Cytoplasmic delivery peptide

Abstract

To provide a cytoplasmic delivery peptide.SOLUTION: Provided is a cytoplasmic delivery peptide comprising, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, or comprising, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain, wherein the acidic amino acid domain comprises 1 to 3 acidic amino acids, the mastoparan domain comprises an amino acid sequence having at least 90% identity to a specific amino acid sequence, and the basic amino acid domain comprises 12 or more basic amino acids.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to cytoplasmic delivery peptides. [Background technology]

[0002] Although the number of macromolecular drug candidate substances such as antibodies is increasing, many of them cannot penetrate cell membranes, and their targets are limited to extracellular molecules or the extracellular domains of membrane proteins. To target molecules inside cells, a technology is required to deliver these substances into the target cells. Existing cytoplasmic delivery peptides are disclosed, for example, in Patent Document 1, Non-Patent Documents 1 and 2, etc. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6202707 [Non-patent literature]

[0004] [Non-Patent Document 1] Azuma, Y. et al. (2018) Angew. Chem. Int. Ed. 57, 12771-12774 [Non-Patent Document 2] Iwasaki, T. et al. (2015) J. Control. Release 210, 115-124 Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present disclosure to provide improved cytoplasmic delivery peptides. [Means for solving the problem]

[0006] In one aspect, the present disclosure provides a cytoplasmic delivery peptide, comprising: comprising, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, or comprising, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain; the acidic amino acid domain is composed of 1 to 3 acidic amino acids; The mastoparan domain consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, The basic amino acid domain is composed of 12 or more basic amino acids. Cytoplasmic delivery peptides are provided.

[0007] In one aspect, the disclosure provides a method of conjugating a cytoplasmic delivery peptide to a protein, the method comprising: (1) Providing the above peptide, in which a cysteine-containing S-S bond sequence is bound to the C-terminus; (2) Peptide (1) and G n contacting a peptide containing a structure of -(linker)-C (n is an integer of 2 or more) to form a disulfide bond; (3) Providing a protein having an amino acid sequence including LPXTG (X is any amino acid) bound to the C-terminus; (4) reacting the peptide obtained in (2) with the protein in (3) using a sortase; The present invention provides a method comprising:

[0008] In one aspect, the disclosure provides a polynucleotide encoding the peptide. In one aspect, the disclosure provides a vector comprising the above-described polynucleotide. In one aspect, the disclosure provides a cell comprising the polynucleotide or the vector. In one aspect, the disclosure provides a composition comprising the above peptide for delivering a substance into a cell. In one aspect, the disclosure provides a method of delivering a substance into a cell, comprising contacting the cell in vitro with the peptide and the substance to be delivered into the cell. Effect of the Invention

[0009] The present disclosure provides improved cytoplasmic delivery peptides. [Brief description of the drawings]

[0010] [Figure 1] Background of the present disclosure. By changing the configuration from MP (SEQ ID NO: 1) to E3MP (SEQ ID NO: 8), toxicity is reduced, but the efficiency of uptake into cells is also reduced. Furthermore, by changing the configuration from E3MP to E3-Dpa-MP (SEQ ID NO: 9), toxicity is increased, but the efficiency of uptake into cells is improved. [Diagram 2] Design of the cytoplasmic delivery peptide of the present disclosure: The cytoplasmic delivery peptide of the present disclosure achieves low toxicity and high intracellular uptake efficiency. [Diagram 3] Cytotoxicity of E3MP and E3MPH16 in HT1080 cells. Cytotoxicity of MP (open diamond; ◇), E3MP (open triangle; △), and E3MPH16 (filled circle; ●) in HT1080 cells treated for 18 h at the indicated concentrations. E3MPH16 showed no cytotoxicity even at 100 μM. [Figure 4] Intracellular uptake of E3MP and E3MPH16 in HT1080 cells. Quantification by flow cytometry of intracellular uptake when HT1080 cells were treated with 10 μM E3MPGGC(Alexa488)(E3MP(Alexa)) and E3MPH16GGC(Alexa488)(E3MPH16(Alexa)) for 1 hour. E3MPH16 was able to promote intracellular uptake 40-fold compared to E3MP. [Diagram 5] Cytoplasmic delivery of Dex-488 and promotion of endosomal escape by E3MPH16. (Left) Confocal microscopy images of HT1080 cells treated with 200 μg / mL Dex-488 and 10 μM of the indicated peptide for 18 h. (Right) Percentage of cells with Dex-488 signal in the cytoplasm in the left image. E3MPH16 delivered fluorescently labeled dextran to the cytoplasm even in the presence of serum. [Figure 6]Confocal microscopy images of various cells treated with 10 μM E3MPH16 together with 200 μg / mL Dex-488 for 18 hours. E3MPH16 delivered dextran to the cytoplasm in various cell types. [Figure 7] Cytoplasmic delivery of NLS-GFP by E3MPH16. Confocal microscopy images of HT1080 cells incubated with 5 μM NLS-GFP and 10 μM E3MPH16 for 18 hours. E3MPH16 enables the introduction of GFP with a nuclear localization signal into the nucleus. [Figure 8] Cytoplasmic delivery of functional proteins by E3MPH16. (Top) Schematic diagram of Cre-loxP recombination assay. (Bottom) Gene recombination efficiency in loxP-reporter HEK293 cells treated with 5 μM Cre or 5 μM Cre and 10 μM E3MP or E3MPH16 for 24 hours. The recombination protein Cre delivered into the cells by E3MPH16 translocated into the nucleus and recognized the loxp sites on the chromosome, causing highly efficient recombination of chromosomal DNA. [Figure 9] Cytoplasmic delivery of functional proteins by E3MPH16. Confocal microscopy images of HT1080 cells treated with 500 μg / mL anti-GFP antibody and E3MP16 for 18 hours, fixed, and immunostained. The anti-GFP antibody delivered into the cells by E3MPH16 specifically bound to GFP within the cells without being degraded or denatured within the cells. [Figure 10] Cytoplasmic delivery of functional proteins by E3MPH16. Cell proliferation suppression effect in HT1080 cells treated with 500μg / mL or 167μg / mL anti-Ras antibody and 10μM E3MPH16 for 24 hours. The anti-Ras antibody introduced into the cells by E3MPH16 bound to the intracellular Ras protein and suppressed cell proliferation. [Figure 11] Schematic diagram of sortase-mediated protein-peptide conjugation. [Figure 12]Preparation of protein-peptide conjugates using sortase. (Top left) Confirmation of protein-peptide conjugates by non-reducing SDS-PAGE. (Bottom) Confocal microscopy images of HT1080 cells after incubating for 18 hours with 4 μM NLS-GFP and E3MPH16 conjugates. NLS-GFP was delivered into the nucleus with high efficiency by the NLS-GFP and E3MPH16 conjugates. [Figure 13] (Left) Confocal microscopy images of HT1080 cells treated with 10 μM of the indicated peptide together with 200 μg / mL Dex-488 for 18 hours. (Right) Percentage of cells with diffuse Dex-488 signals in the cytoplasm based on the image on the left. When the number of histidine residues in the polyhistidine added to the C-terminus of E3MP was 12, the efficiency of delivery into cells increased. [Figure 14] Activity was improved by converting the N-terminus of E3MPH16 to the D-form. Confocal microscopy images of HT1080 cells treated with 10 μM of the indicated peptide together with 200 μg / mL Dex-488 for 18 hours. By converting the glutamic acid (E) residue at the N-terminus of E3MPH16 to the D-form (amino acid residues in the D-form are written in lowercase in the figure), intracellular delivery efficiency was improved. [Figure 15] Activity was improved by converting the N-terminus of E3MPH16 to the D-form. Gene recombination efficiency in loxP-reporter HEK293 cells treated with 5 μM Cre or 5 μM Cre and 10 μM of the specified peptide for 24 hours. Converting the glutamic acid (E) residue at the N-terminus of E3MPH16 to the D-form (amino acid residues in the D-form are written in lowercase in the figure) promoted delivery of the recombinant protein Cre into the cell and subsequent nuclear translocation, resulting in highly efficient recombination of chromosomal DNA via loxp site recognition on the chromosome. [Figure 16]E3MPH16-mediated cytoplasmic delivery of NLS-GFP by local administration in tumor-bearing mice. (Top) Schematic diagram of E3MPH16-mediated delivery of NLS-GFP by local administration to a Colon-26 xenograft tumor model mouse. (Bottom) Twenty-four hours after local administration of 5 μM NLS-GFP, 5 μM NLS-GFP and E3MPH16, or 4 μM NLS-GFP-E3MPH16 conjugate to a Colon-26 xenograft tumor model mouse, tissues were collected and paraffin slides were prepared. Nuclei were stained with DAPI, and the respective fluorescence was observed by confocal microscopy. The white box in the image indicates the enlarged area. E3MPH16 was translocated into the nuclei of cancer cells by NLS-GFP. [Figure 17] Cytoplasmic delivery of Dex-488 and promotion of endosomal escape by E3MPH16. Confocal microscopy images of HT1080 cells treated with 10 μM E3MP16 and 200 μg / mL Dex-488 for the indicated times. E3MPH16 was found to deliver the target substance into the cell via the endosomal pathway. [Figure 18] Confocal microscopy images of HT1080 cells treated with 200 μg / mL Dex-488 and 10 μM E3MPH16, incubated for 1 h and then washed (right panel), and then incubated for an additional 2 h (left panel). E3MPH16 was found to deliver the target substance from the endosome into the cell. [Figure 19] Intracellular uptake pathway involved in cytoplasmic delivery of E3MPH16. In HT1080 cells treated with endocytosis inhibitors for clathrin-dependent endocytosis, caveolae-dependent endocytosis, and macropinocytosis, 10 μM E3MPH16 (Alexa) was added and incubated for 1 hour. It was found that endocytosis is involved in the intracellular delivery of the target substance by E3MPH16. [Figure 20]Intracellular uptake pathway involved in cytoplasmic delivery of E3MPH16. (Left) Confocal microscopy images of HT1080 cells treated with an endocytosis inhibitor for 30 minutes, followed by addition of the endocytosis inhibitor, 10 μM E3MPH16, and 200 μg / mL Dex-488 to HT1080 cells and incubation for 1 hour. (Right) Percentage of cells in which the Dex-488 signal has spread throughout the cytoplasm in the image on the left. It was found that the macropino and caveolae pathways are involved in intracellular delivery of the target substance via endocytosis by E3MPH16. [Figure 21] Cytoplasmic delivery of Dex-488 and promotion of endosomal escape by E3MPH16. (Left) Confocal microscopy images of HT1080 cells treated with 200 μg / mL Dex-488 and 10 μM E3MPH16 for 3 hours at 4°C or 37°C. (Right) Percentage of cells in which the Dex-488 signal is diffused throughout the cytoplasm in the image on the left. Intracellular delivery of the target substance by E3MPH16 was suppressed at 4°C, indicating that E3MPH16 delivers the target substance via the endosomal pathway. [Figure 22] Intracellular uptake pathway involved in cytoplasmic delivery of E3MPH16. (Left) Confocal microscopy images of HT1080 cells treated with 20 nM concanamycin, then incubated for 3 hours with 20 nM concanamycin, 10 μM E3MPH16, and 200 μg / mL Dex-488. (Right) Percentage of cells in the left image with diffuse Dex-488 signal throughout the cytoplasm. Endosome acidification was found to be important for intracellular delivery of the target substance by E3MPH16. [Figure 23]Intracellular uptake pathway involved in the cytoplasmic delivery of E3MPH16. Confocal microscopy images of HT1080 cells expressing various Rab5 mutants, treated with 10μM E3MPH16 and 200μg / mL Dex488. Expression of Rab5Q79L had almost no effect on the cytoplasmic delivery of Dex-488, but expression of Rab5S34N almost completely eliminated the cytoplasmic delivery of Dex-488. It was found that early endosomes are important for intracellular delivery of the delivered substance by E3MPH16. It was found that inhibiting transfer to early endosomes prevented the delivered substance from diffusing within the cytoplasm. [Figure 24] Colocalization of E3MPH16(Alexa) and the lysosomal marker LysoTracker red in HT1080 cells. After incubation with 10 μM E3MPH16(Alexa) for 1 hour, HT1080 cells were stained with Lysotracker red and live cell images were obtained by confocal microscopy. E3MPH16(Alexa) was localized to lysosomes. [Diagram 25] Liposome leakage assay induced by MP (open diamond; ◇), E3MP (open triangle; △), and E3MPH16 (filled circle; ●). CM (left) and EM (right) show liposomes mimicking cellular and endosomal membranes. E3MPH16 was found to be cytotoxic to acidic lipids. [Figure 26] Effect of introducing different acidic amino acids at the N-terminus. Confocal microscopy images of HT1080 cells treated with 10 μM of the indicated peptides together with 200 μg / mL Dex-488 for 18 h. Substitution of the three N-terminal glutamic acids of E3MPH16 with aspartic acid (D) or 2-aminoadipic acid (Aad) maintained the intracellular delivery ability. [Figure 27] Cytotoxicity of H16MPE3 in HT1080 cells. Cytotoxicity when H16MPE3 was treated with the indicated concentrations for 18 hours in HT1080 cells. H16MPE3 did not show cytotoxicity even at 100 μM. [Figure 28]Cytoplasmic delivery of Dex-488 by H16MPE3. (Top) Confocal microscopy images of HT1080 cells treated with 200 μg / mL Dex-488 and 10 μM of the indicated peptide for 18 h. (Bottom) Percentage of cells with Dex-488 signal in the cytoplasm in the top image. H16MPE3 delivered fluorescently labeled dextran to the cytoplasm comparably to E3MPH16. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In this specification, when a numerical value is accompanied by the term "about", it is intended to include a range of ±10% of the value. For example, "about 20" is intended to include "18-22". A range of numerical values ​​includes all numerical values ​​between and at the endpoints. "About" in relation to a range applies to both endpoints of the range. Thus, for example, "about 20-30" is intended to include "18-33".

[0012] The cytoplasmic delivery peptide of the present disclosure comprises, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, or comprises, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain. The cytoplasmic delivery peptide of the present disclosure preferably comprises, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain. Each domain may be directly linked, or may be linked via any linker. Preferably, the acidic amino acid domain is present at the N-terminus of the peptide. Any amino acid sequence may be linked to the C-terminus of the basic amino acid domain.

[0013] The acidic amino acid domain is composed of 1 to 3, preferably 3, acidic amino acids. The acidic amino acids are not limited, but may be selected from, for example, glutamic acid, aspartic acid, and 2-aminoadipic acid. For example, all amino acids in the acidic amino acid domain may be glutamic acid. That is, the acidic amino acid domain may be composed of 1 to 3, preferably 3, glutamic acids.

[0014] The acidic amino acid domain may contain at least one D-amino acid, such as D-glutamic acid, D-aspartic acid, or D-2-aminoadipic acid, preferably D-glutamic acid. For example, the N-terminus of the acidic amino acid domain may be a D-amino acid. All amino acids in the acidic amino acid domain may be D-amino acids. That is, the acidic amino acid domain may consist of 1 to 3, for example, 3 D-amino acids.

[0015] Mastoparan is a membrane damaging peptide derived from bee venom. A known derivative thereof is mastoparan X, which consists of the amino acid sequence INWKGIAAXAKKLL (SEQ ID NO: 1, X represents norleucine). The mastoparan domain of the present disclosure consists of an amino acid sequence that has about 90% or more identity with the amino acid sequence of SEQ ID NO: 1 when calculated using BLAST or the like (e.g., when using the default, i.e., initial condition, parameters of BLAST). For example, the mastoparan domain has about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the mastoparan domain comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0016] The mastoparan domain may comprise a sequence in which one, two or three amino acid residues are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1. The amino acid substitution is preferably a conservative substitution between similar amino acid residues. Depending on the side chain, the amino acid residues can be classified into groups such as acidic amino acids (e.g., glutamic acid, aspartic acid, 2-aminoadipic acid), basic amino acids (e.g., histidine, lysine, arginine), aliphatic amino acids (e.g., methionine, alanine, leucine, isoleucine, valine, 2-aminoheptanoic acid, norleucine), aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine), polar uncharged amino acids (e.g., asparagine, cysteine, glutamine, serine, threonine), etc. Preferably, the amino acid substitution is a conservative substitution between amino acid residues in the same group. For example, in the amino acid sequence of SEQ ID NO: 1, one, two or three, preferably one, amino acid residues may be substituted with other amino acid residues in the same group. For example, the norleucine in SEQ ID NO:1 may be substituted with other aliphatic amino acids, such as methionine, alanine, leucine, isoleucine, valine, or 2-aminoheptanoic acid. For example, a mastoparan domain may comprise or consist of the amino acid sequence of INWKGIAAMAKKLL (SEQ ID NO:2). A mastoparan domain may comprise at least one D-amino acid. All amino acids in a mastoparan domain may be D-amino acids.

[0017] The basic amino acid domain is composed of 12 or more, for example, 12 to 16, and preferably 16 basic amino acids. The basic amino acids are not limited, but are selected from, for example, histidine, lysine, and arginine. For example, all amino acids in the basic amino acid domain may be histidine. That is, the basic amino acid domain may be composed of 12 or more, preferably 12 to 16, and more preferably 16 histidines. For example, the basic amino acid domain may contain 1 to 3, 1 to 4, 1 to 5, or 1 to 6 arginines.

[0018] The basic amino acid domain may contain at least one D-amino acid, such as D-histidine, D-lysine and D-arginine, preferably D-histidine. All amino acids in the basic amino acid domain may be D-amino acids. For example, the basic amino acid domain may consist of 12 or more D-amino acids, such as 12 to 16 D-amino acids, preferably 16 D-amino acids.

[0019] The cytoplasmic delivery peptide of the present disclosure consists of an amino acid sequence that has 90% or more identity with the amino acid sequence of SEQ ID NO: 3 or 4, when calculated using BLAST or the like (e.g., when using the default, i.e., initial condition, parameters of BLAST). SEQ ID NO: 3: EEEINWKGIAAXAKKLLHHHHHHHHHHHHHHH (X represents norleucine) SEQ ID NO: 4: eeeINWKGIAAXAKKLLHHHHHHHHHHHHHHH (e represents D-glutamic acid and X represents norleucine)

[0020] For example, the cytoplasmic delivery peptide has about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more identity to the amino acid sequence of SEQ ID NO: 3 or 4. In some embodiments, the cytoplasmic delivery peptide comprises the amino acid sequence of SEQ ID NO: 3 or 4 or consists of the amino acid sequence of SEQ ID NO: 3 or 4. The cytoplasmic delivery peptide may comprise the amino acid sequence of SEQ ID NO: 3 or 4 in which one or several amino acids have been deleted, substituted, or added.

[0021] Alternatively, the cytoplasmic delivery peptide of the present disclosure consists of an amino acid sequence that has 90% or more identity with, for example, any of the amino acid sequences in the table below, when calculated using BLAST or the like (e.g., when using the default, i.e., initial condition, parameters of BLAST). [Table 1] In the table, X1 represents norleucine, X2 represents 2-aminoadipic acid, and lowercase letters represent D-amino acids.

[0022] For example, the cytoplasmic delivery peptide has about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more identity to the amino acid sequence of any one of SEQ ID NOs: 19 to 27. In one embodiment, the cytoplasmic delivery peptide comprises any one of the amino acid sequences of SEQ ID NOs: 19 to 27, or consists of any one of the amino acid sequences of SEQ ID NOs: 19 to 27. The cytoplasmic delivery peptide may comprise any one of the amino acid sequences of SEQ ID NOs: 19 to 27 in which one or several amino acids have been deleted, substituted, or added.

[0023] In the present disclosure, "several" amino acids means preferably 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Amino acid substitutions are preferably conservative substitutions between similar amino acid residues.

[0024] The cytoplasmic delivery peptide can be produced by a known method. For example, a recombinant expression vector is constructed by known genetic engineering techniques, into which a gene encoding the cytoplasmic delivery peptide is inserted in an expressible manner, and the vector is introduced into an appropriate host cell to express the recombinant protein, followed by purification. Thus, in one aspect, the present disclosure provides a polynucleotide encoding the cytoplasmic delivery peptide, a vector comprising the polynucleotide, or a cell comprising the polynucleotide or vector.

[0025] Alternatively, the cytoplasmic delivery peptide can be produced using organic synthetic chemistry techniques, such as solid-phase peptide synthesis, liquid-phase peptide synthesis, or synthesis using a commercially available automated peptide synthesizer utilizing these techniques.

[0026] The substance can be delivered to the cytoplasm by contacting the cytoplasmic delivery peptide and the substance to be delivered into the cell in vitro or in vivo with the cell. Thus, in one embodiment, a method for delivering a substance into a cell is provided. In one embodiment, a composition comprising the cytoplasmic delivery peptide for delivering a substance into a cell is provided. The cytoplasmic delivery peptide and the substance to be delivered into the cell may be bound to each other.

[0027] For example, a substance can be delivered into cells in vitro by culturing cells in a medium containing a mixture of a cytoplasmic delivery peptide and a substance to be delivered into cells, or a conjugate of a cytoplasmic delivery peptide and a substance to be delivered into cells. The concentration of the cytoplasmic delivery peptide in the medium may be a concentration that can deliver the substance into cells and is not cytotoxic, for example, about 1 μM to about 100 μM, about 2 μM to about 50 μM, about 3 μM to about 30 μM, or about 5 μM to about 20 μM, for example, about 10 μM. The culture time may be a time that can deliver the substance into cells and is not cytotoxic, for example, about 1 hour or more, about 2 hours or more, about 3 hours or more, about 6 hours or more, about 8 hours or more, about 12 hours or more, or about 18 hours or more, and about 48 hours or less, about 24 hours or less, or about 18 hours or less, for example, about 18 hours.

[0028] The cell is a eukaryotic cell, such as an animal cell, a plant cell or a fungal cell. The animal cell is, for example, a mammalian cell. The species of the animal is not limited, and is, for example, human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, and especially human. The tissue from which the cell is derived is not limited. The cell may be a cell established as a cell line, for example, A431, T24, HeLa, Colon-26, HEK293 cell.

[0029] Alternatively, for example, a conjugate of a cytoplasmic delivery peptide and a substance to be delivered into a cell can be administered to a subject to deliver the substance into the cell in vivo. For example, a conjugate of a cytoplasmic delivery peptide and a substance for treating or preventing a disease can be locally administered.

[0030] The substance to be delivered into the cell is not limited and may be an amino acid, a peptide, a protein, a nucleic acid, a lipid, a carbohydrate, a low molecular weight compound, etc., and is preferably a protein. The protein may be an antibody, an enzyme, a signal transduction molecule, a transcription factor, or an inhibitor or regulator thereof. The protein may further be bound to a nucleic acid, a lipid, a carbohydrate, a low molecular weight compound, etc. The substance to be delivered into the cell may be a substance for treating or preventing a disease.

[0031] In one embodiment, the substance to be delivered into a cell is an antibody. In the present disclosure, the term "antibody" includes fragments or derivatives thereof, i.e., molecules that contain a portion of an antibody as a component and retain antigen-binding ability, such as Fab, Fab', F(ab')2, Fv, or single-chain Fv (scFv), Fab3, diabodies, triabodies, tetrabodies, minibodies, nanobodies, Bis-scFv, (scFv)2-Fc, intact IgG, and drug-antibody conjugates. The antibody may be a human antibody or a humanized antibody.

[0032] When the cytoplasmic delivery peptide is bound to a substance to be delivered into a cell, the manner of binding is not particularly limited and may be a covalent bond, an ionic bond, a hydrophobic bond, adsorption, etc., and may be separated inside the cell. Preferably, the substance to be delivered into the cell is bound to the C-terminus of the peptide directly or via an optional linker.

[0033] For example, a cytoplasmic delivery peptide having a cysteine-containing SS-linking sequence bound to its C-terminus and a protein to be delivered into a cell having a cysteine-containing SS-linking sequence bound to its N-terminus, C-terminus, or intramolecularly can be provided, and a substance to be delivered into a cell can be bound to the cytoplasmic delivery peptide by forming a disulfide bond between these cysteines. The SS-linking sequence may be any amino acid sequence as long as it contains cysteine ​​and does not interfere with the disulfide bond.

[0034] The cytoplasmic delivery peptide and the substance to be delivered into the cell may be bound by an enzyme reaction. For example, the cytoplasmic delivery peptide and the protein to be delivered into the cell may be bound by a sortase. Sortase is an enzyme that recognizes the sequence LPXTG (SEQ ID NO: 5, X is any amino acid) in a peptide or protein, cleaves the peptide bond between T and G, and binds to a peptide or protein having at least two Gs at the N-terminus (Figure 11).

[0035] For example, a cytoplasmic delivery peptide can be linked to a protein to be delivered into a cell by a method comprising the following steps, combining a disulfide bond and a sortase reaction. (1) Providing a cytoplasmic delivery peptide having a cysteine-containing S-S bond sequence bound to its C-terminus; (2) Peptide (1) and G n contacting a peptide containing a structure of -(linker)-C (n is an integer of 2 or more) to form a disulfide bond; (3) Providing a protein having an amino acid sequence including LPXTG (SEQ ID NO: 5, where X is any amino acid) bound to the C-terminus; (4) Reacting the peptide obtained in (2) with the protein in (3) using sortase.

[0036] Alternatively, the cytoplasmic delivery peptide and the protein to be delivered into the cell can be conjugated by a method including the following steps. (1) A cytoplasmic delivery peptide with a cysteine-containing S-linkage sequence attached to the C-terminus is n providing a peptide in which a peptide having a structure of -(linker)-C (n is an integer of 2 or more) is bonded via a disulfide bond; (2) providing a protein having an amino acid sequence including LPXTG (SEQ ID NO: 5, where X is any amino acid) bound to the C-terminus; (3) Reacting the peptide of (1) with the protein of (2) using a sortase.

[0037] In these methods, the cysteine-containing S-S binding sequence bound to the C-terminus of the cytoplasmic delivery peptide is, for example, an amino acid sequence that includes or consists of GC. n The linker in -(linker)-C (n is an integer of 2 or more) may have any structure as long as it does not interfere with the S-S bond, and for example, (GS) m (m is an integer equal to or greater than 1), or a non-peptide linker such as polyethylene glycol (PEG). The linker may be absent. For example, a peptide comprising or consisting of the amino acid sequence GGGG(GS)4C (SEQ ID NO:6) may be used.

[0038] For example, the following embodiment is provided. [1] A cytoplasmic delivery peptide comprising: It contains, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, the acidic amino acid domain is composed of 1 to 3 acidic amino acids; The mastoparan domain consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, The basic amino acid domain is composed of 12 or more basic amino acids. Cytoplasmic delivery peptides. [2] The peptide according to 1 above, wherein the acidic amino acid domain consists of three acidic amino acids. [3] The peptide according to 1 or 2 above, wherein the acidic amino acid domain consists of 1 to 3 acidic amino acids selected from glutamic acid, aspartic acid, and 2-aminoadipic acid. [4] The peptide according to any one of 1 to 3 above, wherein the acidic amino acid domain consists of three acidic amino acids selected from glutamic acid, aspartic acid, and 2-aminoadipic acid. [5] The peptide according to any one of 1 to 4 above, wherein the acidic amino acid domain consists of 1 to 3 glutamic acids. [6] The peptide described in any one of 1 to 5 above, wherein the acidic amino acid domain consists of three glutamic acids. [7] The peptide according to any one of 1 to 6 above, wherein the basic amino acid domain consists of 12 to 16 basic amino acids. [8] The peptide according to any one of 1 to 7 above, wherein the basic amino acid domain consists of 16 basic amino acids. [9] The peptide according to any one of 1 to 8 above, wherein the basic amino acid domain consists of 12 or more basic amino acids selected from histidine, lysine and arginine.

[10] The peptide according to any one of 1 to 9 above, wherein the basic amino acid domain consists of 12 or more histidines.

[11] The peptide according to any one of 1 to 10, wherein the basic amino acid domain consists of 16 histidines.

[12] The peptide described in any one of 1 to 11 above, wherein the mastoparan domain comprises the amino acid sequence of SEQ ID NO: 1.

[13] The peptide described in any one of 1 to 12 above, wherein the mastoparan domain consists of the amino acid sequence of SEQ ID NO: 1.

[14] The peptide according to any one of 1 to 13, wherein the acidic amino acid domain contains at least one D-amino acid.

[15] The peptide according to any one of 1 to 14 above, wherein the N-terminus of the acidic amino acid domain is a D-amino acid.

[16] The peptide according to any one of 1 to 15 above, wherein the acidic amino acid domain consists of 1 to 3 D-amino acids.

[17] The peptide according to any one of 1 to 16, wherein the acidic amino acid domain consists of three D-amino acids.

[18] The peptide according to any one of 1 to 17 above, which consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3 or 4.

[19] The peptide described in any one of 1 to 18 above, which comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[20] The peptide described in any one of 1 to 19 above, which consists of the amino acid sequence of SEQ ID NO: 3 or 4.

[21] The peptide according to any one of 1 to 20, wherein a cysteine-containing S-S bond sequence is bound to the C-terminus.

[22] The peptide according to claim 21, wherein an amino acid sequence containing GC is attached to the C-terminus.

[23] The peptide according to claim 21 or 22, wherein the amino acid sequence consisting of GC is bound to the C-terminus.

[24] G n 24. The peptide according to any one of 21 to 23 above, wherein a peptide containing a -(linker)-C (n is an integer of 2 or more) structure is bonded via a disulfide bond.

[25] The linker is absent or (GS) m 25. The peptide according to 24, wherein m is an integer of 1 or more, or polyethylene glycol.

[26] The peptide according to any one of 21 to 25, wherein a peptide containing the amino acid sequence GGGG(GS)4C is bonded via a disulfide bond.

[27] The peptide according to any one of 21 to 26, wherein a peptide consisting of the amino acid sequence GGGG(GS)4C is bonded via a disulfide bond.

[28] The peptide according to any one of 1 to 27 above, which is bound to a substance to be delivered into a cell.

[29] The peptide according to 28 above, wherein the substance to be delivered into a cell is a protein.

[30] The peptide according to claim 29, wherein the protein is an antibody.

[31] The peptide according to 29 or 30, wherein a protein is bound to the C-terminus of the peptide.

[32] The peptide according to any one of 29 to 31 above, wherein the peptide and the protein are linked via an arbitrary amino acid sequence.

[33] The peptide according to any one of 29 to 32, wherein the peptide and the protein are bonded via a disulfide bond.

[34] The peptide according to any one of 29 to 33, wherein a cysteine-containing S-linked sequence is bound to the C-terminus of the peptide, and a cysteine-containing S-linked sequence is bound to the N-terminus, C-terminus or intramolecular portion of a protein, and a disulfide bond is formed between these cysteines.

[35] The peptide according to any one of 29 to 34, wherein a cysteine-containing S-linked sequence is bound to the C-terminus of the peptide, a cysteine-containing S-linked sequence is bound to the C-terminus of a protein, and a disulfide bond is formed between these cysteines.

[36] The SS bond sequence attached to the C-terminus of the protein is LPXTG. n 36. The peptide according to 35 above, comprising a -(linker)-C structure, where X is any amino acid, and n is an integer of 2 or more.

[37] The linker is absent or (GS) m 37. The peptide according to claim 36, wherein m is an integer of 1 or more, or polyethylene glycol.

[38] The peptide according to any one of 35 to 37, wherein the SS bond sequence bound to the C-terminus of the protein comprises the amino acid sequence LPXTGGGG(GS)4C (X is any amino acid).

[39] The peptide according to any one of 35 to 38, wherein the SS bond sequence bound to the C-terminus of the protein consists of the amino acid sequence LPXTGGGG(GS)4C (X is any amino acid).

[40] The peptide according to any one of 35 to 39, wherein the SS bond sequence bound to the C-terminus of the peptide comprises the amino acid sequence of GC.

[41] The peptide according to any one of 35 to 40, wherein the SS bond sequence bound to the C-terminus of the peptide consists of the amino acid sequence of GC.

[42] A method for attaching a cytoplasmic delivery peptide to a protein, comprising: (1) Providing a peptide according to any one of 1 to 23, in which a cysteine-containing S-S bond sequence is bound to the C-terminus, (2) Peptide (1) and G n contacting a peptide containing a structure of -(linker)-C (n is an integer of 2 or more) to form a disulfide bond; (3) Providing a protein having an amino acid sequence including LPXTG (X is any amino acid) bound to the C-terminus; (4) reacting the peptide obtained in (2) with the protein in (3) using a sortase; The method includes:

[43] A method for attaching a cytoplasmic delivery peptide to a protein, comprising: (1) The peptide according to any one of 1 to 23, in which a cysteine-containing S-S bond sequence is bound to the C-terminus, n providing a peptide in which a peptide having a structure of -(linker)-C (n is an integer of 2 or more) is bonded via a disulfide bond; (2) Providing a protein having an amino acid sequence including LPXTG (X is any amino acid) bound to its C-terminus; (3) reacting the peptide of (1) with the protein of (2) using a sortase; The method includes:

[44] (1) The peptide according to any one of 1 to 23, wherein a cysteine-containing S-S bond sequence is bound to the C-terminus, n A peptide comprising the structure -(linker)-C (n is an integer of 2 or more) and a sortase, or (2) The peptide according to any one of 1 to 23, in which a cysteine-containing S-S bond sequence is bound to the C-terminus, is further treated with G n A peptide in which a peptide having a structure of -(linker)-C (n is an integer of 2 or more) is linked via a disulfide bond, and a sortase A kit for conjugating a cytoplasmic delivery peptide to a protein, comprising:

[45] A polynucleotide encoding any one of the peptides described in 1 to 23.

[46] A vector comprising the polynucleotide described in 45.

[47] A cell comprising the polynucleotide described in 45 or the vector described in 46.

[48] ​​A composition for delivering a substance into a cell, comprising the peptide described in any one of 1 to 41.

[49] A method for delivering a substance into a cell, comprising contacting a cell with the peptide according to any one of 1 to 27 and a substance to be delivered into the cell.

[50] The method according to the above 49, wherein the peptide according to any one of the above 1 to 27 is bound to a substance to be delivered into a cell.

[51] A method for delivering a substance into a cell in vitro, comprising culturing cells in a medium containing the peptide according to any one of 1 to 27 and a substance to be delivered into the cell.

[52] The method according to claim 51, wherein the peptide according to any one of claims 1 to 27 is bound to a substance to be delivered into a cell.

[53] A method for delivering a substance into a cell of a subject, comprising administering to the subject the peptide according to any one of 1 to 27 and the substance to be delivered into the cell.

[54] The method according to the above item 53, wherein the peptide according to any one of items 1 to 27 is bound to a substance to be delivered into a cell.

[55] A cytoplasmic delivery peptide, It contains, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, the acidic amino acid domain is composed of 1 to 3 acidic amino acids; The mastoparan domain consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, The basic amino acid domain is composed of four or more basic amino acids. Cytoplasmic delivery peptides.

[56] The peptide according to claim 55, wherein the basic amino acid domain comprises arginine.

[57] The peptide according to 55 or 56, wherein the basic amino acid domain consists of four or more arginines.

[58] The peptide according to any one of 55 to 57, wherein the basic amino acid domain consists of 8 or more arginines.

[0039] Also, for example, the following embodiments are provided. [1] A cytoplasmic delivery peptide comprising: comprising, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, or comprising, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain; the acidic amino acid domain is composed of 1 to 3 acidic amino acids; The mastoparan domain consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, The basic amino acid domain is composed of 12 or more basic amino acids. Cytoplasmic delivery peptides. [2] The peptide according to 1, which comprises, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain. [3] The peptide according to 1, which comprises, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain. [4] The peptide according to any one of 1 to 3 above, wherein the acidic amino acid domain consists of three acidic amino acids. [5] The peptide according to any one of 1 to 4 above, wherein the acidic amino acid domain consists of 1 to 3 acidic amino acids selected from glutamic acid, aspartic acid, and 2-aminoadipic acid. [6] The peptide according to any one of 1 to 5 above, wherein the acidic amino acid domain consists of three acidic amino acids selected from glutamic acid, aspartic acid, and 2-aminoadipic acid. [7] The peptide according to any one of 1 to 6 above, wherein the acidic amino acid domain consists of 1 to 3 glutamic acids. [8] The peptide described in any one of 1 to 7 above, wherein the acidic amino acid domain consists of three glutamic acids. [9] The peptide according to any one of 1 to 8 above, wherein the basic amino acid domain consists of 12 to 16 basic amino acids.

[10] The peptide according to any one of 1 to 9 above, wherein the basic amino acid domain consists of 16 basic amino acids.

[11] The peptide according to any one of 1 to 10 above, wherein the basic amino acid domain consists of 12 or more basic amino acids selected from histidine, lysine and arginine.

[12] The peptide according to any one of 1 to 11 above, wherein the basic amino acid domain consists of 12 or more histidines.

[13] The peptide according to any one of 1 to 12, wherein the basic amino acid domain consists of 16 histidines.

[14] The peptide described in any one of 1 to 13 above, wherein the mastoparan domain comprises the amino acid sequence of SEQ ID NO: 1.

[15] The peptide described in any one of 1 to 14 above, wherein the mastoparan domain consists of the amino acid sequence of SEQ ID NO: 1.

[16] The peptide according to any one of 1 to 15 above, which comprises at least one D-amino acid.

[17] The peptide according to any one of 1 to 16, wherein the acidic amino acid domain contains at least one D-amino acid.

[18] The peptide according to any one of 1 to 17 above, wherein the N-terminus of the acidic amino acid domain is a D-amino acid.

[19] The peptide according to any one of 1 to 18, wherein the acidic amino acid domain consists of 1 to 3 D-amino acids.

[20] The peptide according to any one of 1 to 19, wherein the acidic amino acid domain consists of three D-amino acids.

[21] The peptide according to any one of 1 to 20 above, which consists of an amino acid sequence having 90% or more identity with any one of the amino acid sequences of SEQ ID NOs: 3, 4, and 19 to 27.

[22] The peptide according to any one of 1 to 21 above, comprising any one of the amino acid sequences of SEQ ID NOs: 3, 4, and 19 to 27.

[23] The peptide according to any one of 1 to 22, which consists of an amino acid sequence of any one of SEQ ID NOs: 3, 4, and 19 to 27.

[24] The peptide according to any one of 1 to 23 above, which consists of an amino acid sequence having 90% or more identity with any one of the amino acid sequences of SEQ ID NOs: 3, 4, and 19 to 26.

[25] The peptide according to any one of 1 to 24 above, which comprises an amino acid sequence having 90% or more identity with any one of the amino acid sequences of SEQ ID NOs: 3, 4, and 19 to 26.

[26] The peptide according to any one of 1 to 25, which consists of an amino acid sequence of any one of SEQ ID NOs: 3, 4, and 19 to 26.

[27] The peptide described in any one of 1 to 26 above, which consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3 or 4.

[28] The peptide described in any one of 1 to 27 above, which comprises the amino acid sequence of SEQ ID NO: 3 or 4.

[29] The peptide described in any one of 1 to 28 above, consisting of the amino acid sequence of SEQ ID NO: 3 or 4.

[30] The peptide according to any one of 1 to 29, wherein a cysteine-containing S-S bond sequence is bound to the C-terminus.

[31] The peptide according to claim 30, wherein an amino acid sequence containing GC is attached to the C-terminus.

[32] The peptide according to claim 30 or 31, wherein the amino acid sequence consisting of GC is bound to the C-terminus.

[33] G n 33. The peptide according to any one of 30 to 32 above, wherein a peptide containing a -(linker)-C (n is an integer of 2 or more) structure is bonded via a disulfide bond.

[34] The linker is absent or (GS) m (m is an integer of 1 or more) or polyethylene glycol.

[35] The peptide according to any one of 30 to 34, wherein a peptide containing the amino acid sequence GGGG(GS)4C is bonded via a disulfide bond.

[36] The peptide according to any one of claims 30 to 35, wherein a peptide consisting of the amino acid sequence GGGG(GS)4C is bonded via a disulfide bond.

[37] The peptide according to any one of 1 to 36, which is bound to a substance to be delivered into a cell.

[38] The peptide according to 37 above, wherein the substance to be delivered into a cell is a protein.

[39] The peptide according to claim 38, wherein the protein is an antibody.

[40] The peptide according to 38 or 39, wherein a protein is bound to the C-terminus of the peptide.

[41] The peptide according to any one of 38 to 40, wherein the peptide and the protein are linked via an arbitrary amino acid sequence.

[42] The peptide according to any one of 38 to 41 above, wherein the peptide and the protein are bonded via a disulfide bond.

[43] The peptide according to any one of 38 to 42, wherein a cysteine-containing S-linked sequence is bound to the C-terminus of the peptide, and a cysteine-containing S-linked sequence is bound to the N-terminus, C-terminus or intramolecularly of a protein, and a disulfide bond is formed between these cysteines.

[44] The peptide according to any one of 38 to 43, wherein a cysteine-containing S-linked sequence is bound to the C-terminus of the peptide, a cysteine-containing S-linked sequence is bound to the C-terminus of the protein, and a disulfide bond is formed between these cysteines.

[45] The SS bond sequence attached to the C-terminus of the protein is LPXTG. n 45. The peptide according to 44 above, comprising a structure of -(linker)-C, where X is any amino acid, and n is an integer of 2 or more.

[46] No linker or (GS) m (m is an integer of 1 or more) or polyethylene glycol.

[47] The peptide according to any one of 44 to 46, wherein the SS bond sequence bound to the C-terminus of the protein comprises the amino acid sequence LPXTGGGG(GS)4C (X is any amino acid).

[48] ​​The peptide according to any one of 44 to 47, wherein the SS bond sequence bound to the C-terminus of the protein consists of the amino acid sequence LPXTGGGG(GS)4C (X is any amino acid).

[49] The peptide according to any one of 44 to 48, wherein the SS bond sequence bound to the C-terminus of the peptide comprises the amino acid sequence of GC.

[50] The peptide according to any one of 44 to 49, wherein the SS bond sequence bound to the C-terminus of the peptide consists of the amino acid sequence of GC.

[51] A method for attaching a cytoplasmic delivery peptide to a protein, comprising: (1) Providing a peptide according to any one of 1 to 32, in which a cysteine-containing S-S bond sequence is bound to the C-terminus, (2) Peptide (1) and G n contacting a peptide containing a structure of -(linker)-C (n is an integer of 2 or more) to form a disulfide bond; (3) Providing a protein having an amino acid sequence including LPXTG (X is any amino acid) bound to the C-terminus; (4) reacting the peptide obtained in (2) with the protein in (3) using a sortase; The method includes:

[52] A method for attaching a cytoplasmic delivery peptide to a protein, comprising: (1) The peptide according to any one of 1 to 32, in which a cysteine-containing S-S bond sequence is bound to the C-terminus, n providing a peptide in which a peptide having a structure of -(linker)-C (n is an integer of 2 or more) is bonded via a disulfide bond; (2) Providing a protein having an amino acid sequence including LPXTG (X is any amino acid) bound to its C-terminus; (3) reacting the peptide of (1) with the protein of (2) using a sortase; The method includes:

[53] (1) The peptide according to any one of 1 to 32, wherein a cysteine-containing S-S bond sequence is bound to the C-terminus, n A peptide comprising the structure -(linker)-C (n is an integer of 2 or more) and a sortase, or (2) The peptide according to any one of 1 to 32, in which a cysteine-containing S-S bond sequence is bound to the C-terminus, is further treated with G n A peptide in which a peptide having a structure of -(linker)-C (n is an integer of 2 or more) is linked via a disulfide bond, and a sortase A kit for conjugating a cytoplasmic delivery peptide to a protein, comprising:

[54] A polynucleotide encoding any one of the peptides described in 1 to 32.

[55] A vector comprising the polynucleotide described in 54.

[56] A cell comprising the polynucleotide described in 54.

[57] A cell comprising the vector described in 55.

[58] A composition for delivering a substance into a cell, comprising the peptide described in any one of 1 to 50.

[59] A method for delivering a substance into a cell, comprising contacting a cell in vitro with the peptide described in any one of 1 to 36 and a substance to be delivered into the cell.

[60] The method according to the above item 59, wherein the peptide according to any one of items 1 to 36 is bound to a substance to be delivered into a cell.

[61] A method for delivering a substance into a cell in vitro, comprising culturing cells in a medium containing the peptide according to any one of 1 to 36 and a substance to be delivered into the cell.

[62] The method according to claim 61, wherein the peptide according to any one of claims 1 to 36 is bound to a substance to be delivered into a cell.

[63] A method for delivering a substance into a cell of a subject, comprising administering to the subject the peptide according to any one of 1 to 36 and the substance to be delivered into the cell.

[64] The method according to claim 63, wherein the peptide according to any one of claims 1 to 36 is bound to a substance to be delivered into a cell.

[65] A cytoplasmic delivery peptide, comprising, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, or comprising, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain; the acidic amino acid domain is composed of 1 to 3 acidic amino acids; The mastoparan domain consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, The basic amino acid domain is composed of four or more basic amino acids. Cytoplasmic delivery peptides.

[66] The peptide according to claim 65, which comprises, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain and a basic amino acid domain.

[67] The peptide according to claim 65 or 66, wherein the basic amino acid domain comprises arginine.

[68] The peptide according to any one of 65 to 67, wherein the basic amino acid domain consists of four or more arginines.

[69] The peptide according to any one of 65 to 68, wherein the basic amino acid domain consists of 8 or more arginines.

[0040] All documents cited herein are hereby incorporated by reference. The present invention will be described in more detail below with reference to the following examples, but the present invention is not limited to these examples. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible without departing from the technical spirit of the present invention. EXAMPLES

[0041] Materials and Methods Materials and Reagents All materials, reagents, and cell culture media were obtained from FUJIFILM Wako Pure Chemicals, Nacalai Tesque, Sigma-Aldrich, or Thermo Fisher Scientific unless otherwise stated. Fmoc-protected amino acids were obtained from Peptide Institute or Watanabe Chemicals. Cell culture plates and shake flasks were purchased from IWAKI and violamo.

[0042] cell culture HT1080, A431, T24 and HEK293 cells were obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank. Cells were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) [DMEM(+)] supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) (Gibco). HEK293 Cre reporter cells were transfected with Flp-In (商標) HeLa cells were obtained from The European Collection of Authenticated Cell Cultures (ECACC) and cultured in α-minimal essential medium (α-MEM) [α-MEM(+)] supplemented with 10% (v / v) heat-inactivated bovine serum (BS) (Gibco). Colon-26 cells were obtained from RIKEN BRC and cultured in Roswell Park Memorial Institute 1640 medium (RPMI) [RPMI(+)] supplemented with 10% (v / v) heat-inactivated FBS. The cells were maintained at 37° C. in a humidified 5% CO2 incubator and passaged every 3-4 days. Y13-259 hybridomas obtained from ATCC were cultured in Hybridoma Serum-Free Medium and maintained at 37° C. in a humidified 10% CO2 incubator and passaged every 4 days. Expi293 cells (Invitrogen) were maintained in shake flasks at 37° C. in a humidified 8% CO2 incubator with rotation at 120 rpm in Expi293 Expression Medium (Invitrogen) and passaged every 3-4 days.

[0043] Peptide synthesis The peptides used in the experiment are shown in the table below. In the amino acid sequence, X1 represents norleucine, X2 represents 2-aminoadipic acid, and lowercase letters represent D-amino acids. The peptide chains were assembled on TGS-RAM resin (Shimadzu) using standard Fmoc solid-phase peptide synthesis (SPPS) with a Syro I peptide synthesizer (Biotage) (I. Nakase, et al., Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin tissue and macropinocytosis, Biochemistry 46 (2007) 492-501). Deprotection of the protecting groups and release of the peptides from the resin were carried out at room temperature (approximately 23°C) for 3 hours using trifluoroacetic acid (TFA) / ethanedithiol (95:5). The peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC). As previously reported (Takeuchi, T. et al., Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate, ACS Chem. Biol. 1 (2006) 299-303), E3MP(Alexa) and E3MPH16(Alexa) were chemically synthesized by Fmoc solid-phase peptide synthesis and fluorescently labeled with Alexa Fluor 488 C5 maleimide sodium salt.

[0044] [Table 2]

[0045] Cytotoxicity assessment (WST-8 assay) Cytotoxicity was measured using Cell Counting Kit-8 (CCK8) (Dojindo). HT1080 cells (5 × 10 3(100 μL) were seeded on a 96-well microplate (Iwaki) and cultured for 24 hours until 80-90% confluent. The medium was removed, washed with phosphate-buffered saline (PBS), and 100 μL of each peptide solution (in DMEM(+)) was added and cultured at 37°C for 18 hours. The cells were washed twice with PBS containing 0.5 mg / mL sodium heparin, and 100 μL of DMEM(+) containing 10 μL of WST-8 was added. After incubation at 37°C for 2 hours, the absorbance at 450 nm (A450) was measured and the survival rate was calculated by comparing with that of untreated cells.

[0046] Quantification of peptide cellular uptake HT1080 cells (5 x 10 4 (cells) were seeded in 24-well microplates (Iwaki) and cultured for 24 hours until 80-90% confluent. After washing twice with PBS, cells were treated with 10 μM of each peptide in DMEM(+) for 1 hour at 37°C. Cells were washed twice with PBS containing 0.5 mg / mL sodium heparin and incubated with 0.05% trypsin for 10 minutes at 37°C. Cells were harvested, washed with PBS, and analyzed by flow cytometry using an Attune NxT Flow Cytometer (Invitrogen). Each sample was analyzed 10,000 times. For inhibitor experiments, cells were incubated with 20 μM chlorpromazine (CPZ), 50 μM nystatin, or 40 μM 5-(N-ethyl-N-isopropyl)amiloride (EIPA) in DMEM(+) for 30 minutes before peptide addition. Subsequent incubation with 10 μM E3MPH16(Alexa) was performed in the presence of each inhibitor in DMEM(+) for 1 h.

[0047] Microscopic observation of cellular uptake of Dex10-Alexa488 HT1080 cells (2 x 10 5(10 pieces) were seeded on 35 mm glass-bottom dishes (Iwaki) and cultured for 24 hours until 80-90% confluent. After washing twice with PBS, the cells were treated with a mixture of Alexa Fluor 488-labeled dextran (10 kDa) (Dex10-Alexa488 or Alexa488-Dextran 10 kDa) (Invitrogen) (final concentration 200 μg / mL) and each peptide in DMEM(+) at 37°C for 18 hours. After washing twice with PBS containing 0.5 mg / mL heparin sodium, nuclei were stained with 5 μg / mL Hoechst 33342 (Invitrogen), DMEM(+) was added to the dishes, and the intracellular localization of Dex10-Alexa488 was observed using an FV3000 Olympus confocal microscopy (Olympus). The number of cells with diffuse cytoplasmic dextran signal was automatically counted for each sample out of a total of ≥200 cells using NIH imageJ 1.53 k and Python 3.8.8.

[0048] In experiments using various cells, A431 cells, T24 cells, HeLa cells, Colon-26 cells and HEK293 cells were treated with 200μg / mL Dex10-Alexa488 and 10μM E3MPH16 in serum-containing medium for 18 hours at 37℃. After incubation, the cells were washed twice with PBS containing 0.5mg / mL sodium heparin, the corresponding serum-containing medium was added and observed with FV3000.

[0049] For inhibitor experiments, cells were incubated with 20 μM CPZ, 50 μM nystatin, 40 μM EIPA or 20 nM concanamycin A (ConcA) in DMEM(+) for 30 min before peptide addition, and then incubated with 200 μg / mL Dex10-Alexa488 and 10 μM E3MPH16 in the presence of each inhibitor in DMEM(+) for 1 h at 37°C. After washing with PBS containing 0.5 mg / mL sodium heparin, cells were observed on an FV3000.

[0050] For 4° C. experiments, cells were preincubated for 15 min at 4° C. Cells were washed with cold PBS and incubated with 200 μg / mL Dex10-Alexa488 in the presence of 10 μM E3MPH16 in DMEM(+) for 3 h at 4° C. and observed with FV3000.

[0051] To evaluate the role of endosomal maturation in cytoplasmic delivery by E3MPH16, Rab5 WT-mCherry, Rab5 S34N-mCherry, or Rab5 Q79L-mCherry (M. Akishiba, S. Futaki, Inducible membrane permeabilization by attenuated lytic peptides: a new concept for accessing cell interiors through ruffled membranes, Mol. Pharm. 16 (2019) 2540-2548) plasmids were transfected into HT1080 cells using Lipofectamine LTX (Invitrogen) for 24 h. After washing twice with PBS, cells were treated with 200 μg / mL Dex10-Alexa488 and 10 μM E3MPH16 in DMEM(+) for 3 h at 37 °C. After washing twice with PBS containing 0.5 mg / mL sodium heparin, DMEM(+) was added to the dish, and the cells were observed with FV3000.

[0052] Delivery of NLS-GFP Enhanced green fluorescent protein (GFP) with a nuclear localization signal (NLS) added to its N-terminus and a 6-His tag added to its C-terminus (NLS-GFP) (K. Sakamoto, et al., Optimizing charge switching in membrane lytic peptides for endosomal release of biomacromolecules, Angew. Chem. Int. Ed. 59 (2020) 19990-19998) was expressed in E. coli BL21 (DE3) cells by induction with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 12 h at 18 °C. Cells were resuspended in lysis buffer [20 mM tris(hydroxymethyl)aminomethane (Tris), 300 mM NaCl, 10 mM imidazole (pH 8.0)], sonicated, and centrifuged at 18,000 rpm for 30 min. The NLS-GFP protein was purified from the soluble fraction using Ni-NTA agarose (QIAGEN) and concentrated using Amicon Ultra-4 (10 kDa cutoff) centrifugal filter units (Merck) in PBS or 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5), and the purity was confirmed by SDS-PAGE.

[0053] NLS-GFP-E3MPH16 conjugate was produced. The outline is shown in Figure 11. The transpeptide transfer reaction was carried out by adding 20 μM sortase A, 20 μM NLS-EGFP-srtA tag (LPETGG), and 100 μM E3MPH16(SS)-G4 to a transfer buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2) and reacting for 18 hours at 4°C. After that, the conjugate was eluted with PBS(-) using a Biospin column (Bio-Rad).

[0054] Colon-26 cells (2 × 10 5(10 pieces) were seeded in a 24-well microplate and cultured for 24 hours until 80-90% confluent. After washing twice with PBS, the cells were treated with 5 μM NLS-GFP and 10 μM E3MPH16 or 4 μM NLS-GFP-E3MPH16 conjugate in DMEM(+) at 37°C for 18 hours. After washing twice with PBS containing 0.5 mg / mL sodium heparin, the nuclei were stained with 5 μg / mL Hoechst 33342, DMEM(+) was added to the dish, and the intracellular localization of Dex10-Alexa488 was observed with FV3000.

[0055] Delivery of Cre recombinase Cre recombinase protein with a 6-His tag added to its C-terminus (Y. Kawaguchi, et al., supra) was expressed in E. coli BL21 (DE3) cells by induction with 1 mM IPTG for 4 h at 37°C. The cells were harvested by centrifugation, suspended in binding buffer [10 mM Tris, 100 mM NaH2PO4, 500 mM NaCl, 10 mM imidazole (pH 8.0)] containing Complete, Mini, EDTA-free protease inhibitor (Roche), lysozyme, DNaseI, and RNaseA, sonicated, centrifuged, and purified on a His-trap FF column (GE Healthcare) using Akta Start (GE Healthcare). The protein bound to the column was eluted with elution buffer [10 mM Tris, 100 mM NaH2PO4, 500 mM NaCl, 500 mM imidazole (pH 8.0)]. The sample was dialyzed overnight at 4°C in HEPES buffer [20 mM HEPES, 1 M NaCl (pH 7.4)], and the purity was confirmed by SDS-PAGE.

[0056] Cre-loxP recombination assay The HEK293 Flp-In system (Invitrogen) was used to generate Cre reporter cells (Y. Kawaguchi, et al., supra). HEK293 Cre reporter cells (7 × 10 4(1000 pieces) were seeded in a 24-well microplate and cultured in DMEM(+) for 24 hours. HEK293 Cre reporter cells were treated with 5 μM Cre in DMEM(+) for 24 hours at 37°C in the presence or absence of peptide. The number of Cre reporter cells expressing GFP was analyzed by flow cytometry, and recombination efficiency was calculated.

[0057] Preparation of antibodies Expi293 (商標) Anti-GFP-IgG (IgG = immunoglobulin G) was prepared using the Expression System (Thermo Fisher Scientific). A plasmid (Addgene, 114492) (25 μg) encoding anti-GFP-IgG was mixed with ExpiFectamine 293 (Thermo Fisher Scientific) and transfected onto Expi293 cells (3 × 10 6 The cells were transfected into a 100-well plate (25 mL, 150 μL of ExpiFectamine293 Transfection Enhancer 1 and 1.5 mL of Enhancer 2). 24 hours after transfection, 150 μL of ExpiFectamine293 Transfection Enhancer 1 and 1.5 mL of Enhancer 2 were added and incubated for 5 days. After centrifugation, the supernatant containing the antibody was sterilized by filtration through a 0.45 μm filter. The solution was applied to a HiTrap Protein A High Performance (GE Healthcare). IgG was released from the column by a pH gradient of elution buffer (0.1 M glycine buffer, pH = 2.7) and the solution was neutralized by adding 1 M Tris-HCl. The fraction containing anti-GFP-IgG was exchanged into PBS by dialysis and the purity was confirmed by SDS-PAGE. The anti-GFP-IgG thus obtained was stored at 4 °C.

[0058] Anti-Ras IgG was purified from the supernatant of the Y13-259 hybridoma culture medium. Y13-259 hybridoma cells (1.5 × 10 5The cells were seeded in a 10 cm non-treated dish (10 mL) and cultured at 37°C for 5 days. The supernatant was collected by centrifugation and filtered. The solution was applied to a HiTrap Protein G High Performance (GE Healthcare), and the IgG was released from the column by a pH gradient of elution buffer (0.1 M glycine buffer, pH = 2.7). The solution was neutralized and dialyzed against PBS, and the purity was confirmed by SDS-PAGE. The anti-Ras-IgG thus obtained was stored at 4°C.

[0059] The antigen recognition ability of the purified antibodies was confirmed by immunostaining. HeLa or HT1080 cells were transfected with a plasmid encoding HRas(G12V) fused to GFP at the N-terminus [GFP-HRas(G12V)] using Lipofectamine LTX (Thermo Fisher Scientific). This plasmid was kindly provided by Karel Svoboda (Addgene, 18666) (R. Yasuda, et al., Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging, Nat. Neurosci. 9 (2006) 283-291). After 24 hours of incubation, the cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100, and treated with primary antibodies anti-GFP-IgG or anti-Ras-IgG. Cells were treated with anti-mouse IgG-Alexa594, nuclei were stained with 5 μg / mL Hoechst 33342, and colocalization of anti-GFP-IgG or anti-Ras-IgG with GFP-HRas(G12V) was observed using FV3000.

[0060] Intracellular delivery of antibodies HT1080 cells were transfected with a plasmid encoding GFP-HRas(G12V) using Lipofectamine LTX (Thermo Fisher Scientific). After washing twice with PBS, cells were treated with 400 μg / mL anti-GFP-IgG in DMEM(+) with or without 10 μM E3MPH16 for 18 h at 37°C. After washing twice with PBS containing 0.5 mg / mL sodium heparin, cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100, and treated with secondary antibody anti-mouse IgG-Alexa594. Nuclei were stained with 5 μg / mL Hoechst 33342, and colocalization of anti-GFP-IgG and GFP-HRas(G12V) was observed with FV3000.

[0061] Cell proliferation inhibition assay Cell viability assays were performed using Cell Counting Kit-8 (Dojinjo). HT1080 cells (5 × 10 3 (100 μL) were seeded in 96-well microplates and cultured for 24 hours until 80-90% confluent. The cells were washed twice with PBS and treated with 500 μg / mL or 167 μg / mL human serum IgG or anti-Ras-IgG in DMEM(+) with or without 10 μM E3MPH16 for 24 hours at 37°C, washed twice with PBS containing 0.5 mg / mL sodium heparin, and 100 μL of DMEM(+) containing 10 μL of WST-8 solution was added. After incubation for 2 hours at 37°C, the absorbance at 450 nm was measured and proliferation was calculated relative to untreated cells.

[0062] In vivo delivery BALB / c mice (5 weeks old; female) were inoculated with CT26 cells (1 × 10 6 The average tumor volume was 100 mm 3Once the tumor reached the endothelial cell mass, samples (5 μM NLS-GFP, 5 μM NLS-GFP and 10 μM E3MPH16, or 4 μM NLS-GFP-E3MPH16 conjugate in 50 μL PBS) were injected locally. 24 hours after injection, mice were sacrificed and tumor samples were collected. Fresh tumors were embedded in TissueTek OCT compound (SAKURA finetek) and rapidly frozen in a deep freezer. Frozen samples were sectioned at 6-8 μm thickness using a cryostat. Sections were fixed in 10% formalin in 0.1 M cacodylate buffer, stained with 4',6-diamidino-2-phenylindole (DAPI), and observed with an FV3000.

[0063] Subcellular localization of peptides HT1080 cells (2 x 10 5 (10 pieces) were seeded on 35 mm glass-bottom dishes (Iwaki) and cultured for 24 hours until 80-90% confluent. After washing twice with PBS, cells were treated with 5 μM E3MP(Alexa), E3MPH16(Alexa) or MP(Alexa) in DMEM(+) at 37°C for 1 hour. After washing twice with PBS containing 0.5 mg / mL sodium heparin, cells were added to the dishes and the intracellular distribution of fluorescently labeled peptides was analyzed with FV3000 without fixation. For analysis of colocalization of E3MPH16(Alexa) with endosomes, cells were treated with 10 μM E3MPH16(Alexa) for 1 hour and washed with PBS containing 0.5 mg / mL sodium heparin, after which endosomes were labeled with 500 nM LysoTracker Red DND-99 (Invitrogen) in DMEM(+) and cells were visualized with FV3000.

[0064] Preparation of liposomes Phospholipids were purchased from Avanti Polar Lipids. Neutral and acidic large unilamellar liposomes (LUVs) were prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) at molar ratios of 1:0 and 3:1, respectively (M. Akishiba, et al., Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide, Nat. Chem. 9 (2017) 751-761). Chloroform solutions of each phospholipid were mixed in the appropriate ratio, volatile solvents were removed by rotary evaporation, and the mixture was completely dried under high vacuum overnight. A dye buffer containing 12.5 mM 8-aminonaphthalene 1,3,6-trisulfonic acid (ANTS), 45 mM p-xylene-bis-pyridinium bromide (DPX), 150 mM NaCl, and 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.0 or 7.0) was added to the residue, vortexed, and the lipid suspension was subjected to five consecutive cycles of freeze-thaw (M. Smolarsky, et al., A simple fluorescent method to determine complement-mediated liposome immune lysis, J. Immunol. Methods 15 (1977) 255-265). The resulting suspension was extruded through a polycarbonate filter (100 nm) using an extruder (Avanti Polar Lipids). LUVs were separated by gel filtration [column: PD-10 (GE Healthcare); eluent: 10 mM MES (pH 5.0 or 7.0) containing 150 mM NaCl] to remove the outer fluorescent dye. Total lipid concentration was determined by an enzymatic method (LabAssay (商標) The PC was calculated by measuring the PC using a phospholipid (FUJIFILM Wako Chemicals).

[0065] Leakage assay The membrane-perturbing activity of the peptides was monitored by the increase in fluorescence induced by leakage of ANTS and DPX from the LUVs (M. Akishiba, et al., supra). Peptide solutions in 10 mM MES (pH 5.0 or 7.0) containing 150 mM NaCl were added to LUVs containing ANTS / DPX solutions (final 200 μM with respect to phospholipids) in the same buffer. After shaking at 25°C for 1 h, the change in the fluorescence signal was measured using 315 nm excitation and 455 nm emission filters in a VICTOR Nivo multimode plate reader (PerkinElmer Life Sciences). The leakage (%) was calculated as follows: leakage (%) = (Fobs-Fblank) / (Fmax-Fblank) x 100, where Fmax is the maximum fluorescence intensity after addition of TritonX-100 (0.1% (v / v)).

[0066] statistical analysis All statistical analyses were performed using JMP Pro (ver. 17.0.0; SAS Institute). For comparison of two groups, an unpaired Student's t test was used after verifying equal variances with the F test. For multiple comparison analysis, one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test or Tukey-Kramer honestly significant difference test was used. A calculated p value of <0.05 was considered to indicate a significant difference.

[0067] [result] Peptide cytotoxicity The cytotoxicity of MP, E3MP and E3MPH16 was evaluated in HT1080 cells. The results are shown in Figure 3. E3MPH16, like E3MP, showed no cytotoxicity even at 100 μM. In contrast, when treated with MP, cell viability was reduced to approximately 80% at 10 μM.

[0068] Cellular uptake of peptides To evaluate the cellular uptake of E3MP and E3MPH16, the C-terminus of E3MP and E3MPH16 was labeled with Alexa Fluor 488 via a Gly-Gly-Cys linker (E3MP(Alexa) and E3MPH16(Alexa)). HT1080 cells were incubated with 10 μM of each peptide for 1 h, and total cellular uptake was analyzed by flow cytometry. The results are shown in Figure 4. The amount of E3MPH16(Alexa) uptake was 40-fold higher compared to E3MP(Alexa).

[0069] Peptide-mediated cytoplasmic delivery of macromolecules The ability of E3MPH16 to deliver macromolecules to the cytoplasm was evaluated using Alexa Fluor 488-labeled dextran (10 kDa) (Dex10-Alexa488) as a model for macromolecules. HT1080 cells were treated with a mixture of 200 μg / mL Dex10-Alexa488 and 10 μM E3MP, E3MPH16 or H16 for 18 h and observed by CLSM. The results are shown in Figure 5. Granular fluorescent signals indicate that Dex10-Alexa488 has accumulated in endosomes, while diffuse fluorescent signals indicate that Dex10-Alexa488 has diffused into the cytoplasm. Diffusion into the cytoplasm was seen in cells treated with E3MPH16 but not in cells treated with E3MP or H16. Similar experiments were performed in various cell types, and E3MPH16 delivered dextran to the cytoplasm in all cells (Figure 6).

[0070] Peptide-mediated cytoplasmic delivery of proteins (1)NLS-GFP We evaluated the intracellular delivery of GFP with a nuclear localization signal conjugated by E3MPH16. HT1080 cells were treated with 5 μM NLS-GFP and 10 μM E3MPH16 for 18 hours and observed by CLSM. The results are shown in Figure 7. Accumulation of NLS-GFP in the nucleus was confirmed. This indicates that NLS-GFP was delivered to the cytoplasm by E3MPH16 while maintaining its functionality, and then translocated into the nucleus.

[0071] (2) Cre We evaluated the cytoplasmic delivery of functional proteins by E3MPH16 using a Cre-loxP recombination assay. We used a previously developed Cre reporter cell line (Y. Kawaguchi, et al., supra). The gene encoding loxP-DsRed-loxP-EGFP was introduced into the HEK293 cell line. The reporter cell constitutively expresses red fluorescent protein (DsRed), but when Cre recombines between the loxP sequences, it produces green fluorescent protein (GFP). Thus, the intracellular activity of Cre can be monitored by the change in fluorescence color from red to green. The cells were incubated with 5 μM Cre and 10 μM E3MPH16 for 24 hours, and the recombination efficiency was evaluated by flow cytometry. The results are shown in Figure 8. Recombination occurred in approximately 25% of the cells treated with E3MPH16. This indicates that E3MPH16 can efficiently deliver Cre to the cytoplasm while maintaining its function.

[0072] (3) Antibodies We examined whether E3MPH16 could deliver exogenous antibodies into cells and whether the antibodies could recognize intracellular antigens by immunostaining. HT1080 cells were transiently expressed with GFP-HRas(G12V). GFP-HRas(G12V) is a mutant Ras protein that activates Raf1 and Rac, which is suggested to be involved in cancer cell survival. Prior to the delivery experiment, we confirmed that GFP-HRas(G12V) was expressed and localized along the cell membrane. Cells were incubated with anti-GFP-IgG in the presence of E3MPH16 for 24 hours. The cells were fixed and stained with a secondary antibody labeled with Alexa594. The results are shown in Figure 9. Almost no secondary antibody signal was observed in cells treated with anti-GFP-IgG alone, but significant colocalization of GFP and Alexa594 fluorescent signals was observed in cells treated with anti-GFP-IgG in the presence of E3MPH16. This result indicates that E3MPH16 can deliver IgG to the cytoplasm and that the delivered IgG can bind to target proteins within the cells.

[0073] It is known that microinjection of anti-Ras-IgG, which recognizes various Ras protein mutants, into cells inhibits the interaction of Ras with Raf-1 and inhibits cell proliferation. To examine whether delivery of anti-Ras-IgG by E3MPH16 leads to cell proliferation inhibition, anti-Ras-IgG was prepared and cell proliferation inhibition was evaluated by WST-8 assay. The results are shown in Figure 10. No cell proliferation inhibition was observed in samples treated with control IgG or anti-Ras-IgG alone, or in samples treated with control IgG (polyclonal IgG derived from human serum) in the presence of E3MPH16, but significant proliferation inhibition was observed (approximately 20%) when treated with anti-Ras-IgG in the presence of E3MPH16. This inhibitory effect was comparable to the effect of intracellular expression of scFv derived from the variable region of anti-Ras-IgG (H. Kabayama, et al., An ultra-stable cytoplasmic antibody engineered for in vivo applications, Nat. Commun. 11 (2020) 336). These results indicate that E3MPH16 can efficiently deliver various proteins, such as antibodies, to the cytoplasm to exert their functions.

[0074] Cytoplasmic delivery of peptide-conjugated proteins Using sortase, we prepared a conjugate of NLS-GFP and E3MPH16 (Figures 11 and 12). The conjugate was confirmed to have been obtained by non-reducing SDS-PAGE. HT1080 cells were treated with a mixture of 4 μM NLS-GFP and 10 μM E3MPH16 or 4 μM NLS-GFP-E3MPH16-conjugate for 18 hours and observed by CLSM. The results are shown in Figure 12. The conjugate delivered NLS-GFP into the nucleus with high efficiency.

[0075] Cytoplasmic delivery of various peptides (1) Number of histidine residues HT1080 cells were treated with a mixture of 200 μg / mL Dex10-Alexa488 and 10 μM E3MPH10, E3MPH12, E3MPH14 or E3MPH16 for 18 hours and observed by CLSM. The results are shown in Figure 13. Diffusion of fluorescence into the cytoplasm was observed in cells treated with E3MPH12, E3MPH14 and E3MPH16, but was hardly observed in cells treated with E3MPH10. This result indicates that the delivery efficiency increases when there are 12 or more histidines at the C-terminus.

[0076] (2) N-terminal D amino acid HT1080 cells were treated with a mixture of 200 μg / mL Dex10-Alexa488 and 10 μM E3MPH16, e3MPH16E3, e3MPH16 or e3MPh16 for 18 hours and observed by CLSM. The results are shown in Figure 14. The delivery efficiency to the cytoplasm was improved by changing the N-terminal glutamic acid (E) residue of E3MPH16 to the D form.

[0077] Cre reporter cells were incubated with 5 μM Cre recombinase and 10 μM E3MPH16, E3MPH16E3, e3MPH16, e3MPH16 or e3mph16 for 24 hours, and the recombination efficiency was evaluated by flow cytometry. The results are shown in Figure 15. By converting the N-terminal glutamic acid (E) residue of E3MPH16 to the D-form, the delivery of the recombinant protein Cre into the cells and the subsequent nuclear translocation were promoted, suggesting that the recombination of chromosomal DNA via loxp site recognition on the chromosome was induced with high efficiency.

[0078] In vivo cytoplasmic delivery We investigated whether peptides can deliver proteins in vivo. 5 μM NLS-GFP, 5 μM NLS-GFP and E3MPH16, or 4 μM NLS-GFP-E3MPH16 conjugate was administered locally to xenograft tumor model mice. After 24 hours, tissues were excised, frozen sections were prepared, and observed under a confocal microscope. The results are shown in Figure 16. In the xenografts of mice treated with NLS-GFP and E3MPH16, or NLS-GFP-E3MPH16 conjugate, a significant GFP fluorescent signal was detected, and colocalization of the NLS-GFP signal with DAPI (nuclear staining) was also observed, but no signal was observed in mice treated with NLS-GFP alone. This result indicates that E3MPH16 can deliver macromolecules such as proteins to the cytoplasm in vivo.

[0079] Peptide internalization pathway Using Dex10-Alexa488 as a model for macromolecules, we investigated the pathway by which E3MPH16 delivers macromolecules to the cytoplasm. HT1080 cells were treated with a mixture of 200 μg / mL Dex10-Alexa488 and 10 μM E3MPH16 and observed by CLSM. The results are shown in Figures 17 and 18. After 1 hour of incubation, granular fluorescent signals were observed, indicating accumulation in endosomes. After 3 hours, the percentage of cells with fluorescent signals spreading to the cytoplasm increased to about 50%. By 6 hours, this percentage increased to 85%, and reached 100% after 18 hours. This result indicates that E3MPH16 delivers materials to the cytoplasm from the endosomal pathway.

[0080] To determine the endocytic pathway involved in the cytoplasmic delivery of E3MPH16, experiments were performed with various endocytosis inhibitors. Chlorpromazine (CPZ) was used as an inhibitor of clathrin-mediated endocytosis, nystatin as an inhibitor of caveolae-mediated endocytosis, and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) as an inhibitor of macropinocytosis. HT1080 cells were first incubated with these endocytosis inhibitors, and then incubated with the inhibitors and 10 μM E3MPH16(Alexa). Total cellular uptake was assessed by flow cytometry. The results are shown in Figure 19. CPZ treatment reduced the total cellular uptake of E3MPH16(Alexa) by about 20%, whereas treatment with nystatin and EIPA reduced it by about 50% and about 90%, respectively. Microscopic observation revealed that treatment with nystatin and EIPA resulted in a dot-like reduction in the fluorescent signal. This result indicates that caveolae-mediated endocytosis and macropinocytosis are important for the cellular uptake of E3MPH16(Alexa). The cytoplasmic release of Dex10-Alexa488 by E3MPH16 was also significantly impaired by nystatin and EIPA (Fig. 20), consistent with the results in Fig. 19.

[0081] Endocytosis is energy dependent, so it does not function under conditions that suppress ATP production, such as low temperature. Cells were incubated at 4°C for 15 min, then treated with E3MPH16 and Dex10-Alexa488 at 4°C for 3 h, and observed by CLSM. The results are shown in Figure 21. No significant cytoplasmic release of Dex10-Alexa488 was observed.

[0082] We investigated the necessity of endosome maturation for E3MPH16-induced release of endosome-trapped cargo into the cytoplasm. We used concanamycin A (ConcA), an inhibitor of endosomal pH acidification. The results are shown in Figure 22. In the presence of ConcA, the Dex10-Alexa488 signal in the cytoplasm was significantly reduced.

[0083] To investigate whether endosomal escape occurs in the early or late endosomal compartment, we utilized mutants of the Rab5 protein that regulates endosomal maturation. The Rab5 S34N mutant inhibits conversion to early endosomes, and the Rab5 Q79L mutant inhibits maturation to late endosomes. For visualization, wild-type Rab5 (Rab5 WT) or fusion proteins of these mutants with mCherry (Rab5 WT-mCherry, Rab5 S34N-mCherry, and Rab5 Q79L-mCherry) were transiently expressed in HT1080 cells. The cells were treated with E3MPH16 and Dex10-Alexa488 for 3 hours. The results are shown in Figure 23. In cells expressing Rab5 WT-mCherry and Rab5 Q79L-mCherry, approximately 40% of the cells had diffuse Dex10-Alexa488 signals in the cytoplasm, suggesting cytoplasmic release of Dex10-Alexa488. Cells expressing Rab5 S34N-mCherry had granular Dex10-Alexa488 signals without cytoplasmic distribution, suggesting that Dex10-Alexa488 was retained in endosomes. The efficiency of cytoplasmic release of Dex10-Alexa488 in cells expressing Rab5 WT-mCherry and Rab5 Q79L-mCherry was comparable to the results in Figure 17. These results indicate that early endosomes are important for intracellular delivery by E3MPH16.

[0084] The intracellular localization of E3MPH16(Alexa) was analyzed. HT1080 cells were incubated with 10 μM E3MPH16(Alexa) for 1 hour and then stained with LysoTracker Red. The results are shown in FIG. 24. E3MPH16(Alexa) showed strong dot-like signals and colocalized with LysoTracker Red. This result indicates that E3MPH16 is taken up into endosomes by endocytosis and trapped in lysosomes.

[0085] Liposome leakage assay Liposome leakage assay was used to analyze the lipid composition and membrane disruption tendency of E3MPH16, MP, and E3MP depending on pH. Large unilamellar liposomes (LUVs) were prepared by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a model of cell membrane at pH 7.4, and POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) in a 3:1 ratio as a model of endosomal membrane at pH 5. These LUVs contained 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and its quencher p-xylene-bis-pyridinium bromide (DPX). In these LUVs, the fluorescence of ANTS is quenched by DPX. When the dye is released by peptide-induced LUV rupture, the fluorescence of ANTS is restored, and the membrane-disrupting activity of the peptide can be evaluated from the increase in fluorescence intensity. The results are shown in Figure 25. Although almost no pH / lipid-dependent changes were observed in the lytic activity of MP, a significant decrease in the lytic activity against LUVs of the cell membrane model was observed for E3MPH16 and E3MP. In particular, E3MPH16 did not show lytic activity against LUVs of the cell membrane model even at a P / L ratio (molar ratio of peptide / lipid) of 0.1. Conversely, in the endosomal membrane model, E3MPH16 showed lytic activity almost comparable to that of MP. By introducing three Glu residues into the N-terminus of MP, the lytic activity against LUVs of the cell membrane model was significantly attenuated. The addition of the H16 segment further attenuated the lytic activity of E3MP against LUVs of the cell membrane model. At a P / L ratio of 0.5, almost complete switching of the lytic activity of E3MPH16 was achieved between the endosomal membrane model and the cell membrane model, indicating that the design of E3MPH16 is valid.

[0086] Replacement of N-terminal glutamic acid with an acidic amino acid HT1080 cells were treated with a mixture of 200 μg / mL Dex10-Alexa488 and 10 μM D3MPH16, E3MPH16 or Aad3MPH16 for 18 hours and observed by CLSM. The results are shown in Figure 26. In all cases, Dex10-Alexa488 was diffused into the cytoplasm. In other words, even when the three glutamic acids at the N-terminus of E3MPH16 were replaced with aspartic acid (D) or 2-aminoadipic acid (Aad), the intracellular delivery ability was maintained.

[0087] Evaluation of H16MPE3 The cytotoxicity of H16MPE3 in HT1080 cells was evaluated by WST-8 assay. The results are shown in Figure 27. H16MPE3 did not show cytotoxicity even at 100 μM. HT1080 cells were treated with a mixture of 200 μg / mL Dex10-Alexa488 and 10 μM H16MPE3 for 18 hours and observed by CLSM. The results are shown in Figure 28. H16MPE3 delivered Dex10-Alexa488 to the cytoplasm as well as E3MPH16. These results indicate that the function of E3MPH16 is maintained even when the three glutamic acid residues at the N-terminus and the 16 histidine residues at the C-terminus of E3MPH16 are replaced.

[0088] The above experiments revealed at least the following points. By adding various functional peptide sequences, we created the low-toxicity, highly active and versatile polymeric cytoplasmic delivery peptide E3MPH16. Using E3MPH16, we were able to successfully deliver proteins and antibodies that normally do not penetrate the membrane into the cytoplasm of various cells and allow them to exert their functions. It has been shown that E3MPH16 may be delivered more efficiently by conjugating it to a protein or antibody. NLS-GFP could be delivered into the nucleus by conjugation and mixing with E3MPH16 in vivo. [Industrial Applicability]

[0089] The present disclosure can be used in the medical field.

Claims

1. 1. A cytoplasmic delivery peptide, comprising: comprising, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain, or comprising, in order from the N-terminus, a basic amino acid domain, a mastoparan domain, and an acidic amino acid domain; the acidic amino acid domain is composed of 1 to 3 acidic amino acids; The mastoparan domain consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:1, The basic amino acid domain consists of 12 or more basic amino acids; Cytoplasmic delivery peptides.

2. The peptide according to claim 1 , comprising, in order from the N-terminus, an acidic amino acid domain, a mastoparan domain, and a basic amino acid domain.

3. The peptide according to claim 1 , wherein the basic amino acid domain consists of 12 or more basic amino acids selected from histidine, lysine and arginine.

4. The peptide of claim 1 , wherein the mastoparan domain comprises the amino acid sequence of SEQ ID NO:

1.

5. The peptide of claim 1, wherein the acidic amino acid domain comprises at least one D-amino acid.

6. The peptide according to claim 1, which consists of an amino acid sequence having 90% or more identity with any of the amino acid sequences of SEQ ID NOs: 3, 4, and 19 to 27.

7. The peptide according to claim 1, which consists of any one of the amino acid sequences of SEQ ID NOs: 3, 4 and 19 to 27.

8. The peptide according to claim 1, which consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 3 or 4.

9. The peptide according to claim 1, consisting of the amino acid sequence of SEQ ID NO: 3 or 4.

10. The peptide according to claim 1 , wherein a cysteine-containing SS bond sequence is attached to the C-terminus.

11. The peptide of claim 1 , which is linked to a substance to be delivered into a cell.

12. The peptide according to claim 11, wherein the substance to be delivered into the cell is a protein, a cysteine-containing SS bond sequence is bound to the C-terminus of the peptide, a cysteine-containing SS bond sequence is bound to the C-terminus of the protein, and a disulfide bond is formed between these cysteines.

13. 1. A method for conjugating a cytoplasmic delivery peptide to a protein, comprising: (1) Providing a peptide according to any one of claims 1 to 10, in which a cysteine-containing SS bond sequence is bound to the C-terminus; (2) Peptide (1) and G ｎ contacting a peptide containing a structure of -(linker)-C (n is an integer of 2 or more) to form a disulfide bond; (3) Providing a protein having an amino acid sequence including LPXTG (wherein X is any amino acid) linked to the C-terminus; (4) reacting the peptide obtained in (2) with the protein in (3) using a sortase; The method includes:

14. A polynucleotide encoding the peptide according to any one of claims 1 to 10.

15. A vector comprising the polynucleotide of claim 14.

16. A cell comprising the polynucleotide of claim 14.

17. A composition comprising the peptide according to any one of claims 1 to 12 for delivering a substance into a cell.

18. A method for delivering a substance into a cell, comprising contacting a cell in vitro with the peptide according to any one of claims 1 to 10 and the substance to be delivered into the cell.

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