Peptide having cell membrane permeability and screening method therefor

Abstract

Provided is a peptide having cell membrane permeability that includes an amino acid sequence X satisfying the following conditions (1)-(4), or a salt of the peptide. (1) The amino acid sequence X includes at least one type of amino acid residue selected from among R, K, and H, and the total number amino acid residues of these three types is 1-7. (2) The amino acid sequence X includes at least one type of amino acid residue selected from among Y, W, and F, and the total number of amino acid residues of these three types is 1-5. (3) The amino acid sequence X includes at least one type of amino acid residue selected from among L, V, and M, and the total number of amino acid residues of these three types is 1-4. (4) The number of amino acid residues is 6-15. Also provided is a screening method for a peptide having cell membrane permeability, said method comprising: a step A for creating a cDNA display library that includes a cDNA display molecule; a step B for bringing a cell into contact with the cDNA display library and performing incubation; and a step C for recovering the cDNA display molecule from the contents of the cell or from permeated matter.

Description

Cell membrane permeable peptides and screening method thereof

[0001] The present invention relates to a peptide having cell membrane permeability and a screening method thereof.

[0002] In recent years, molecular targeted drugs that act by targeting specific proteins or genes have attracted attention, and proteins and genes present in cells are one of the attractive targets for drug discovery. As a method for delivering drugs into cells, a method is known in which a peptide having cell membrane permeability (hereinafter also referred to as "membrane-permeable peptide") is bound to the drug or its carrier to impart cell membrane permeability (e.g., Patent Document 1).

[0003] Special Publication No. 2021-531274

[0004] There are few membrane-permeable peptides that have excellent intracellular delivery and are actually used clinically, so there is a demand for novel membrane-permeable peptides. Furthermore, while existing membrane-permeable peptides can be introduced into many cells, they are thought to lack cell selectivity. Therefore, cell-selective membrane-permeable peptides are desired to reduce side effects.

[0005] In view of the above, the main object of the present invention is to provide a novel peptide having excellent cell membrane permeability, and a further object is to provide a novel cell-selective membrane-permeable peptide.

[0006] [1] According to one aspect of the present invention, there are provided a peptide or a salt thereof having cell membrane permeability, which comprises an amino acid sequence X that satisfies the following (1) to (4): (1) comprising at least one amino acid residue selected from R, K, and H, and the total number of amino acid residues of these three types is 1 to 7; (2) comprising at least one amino acid residue selected from Y, W, and F, and the total number of amino acid residues of these three types is 1 to 5; (3) comprising at least one amino acid residue selected from L, V, and M, and the total number of amino acid residues of these three types is 1 to 4; and (4) comprising 6 to 15 amino acid residues. [2] In the peptide or salt thereof according to [1] above, the amino acid sequence X may be linear, and in the amino acid sequence X, the number of consecutive D, F, L, Q, R, S, V, and Y may each be 2 or less, and the number of consecutive other amino acid residues may be 1; alternatively, the amino acid sequence X may form a cyclic structure, and in the amino acid sequence X, the number of consecutive F, L, and Y may each be 2 or less, the number of consecutive R may be 3 or less, and the number of consecutive other amino acid residues may be 1. [3] In the peptide or salt thereof according to [1] or [2] above, the amino acid sequence X may be linear, and the total number of eight amino acid residues, E, M, A, L, Q, K, R, and H, may account for 25% to 60% of the total number of all amino acid residues constituting the amino acid sequence X, or the amino acid sequence X may form a cyclic structure via a disulfide bond between two cysteine ​​residues, and the total number of eight amino acid residues, E, M, A, L, Q, K, R, and H, may account for 25% to 60% of the total number of all amino acid residues constituting the amino acid sequence X excluding the two cysteine ​​residues forming the disulfide bond. [4] The peptide or salt thereof according to any of [1] to [3] above may have cell-selective cell membrane permeability. [5] The peptide or salt thereof according to any one of [1] to [4] above may have selective cell membrane permeability for at least one selected from macrophage cells, cancer cells, neutrophil cells, mast cells, T cells, B cells, gastrointestinal epithelial cells, the blood-brain barrier, and skin tissue.[6] The peptide or salt thereof according to any one of [1] to [5] above may have 6 to 30 amino acid residues. [7] The peptide or salt thereof according to any one of [1] to [6] above may have a GRAVY value of -2.6 to 1.9 and may be linear. [8] The peptide or salt thereof according to any one of [1] to [7] above may comprise an amino acid sequence represented by any one of SEQ ID NOS: 1 to 40 or a homologous sequence thereof, wherein the homologous sequence may include a substitution, insertion, or deletion of 1 to 5 amino acid residues in the amino acid sequence represented by any one of SEQ ID NOS: 1 to 40 and may have 60% or more sequence identity with the amino acid sequence. [9] According to another aspect of the present invention, there is provided a drug-peptide conjugate comprising a drug and the peptide or salt thereof according to any one of [1] to [8] above bound to the drug.

[10] In the drug-peptide conjugate according to [9] above, the drug may be a protein or a nucleic acid.

[11] Another aspect of the present invention provides use of the peptide or salt thereof according to any one of [1] to [8] above in drug delivery.

[12] Another aspect of the present invention provides a method for screening for peptides with cell membrane permeability, comprising: step A: preparing a cDNA display library containing cDNA display molecules; step B: contacting and incubating cells with the cDNA display library; and step C: recovering the cDNA display molecules from the contents or permeate of the cells.

[13] The screening method according to

[12] above may further comprise preparing a sub-cDNA display library using the recovered cDNA display molecules.

[14] In the screening method according to

[12] or

[13] above, selection comprising steps A, B, and C in this order may be repeated two or more times.

[0007] According to an embodiment of the present invention, a novel membrane-permeable peptide is provided. Furthermore, according to a screening method for membrane-permeable peptides according to an embodiment of the present invention, novel membrane-permeable peptides can be suitably screened for in many cell types by using a cDNA display method.

[0008] 1 is a schematic diagram illustrating an example of a method for preparing a cDNA display library. 2 is a schematic diagram illustrating the structure of an example of a cDNA display molecule. 3 is a schematic diagram illustrating a screening method according to one embodiment of the present invention. 4 is a schematic diagram illustrating a screening method according to one embodiment of the present invention. 5 is a diagram illustrating a template DNA sequence for protein expression in a cell-free system. 6 is a diagram illustrating a template DNA sequence for protein expression in a cell-free system. 7 is a diagram illustrating a template DNA sequence for protein expression in a cell-free system. 8 is a diagram illustrating a template DNA sequence for protein expression in a cell-free system. 9 is a photograph showing the results of an intracellular internalization test of eGFP-peptide fusion proteins. 10 is a photograph showing the results of an intracellular internalization test of eGFP-peptide fusion proteins. 11 is a photograph showing the results of an intracellular internalization test of IgG-peptide conjugates in macrophage cells. 12 is a diagram showing the ratio of the intracellular internalization amount of IgG-peptide conjugates to the intracellular internalization amount of IgG in macrophage cells. 13 is a diagram showing the ratio of the intracellular internalization amount of IgG-peptide conjugates to the intracellular internalization amount of IgG in macrophage cells. 1 is a diagram showing the ratio of the amount of cellular internalization of an IgG-peptide conjugate to the amount of IgG internalized in A549 cells. 2 is a diagram showing the ratio of the amount of cellular internalization of an IgG-peptide conjugate to the amount of IgG internalized in macrophage cells and A549 cells. 3 is a diagram showing the luciferase activity inhibitory effect of siRNA (LUC)-encapsulated lipid nanoparticles in A549-Luc cells. 4 is a diagram showing the luciferase activity inhibitory effect of siRNA (LUC)-encapsulated lipid nanoparticles in BxPC-3-Luc#2 cells. 5 is a diagram showing the luciferase expression effect of mRNA (LUC)-encapsulated lipid nanoparticles in A549 cells. 6 is a diagram showing the luciferase expression effect of mRNA (LUC)-encapsulated lipid nanoparticles in Panc-1 cells.

[0009] Preferred embodiments of the present invention will be described below, but the present invention is not limited to these embodiments. Furthermore, unless inappropriate in the context, the respective embodiments can be combined as appropriate. Furthermore, in this specification, the term "to" indicating a numerical range includes the upper and lower limits.

[0010] As used herein, amino acids or amino acid residues may be represented by the following single-letter symbols: alanine (A), leucine (L), arginine (R), lysine (K), asparagine (N), methionine (M), aspartic acid (D), phenylalanine (F), cysteine ​​(C), proline (P), glutamine (Q), serine (S), glutamic acid (E), threonine (T), glycine (G), tryptophan (W), histidine (H), tyrosine (Y), isoleucine (I), and valine (V).

[0011] A. Peptides with Cell Membrane Permeability One aspect of the present invention provides a peptide or a salt thereof having cell membrane permeability, the peptide comprising an amino acid sequence X satisfying the following conditions (1) to (4): (1) comprising at least one amino acid residue selected from R, K, and H, and the total number of amino acid residues of these three types is 1 to 7; (2) comprising at least one amino acid residue selected from Y, W, and F, and the total number of amino acid residues of these three types is 1 to 5; (3) comprising at least one amino acid residue selected from L, V, and M, and the total number of amino acid residues of these three types is 1 to 4; or (4) comprising 6 to 15 amino acid residues. As described above, a peptide comprising an amino acid sequence X consisting of 6 to 15 amino acid residues, which contains a specific number of at least one basic amino acid residue selected from R, K, and H, at least one aromatic amino acid residue selected from Y, W, and F, and at least one amino acid residue having a hydrophobic linear side chain selected from L, V, and M, can exhibit excellent cell membrane permeability. In the amino acid sequence X satisfying the above (1), the total number of the three amino acid residues, R, K, and H, may be 1 to 6. The proportion of the total number of the three amino acid residues, R, K, and H, to the total number of amino acid residues constituting the amino acid sequence X may be, for example, 7% to 54%, or may be 7% to 42%. The proportion of the total number of the three amino acid residues, Y, W, and F, to the total number of amino acid residues constituting the amino acid sequence X may be, for example, 7% to 50%, or may be 8% to 50%. The proportion of the total number of the three amino acid residues, L, V, and M, to the total number of amino acid residues constituting the amino acid sequence X may be, for example, 7% to 40%. In one embodiment, the amino acid sequence X may contain at least one amino acid residue selected from K, L, and V. The number of amino acid residues constituting the amino acid sequence X may be, for example, 7 to 15, or for example, 8 to 14.

[0012] The membrane-permeable peptide may be linear or may have a cyclic structure. The cyclic structure may be formed, for example, by binding between the N-terminal amino acid residue and the C-terminal amino acid residue of the peptide, by binding between either terminal amino acid residue and an amino acid residue in a non-terminal portion, or by binding between amino acid residues in a non-terminal portion. The cyclic structure may be formed, for example, by a disulfide bond between two cysteine ​​residues (C). For example, the amino acid sequence X may be linear or may form a cyclic structure. In one embodiment, the amino acid sequence X is linear; and when amino acid residues selected from D, F, L, Q, R, S, V, and Y are present in the amino acid sequence X, the number of consecutive amino acid residues for each of these amino acid residues is 2 or less, and the number of consecutive amino acid residues for the other amino acid residues may be 1. In another embodiment, amino acid sequence X forms a cyclic structure; and when amino acid residues selected from F, L, and Y are present in amino acid sequence X, the number of consecutive occurrences of each of these amino acid residues is 2 or less, the number of consecutive occurrences of R is 3 or less, and the number of consecutive occurrences of other amino acid residues may be 1. Here, the number of consecutive occurrences of each amino acid residue represents the number of consecutive occurrences of that amino acid residue in amino acid sequence X. Thus, when the number of consecutive occurrences is 1, that amino acid residue does not occur consecutively (in other words, adjacently) in amino acid sequence X.

[0013] In one embodiment, amino acid sequence X may contain 25% to 60% amino acid residues having a tendency to form an α-helix structure selected from E, M, A, L, Q, K, R, and H (P.Y. Chou, G.D. Fasmann Adv. Enzymol. Relat. Areas Mol. Biol. 47, 45-148 (1978)). Here, the above percentage refers to the ratio of the total number of amino acid residues having a tendency to form an α-helix structure to the total number of amino acid residues constituting amino acid sequence X in the case of a linear peptide. Furthermore, the above percentage refers to the ratio of the total number of amino acid residues having a tendency to form an α-helix structure to the total number of amino acid residues constituting amino acid sequence X excluding two Cs that form disulfide bonds. In this embodiment, the above percentage is preferably 30% to 50%.

[0014] In one embodiment (embodiment a), amino acid sequence X is linear, contains at least H as a basic amino acid residue, and satisfies the following (i) and / or (ii): (i) at least one H residue is contained as two consecutive amino acid residues, i.e., HV, VH, HY, or YH; and (ii) when H is located at the C-terminus, amino acid sequence X contains one or more residues of each of six amino acids: V, T, R, E, Y, and K.

[0015] In one embodiment (embodiment b), the amino acid sequence X is linear, contains only R as a basic amino acid residue, and satisfies the following (iii) and / or (iv): (iii) at least one R residue located other than at either end is included as two consecutive amino acid residues, AR, VR, or RL; and (iv) R located at the N-terminus is included as two consecutive amino acid residues, RV or RW.

[0016] In one embodiment (embodiment c), amino acid sequence X is linear and contains only K as a basic amino acid residue, and at least two or more types of amino acid residues selected from F, V, L, and I, such that the total number of these amino acid residues accounts for 40% to 50% of the total number of amino acid residues constituting amino acid sequence X.

[0017] In one embodiment (embodiment d), the amino acid sequence X is linear and does not contain any H as basic amino acid residues, but contains one or more K and one or more R residues, and the fourth and eighth amino acid residues from the N-terminus to the C-terminus are K or R.

[0018] In one embodiment, the membrane-permeable peptide (e.g., amino acid sequence X) has a GRAVY (Grand Average of Hydropathy) value of -2.6 to 1.9. The GRAVY value is an index of the hydrophilicity or hydrophobicity of an entire peptide or protein calculated from its amino acid composition, with positive values ​​indicating a tendency toward hydrophobicity. A peptide (e.g., a linear peptide) containing at least one basic amino acid residue selected from R, K, and H, at least one aromatic amino acid residue selected from Y, W, and F, and at least one amino acid residue having a hydrophobic linear side chain selected from L, V, and M, and having a GRAVY value within the above range, may have excellent cell membrane permeability. The GRAVY value of the membrane-permeable peptide (e.g., amino acid sequence X) is preferably -2.6 to 0.7, and may be, for example, -2.6 to 0.3. The GRAVY value can be calculated according to the method of Kyte et al. (J. Kyte, R.F. Doolittle, J. Mol. Biol. 152 (1982) 105). When the amino acid sequence X is linear and has 10 or more amino acid residues, the GRAVY value of any 10 consecutive amino acid residues is preferably 1.0 or less, more preferably 0.7 or less, and even more preferably 0.3 or less.

[0019] In one embodiment, the membrane-permeable peptide contains an amino acid residue having a functional group in its side chain, more preferably at the N-terminus and / or C-terminus. Such membrane-permeable peptides can be suitably bound to drugs (e.g., proteins, nucleic acids) and their carriers by reactions utilizing the functional groups. Examples of amino acids having a functional group in their side chains include cysteine, serine, threonine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, etc. Among these, cysteine, aspartic acid, glutamic acid, and lysine are preferred.

[0020] The cells for which the membrane-permeable peptide exhibits cell membrane permeability may be any cells, including, for example, cancer cells, immune cells such as macrophages, neutrophils, T cells, and B cells, mast cells, cells or cell groups constituting skin tissues such as the epidermis and dermis, gastrointestinal epithelial cells, vascular epithelial cells, and cells or cell groups constituting the blood-brain barrier.

[0021] In one embodiment, the membrane-permeable peptide has cell-selective membrane permeability. Specifically, the peptide may exhibit higher cell membrane permeability for a specific cell (typically, the cell used in screening) than for other cells. For example, in the screening method described below, a peptide screened using lung cancer cells may exhibit higher cell membrane permeability for lung cancer cells than for other cells. Furthermore, a peptide screened using macrophage cells may exhibit higher cell membrane permeability for macrophage cells than for other cells. In the present specification, a peptide can be determined to have cell membrane permeability when it is labeled with a fluorescent dye such as Alexa Fluor fluorescent dye (Molecular Probes), Cy fluorescent dye, or FITC, or when a fusion protein of a peptide with eGFP or GFP is prepared and incubated with cells at a concentration of 5 μM for 1 hour, and fluorescence from the labeled peptide or fusion protein is confirmed within the cells by observation under a fluorescence microscope, preferably when fluorescence of higher intensity than that of a control without the peptide is confirmed; however, the determination of cell membrane permeability is not limited thereto. In one embodiment, the membrane-permeable peptide can improve the intracellular internalization of a drug by binding to the drug.

[0022] In the above-mentioned membrane-permeable peptides, one or more amino acid residues may be added to the N-terminus and / or C-terminus of amino acid sequence X. For example, peptides in which desired amino acid residues have been added to the N-terminus and / or C-terminus of amino acid sequence X from the standpoint of imparting reactivity with a drug or its carrier, adding a linker, imparting hydrophilicity or hydrophobicity, facilitating purification, etc., may also be included in the membrane-permeable peptides according to embodiments of the present invention, so long as they have cell membrane permeability. The number of added amino acid residues is not limited, as long as cell membrane permeability is maintained, and may be, for example, 1, 2, or 3 or more, and may be, for example, 24 or less, 15 or less, 10 or less, 8 or less, or 6 or less.

[0023] The lower limit of the number of amino acid residues constituting the membrane-permeable peptide may be, for example, 6 or more, 7 or more, or 8 or more. The upper limit is not particularly limited, but may be, for example, 30 or less, 25 or less, 20 or less, or 15 or less, from the viewpoint of ease of synthesis, etc.

[0024] Specific examples of the amino acid sequence X include the amino acid sequences represented by any one of SEQ ID NOS: 1 to 40 shown in Table 1, or homologous sequences thereof. In one embodiment, the membrane-permeable peptide is a peptide consisting of the amino acid sequence represented by any one of SEQ ID NOS: 1 to 40. In another embodiment, the membrane-permeable peptide is a peptide consisting of a sequence homologous to the amino acid sequence represented by any one of SEQ ID NOS: 1 to 40 (however, preferably, the homologous sequence satisfies one or more, more preferably all, of the above-mentioned (1) to (4) described with respect to amino acid sequence X, the percentage of basic amino acid residues, aromatic amino acid residues, and / or amino acid residues having a hydrophobic linear side chain, the predetermined number of consecutive amino acid residues, the percentage of amino acid residues that tend to form an α-helical structure, and the predetermined GRAVY value range). As used herein, a "homologous sequence" refers to a sequence that has, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and even more preferably 95% or more identity to the original amino acid sequence. In one embodiment, a homologous sequence may contain 1, 2, 3, 4, or 5, preferably 1, 2, or 3, more preferably 1 or 2 amino acid residues substituted, inserted, or deleted relative to the original amino acid sequence.The substitution of the amino acid residue may be, for example, a conservative substitution.Conservative substitution means replacing one amino acid residue with another amino acid residue having similar structural and / or chemical properties.For example, nonpolar amino acids include G, A, L, I, V, P, W, and M; polar neutral amino acids include S, T, C, Y, N, and Q; basic amino acids include R, K, and H; and acidic amino acids include D and E. Furthermore, for example, the substitution of the amino acid residues can be carried out so that the proportion of the total number of amino acid residues (specifically, E, M, A, L, Q, K, R, and H) that have a tendency to form the α-helical structure in the peptide sequence after substitution (i.e., the homologous sequence) is, for example, 25% to 60%, preferably 30% to 50%.Such substitution of amino acid residues can be carried out by substituting at least one amino acid residue selected from E, M, A, L, Q, K, R, and H with an amino acid residue selected from naturally occurring amino acid residues other than these and C (specifically, V, I, Y, W, F, T, G, N, P, S, and D), or by substituting at least one naturally occurring amino acid residue among the amino acid residues having a tendency to form an α-helical structure with an amino acid residue selected from the amino acid residues having a tendency to form an α-helical structure. In yet another embodiment, the membrane-permeable peptide is a peptide in which one or more amino acid residues have been added to the N-terminus and / or C-terminus of an amino acid sequence represented by any of SEQ ID NOS: 1 to 40 or a homologous sequence thereof. For example, peptides in which desired amino acid residues have been added to the N-terminus and / or C-terminus of an amino acid sequence represented by any of SEQ ID NOS: 1 to 40 or a homologous sequence thereof from the viewpoint of imparting reactivity with a drug or its carrier, providing a linker, imparting hydrophilicity or hydrophobicity, facilitating purification, etc., can also be included in the membrane-permeable peptides according to embodiments of the present invention, as long as they have cell membrane permeability. The number of added amino acid residues may be, as described above, for example, 1, 2, or 3 or more, and may be, for example, 24 or less, 15 or less, 10 or less, 8 or less, or 6 or less.

[0025]

[0026] Among the amino acid sequences represented by SEQ ID NOs: 1 to 40, the amino acid sequences represented by SEQ ID NOs: 5, 7, 8, 9, 11, 15, 16, 17, 19, 26, 28, 29, 30, 31, 34, 35, 36, 38, and 39 correspond to amino acid sequence X according to embodiment a, the amino acid sequences represented by SEQ ID NOs: 14, 18, 20, 21, 24, 25, 37, and 40 correspond to amino acid sequence X according to embodiment b, the amino acid sequences represented by SEQ ID NOs: 4, 22, 23, and 27 correspond to amino acid sequence X according to embodiment c, and the amino acid sequences represented by SEQ ID NOs: 6 and 10 correspond to amino acid sequence X according to embodiment d. Specific examples of amino acid sequence X that can suitably exhibit the effects of the present invention include the amino acid sequences represented by SEQ ID NOs: 8, 15, 19, 23, and 37, and sequences homologous thereto.

[0027] Examples of methods for producing membrane-permeable peptides include chemical synthesis methods such as solid-phase synthesis, stepwise elongation, and liquid-phase synthesis, as well as fermentation and enzymatic methods. Among these, solid-phase synthesis is preferred. Examples of solid-phase synthesis include Fmoc synthesis and Boc synthesis.

[0028] The membrane-permeable peptide may be in the form of a derivative or a salt, as long as the effects of the present invention are obtained. Thus, in this specification, unless clearly inappropriate in the context, the term "peptide" may include a derivative of the peptide or a salt of the peptide or its derivative.

[0029] Peptide derivatives include those in which functional groups such as the N-terminal amino group, C-terminal carboxyl group, side chain carboxyl group, amino group, guanidino group, hydroxyl group, and thiol group of the peptide are substituted with various substituents. The substituents are not particularly limited, and examples include alkyl groups, acyl groups, hydroxyl groups, amino groups, alkylamino groups, nitro groups, amide groups, sulfonyl groups, halogens, and various protecting groups. These substituents may be further substituted with halogens such as fluorine. Furthermore, the substitution may be the introduction of labels such as fluorescent labels and biotin labels.

[0030] The peptide salt is preferably a pharmacologically acceptable salt. Pharmacologically acceptable salts include acid addition salts and base addition salts. Acid addition salts include, for example, inorganic acid salts and organic acid salts. Inorganic acid salts include, for example, hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate. Organic acid salts include, for example, citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate. Base addition salts include, for example, inorganic base salts and organic base salts. Inorganic base salts include, for example, sodium salt, potassium salt, calcium salt, magnesium salt, and ammonium salt. Organic base salts include, for example, triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt.

[0031] B. Drug-Peptide Conjugates (Conjugates) A ​​drug-peptide conjugate according to an embodiment of the present invention comprises a drug and the membrane-permeable peptide described in Section A bound to the drug. Due to the cell membrane permeability of the membrane-permeable peptide, a drug-peptide conjugate according to an embodiment of the present invention may also have cell membrane permeability.

[0032] The drug is not particularly limited, and examples thereof include proteins (e.g., antibodies or functional fragments thereof, hormones, and enzymes), nucleic acids (e.g., high molecular weight nucleic acids such as plasmid DNA and mRNA, and low molecular weight nucleic acids such as siRNA, miRNA, antisense nucleic acids, and aptamers), other physiologically active substances (e.g., antitumor agents, signal transduction inhibitors, antimetabolites, analgesics, anti-inflammatory agents, and antibacterial agents), fluorescent dyes, and contrast agents. The mass (molecular weight) of the drug is not particularly limited. Specifically, the mass of the drug may be less than 500 Da or 500 Da or more, for example, 1,000 Da to 150,000 Da, or for example, 3,000 Da to 150,000 Da. The membrane-permeable peptide according to an embodiment of the present invention can also confer membrane permeability to biopolymers such as proteins and nucleic acids.

[0033] The number of membrane-permeable peptides bound to a drug can be appropriately set depending on the mass or molecular weight of the drug, the membrane permeability level of the membrane-permeable peptide, etc. The number of membrane-permeable peptides bound to a drug is, for example, 1 to 10, and may be 1 to 5 or 1 to 3 per drug molecule.

[0034] In a drug-peptide conjugate, the drug and the membrane-permeable peptide may be directly bonded or may be bonded via a linker. In the case of direct bond, a drug-peptide conjugate can be formed by reacting a functional group of the drug with a functional group of the membrane-permeable peptide. In the case of bond via a linker, a drug-peptide conjugate can be formed, for example, by reacting a functional group located at one end of the linker with a functional group of the drug, and simultaneously reacting a functional group located at the other end of the linker with a functional group of the membrane-permeable peptide. Between the functional groups located at both ends of the linker, an alkylene group having 2 to 10 carbon atoms (e.g., a polyoxyethylene group) which may contain an ether bond, an amide bond, an ester bond, or the like may be present. When the drug is a protein, a drug-peptide conjugate can also be obtained as a fusion protein in which the membrane-permeable peptide is linked to the N-terminus and / or C-terminus of the protein by genetic engineering techniques.

[0035] Examples of combinations of functional groups in the above reaction include an azide group and an alkyne, a thiol group and a (meth)acryloyl group, a thiol group and a maleimide group, a thiol group and a thiol group, a thiol group and a carboxyl group, a (meth)acryloyl group and a hydroxyl group, a (meth)acryloyl group and an amino group, a carboxyl group and an amino group, a carboxyl group and a hydroxyl group, and an amino group and a hydroxyl group.

[0036] C. Use of Peptides with Cell Membrane Permeability The membrane-permeable peptides described in Section A can be used in drug delivery. In one embodiment, the membrane-permeable peptides can be used as targeting moieties in drug delivery to intracellular targets. In this case, it is preferable that the membrane-permeable peptide has selective membrane permeability for specific cells. In another embodiment, the membrane-permeable peptides can be used in blood or oral administration of drugs to improve the transfer of drugs from blood vessels to tissues or from the gastrointestinal tract to tissues. In this case, it is preferable that the membrane-permeable peptide has membrane permeability for vascular epithelial cells or gastrointestinal epithelial cells, and more preferably has selective membrane permeability. In yet another embodiment, the membrane-permeable peptides can be used in drug delivery to the brain to improve the transfer of drugs into the brain. In this case, it is preferable that the membrane-permeable peptide has brain-barrier permeability. In yet another embodiment, the membrane-permeable peptides can be used in drug delivery through the skin to improve transfer from the skin to the bloodstream. In this case, it is preferable that the membrane-permeable peptide has skin permeability. Alternatively, when it is desired to act on the skin, it is preferable that the compound has the ability to migrate into epidermal or dermal cells.

[0037] When using the membrane-permeable peptide in drug delivery, the membrane-permeable peptide can be bound to the drug or its carrier (specifically, a component of the carrier). Thus, the present invention provides a method for increasing the membrane permeability of a drug or drug delivery carrier, comprising binding the membrane-permeable peptide to the drug or drug delivery carrier or a component thereof, and a method for producing a drug or drug delivery carrier with enhanced or imparted membrane permeability, comprising binding the membrane-permeable peptide to the drug or drug delivery carrier or a component thereof. The binding may be covalent or non-covalent. The complex (conjugate) in which the drug and the membrane-permeable peptide are covalently bound is as described in Section B. The carrier to which the membrane-permeable peptide is bound is not particularly limited. Preferred examples of drug carriers include nanoparticles such as liposomes, micelles, and vesicles. These nanoparticles are generally composed of hydrophobic compounds or polymers, hydrophilic compounds or polymers, and / or amphipathic compounds or polymers, including polyamino acids, lipids, polysaccharides, and other polymers (e.g., polyethylene glycol). In one embodiment, the membrane-permeable peptide can be attached to the end of these components that make up the nanoparticle so that they are exposed on the nanoparticle surface. The attachment method can be selected appropriately depending on the purpose. In the case of covalent attachment, it can be performed in the same manner as the attachment of a drug to a membrane-permeable peptide, and a combination of mutually reactive functional groups as described above can be used. In the case of non-covalent attachment, attachment (complexation) can be achieved by utilizing electrostatic interaction, hydrophobic interaction, hydrogen bonding, van der Waals force, etc.

[0038] D. Screening Method for Peptides with Cell Membrane Permeability A screening method for peptides with cell membrane permeability according to an embodiment of the present invention includes: step A: preparing a cDNA display library containing cDNA display molecules; step B: contacting and incubating cells with the cDNA display library; and step C: recovering the cDNA display molecules from the cell contents or permeate. The screening method utilizes a cDNA display method. The cDNA display method is a genotype-phenotype matching technique that can establish a one-to-one correspondence between the function and / or phenotype of a gene expression product, such as a protein, and the cDNA encoding the corresponding gene. Specifically, the cDNA display molecules recovered in step C are subjected to PCR to amplify and identify the gene encoding the peptide carried by the molecule, thereby identifying the amino acid sequence of the membrane-permeable peptide. Because the screening method utilizes a cDNA display method using highly stable cDNA display molecules, desired peptides can be efficiently obtained even in a system using live cells. Furthermore, by selecting the cells to be used for screening, peptides with membrane permeability for desired cells can be efficiently obtained.

[0039] In the above screening method, a sub-cDNA display library can be prepared using the recovered cDNA display molecules. In this case, the preparation of the sub-cDNA display library can be considered as step A, followed by steps B and C. That is, in the above screening method, steps A, B, and C are considered as one selection round, and step A is performed again using the selection product (recovered cDNA display molecules) to initiate the next selection round. As a result, the above selection can be repeated two or more times. By performing two or more rounds of selection, peptides with relatively low membrane permeability can be eliminated, allowing peptides with higher membrane permeability to be efficiently obtained. Selection can be repeated until the cDNA sequences of the recovered cDNA display molecules are sufficiently converged (e.g., until the sequence with the most reads accounts for 0.1% or more, preferably 1% or more of the total number of reads). The number of selection rounds is, for example, one or more, preferably two or more, and may be three or more or four or more, and for example, six or less, preferably five or less.

[0040] D-1. Step A In step A, a cDNA display library containing cDNA display molecules is prepared. Methods for preparing a cDNA display library are known, and any method can be used. The cDNA display library can be prepared, for example, as shown in FIG. 1 , by a method including: preparing a DNA library containing DNA encoding random amino acid sequences (step a); transcribing the DNA of this DNA library into mRNA to obtain an mRNA library (step b); linking a puromycin-bound linker X to the 3' end of the mRNA in the mRNA library to obtain an mRNA-linker conjugate (step c); translating the mRNA of the mRNA-linker conjugate in a cell-free translation system to generate a peptide, which is then linked to puromycin to obtain an mRNA-linker-peptide conjugate (step d); and reverse transcribing the mRNA of the mRNA-linker-peptide conjugate to generate cDNA and obtain a cDNA display molecule (step e).

[0041] In one embodiment, as shown in the enlarged view of a key portion in FIG. 1( c), linker X comprises a main backbone m containing single-stranded DNA and / or peptide nucleic acid (PNA) to which puromycin P is linked, and the main backbone may have a ligation site at the 5' end that hybridizes with mRNA and can be linked to its 3' end, and a reverse transcription primer site at the 3' end. Having such a structure allows efficient ligation with mRNA in step c to obtain an mRNA-linker conjugate. Furthermore, reverse transcription from the reverse transcription primer site in step e allows reliable association of the peptide with the cDNA.

[0042] The main backbone preferably has a length of 10-mer to 60-mer, more preferably 10-mer to 45-mer, and even more preferably 15-mer to 30-mer.

[0043] Puromycin is a compound that functions as an analog of the 3'-terminus of aminoacyl-tRNA and can bind to a peptide chain elongating within a ribosome. Puromycin may be a puromycin-like compound that has a structure similar to the 3'-terminus of aminoacyl-tRNA and has the ability to bind to the C-terminus of a synthesized protein when the protein is synthesized in a translation system.

[0044] Preferably, the linker X further has, in the main backbone m, a solid-phase binding site b capable of binding to a solid-phase site, and a pair of cleavage sites c1 and c2 located on either side of the solid-phase binding site b. By having such a structure, the mRNA-linker-peptide conjugate obtained in step d can be purified and reverse-transcribed (step e) in a state where it is bound (immobilized) to a solid phase via the linker, and by cleaving it at the pair of cleavage sites c1 and c2 after reverse transcription, a cDNA display molecule can be suitably produced.

[0045] The solid phase binding site may be any site capable of binding the mRNA-linker-peptide conjugate to the solid phase via a linker, and specific examples thereof include a base capable of binding biotin (e.g., deoxythymine (dT)), a base to which biotin is bound (e.g., biotin-deoxythymine (Biotin-dT)), a base modified with an amino group (e.g., amino-modified deoxythymine), a base modified with a carboxyl group (e.g., carboxyl-modified deoxythymine), a base modified with a thiol group (e.g., thiol-modified deoxythymine), etc. Of these, biotin-deoxythymine is preferred from the viewpoint of affinity with avidin.

[0046] The pair of cleavage sites are, for example, enzyme cleavage sites. This allows the mRNA-linker-peptide conjugate bound to the solid phase via the solid phase binding sites to be cleaved with an enzyme or the like that cleaves the cleavage sites, thereby allowing the cDNA display molecule to be removed from the solid phase. The enzyme cleavage sites can be appropriately selected depending on the type of enzyme used. A specific example of the enzyme cleavage site is ribo G (Guanosine).

[0047] 2 is a schematic diagram illustrating the structure of an example of a cDNA display molecule. The cDNA display molecule 10 is a cleavage residue of linker X, and includes linker residue 1 to which puromycin 3 is linked, cDNA 5 linked to one end of linker residue 1, mRNA 2 linked to the other end and hybridized with cDNA 5, and peptide 4 bound to puromycin 3.

[0048] For details of the structure and method of producing the cDNA display molecule 10 and the method of producing a cDNA display library, see, for example, WO2006 / 041194.

[0049] The diversity of the cDNA display library used in the initial selection (the diversity of random amino acid sequences displayed in the library) is, for example, 1.0 × 10 １０ ~1.0 x 10 １３ , preferably 1.0×10 １１ ~1.0 x 10 １３, more preferably 1.0 × 10 １１ ~1.0 x 10 １２ It could be.

[0050] D-2. Step B In step B, cells are contacted with the cDNA display library and incubated. The cells to be contacted can be selected appropriately depending on the purpose, and may be one type or two or more types. The cells may also form tissue. Therefore, an embodiment in which tissue is contacted with the cDNA display library and incubated in step B is also included in the screening method according to this embodiment of the present invention.

[0051] The incubation temperature and incubation time can be appropriately set depending on the cells to be contacted, the application of the membrane-permeable peptide, etc. For example, the incubation temperature can be the culture temperature of the cells to be contacted. The incubation time can be, for example, 5 minutes to 72 hours, and may be 30 minutes to 24 hours or 10 minutes to 4 hours.

[0052] D-3. Step C In step C, the cDNA display molecules are recovered from the contents or permeate of the cells after the incubation. The recovery of the cDNA display molecules can be carried out using a commercially available kit or the like.

[0053] The recovered cDNA display molecules are subjected to PCR to identify the cDNA base sequence and the encoded amino acid sequence. If sequence convergence is insufficient, a sub-cDNA display library can be prepared using the PCR product (Step A) and subjected to selection again. If sequence convergence is sufficient, selection can be terminated without performing Step A, and a peptide consisting of the identified amino acid sequence can be obtained as a membrane-permeable peptide.

[0054] The sub-cDNA display library can be prepared from the recovered cDNA display molecules in the same manner as the method for preparing the cDNA display library in step A, except that the PCR products of the recovered cDNA display molecules are used as a DNA library to prepare an mRNA library.

[0055] 3 and 4 are each a schematic diagram illustrating a screening method according to one embodiment of the present invention. In the embodiment shown in FIG. 3, the contact and incubation of cells with the cDNA display library in step B is performed by adding the cDNA display library 20 to a culture vessel 30 containing cells 32 and a culture medium 34, followed by incubation. The amount of the cDNA display library added (more specifically, the amount of cDNA display molecules added) can be, for example, 100 fmol to 1000 fmol, and preferably 200 fmol to 400 fmol. In one embodiment, the amount of the cDNA display library added is such that the number of cDNA display molecules per cell at the time of display is, for example, 1.0 x 10 ５ ~1.0 x 10 ８ , preferably 1.0×10 ６ ~1.0 x 10 ８ , more preferably 1.0 × 10 ７ ~1.0 x 10 ８ The incubation time is appropriately set depending on the purpose of the test, and may be a relatively short time (for example, 5 minutes to 4 hours, preferably 30 minutes to 2 hours) or a relatively long time (for example, 20 hours to 72 hours, preferably 24 hours to 48 hours).

[0056] After incubation, the cells 32 are recovered and washed as necessary, and then the cell contents are recovered by hypotonic treatment, disruption, or the like, and the cDNA display molecules 10 are then recovered from the cell contents using a nucleic acid purification kit or the like (Step C).The recovered cDNA display molecules 10 are subjected to PCR, and if the identified amino acid sequences are not sufficiently converged, a sub-cDNA display library 20' is prepared (Step A), and the subsequent Steps B and C can be performed using the sub-cDNA display library 20'.

[0057] Any appropriate cells can be used as the cells depending on the purpose. Examples include cancer cells; immune cells such as macrophages, neutrophils, T cells, and B cells; mast cells; and cells or cell groups that constitute skin tissues such as the epidermis and dermis. According to the embodiment shown in Figure 3, peptides that can permeate cell membranes and remain in cells (in other words, peptides that have cytoplasmic translocation properties) can be suitably screened.

[0058] In the embodiment shown in FIG. 4 , contact and incubation of the cells with the cDNA display library in step B is performed by adding the cDNA display library 20 to a cell culture insert 42 inserted into a culture well 40 and containing cells 44 and a culture medium 46, followed by incubation. The bottom surface 42 a of the cell culture insert 42 is preferably confluent and completely covered with a single or multiple layer of cells. The amount of the cDNA display library added (more specifically, the amount of cDNA display molecules added) can be, for example, 100 fmol to 1000 fmol, preferably 200 fmol to 400 fmol. The incubation time can be, for example, 5 minutes to 24 hours, preferably 15 minutes to 4 hours, and more preferably 30 minutes to 2 hours.

[0059] Because the bottom surface 42a of the cell culture insert 42 is formed of a porous membrane, the culture medium 46 outside the cell culture insert 42 may contain permeants that have permeated the cells 44 from the inside to the outside of the cell culture insert. Therefore, the cDNA display molecules 10 that have permeated the cells 44 can be recovered from the culture medium 46 outside the cell culture insert (Step C). Recovery of the cDNA display molecules 10 from the culture medium can be performed using a nucleic acid purification kit or the like. If the recovered cDNA display molecules 10 are subjected to PCR, and the identified amino acid sequences are not sufficiently converged, a sub-cDNA display library 20' can be prepared (Step A), and the subsequent Steps B and C can be performed using the sub-cDNA display library 20'.

[0060] Alternatively, the cDNA display library may be added to the culture medium outside the cell culture insert, contacting the cells through the porous membrane on the bottom of the cell culture insert, and the cDNA display molecules that have permeated the cells may be recovered from the culture medium inside the cell culture insert.

[0061] Any appropriate cells can be used as the cells depending on the purpose. Examples include gastrointestinal epithelial cells; vascular epithelial cells; cells or cell groups that constitute the blood-brain barrier; and cells or cell groups that constitute skin tissues such as the epidermis and dermis. According to the embodiment shown in FIG. 4, peptides that can permeate from one side of a cell layer to the other (e.g., peptides that can permeate from the gastrointestinal tract to the blood, from the blood to tissues, or from the blood to the brain, or that can be absorbed transdermally) can be suitably screened.

[0062] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0063] [Experimental Example 1: Preparation of a cDNA display library] A cDNA display library containing random peptide regions was prepared by the in vitro virus method using the linker described in WO2006 / 041194. The details are as follows.

[0064] An mRNA library was prepared using the RiboMAX Large Scale RNA Production System-SP6 kit (Promega) based on a DNA library containing DNA encoding random peptides of 8 to 15 amino acid residues. 1 pmol to 2 pmol of cDNA, 4 μL of rNTP mixture, 2 μL of transcriptase, and 4 μL of reaction buffer were mixed and then adjusted to 20 μL with purified water (all reagents except cDNA were included in the kit). The transcription solution was incubated at 37°C for 2.5 hours, after which 1 μL of RQ1 RNase-Free DNase (1 U / 1 μL) was added and incubated at 37°C for 20 minutes. The mRNA was then purified using the RNeasy Mini kit (QIAGEN) according to the kit's instructions.

[0065] Next, a conjugate of mRNA and the puromycin linker (hereinafter referred to as the linker) described in WO 2006 / 041194 was prepared. Details are as follows. 20 pmol of mRNA and 20 μM of linker were mixed, and then a solution was prepared with purified water (Ambion) to make a 20 μL solution. The solution was incubated at 90°C for 30 seconds, cooled to 70°C at 0.12°C / sec, incubated for 30 seconds, and cooled to 4°C at 0.06°C / sec. After cooling, 1 μL of T4 polynucleotide kinase (Takara Bio), 1 μL of T4 RNA ligase (Takara Bio), 2 μL of T4 RNA ligase buffer (included with T4 RNA ligase), and 1.5 μL of 0.1% BSA (included with T4 RNA ligase) were added to the cooled solution, and the mixture was incubated at 25°C for 1 hour to prepare an mRNA-linker conjugate.

[0066] Next, peptide synthesis was carried out by mixing the mRNA-linker conjugate with a cell-free translation system. 20 μL of the conjugate, 50 μL of Wheat Germ Extract (Promega), 8 μL of amino acid mixture (Promega), 5 μL of 1 M KOAc (Ambion), 5 μL of RNase inhibitor (Thermo Fisher Scientific), and 12 μL of purified water were mixed to prepare a translation reaction solution, which was then incubated at 25°C for 15 minutes. Then, 18.25 μL of salt mix (3 M KCl, 1 M MgCl ２　 46 μL of 0.5 M EDTA pH 8.0 (Thermo Fisher Scientific) was then added, and the mixture was incubated at 25° C. for 1 hour. 28 μL of 0.5 M EDTA pH 8.0 (Thermo Fisher Scientific) was then added. This yielded an mRNA-linker-peptide conjugate (post-translation product).

[0067] Using biotin contained in the linker in the post-translational product, purification was performed using DynaBeads MyOne Streptavidin C1 (Thermo Fisher Scientific) (hereinafter referred to as magnetic beads). After thoroughly suspending 150 μL of magnetic beads, the beads were washed twice with an equal volume of 2x Binding buffer (buffer composition: 20 mM Tris-HCl (pH 7.5), 2 M NaCl, 2 mM EDTA, 0.2% Polyoxyethylene (20) Sorbitan Monolaurate) (after addition and suspension, the supernatant was discarded). Subsequently, 348 μL of the post-translational product was added and incubated at 25 ° C for 30 minutes while rotating on a rotator. The supernatant was then discarded and the beads were washed twice with 400 μL of 1x Binding buffer. Further, the plate was washed once with 200 μL of 1×RT buffer (Promega).

[0068] After washing, 8 μL of 2.5 mM dNTP mixture (Takara Bio), 4 μL of RNase inhibitor (Thermo Fisher Scientific), 20 μL of 10x RT buffer (Promega), and 166 μL of purified water were added to the magnetic beads and mixed by tapping. Then, 2 μL of M-MLV Reverse Transcriptase (Promega) was added and the mixture was incubated at 42°C for 30 minutes while rotating on a rotator.

[0069] Next, the supernatant of the magnetic beads was discarded, and the beads were washed once with 100 μL of 1x Binding buffer, followed by washing once with 50 μL of equilibration buffer (composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM imidazole, 0.05% Polyoxyethylene (20) Sorbitan Monolaurate). 99.5 μL of equilibration buffer and 0.5 μL of RNase T1 (Thermo Fisher Scientific) were added, and the mixture was incubated at 37 °C for 20 minutes while rotating on a rotator, after which the supernatant was recovered. The solution recovered in this way is an unpurified cDNA display library containing cDNA display molecules.

[0070] 100 μL of HisPur Ni-NTA Magnetic Beads (Thermo Fisher Scientific) were washed once with 100 μL of equilibration buffer. The recovered cDNA display library was added and shaken at room temperature for 30 minutes. After shaking, the supernatant was discarded and the beads were washed twice with 100 μL of equilibration buffer. 20 μL of elution buffer (composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 250 mM imidazole, 0.05% Polyoxyethylene (20) Sorbitan Monolaurate) was then added and mixed by tapping. After collecting the supernatant, 20 μL of elution buffer was added again and mixed by tapping. The supernatant was collected in the same manner, and the combined volume was adjusted to 40 μL.

[0071] The imidazole contained in the recovered solution was removed by dialysis. Dialysis was performed using a Slide-A-Lyzer MINI (3.5 kDa MWCO) (Thermo Fisher Scientific). After stirring against 60 mL of dialysate (PBS) at 4°C for 60 minutes, the dialysate was replaced and the mixture was stirred at 4°C for an additional 60 minutes. The solution recovered by dialysis was used as a cDNA display library for subsequent screening.

[0072] [Experimental Example 2: Screening of peptides that permeate gastrointestinal epithelial cells] <Cell culture> Ad-MED vitrigel was placed on a 24-well plate. （Ｒ） 2 Culture Insert (Kanto Chemical Co., Ltd.) was set and F-hiSIEC （Ｒ） Human iPS cell-derived intestinal epithelial cells "F-hiSIEC™ (Fujifilm Corporation)" suspended in Seeding Medium (Fujifilm Corporation) were used to prepare 1.0 x 10 ５ The cells were seeded at 1000 cells / well. ２ The cells were cultured under a 50°C / 100°F (200°C) ambient temperature, and the medium was changed according to the schedule in Example 2 of the medium change manual (F-hiSIEC™ Culture Medium (FUJIFILM Corporation) was used during the culture period). With the start of culture considered as day 1, the first permeation test (round 1) was conducted on day 12, and the second permeation test (round 2) was conducted on day 14.

[0073] First penetration test: Random linear peptides consisting of 10, 12, and 15 amino acids, and random cyclic peptides consisting of 10, 11, and 12 amino acids with cysteines at both ends were used as peptide sequences in the variable regions of the cDNA display library. Three cDNA display libraries displaying linear peptides with different numbers of amino acid residues or three cDNA display libraries displaying cyclic peptides with different numbers of amino acid residues were mixed to prepare two cDNA display libraries: a linear mixed library and a cyclic mixed library. These libraries were then subjected to penetration experiments in parallel and separately. Each mixed library was suspended in PBS and then diluted with RPMI 1640 medium without fetal bovine serum (FBS). After removing the culture inserts and the medium from the wells, 150 μL of each diluted library solution (approximately 100 fmol to 300 fmol as the amount of cDNA display molecule added) was added to separate culture inserts (apical side), and 600 μL of RPMI 1640 medium was added to each well (basolateral side), and the permeation test was initiated. At 37°C, 5% CO ２ After incubation under 5% CO₂ for 1 hour, the medium on the receptor (basolateral) side was completely collected into 1.5 mL tubes and stored at −80°C until quantitative analysis of nucleic acids and the next round of display library construction.

[0074] <Purification and amplification of permeation samples> Approximately 500 μL of the sample recovered in the permeation test was placed in an ultrafiltration centrifugal filter (Amicon Ultra 10 kDa MWCO, Millipore UFC5010) and centrifuged at 4 ° C and 14,000 rpm for 10 minutes (TOMY TMP-24, rotation radius Rmax 87 mm), and the filtrate was discarded. The filter membrane was inverted and placed in a new tube, and centrifuged at 4 ° C and 1,000 g for 2 minutes, and the solution (70 μL) was recovered. DNA purification was performed using a DNA purification kit (QIA quick PCR Purification Kit QIAGEN 28104), and 25 μL of DNA sample was recovered. The DNA sample was PCR amplified according to standard methods, and a two-round cDNA display library was prepared.

[0075] <Second permeation test and purification and amplification of permeation samples> The second permeation test was performed in the same manner as the first permeation test, except that the cDNA display library for the second round was used. The entire medium on the receptor (basolateral) side was collected into a 1.5 mL tube. A cDNA display library for the third round was prepared from the collected solution in the same manner as above.

[0076] <Third and Fourth Permeation Tests and Purification and Amplification of Permeation Samples> For the third and fourth permeation tests, new human iPS cell-derived intestinal epithelial cells F-hiSIEC™ were prepared in the same manner as for the first and second permeation tests. Using the cDNA display library for the third round, permeation tests and purification and amplification of permeation samples were performed in the same manner as above to prepare a cDNA display library for the fourth round. Using the cDNA display library for the fourth round, permeation tests and purification and amplification of permeation samples were performed in the same manner as above.

[0077] <Next-generation sequencing (NGS) analysis> Samples collected in each permeation test were subjected to a standard PCR method using adapter sequences, with reference to Illumina Inc.'s published documentation on the workflow of the Tailed-PCR method. A library for next-generation sequencing was then prepared and analyzed using a MiniSeq (Illumina) next-generation sequencer. The resulting paired-end FastQ files (two FastQ files) were analyzed using Flash2 to determine the amino acid sequences of peptides that permeate the cell membrane. The sequences with the highest read counts in the fourth permeation test are listed below.

[0078]

[0079] Experimental Example 3: Screening of peptides that translocate into lung cancer A549 cells. A549 cells (RIKEN BioResource Research Center) were cultured in RPMI1640 medium (ATCC modification, Thermo-Fisher Scientific, product number A10491-01) + 10% FBS (containing penicillin-streptomycin, Thermo-Fisher Scientific, product number "15140-122", 1 / 100 volume), and plated at 0.5 × 10 ５ The cells were seeded at 1000 x g / well. ２ After culturing for 72 hours under the conditions of 37°C and 5% CO, the medium in each well was removed. A cDNA display library equivalent to that in Experimental Example 2 (a total of six types of cDNA display libraries with different numbers of amino acid residues in the peptides displayed (three types of linear peptides and three types of cyclic peptides)) was prepared. These were mixed and diluted to 310 μL with RPMI 1640 medium without FBS or antibiotics, and 300 μL of this mixture (approximately 100 fmol to 300 fmol of cDNA display molecules added) was added to the wells. ２ Below, after 1 hour of uptake, the spiked library was collected. Then, 300 μL of fresh medium was added, and the culture was continued for 23 hours, after which the cells were washed as follows.

[0080] After washing three times with 300 μL of PBS, the cells were washed three times with 300 μL of PBS + 0.05% Tween 20, and once with 300 μL of 10 mM HCl + 150 mM NaCl. Then, 300 μL of 10 mM HEPES buffer (pH 7.0) was added on ice and the cells were subjected to hypotonic treatment for 15 minutes. After confirming cell rupture under a microscope, the entire sample containing the cell contents was collected in a 1.5 mL tube. The supernatant was obtained by centrifugation at 1000 g for 3 minutes. Nucleic acids were purified and amplified from this supernatant using the same method as in Experimental Example 2, and a cDNA display library for the next round was prepared.

[0081] The above test procedure was repeated two more times (a total of three selections), and the samples collected as cell contents were subjected to next-generation sequencing (NGS) analysis. The resulting paired-end fastq file was analyzed using flash2 to determine the amino acid sequence of the peptide translocated into the cells. The sequences with the highest read counts were listed as follows:

[0082]

[0083] [Experimental Example 4: Screening of peptides that translocate into pancreatic cancer Panc-1 cells] Panc-1 cells (RIKEN BioResource Research Center) were cultured in the same RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) as in Experimental Example 3, and 0.5 × 10 cells were plated on a 24-well plate. ５ The cells were seeded at 1000kJ / well. A total of two selections were performed in the same manner as in Experimental Example 3 to determine the amino acid sequences of peptides that translocated into the cells. The sequences with the highest read counts in the NGS analysis were listed as follows:

[0084]

[0085] In Experimental Example 3, a similar experiment was performed using an mRNA display library instead of a cDNA display library. The residual rates of nucleic acids one hour after cell contact were both over 90% for linear peptide display molecules and cyclic peptide display molecules in the cDNA display library, whereas the residual rate of linear peptide display molecules in the mRNA display library was 0.47%. In Experimental Example 4, a similar experiment was performed using an mRNA display library instead of a cDNA display library. The residual rates of nucleic acids one hour after cell contact were 23% and 51%, respectively, for linear peptide display molecules and cyclic peptide display molecules in the cDNA display library, whereas the residual rate of linear peptide display molecules in the mRNA display library was 0.76%. This demonstrates that highly stable screening is possible even in systems using living cells using the cDNA display method.

[0086] Experimental Example 5: Screening of peptides that translocate into neutrophil cells HL-60 cells (RIKEN BioResource Research Center) were cultured in RPMI 1640 medium + 10% FBS (containing penicillin-streptomycin) and plated at 1.0 × 10 ５ Dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical Industries, Ltd., 041-29351) was added to a final concentration of 1.3%, and the cells were incubated at 37°C and 5% CO ２ Bottom, HL-60 cells were cultured for 72 hours to differentiate into neutrophils.

[0087] The medium was removed from each well, and a cDNA display library equivalent to that in Experimental Example 3 was diluted with RPMI 1640 medium without FBS or antibiotics to a total volume of 310 μL, and 300 μL of the diluted solution was added to the wells. ２After 1 hour of uptake, the loaded library was recovered. Then, 300 μL of PBS was added, and the cells were recovered by pipetting. The cells were then washed by centrifugation at 200 g for 3 minutes. The cells were then washed with 300 μL of PBS + 0.05% Tween 20 and similarly recovered by centrifugation. Subsequently, 300 μL of 10 mM HEPES buffer (pH 7.0) was added on ice, and the cells were subjected to hypotonic treatment for 15 minutes. After confirming cell rupture under a microscope, the entire sample containing the cell contents was collected in a 1.5 mL tube. The supernatant was obtained by centrifugation at 1000 g for 3 minutes. This supernatant was purified and amplified as in Experimental Example 2 to prepare the cDNA display library for the next round.

[0088] The above test procedure was repeated four more times (a total of five selections), and the samples collected as cell contents were subjected to NGS analysis. The resulting paired-end fastq file was analyzed using flash2 to determine the amino acid sequence of the peptide translocated into the cells. The sequences with the highest read counts were listed as follows:

[0089]

[0090] [Experimental Example 6: Screening of peptides that translocate into macrophage cells] THP-1 cells (RIKEN BioResource Research Center) were cultured in RPMI 1640 medium + 10% FBS (containing penicillin-streptomycin) and plated in a 24-well plate at 1.0 × 10 ５ A 1 / 1000 volume of DMSO stock solution (10 μg / mL) of Phorbol 12-myristate 13-acetate (PMA, Funakoshi, AG-CN2-0010-M001) was added, and the cells were incubated at 37°C, 5% CO ２The medium was removed, and 300 μL of fresh RPMI 1640 medium + 10% FBS was added per well, followed by further incubation for 24 hours. IFNγ (Cosmo Bio (Proteintech), HZ-1301) and LPS (Cosmo Bio (Santa Cruz Biotechnology), SC-3535) were added to a final concentration of 20 ng / mL and 250 ng / mL, respectively, and the plates were incubated at 37°C and 5% CO ２ Bottom, THP-1 cells were cultured for 24 hours to differentiate into macrophages.

[0091] The medium was removed from each well, and a cDNA display library equivalent to that in Experimental Example 3 was diluted with RPMI 1640 medium without FBS or antibiotics to a total volume of 310 μL, and 300 μL of the diluted solution was added to the wells. ２ Bottom, After 1 hour of uptake, cells were washed as follows.

[0092] After washing three times with 300 μL of PBS, the cells were washed three times with 300 μL of PBS + 0.05% Tween 20, and once with 300 μL of 10 mM HCl + 150 mM NaCl. Then, 300 μL of 10 mM HEPES buffer (pH 7.0) was added on ice and the cells were subjected to hypotonic treatment for 15 minutes. After confirming cell rupture under a microscope, the entire sample containing the cell contents was collected in a 1.5 mL tube. The supernatant was obtained by centrifugation at 1000 g for 3 minutes. Nucleic acids were purified and amplified from this supernatant using the same method as in Experimental Example 2, and used as a cDNA display library for the next round.

[0093] The above test procedure was repeated three more times (a total of four selections), and the samples collected as cell contents were subjected to NGS analysis. The resulting paired-end fastq file was analyzed using flash2 to determine the amino acid sequence of the peptide translocated into the cells. The sequences with the highest read counts were listed as follows:

[0094]

[0095] Experimental Example 7: Screening of peptides that permeate the BBB model Cell culture A monkey-type BBB kit, 6 inserts / 24 wells (Pharmacocell Co., Ltd., PCC-MBT-24Q) was cultured according to the manufacturer's instructions. DMEM / F-12 medium (Gibco, 11330-032) + 0.5% BSA (Fujifilm Wako Pure Chemical Industries, 017-22231) + 500 nM hydrocortisone was used as the assay buffer. The first day of culture was considered to be day 1, and the first (round 1) permeation test was performed on day 5, and the second (round 2) on day 7.

[0096] First penetration test: Random linear peptides consisting of 8, 10, and 12 amino acids and random cyclic peptides consisting of 9, 10, and 11 amino acids with cysteines at both ends were used as peptide sequences for the variable regions in the cDNA display library. Three cDNA display libraries displaying linear peptides with different numbers of amino acid residues or three cDNA display libraries displaying cyclic peptides with different numbers of amino acid residues were mixed to prepare two cDNA display libraries: a linear mixed type and a cyclic mixed type. These were then subjected to parallel penetration experiments. Each mixed type cDNA display library was suspended in PBS solution to obtain two mixed cDNA display library solutions. On day 5 after the start of culture, the inserts in the BBB culture were temporarily transferred to a washing well. The medium inside the inserts was carefully removed using an aspirator and then transferred to a test well containing 900 μL of Assay Buffer. 20 μL of each cDNA display library solution was mixed and diluted with 200 μL of Assay Buffer, and 200 μL of each was added to each insert to initiate the permeation test. ２ After 2 hours of incubation, each receptor solution was collected.

[0097] <Purification and Amplification of Permeated Sample> The collected solution was purified and amplified in the same manner as in Experimental Example 2, and then used as a cDNA display library for the next round.

[0098] <Second permeation test and purification and amplification of permeation samples> On day 7 after the start of culture, a second permeation test was performed in the same manner as the first permeation test, except that the cDNA display library for round 2 was used. Each receptor was recovered, and the recovered solution was purified and amplified in the same manner as in Experimental Example 2 to prepare a cDNA display library for round 3.

[0099] <Third and Fourth Permeation Tests and Purification and Amplification of Permeation Samples> For the third and fourth permeation tests, a new monkey-type BBB kit 6 inserts / 24 wells (Pharmacocell Co., Ltd.) was prepared in the same manner as for the first and second permeation tests. Using the cDNA display library for the third round, permeation tests and purification and amplification of permeation samples were performed in the same manner as above to prepare a cDNA display library for the fourth round. Using the cDNA display library for the fourth round, permeation tests and purification and amplification of permeation samples were performed in the same manner as above.

[0100] The samples collected in each permeation test are analyzed by next-generation sequencing (NGS) to confirm the degree of convergence of the amino acid sequences, and the permeation test is repeated until sufficient convergence is achieved. Once sufficient convergence is confirmed, the sequence with the largest read count is identified as the amino acid sequence of the membrane-permeable peptide.

[0101] Experimental Example 8: Screening of peptides that permeate the skin model A three-dimensional skin model similar to human skin, EpiDerm Full Thickness 400 (Kurabo Industries, Ltd., #EFT-400), was cultured using the medium provided with the kit according to the manufacturer's manual. This resulted in the construction of a skin model on the donor side (inside) of a collagen-coated culture cup equivalent in size to a 24-well plate. Screening was initiated by adding 100 μL of the peptide library to the donor side of this culture cup. After 24 hours of culture from the addition, 2.5 mL of the culture medium on the receptor side was collected. The mixture was placed in an ultrafiltration centrifugal filter (Amicon Ultra 10 kDa MWCO, Millipore UFC5010) and centrifuged at 4 ° C. and 14,000 rpm for 10 minutes (TOMY TMP-24, rotation radius Rmax 87 mm) and concentrated to approximately 100 μL. DNA purification was performed using a DNA purification kit (QIA quick PCR Purification Kit QIAGEN 28104), and 25 μL of DNA sample was recovered. The DNA sample was PCR amplified according to standard methods, and a cDNA display library for two rounds was prepared. Similarly, the rounds of permeation test were repeated, up to a total of four rounds.

[0102] The samples collected in each permeation test were analyzed by next-generation sequencing (NGS), and the resulting paired-end fastq files were analyzed using flash2 to determine the amino acid sequences of the peptides that permeate the skin model. The sequence with the highest read count was "RWNCVSCAFL" (SEQ ID NO: 40).

[0103] Experimental Example 9: Peptide Skin Permeability Test As in Experimental Example 8, a three-dimensional skin model, EpiDerm Full Thickness 400, was prepared according to the manufacturer's instructions. Meanwhile, the peptide of SEQ ID NO: 40 was modified at the N-terminus with fluorescein amidite (FAM) via a PEG8 linker (synthesized by Eurofins Genomics, Inc.; theoretical molecular weight: 1918.1). As a control, a known membrane-permeable peptide, the TAT sequence (a sequence obtained by removing the N-terminal C from the amino acid sequence of SEQ ID NO: 51), was used, and its N-terminus was modified with FAM (also synthesized by Eurofins Genomics, Inc.; theoretical molecular weight: 1979.2). 100 μL of a 1% DMSO-containing PBS solution of each FAM-modified peptide was added to the center of the donor side of the cup in which the skin model was constructed, and the permeability test was initiated (n=3). The incubation temperature was 37°C, 5% CO ２ After 24 hours of incubation, the culture medium outside the cup was collected, and the peptide concentration that had permeated the skin model was quantified based on fluorescence using a microplate reader (TECAN Spark) (excitation wavelength: 494 nm, emission wavelength: 521 nm). As a result, the percentage of the peptide permeated outside the cup relative to the amount of FAM-modified peptide of the TAT sequence added, i.e., the transmittance, was 21.4 ± 3.3% (mean ± standard deviation of three cases). The transmittance of the FAM-modified peptide of SEQ ID NO: 40 was 49.8 ± 10.0% (mean ± standard deviation of three cases), which was significantly higher than the TAT sequence (p < 0.01).

[0104] Experimental Example 10: Preparation of eGFP-peptide fusion protein Synthesis of template DNA necessary for the expression of eGFP or eGFP-peptide fusion protein, including DNA necessary for protein expression in a cell-free system, was requested from Eurofins, Inc. The synthesized template DNA is as follows, and its DNA sequence is shown in Figures 5 to 9.

[0105] Approximately 5 μg of each DNA fragment was obtained. A portion of this (0.25 μg to 1 μg) was used to prepare the "Cell-free Kun" （Ｒ） eGFP and eGFP-peptide fusion proteins were prepared and purified using the following procedure:

[0106] The template DNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (approximately 100 μg / mL), further diluted with purified water to the specified concentration according to the manufacturer's manual, and mixed with the reaction solution premix provided with the kit to make a final total of 100 μL (reaction solution). Subsequently, 1 mL of the dialysis solution provided with the kit was placed outside the dialysis unit, and the reaction solution was placed in the dialysis unit. After shaking at 30°C for 16 hours (amplitude 25 mm, 80 rpm, Bioshaker BR-53FP, Taitec Co., Ltd.), the reaction solution was collected in a new tube and left to stand on ice for 5 minutes to terminate the reaction. Subsequently, the volume was adjusted to approximately 1000 μL with PBS + 0.05% Tween 20, and HisPur （Ｒ） , Ni-NTA Magnetic Beads (Thermo Scientific （Ｒ） Purification was performed using a 100 μL Magnetic Beads Slurry Kit (Product Number: 88831). The beads were equilibrated in advance using the following procedure. 100 μL of the magnetic bead slurry was transferred to a new 1.5 mL tube, and 400 μL (4 volumes) of equilibration buffer (PBS + 0.05% Tween 20) was added, followed by suspension with a vortex (low) for 10 seconds. The beads were collected using a magnetic stand, the supernatant was removed, and an additional 1000 μL (10 volumes) of equilibration buffer was added, followed by suspension with a vortex (low) for 10 seconds. The supernatant was similarly removed, and the beads were recovered and equilibrated.

[0107] The reaction solution was added to the equilibrated beads and suspended. After rotating and mixing for 1 hour at 4°C, the beads were collected using a magnetic stand. They were washed three times with approximately 1 mL of PBS + 0.05% Tween 20 + 5 mM imidazole. Subsequently, the beads were eluted with the following buffers, and the yellow-green fraction was collected: two times with 20 μL of PBS + 250 mM imidazole, two times with 20 μL of PBS + 250 mM imidazole + 1 mM EDTA, and two times with 20 μL of PBS + 250 mM imidazole + 10 mM EDTA.

[0108] The recovered eGFP and eGFP-peptide fusion proteins were used in the following cell experiments after replacing the medium with 20 mM HEPES (pH 7.0) + 150 mM NaCl using an ultrafiltration membrane (Amicon Ultra 10 kDa 0.5 mL Millipore UFC501024). The concentrations were calculated based on the extinction coefficient at 280 nm calculated from the amino acid sequence.

[0109] Experimental Example 11: Translocation test of eGFP-peptide fusion protein into A549 lung cancer cells. A549 cells suspended in RPMI 1640 medium + 10% FBS (containing penicillin-streptomycin) were seeded at 5000 cells / well on a 96-well plate and incubated at 37°C, 5% CO ２ After removing the medium, the cells were washed once with 200 μL of RPMI 1640 medium without FBS or antibiotics, and the protein solutions (eGFP, eGFP-TAT, eGFP-L10#1) were diluted with the same RPMI 1640 medium and added to the wells (final concentration 5 μM, 30 μL / well). ２ After incubation for 30 minutes under LYSO-ID （Ｒ） ReD detection kit reagent (Cosmo Bio, product number ENZ-51005-0100) was diluted 500-fold with RPMI 1640 medium, and 10 μL of the diluted solution was added. After further incubation for 15 minutes, NucBlue （Ｒ） 3 μL of reagent (Thermo-Fisher Scientific) was added. After continuing the incubation for 15 minutes, the plate was cooled on ice and washed three times with 200 μL of PBS containing 0.5 mg / mL heparin sulfate cooled to 4°C. After washing twice with 100 μL of Live Cell Imaging solution (Thermo-Fisher Scientific) cooled to 4°C, 50 μL of the same solution was added and observed under a fluorescence microscope. The results of photographs taken with an all-in-one fluorescence microscope "BZ-X810" (Keyence Corporation) are shown in Figure 10.

[0110] As shown in Figure 10, in the sample using eGFP-L10#1, which is a fusion protein of a peptide containing the amino acid sequence of SEQ ID NO: 7 and eGFP, green fluorescence derived from eGFP was observed surrounding blue fluorescence derived from the nucleus, which indicates that GFP-L10#1 was efficiently translocated into the cytoplasm. Here, the mass of eGFP is 27 kDa, and the mass of the peptide added to eGFP is 1.2 kDa. On the other hand, in the sample using eGFP and the sample using eGFP-TAT, which is a fusion protein of the known membrane-permeable peptide TAT and eGFP, only a small amount of eGFP-derived fluorescence was observed around the nucleus, and as a result, translocation of eGFP or eGFP fusion protein into the cytoplasm was hardly confirmed.

[0111] Experimental Example 12: Translocation test of eGFP-peptide fusion protein into THP-1 macrophages. THP-1 macrophage cells were seeded at 5,000 cells / well in a 96-well plate and activated with PMA, IFNγ, and LPS. After removing the medium from the wells, the wells were washed once with 200 μL of RPMI 1640 medium without FBS or antibiotics. Protein solutions (eGFP, eGFP-L10#2) were diluted with the same RPMI 1640 medium and added to the wells (final concentration: 3 μM, 30 μL / well). Cells were observed 1 hour after addition, following the same procedure as in Experimental Example 11. The results are shown in Figure 11.

[0112] As shown in Figure 11, in the sample using eGFP-L10#2, which is a fusion protein of eGFP and a peptide comprising the amino acid sequence of SEQ ID NO: 37, green fluorescence derived from eGFP was observed along with blue fluorescence derived from the nucleus, indicating that eGFP-L10#2 had translocated into macrophage cells. Here, the mass of eGFP is 27 kDa, and the mass of the peptide attached to eGFP is 1.5 kDa. On the other hand, in the sample using GFP, no fluorescence derived from eGFP was observed, and as a result, translocation of eGFP into macrophage cells was not confirmed.

[0113] Experimental Example 13: Macrophage intracellular internalization test of Alexa647-labeled IgG Alexa647-labeling of IgG AlexaFluor647 NHS Ester (Thermo, #A20006) was dissolved in DMSO to a concentration of 10 mM. hIgG (normal human IgG, whole molecule, purified product) (Takara, #143-09501) was dissolved in PBS to a concentration of 10 mg / mL. 1 mg of hIgG was added to a 1.5 mL tube (total volume 200 μL) to a final concentration of 5 mg / mL, and AlexaFluor647 NHS Ester was added at a 10-fold molar concentration relative to hIgG. The mixture was then stirred by end-over-end at 25°C for 30 minutes. The reaction mixture was applied to a Zeba spin desalting column (7K MWCO) (Thermo, #89882) equilibrated with PBS, and the unreacted reagents were removed to recover the Alexa647-labeled IgG. Based on absorbance measurements at 280 nm and 650 nm as described in the manufacturer's manual, it was calculated that approximately three Alexa647 molecules were labeled per recovered IgG molecule.

[0114] <Peptide addition to Alexa647-labeled IgG> To approximately 1 mg of Alexa647-labeled IgG (PBS solution), a 10 mM DMSO solution of Maleimide-PEG2-NHS Ester (Tokyo Chemical Industry, product code M3339) as a crosslinker containing a hydrophilic linker or a 10 mM PBS solution of Sulfo-SMCC crosslinker was added in a 20-fold molar amount, and the mixture was stirred end-over-end at 25°C for 1 hour. The reaction solution was applied to a Zeba spin desalting column (7K MWCO) equilibrated with PBS, and the unreacted crosslinker was removed to recover the IgG fraction. Peptides containing the amino acid sequences obtained in the above screening and having a C at the N-terminus (synthesized by Eurofins Genomics, Inc.; the amino acid sequences of the peptides conjugated to IgG are shown in the table below) were dissolved in DMSO to give a 10 mM DMSO solution. The DMSO peptide solution was added to the recovered IgG fraction in a molar amount five times that of IgG, and EDTA was added to a final concentration of 1 mM. The mixture was then stirred end-over-end at 25°C for 1 hour. Subsequently, a five-fold molar amount of cysteine ​​was added, and the mixture was stirred end-over-end at 25°C for 30 minutes. The reaction solution was applied to a Zeba spin desalting column (7K MWCO) equilibrated with PBS, and the unreacted peptide, cysteine, and EDTA were removed to recover the IgG-peptide conjugate. For example, when the peptide was T001, it was calculated that 3 to 4 molecules of the peptide were attached per IgG molecule based on the absorbance measurements at 280 nm and 650 nm and the measured values ​​of the fluorescence intensity derived from tryptophan (excitation wavelength: 280 nm, emission wavelength: 350 nm).

[0115]

[0116] <Intracellular delivery of IgG into THP-1 macrophages via peptide addition> A test was performed using THP-1 macrophage cells seeded at 20,000 cells / well in a 96-well plate and activated with PMA, IFNγ, and LPS. After removing the medium from the wells and washing once with 200 μL of RPMI 1640 medium without FBS or antibiotics, a protein solution (IgG or IgG-peptide conjugate) was diluted with the same RPMI 1640 medium and added to the wells (final concentration 5 μM, 30 μL / well). Cells were observed 1 hour after addition, following the same procedure as in Experimental Example 11. The results are shown in Figure 12. Images taken with an all-in-one fluorescence microscope BZ-X810 (Keyence Corporation) were analyzed using the accompanying software, BZ-X800 Analyzer. The uptake ratios after 1 hour of uptake were compared based on the ratio of red luminance (integrated) to blue luminance (integrated). The results are shown in Figure 13 (in the figure, the height of the bars indicates the average value of n = 3, and the error bars indicate the standard deviation).

[0117] As shown in Figures 12 and 13, the amount of IgG translocated into cells was increased by binding peptides (T001, T005, and T006) containing the amino acid sequences obtained by screening using macrophage cells. On the other hand, no increase in the amount of IgG translocated into cells was confirmed for TAT-conjugated IgG.

[0118] Experimental Example 14: Evaluation of cell specificity of cell membrane permeability 1. The same procedure as in Experimental Example 13 was repeated, except that the protein solution was added to the wells so that the final concentration of IgG or IgG-peptide conjugate was 1 μM. One hour after addition, the cells were observed and photographed using an all-in-one fluorescence microscope BZ-X810 (Keyence Corporation), and the images were analyzed using the accompanying software, BZ-X800 Analyzer. The uptake ratio after one hour of uptake was compared based on the ratio of red brightness (integrated) to blue brightness (integrated). The results are shown in Figure 14 (in the figure, the bar height indicates the average value of n = 2 to 3, and the error bars indicate the standard deviation).

[0119] As shown in Figure 14, the IgG bound to peptides (T001, T006) containing the amino acid sequences obtained by screening using macrophage cells showed a greater increase in the amount of migration into macrophage cells than the IgG bound to peptides (R001, R005) containing the amino acid sequences obtained by screening using A549 cells.

[0120] Experimental Example 15: Evaluation of cell specificity of cell membrane permeability 2. One hour after addition, the cells were observed and photographed using an all-in-one fluorescence microscope BZ-X810 (Keyence Corporation) in the same manner as in Experimental Example 14, except that A549 cells cultured in the same manner as in Experimental Example 11 were used. Images were analyzed using the accompanying software, BZ-X800 Analyzer. The uptake ratios over a one-hour uptake period were compared based on the ratio of red luminance (integrated) to blue luminance (integrated). The results are shown in Figure 15 (in the figure, the bar height indicates the average value for n = 2 to 3, and the error bars indicate the standard deviation).

[0121] As shown in Figure 15, the IgG bound to peptides (R001, R005) containing the amino acid sequences obtained by screening using A549 cells showed a greater increase in the amount of translocation into A549 cells than the IgG bound to peptides (T001, T006) containing the amino acid sequences obtained by screening using macrophage cells.

[0122] The results of Experimental Examples 14 and 15 are shown together in Figure 16 (in the figure, the height of the bars indicates the average value of n = 2 to 3, and the error bars indicate the standard deviation). As shown in Figure 16, it can be seen that peptides R001 and R005, which contain amino acid sequences obtained by screening using A549 cells, have selective cell membrane permeability for A549 cells, and peptides T001 and T006, which contain amino acid sequences obtained by screening using macrophage cells, have selective cell membrane permeability for macrophage cells.

[0123] Experimental Example 17: Preparation of lipid nanoparticles (LNP) encapsulating siRNA and peptide modification Using a Lipid Nanoparticle (LNP-102) Exploration Kit (Cayman Chemical), LNP encapsulating siRNA was prepared according to the manual. However, 1,2-Distearoyl-rac-glycero-3-(2'-maleimidoethyl)polyoxyethylene (DSG-PEG(2000)-Maleimide) (Sunbright GS-020MA, Nippon Oil & Fats) was added as a lipid having a maleimide group for peptide modification. The siRNA was prepared by outsourcing the synthesis of the following sequence (siRNA (LUC)) against luciferase to Ajinomoto Bio-Pharma Services Gene Design Co., Ltd. ・ siRNA (LUC) Sense strand: 5'-CUUACGCUGAGUACUUCGAdTdT (SEQ ID NO: 52) Antisense strand: 5'-UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO: 53)

[0124] 1,2-Distearoyl-sn-Glycero-3-Phosphatidylcholine (DSPC), cholesterol, and DMG-PEG(2000) included in the kit were dissolved in ethanol. DSG-PEG(2000)-Maleimide was dissolved in DMF. The siRNA (LUC) was dissolved in 50 mM sodium acetate buffer (pH 5.0) (autoclaved). The lipid molar ratio recommended by the manufacturer's manual (ionized lipid SM-102: neutral phospholipid DSPC: cholesterol: PEGylated lipid DMG-PEG (2000) ratio = 50: 10: 38.5: 1.5) was based on the PEGylated lipid DMG-PEG (2000) ratio was reduced by 0.5, and instead DSG-PEG (2000) -Maleimide was added at 0.5 molar ratio. SM-102 was 100 mg / mL, DSPC was 25 mg / mL, cholesterol was 5 mg / mL, DMG-PEG (2000) was 1 mg / mL, and DSG-PEG (2000) -Maleimide was 5 mg / mL stock solution was used. To achieve the molar ratio and siRNA content recommended by the manual, the stock solution of each lipid was added to a 50 mM sodium acetate (pH 5.0) solution of siRNA and mixed for 15 seconds by rapid pipetting. The prepared sample (approximately 60 μL) was injected into a microdialysis cartridge Xpress Micro Dialyzer MD100 (molecular weight cutoff 140 kDa) (Scienova) (Funakoshi (SCI), code: 40931) set in a 2 mL tube. The sample was dialyzed against 1.2 mL of PBS (20 minutes, 3 times), and the recovered sample was used as maleimide group-containing LNP. Next, a 10 mM DMSO solution of Cys-attached membrane-permeable peptide 1 (CTVRGERYKH (SEQ ID NO: 54)) with a 5-fold molar amount of cysteine ​​at the N-terminus was added relative to the maleimide group, and the mixture was incubated at room temperature for 60 minutes. Cys-added membrane-permeable peptide 1 has the amino acid sequence of No. 2-6 (SEQ ID NO: 15) with cysteine ​​added to the N-terminus. Then, 5-fold molar amount of cysteine ​​(10 mM PBS solution) was added, and the mixture was incubated at room temperature for 15 minutes.After dialysis in the same manner as above, the recovered LNP was designated as peptide-modified LNP (abbreviation: A001) and stored at 4°C until use.

[0125] Peptide-modified LNP (abbreviation: B002) was prepared in the same manner as above, except that Cys-added membrane-permeable peptide 2 (CMNYHKSNRHN (SEQ ID NO: 55)) having an N-terminal cysteine ​​was used instead of Cys-added membrane-permeable peptide 1. Cys-added membrane-permeable peptide 2 has the amino acid sequence of No. 1-8 (SEQ ID NO: 8) with a cysteine ​​added to the N-terminus.

[0126] As a control, LNP in which cysteine ​​was bound to the maleimide group (Cys (control)) was prepared in the same manner as above, except that the Cys-attached membrane-permeable peptide was not used.

[0127] In this manner, the following siRNA (LUC)-encapsulating LNPs were obtained: peptide-modified LNP modified with Cys-added membrane-permeable peptide 1 (abbreviation: A001), peptide-modified LNP modified with Cys-added membrane-permeable peptide 2 (abbreviation: B002), and control LNP (Cys (control)) to which cysteine ​​was bound.

[0128] Experimental Example 18: Luciferase activity inhibitory effect of siRNA (LUC)-encapsulated LNPs on luciferase-expressing cells A549-Luc cells and BxPC-3-Luc#2 cells (both obtained from the RIKEN BioResource Research Center) were cultured in RPMI 1640 medium + 10% FBS (containing penicillin-streptomycin) and seeded at 2000 cells / 0.1 mL / well in a Corning 96-well white plate (product number 3610). At 37°C and 5% CO ２ After 24 hours of incubation, 10 μL of siRNA (LUC)-encapsulated LNP (specifically, Cys (control), A001, or B002) was added to each well so that the final siRNA concentration was 100 nM and then diluted three-fold. ２The cells were cultured for 24 hours under 500 saturation conditions, and the luciferase activity of each well was assessed using the ONE-Glo EX Luciferase Assay System (Promega, E8110). Specifically, after removing the medium from each well, 50 μL of a mixture of equal volumes of PBS and ONE-Glo EX was added to each well and stirred at 300 rpm for 3 minutes in the dark. Luminescence was then measured using a microplate reader (TECAN Spark) (imaging time: 1 second). Luciferase activity was calculated from the ratio of the luminescence intensity of each well to that of wells containing PBS instead of siRNA (LUC)-encapsulated LNP, and plotted against siRNA concentration. The measurement results for A549-Luc cells are shown in Figure 17, and the measurement results for BxPC-3-Luc#2 cells are shown in Figure 18 (in the figures, points indicate the average value of n = 3, and error bars indicate the standard deviation. However, when there is no error bar, the standard deviation is the size within the points).

[0129] As shown in Figures 17 and 18, in both cells, luciferase activity was suppressed depending on the concentration of siRNA added. When the concentration of siRNA added was the same, the peptide-modified LNPs (A001 and B002) suppressed luciferase activity more strongly than LNPs (Cys (control)) to which cysteine ​​was bound instead of peptide. This suggests that siRNA (LUC)-encapsulated LNPs could be efficiently delivered to the cytoplasm by modifying them with a membrane-permeable peptide.

[0130] Experimental Example 19: Preparation of mRNA-encapsulating lipid nanoparticles (LNP) and peptide modification As in Experimental Example 17, DSG-PEG(2000)-Maleimide was added to the Lipid Nanoparticle (LNP-102) Exploration Kit to prepare mRNA-encapsulating LNP. The mRNA used was CleanCap FLuc mRNA (TriLink BioTechnologies, L-7602-100) (mRNA(LUC)).

[0131] To achieve the mRNA content recommended by the manual, the lipid stock solution was added to a 50 mM sodium acetate (pH 5.0) solution of mRNA and mixed for 15 seconds while rapidly pipetting. The prepared sample was dialyzed in the same manner as in Experimental Example 17, and the collected sample was used as maleimide group-containing LNP. Subsequently, a 10 mM DMSO solution of Cys-added membrane-permeable peptide 1 was added in a 5-fold molar amount relative to the maleimide group, and the mixture was incubated at room temperature for 60 minutes. Then, a 5-fold molar amount of cysteine ​​(10 mM PBS solution) was added, and the mixture was incubated at room temperature for 15 minutes. After similar dialysis, the collected LNP was used as peptide-modified LNP (abbreviation: AM01) and stored at 4 ° C.

[0132] Peptide-modified LNP (abbreviation: BM02) was prepared in the same manner as above, except that Cys-added membrane-permeable peptide 2 was used instead of Cys-added membrane-permeable peptide 1.

[0133] Peptide-modified LNP (abbreviation: PM02) was prepared in the same manner as above, except that Cys-added membrane-permeable peptide 3 (CVRVKTYWENH (SEQ ID NO: 56)) having an N-terminal cysteine ​​was used instead of Cys-added membrane-permeable peptide 1. Cys-added membrane-permeable peptide 3 has the amino acid sequence of No. 3-5 (SEQ ID NO: 19) with a cysteine ​​added to the N-terminus.

[0134] As a control, LNP in which cysteine ​​was bound to the maleimide group (Cys (control)) was prepared in the same manner as above, except that the Cys-attached membrane-permeable peptide was not used.

[0135] In this manner, the following mRNA (LUC)-encapsulating LNPs were obtained: peptide-modified LNP modified with Cys-added membrane-permeable peptide 1 (abbreviation: AM01), peptide-modified LNP modified with Cys-added membrane-permeable peptide 2 (abbreviation: BM02), peptide-modified LNP modified with Cys-added membrane-permeable peptide 3 (abbreviation: PM02), and a cysteine-linked control LNP (Cys(control)).

[0136] Experimental Example 20: Effect of peptide modification on luciferase expression of mRNA (LUC)-encapsulated LNPs A549 cells and Panc-1 cells were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and seeded at 10,000 cells / 0.1 mL / well on a Corning 96-well white plate. Incubated at 37°C and 5% CO ２ After 24 hours of culture, 10 μL of mRNA (LUC)-encapsulated LNP (specifically, Cys (control), AM01, BM02, or PM02) was added to each well so that the final amount of mRNA added was 40 ng / well. ２ The cells were cultured under 5% CO₂ for 24 hours, and the luciferase activity of each well was evaluated using the ONE-Glo EX Luciferase Assay System in the same manner as in Experimental Example 18. The measurement results of the luminescence intensity of each well were plotted. The measurement results for A549 cells are shown in Figure 19, and the measurement results for Panc-1 cells are shown in Figure 20 (in the figures, the bars indicate the average value of n=3, and the error bars indicate the standard deviation).

[0137] As shown in Figures 19 and 20, for both A549 cells and Panc-1 cells, stronger luminescence was observed in cells treated with peptide-modified LNPs (AM01, BM02, and PM02) than in cells treated with LNPs to which cysteine ​​was bound instead of peptide (Cys (control)). This suggests that modifying mRNA (LUC)-encapsulated LNPs with a membrane-permeable peptide enabled efficient delivery of mRNA into the cytoplasm.

[0138] The membrane-permeable peptides according to the embodiments of the present invention can be suitably used in the production of, for example, DDS systems, pharmaceutical compositions, and the like.

Claims

1. A peptide or a salt thereof which contains an amino acid sequence X that satisfies the following (1) to (4) and has cell membrane permeability: (1) containing at least one amino acid residue selected from R, K, and H, and the total number of these three amino acid residues is 1 to 7; (2) containing at least one amino acid residue selected from Y, W, and F, and the total number of these three amino acid residues is 1 to 5; (3) containing at least one amino acid residue selected from L, V, and M, and the total number of these three amino acid residues is 1 to 4; and (4) The number of amino acid residues is 6 to 15; The amino acid sequence X is an amino acid sequence represented by any one of SEQ ID NOs: 1 to 40 or a homologous sequence thereof; The homologous sequence is a peptide or a salt thereof, which contains a substitution, insertion, or deletion of 1 to 5 amino acid residues in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 40 and has a sequence identity of 60% or more with the amino acid sequence.

2. The peptide or salt thereof described in claim 1, wherein the homologous sequence comprises a substitution, insertion, or deletion of 1 to 3 amino acid residues in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 40.

3. The peptide or salt thereof described in claim 1, wherein the homologous sequence comprises a substitution of one or two amino acid residues in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 40.

4. The peptide or salt thereof described in claim 3, wherein the substitution of the amino acid residue is a conservative substitution.

5. The peptide or salt thereof according to claim 1, which has cell-selective cell membrane permeability.

6. The peptide or salt thereof according to claim 1, which has selective cell membrane permeability to at least one selected from macrophage cells, cancer cells, neutrophil cells, mast cells, T cells, B cells, gastrointestinal epithelial cells, the blood-brain barrier, and skin tissue.

7. 2. The peptide or salt thereof according to claim 1, which has 6 to 30 amino acid residues.

8. The peptide or salt thereof according to claim 1, which has a GRAVY value of -2.6 to 1.9 and is linear.

9. A drug-peptide conjugate comprising a drug and the peptide or salt thereof according to claim 1 bound to the drug.

10. The drug-peptide conjugate of claim 9, wherein the drug is a protein or a nucleic acid.

11. 13. Use of the peptide or salt thereof according to claim 1 in drug delivery.

12. Step A of generating a cDNA display library comprising cDNA display molecules; A step B of incubating the cells in contact with the cDNA display library; and C. recovering the cDNA display molecule from the contents of the cells; A method for screening for a peptide having cytoplasmic transportability, comprising:

13. The method of claim 12, further comprising generating a sub-cDNA display library using the recovered cDNA display molecules.

14. The screening method according to claim 12, wherein the selection comprising the steps A, B, and C in this order is repeated two or more times.

15. The screening method described in claim 12, wherein the incubation time in step B is 20 hours to 72 hours.