less, more preferably 40% by mass or less, and even more preferably 20% by mass or less.

[0032] The pharmaceutical composition of the present invention can be administered to humans, but may also be administered to animals other than humans, including pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; livestock such as cows and pigs; laboratory animals such as mice and rats; and animals kept in zoos, etc.

[0033] <<Cancer Treatment Method>> The cancer treatment method of the present invention comprises the step of administering an effective amount of the polypeptide to a subject in need of treatment. That is, the polypeptide of the present invention can be used in a cancer treatment method. Cancer can be treated by administering an effective amount of the pharmaceutical composition to a human or animal.

[0034] <Polypeptide for treating cancer> The polypeptide of the present invention is for treating cancer. The polypeptide can be used in a method for treating cancer. That is, the present specification discloses a polypeptide for treating cancer.

[0035] Use of Polypeptide in the Production of a Pharmaceutical Composition The polypeptide can be used in the production of a pharmaceutical composition. That is, the present specification discloses the use of the polypeptide in the production of a pharmaceutical composition. The pharmaceutical composition is, but is not limited to, a pharmaceutical composition for cancer treatment.

[0036] Antiviral Agents The antiviral agents of the present invention comprise, as an active ingredient, (A) the polypeptide of the present invention; or (B) (b1) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 8, (b2) a polypeptide comprising an amino acid sequence represented by SEQ ID NOs: 1 to 8 in which 1 to 4 amino acids have been deleted, substituted, inserted, and / or added, and which has cell fusion activity, (b3) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 8, which has a methyl group at its N-terminus, or (b4) a polypeptide comprising an amino acid sequence represented by SEQ ID NOs: 1 to 8 in which 1 to 4 amino acids have been deleted, substituted, inserted, and / or added, and which has cell fusion activity, which has a methyl group at its N-terminus. The polypeptides are capable of fusing viral envelopes and exhibit antiviral activity. That is, they can be used as antiviral agents for enveloped viruses.

[0037] Examples of enveloped viruses include, but are not limited to, viruses of the Poxviridae, Baculoviridae, Rhabdoviridae, Bunyaviridae, Togaviridae, Herpesviridae, Paramyxoviridae, Orthomyxoviridae, Retroviridae, Arenaviridae, and Coronaviridae families; and more specific examples include influenza viruses such as avian influenza virus, human influenza virus, and swine influenza virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, varicella-zoster virus, herpes simplex virus, human herpes virus, mumps virus, respiratory syncytial virus, Ebola virus, rubella virus, coronavirus, measles virus, arbovirus, SARS virus, hepatitis A virus, hepatitis D virus, hepatitis E virus, yellow fever virus, adult T-cell leukemia virus, rabies virus, hantavirus, dengue virus, Nipah virus, and lyssavirus.

[0038] The polypeptide can be used in a method for treating a viral disease. The polypeptide can be used as a polypeptide for treating a viral disease. The polypeptide can be used to produce a pharmaceutical composition for treating a virus.

[0039] <<Action>> The mechanism by which the polypeptides of the present invention have cell fusion activity has not been fully elucidated, but is presumed as follows. However, the present invention is not limited by the following presumption. It is believed that the polypeptides of the present invention exhibit cell fusion activity due to a structure commonly present in the amino acid sequences of SEQ ID NOs: 1 to 8. Although not limited thereto, proline at position 1 is thought to be relatively important. On the other hand, leucine or isoleucine at positions 2 and 9 exhibit cell fusion activity even when substituted with each other, therefore, it is believed that the amino acids at positions 2 and 9 are substitutable and highly likely to exhibit cell fusion activity even when substituted with other amino acids (e.g., valine). Furthermore, threonine and glutamine at positions 5 and 6 also exhibit cell fusion activity even when substituted with each other, therefore, it is believed that the amino acids at positions 5 and 6 are substitutable and highly likely to exhibit cell fusion activity even when substituted with other amino acids with similar properties, including serine at position 4 and threonine at position 7. Furthermore, it is believed that alanine at positions 8 and 10 may also exhibit cell fusion activity even when substituted with amino acids with similar properties, such as glycine. Furthermore, methylation of the N-terminal proline is not essential for the cell fusion ability of each peptide and the induction of apoptosis in cancer cells. Therefore, peptides in which one or more amino acids have been added to the N-terminal proline can also exhibit cell fusion and apoptosis induction abilities. Furthermore, the mechanism by which the polypeptides of the present invention have anticancer activity has not been fully elucidated, but is presumed as follows. However, the present invention is not limited by the following presumption. It is presumed that the polypeptides of the present invention can fuse cancer cells and induce apoptosis in the cells, thereby killing the cancer cells. Furthermore, the cell fusion is induced regardless of the type of cancer. Therefore, the polypeptides of the present invention are considered to be effective against many types of cancer. Furthermore, the excellent water solubility of the polypeptides of the present invention is presumed to be due to the presence of the group represented by formula (I) at the C-terminus of the amino acid sequences represented by SEQ ID NOs: 1 to 8.The amino acid sequences represented by SEQ ID NOs: 1 to 8 contain many hydrophobic amino acids, and therefore, by linking some hydrophilic amino acids via hydrophilic linkers, it is believed that the sequences exhibit excellent water solubility as a whole.

[0040] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0041] Example 1 In this synthesis example, peptides were synthesized in which the N-terminal proline of each of the amino acid sequences shown in SEQ ID NOs: 1 to 8 was methylated, a peptide in which the amino acid sequence shown in SEQ ID NO: 1 (proline unmethylated), and a peptide in which the N-terminal proline of each of the amino acid sequences shown in SEQ ID NO: 9 was methylated. Peptide synthesis was outsourced to Greiner / Fasmax. The amino acid sequence shown in SEQ ID NO: 9 is an amino acid sequence in which threonine and alanine have been added to the C-terminus of the amino acid sequence shown in SEQ ID NO: 1. CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 1; SEQ ID NO: 1) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 2; SEQ ID NO: 2) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 3; SEQ ID NO: 3) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 4; SEQ ID NO: 4) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 5; SEQ ID NO: 5) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 6; SEQ ID NO: 6) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 7; SEQ ID NO: 7) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala (hereinafter referred to as peptide 8; SEQ ID NO: 8) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala-Thr-Ala (hereinafter referred to as peptide 9; SEQ ID NO: 9) Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala (hereinafter referred to as peptide 10; SEQ ID NO: 1) Amino acids were synthesized according to the standard 9-fluorenylmethoxycarbonyl (Fmoc) method.Specifically, Fmoc amino acids were activated with an HBTU / HOBT solution (HBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBT: 1-Hydroxybenzotriazole), and then condensed with N,N'-Diisopropylethylamine (DIEA). The synthesized amino acids were cleaved from the resin as follows: A TFA (trifluoroacetic acid) solution (4.125 mL TFA, 0.25 mL HO, 0.375 g phenol, 0.125 mL ethanedithiol, and 0.25 mL thioanisole) was prepared and added to the resin. The mixture was incubated at room temperature for 2 hours, and then precipitated with cold ether to obtain crude peptides. The resulting crude peptides were purified using RP-HPLC and lyophilized. The purity of the purified peptides was examined by HPLC and MS under the following conditions.・HPLC conditions A Buffer: 0.1% TFA / H2O, B Buffer: 0.1% TFA / Acetonitrile Column: SunFire C18 Column, 5 μm, 4.6 x 150 mm Flow rate: 1 mL / min Wavelength: 220 nm ・MALDI-TOF-MS.

[0042] In this synthesis example, polypeptides were synthesized in which miniPEG-D-Lys-D-Lys-D-Lys-NH was bound to the N-terminus of each of the amino acid sequences represented by SEQ ID NOS: 1 to 8. Peptide synthesis was outsourced to Peptide Institute, Inc. CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 11) CH3-Pro-Leu-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 12) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 13) CH3-Pro-Leu-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 14) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Ile-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 15) CH3-Pro-Ile-Val-Ser-Thr-Gln-Thr-Ala-Leu-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 16) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 17) CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Leu-Ala-miniPEG-D-Lys-D-Lys-D-Lys-NH2 (hereinafter referred to as peptide 18) The "miniPEG" is -NH-(CH ２ CH ２ O) ２ The procedure for the preparation of peptide 16 is described below.

[0043] Starting from commercially available Fmoc-Rink Amide resin (0.46 g, 0.25 mmol), the peptide chain was sequentially elongated by Fmoc solid-phase synthesis according to the standard method for ABI-433A to construct the desired protected peptide resin. The amino acid corresponding to miniPEG was the following formula (II): The resulting resin was then treated with trifluoroacetic acid / H ２ The mixture was treated with 0 / triisopropylsilane / 1,3-dimethoxybenzene (18 mL / 0.5 mL / 0.5 mL / 1 mL) at room temperature for 1.5 hours, the resin was removed by filtration, and the mixture was concentrated under reduced pressure. The crude peptide was solidified with ether (50 mL) and purified on a reverse-phase HPLC ODS column (YMC-Pack ODS-A φ30 x 250 mm) using 0.1% trifluoroacetic acid in water and acetonitrile as eluents. Lyophilization yielded 180 mg of the peptide trifluoroacetate salt. The resulting peptide trifluoroacetate salt (180 mg) was then salt-exchanged on an ion-exchange column (DOWEX 1x2 100-200 Mesh Anion Exchange Resin CH3COO type) using 5% acetic acid in water as eluent, and lyophilized to yield 120 mg of the target compound as a white lyophilized powder of acetate. Other peptides were also produced according to the procedure for producing peptide 16.

[0044] Example 3 In this example, peptides 11 to 18 were allowed to act on RFL cells (cells derived from fetal rat lung) or RM4 cells, and the cell fusion activity of the peptides was examined. ６ 8 × 10 cells were suspended in 6 mL of RPMI-1640 (Wako, 189-02025) medium supplemented with 5% FBS (Biosera, Cat No. 015BS493), and placed in each well of a 24-well plate (Iwaki, 2820-024). ４0.25 mL of each cell was dispensed and cultured. The medium was removed, and fresh medium (20 μL) and peptides 1 to 18 (1 μg / mL) were dispensed, followed by further culture for 24 to 36 hours. After culture was completed, the cells were fixed with methanol (Wako), and nuclear staining was performed with Giemsa stain (Muto Chemical 15003) before microscopic examination. Figures 1 to 8 show micrographs of RFL cells (A) or RM4 cells (B). Cell fusion occurred in RFL cells or RM4 cells, and fused cells with multiple nuclei were observed. Figure 9 shows an electron micrograph of RFL cells treated with peptide 16.

[0045] Example 4 In this example, the apoptotic activity of peptide 16 was examined using RFL cells and RM4 cells. Caspase-3 / 7 activity, an indicator of apoptosis, was measured using an IncuCyte S3 live cell analysis system (Essen Biosciences). Caspase-3 / 7 activity is measured using an inactive, non-fluorescent (DEVD) substrate that can pass through the cell membrane. Activated caspase-3 / 7 cleaves the substrate, releasing a DNA-bound green fluorescent label, and caspase-3 / 7 activity is measured based on the intensity of the green fluorescence.

[0046] RFL cells or RM4 cells were seeded in a 96-well plate and 0.06 μg / mL of peptide 16 was added. Caspase-3 / 7 Green Reagent (unit size: 20 μl, 5 mM / vial) was diluted 500-fold with Ham's F-12K and added. Using an IncuCyte S3 live cell analysis system, measurements were taken with continuous scanning every hour for 3 days at a 10x objective lens magnification and 4 fields of view. RFL cells or RM4 cells not treated with peptide 16 were used as controls. Figure 10 shows caspase-3 / 7 activity in RFL cells, and Figure 11 shows caspase-3 / 7 activity in RM4 cells. In both RFL and RM4 cells, activity increased rapidly between 10 and 20 hours, and then gradually increased thereafter. On the other hand, in RFL cells and RM4 cells not treated with peptide 16, the activity of caspase-3 / 7 was not increased.

[0047] Example 5 In this example, peptide 16 was applied to HVJ virus (Sendai virus) to examine its ability to fuse with the virus. An electron microscope image is shown in Figure 12. Fusion of the HVJ virus envelope was observed.

[0048] Example 6 In this example, the anticancer effect of peptide 16 on A549 cells (human alveolar adenocarcinoma cells) was investigated in vivo. nu / CrlCrlj nude mice, 1 × 10 ８ A549 cells suspended at a concentration of 18.75 or 37.5 mg / kg were subcutaneously implanted in 0.1 mL into the right flank of each mouse. After tumor implantation, peptide 16 was administered intravenously via the tail vein at a dose of 18.75 mg / kg or 37.5 mg / kg twice weekly for 5 weeks (peptide 16 group-1 or peptide 16 group-2). The control group received PBS alone. Thirty-five days after tumor implantation, mice were necropsied and tumors were excised. HE-stained photographs of the excised tumors are shown in Figure 13. Figures 13A (x100) and B (x400) show photographs of tumor masses of A549 cells treated with peptide 16, while Figures 13C (x100) and D (x400) show the control. While the tumor mass in the control mouse was filled with cells, the tumors in the mice treated with peptide 16 underwent necrosis, suggesting that peptide 16 exerted an antitumor effect.

[0049] The polypeptide of the present invention can be used for cell fusion of plant cells and animal cells, and the pharmaceutical composition of the present invention can be used for cancer treatment.

Claims

1. (1) An amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 8, wherein the N-terminus of the amino acid sequence has the following formula (I): -Z-X ｍ -Y (I) (wherein Z is a hydrophilic linker, X is a hydrophilic amino acid residue selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, tyrosine, and cysteine, Y is a carboxyl group or an amino group, m is an integer of 1 to 5, and when m is 2 to 5, the hydrophilic amino acid residues may be the same amino acid residue or a combination of different amino acid residues), or (2) a polypeptide comprising an amino acid sequence having a group represented by formula (I) at the N-terminus, which is an amino acid sequence represented by SEQ ID NOs: 1 to 8, in which 1 to 4 amino acids are deleted, substituted, inserted, and / or added, and which has cell fusion activity.

2. The polypeptide according to claim 1, wherein the amino acid sequences represented by SEQ ID NOs: 1 to 8 have a methyl group at the N-terminus.

3. The Z is -NH-(CH ２ CH ２ O) ｎ The polypeptide of claim 1 or 2, wherein n is an integer of 1 to 4.

4. An antibody or an antigen-binding fragment thereof that binds to the polypeptide described in any one of claims 1 to 3.

5. A cell fusion agent comprising the polypeptide according to any one of claims 1 to 3 as an active ingredient.

6. A pharmaceutical composition comprising as an active ingredient a polypeptide according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, which is for treating cancer.

8. An antiviral agent against enveloped viruses comprising as an active ingredient: (A) a polypeptide according to any one of claims 1 to 3; or (B) (b1) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8; (b2) a polypeptide comprising an amino acid sequence represented by SEQ ID NOs: 1 to 8 in which 1 to 4 amino acids have been deleted, substituted, inserted and / or added, and having cell fusion activity; (b3) a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 1 to 8, and having a methyl group at the N-terminus; or (b4) a polypeptide comprising an amino acid sequence represented by SEQ ID NOs: 1 to 8 in which 1 to 4 amino acids have been deleted, substituted, inserted and / or added, and having cell fusion activity, and having a methyl group at the N-terminus.

9. A method for treating cancer, comprising the step of administering to a subject in need of treatment an effective amount of the polypeptide described in any one of claims 1 to 3.

10. A polypeptide according to any one of claims 1 to 3 for use in the treatment of cancer.

11. Use of a polypeptide according to any one of claims 1 to 3 in the manufacture of a pharmaceutical composition for treating cancer.