Cell membrane permeable peptides and screening method thereof

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

[Technical Field]

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

[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 cell membrane permeability is imparted to a drug or its carrier by binding to a peptide having cell membrane permeability (hereinafter also referred to as "membrane-permeable peptide") (for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2021-531274 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[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; alternatively, 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 one 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, and the homologous sequence may comprise 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 a sequence identity of 60% or more 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 described in [9] above, the drug may be a protein or a nucleic acid.

[11] According to another aspect of the present invention, there is provided use of the peptide or salt thereof according to any one of the above [1] to [8] in drug delivery.

[12] According to another aspect of the present invention, there is provided a method for screening for peptides having 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 described in

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

[14] In the screening method described in

[12] or

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

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

[0008] [Figure 1] FIG. 1 is a schematic diagram illustrating an example of a method for preparing a cDNA display library. [Figure 2] FIG. 1 is a schematic diagram illustrating the structure of an example of a cDNA display molecule. [Figure 3] FIG. 1 is a schematic diagram illustrating a screening method according to one embodiment of the present invention. [Figure 4] FIG. 1 is a schematic diagram illustrating a screening method according to one embodiment of the present invention. [Figure 5] FIG. 1 shows template DNA sequences for protein expression in a cell-free system. [Figure 6] FIG. 1 shows template DNA sequences for protein expression in a cell-free system. [Figure 7] FIG. 1 shows template DNA sequences for protein expression in a cell-free system. [Figure 8] FIG. 1 shows template DNA sequences for protein expression in a cell-free system. [Figure 9] FIG. 1 shows template DNA sequences for protein expression in a cell-free system. [Figure 10] 1 shows photographs showing the results of an intracellular translocation test of eGFP-peptide fusion proteins. [Figure 11]1 shows photographs showing the results of an intracellular translocation test of eGFP-peptide fusion proteins. [Figure 12] 1 shows photographs depicting the results of an intracellular internalization test of IgG-peptide conjugates in macrophage cells. [Figure 13] FIG. 1 shows the ratio of the amount of IgG-peptide conjugate internalized in macrophage cells to the amount of IgG internalized in the cells. [Figure 14] FIG. 1 shows the ratio of the amount of IgG-peptide conjugate internalized in macrophage cells to the amount of IgG internalized in the cells. [Figure 15] FIG. 10 also shows the ratio of the amount of IgG-peptide conjugates internalized in A549 cells to the amount of IgG internalized in the cells. [Figure 16] FIG. 10 also shows the ratio of the amount of IgG-peptide conjugate internalized into macrophage cells and A549 cells to the amount of IgG internalized into cells. [Figure 17] FIG. 1 shows the luciferase activity inhibitory effect of siRNA (LUC)-encapsulated lipid nanoparticles in A549-Luc cells. [Figure 18] FIG. 1 shows the luciferase activity inhibitory effect of siRNA (LUC)-encapsulated lipid nanoparticles on BxPC-3-Luc#2 cells. [Figure 19] FIG. 1 shows the luciferase expression effect of mRNA (LUC)-encapsulated lipid nanoparticles in A549 cells. [Figure 20] FIG. 1 shows the luciferase expression effect of mRNA (LUC)-encapsulated lipid nanoparticles in Panc-1 cells. DETAILED DESCRIPTION OF THE INVENTION

[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, each embodiment can be combined as appropriate. Furthermore, in this specification, the symbol "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. Cell membrane-permeable peptides According to one aspect of the present invention, there is provided 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) It contains 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) It contains 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) It contains 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. (4) The number of amino acid residues is 6 to 15. As described above, a peptide having an amino acid sequence X consisting of 6 to 15 amino acid residues, which contains a specific number of each 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 chain-like side chain selected from L, V, and M, can exhibit excellent cell membrane permeability. In the amino acid sequence X that satisfies the above (1), the total number of the three types of amino acid residues, R, K, and H, may be 1 to 6. The proportion of the total number of the three types of amino acid residues R, K, and H relative to the total number of amino acid residues constituting the amino acid sequence X is, for example, 7% to 54%, and may be 7% to 42%. The proportion of the total number of the three amino acid residues Y, W, and F relative to the total number of amino acid residues constituting the amino acid sequence X is, for example, 7% to 50%, and may be 8% to 50%. The proportion of the total number of the three types of amino acid residues L, V, and M relative to the total number of amino acid residues constituting the amino acid sequence X can be, for example, 7% to 40%. In one embodiment, the amino acid sequence X may comprise at least one amino acid residue selected from K, L, and V. The number of amino acid residues constituting the amino acid sequence X can 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 bonding between the N-terminal amino acid residue and the C-terminal amino acid residue of the peptide, by bonding between either terminal amino acid residue and an amino acid residue in a non-terminal portion, or by bonding 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, 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 amino acid sequence X, the number of consecutive occurrences of each of these amino acid residues is 2 or less, and the number of consecutive occurrences of the other amino acid residues may be 1. In another embodiment, the amino acid sequence X forms a cyclic structure; and when amino acid residues selected from F, L, and Y are present in the 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 the other amino acid residues may be 1. Here, the number of consecutive amino acid residues represents the number of consecutive amino acid residues that can occur in amino acid sequence X. Thus, when the number of consecutive amino acid residues is 1, the amino acid residues do 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, in the case of a linear peptide, 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 addition, in the case of a cyclic peptide, 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 the two Cs that form disulfide bonds. In this embodiment, the above percentage is preferably 30% to 50%.

[0014] In one embodiment (embodiment a), the 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 included as two consecutive amino acid residues, such as HV, VH, HY, or YH. (ii) When H is located at the C-terminus, the amino acid sequence X contains one or more residues of each of six amino acids consisting of 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. (iv) R located at the N-terminus is included as two consecutive amino acid residues, RV or RW.

[0016] In one embodiment (embodiment c), the amino acid sequence X is linear, contains only K as a basic amino acid residue, and contains at least two or more amino acid residues selected from F, V, L, and I, the total number of which accounts for 40% to 50% of the total number of amino acid residues constituting the 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, a 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 a peptide or protein as a whole, 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 a 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 reaction utilizing the functional group. Examples of amino acids having a functional group in their side chain 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 cells 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. Herein, a peptide may be labeled with a fluorescent dye such as Alexa Fluor fluorescent dye (Molecular Probes), Cy fluorescent dye, or FITC, or a fusion protein of a peptide with eGFP or GFP may be prepared and incubated with cells at a concentration of 5 μM for 1 hour. When fluorescence from the labeled peptide or fusion protein is observed within the cells under a fluorescent microscope, preferably when fluorescence intensity is higher than that of a control without the peptide, the peptide can be determined to have cell membrane permeability; however, the determination of cell membrane permeability is not limited thereto. In one embodiment, the membrane-permeable peptide can be conjugated to a drug to improve the intracellular internalization of 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, or facilitating purification 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 of SEQ ID NOS: 1 to 40 shown in Table 1, or sequences homologous thereto. In one embodiment, the membrane-permeable peptide is a peptide consisting of an 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 an 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 (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 having a tendency to form an α-helical structure, and the predetermined GRAVY value range). As used herein, the term "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, the homologous sequence may include substitutions, insertions, or deletions of 1, 2, 3, 4, or 5, preferably 1, 2, or 3, and more preferably 1 or 2, amino acid residues relative to the original amino acid sequence. The substitution of the amino acid residues may be, for example, conservative substitution. Conservative substitution refers to the substitution of 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 may be carried out so that the total proportion of amino acid residues that tend to form the α-helical structure (specifically, E, M, A, L, Q, K, R, and H) in the peptide sequence (i.e., homologous sequence) after substitution 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 natural 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 natural amino acid residue of 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 the amino acid sequence shown in any one 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 the amino acid sequence shown in any one of SEQ ID NOS: 1 to 40 or a homologous sequence thereof from the viewpoint of imparting reactivity with a drug or its carrier, adding a linker, imparting hydrophilicity or hydrophobicity, or facilitating purification 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 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] [Table 1]

[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 the amino acid sequence X of embodiment a, the amino acid sequences represented by SEQ ID NOs: 14, 18, 20, 21, 24, 25, 37, and 40 correspond to the amino acid sequence X of embodiment b, the amino acid sequences represented by SEQ ID NOs: 4, 22, 23, and 27 correspond to the amino acid sequence X of embodiment c, and the amino acid sequences represented by SEQ ID NOs: 6 and 10 correspond to the amino acid sequence X of embodiment d. Specific examples of the 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 homologous sequences thereof.

[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 A drug-peptide conjugate according to an embodiment of the present invention comprises a drug and a membrane-permeable peptide described in Section A conjugated 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 the drug-peptide conjugate, the drug and the membrane-permeable peptide may be bonded directly or via a linker. In the case of direct bond, the 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, the drug-peptide conjugate can be formed, for example, by reacting a functional group at one end of the linker with a functional group of the drug, and simultaneously reacting a functional group at the other end of the linker with a functional group of the membrane-permeable peptide. Between the functional groups 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, the 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 Cell Membrane-Permeable Peptides 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, the membrane-permeable peptide preferably 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, the membrane-permeable peptide preferably 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, the membrane-permeable peptide preferably 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, the membrane-permeable peptide preferably has skin permeability. Alternatively, if the drug is intended to act on the skin, it is preferably capable of transferring into epidermal or dermal cells.

[0037] When the membrane-permeable peptide is used in drug delivery, the membrane-permeable peptide can be bound to a drug or its carrier (specifically, a component of the carrier). Thus, the present invention can provide a method for increasing the membrane permeability of a drug or drug delivery carrier, which comprises 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 imparted or increased membrane permeability, which comprises binding the membrane-permeable peptide to the drug or drug delivery carrier or a component thereof. The bond may be a covalent bond or a non-covalent bond. A complex (conjugate) in which a drug and a membrane-permeable peptide are covalently bonded 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 amphiphilic compounds or polymers, including polyamino acids, lipids, polysaccharides, and other polymers (e.g., polyethylene glycol). In one embodiment, the membrane-permeable peptide can be bound to the end of these components that make up the nanoparticle so that it is exposed on the nanoparticle surface. The binding method can be appropriately selected depending on the purpose. In the case of a covalent bond, it can be carried out in the same manner as in the case of binding a drug and a membrane-permeable peptide, and a combination of mutually reactive functional groups as described above can be used. In the case of a non-covalent bond, binding (complexation) can be achieved by utilizing electrostatic interactions, hydrophobic interactions, hydrogen bonds, van der Waals forces, etc.

[0038] D. Screening method for peptides with cell membrane permeability A method for screening a peptide having cell membrane permeability according to an embodiment of the present invention includes the steps of: Step A, generating a cDNA display library containing cDNA display molecules; Step B: incubating the cells in contact with the cDNA display library; and C. recovering the cDNA display molecule from the contents or permeate of the cells; Includes: The above screening method utilizes the cDNA display method. The cDNA display method is a genotype-phenotype matching technique that allows for a 1:1 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 above screening method utilizes the cDNA display method using highly stable cDNA display molecules, it is possible to efficiently obtain the desired peptide even in a system using living cells. Furthermore, by selecting the cells to be used for screening, it is possible to efficiently obtain peptides that have membrane-permeability for the desired cells.

[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 repeated using the selection products (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, and peptides with higher membrane permeability can 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 can be three or four or more, and for example, six or less, preferably five or less.

[0040] D-1. Process 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. A cDNA display library can be produced by a method including, for example, preparing a DNA library containing DNAs encoding random amino acid sequences (step a), as shown in FIG. 1; transcribing the DNA of this DNA library into mRNA to obtain an mRNA library (step b); linking a puromycin-conjugated linker X to the 3' end of the mRNA of 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 backbone m containing single-stranded DNA and / or peptide nucleic acid (PNA) to which puromycin P is linked, a ligation site at the 5' end of the backbone 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 ensures reliable association of the peptide with the cDNA.

[0042] The main skeleton 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 protein synthesized in a translation system.

[0044] Preferably, 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. Among 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, 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 cleaved residue of the linker X, and includes a linker residue 1 to which puromycin 3 is linked, a cDNA 5 linked to one end of the linker residue 1, an mRNA 2 linked to the other end and hybridized with the cDNA 5, and a peptide 4 bound to the puromycin 3.

[0048] For details of the structure and production method of the cDNA display molecule 10 and the production method of the 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 ~1.0×10 13 , preferably 1.0 x 10 11 ~1.0×10 13 , more preferably 1.0 × 10 11 ~1.0×10 12 It could be.

[0050] D-2. Process 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.Process 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 peptides consisting of the identified amino acid sequence can be obtained as membrane-permeable peptides.

[0054] The sub-cDNA display library can be prepared from the recovered cDNA display molecules in the same manner as the cDNA display library preparation method 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, contact and incubation of cells with a cDNA display library in step B is performed by adding a 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 × 10 5 ~1.0×10 8 , preferably 1.0 x 10 6 ~1.0×10 8 , more preferably 1.0 × 10 7 ~1.0×10 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 the cell contents are recovered by hypotonic treatment, disruption, or the like. 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 FIG. 3, peptides that can permeate cell membranes and remain in cells (in other words, peptides that can translocate to the cytoplasm) 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 42a 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, and 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). The cDNA display molecules 10 can be recovered from the culture medium using a nucleic acid purification kit or the like. 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' can be prepared (Step A), and the sub-cDNA display library 20' can be used to perform the subsequent Steps B and C.

[0060] Alternatively, the cDNA display library can 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 can 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. [Example]

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

[0064] An mRNA library was constructed 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. 1–2 pmol of cDNA, 4 μL of rNTP mixture, 2 μL of transcriptase, and 4 μL of reaction buffer were mixed and then diluted 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 linker) described in WO2006 / 041194 was prepared as follows. 20 pmol of mRNA and 20 μM of linker were mixed and then diluted with purified water (Ambion) to a volume of 20 μL. The solution was incubated at 90°C for 30 seconds, cooled to 70°C at 0.12°C / s, incubated for 30 seconds, and then cooled to 4°C at 0.06°C / s. To the cooled solution, 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 and incubated at 25°C for 1 hour to produce mRNA-linker conjugates.

[0066] Next, peptide synthesis was performed by mixing the mRNA-linker conjugate with a cell-free translation system. A translation reaction solution was prepared by mixing 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, and then incubated at 25°C for 15 minutes. Then, 46 μL of salt mix (18.25 μL of 3 M KCl, 4.75 μL of 1 M MgCl) was added, and the mixture was incubated at 25°C for 1 hour. Furthermore, 28 μL of 0.5 M EDTA pH 8.0 (Thermo Fisher Scientific) was added. This resulted in the production of the mRNA-linker-peptide conjugate (post-translation product).

[0067] The biotin contained in the linker in the post-translation product was utilized for purification using DynaBeads MyOne Streptavidin C1 (Thermo Fisher Scientific) (hereafter referred to as magnetic beads). After thoroughly suspending 150 μL of magnetic beads, the beads were washed twice with an equal volume of 2× Binding buffer (buffer composition: 20 mM Tris-HCl (pH 7.5), 2 M NaCl, 2 mM EDTA, 0.2% Polyoxyethylene (20) Sorbitan Monolaurate) (the supernatant was discarded after addition and suspension). Next, 348 μL of the post-translation product was added, and the mixture was incubated at 25°C for 30 minutes while rotating on a rotator. The supernatant was then discarded, and the mixture was washed twice with 400 μL of 1× Binding buffer. It was then 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 10× RT buffer (Promega), and 166 μL of purified water were added to the magnetic beads, and the mixture was mixed by tapping. 2 μL of M-MLV Reverse Transcriptase (Promega) was then 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 1× binding buffer, followed by 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 way, and the combined solution was brought 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 dialysis solution (PBS) at 4°C for 60 minutes, the dialysis solution was replaced and the solution was stirred at 4°C for another 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 in 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-hiSIECTM (Fujifilm Corporation)" suspended in Seeding Medium (Fujifilm Corporation) were added to a 1.0 x 10 5 Cells were seeded at 1000 cells / well. Following the Fujifilm manual, the cells were cultured at 37°C under 5% CO2, and the medium was changed according to the schedule in Example 2 of the 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 performed on day 12, and the second permeation test (round 2) was performed on day 14.

[0073] <First penetration test> The variable region peptide sequences in the cDNA display libraries were 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. Three cDNA display libraries displaying linear peptides with different amino acid residue numbers or three cDNA display libraries displaying cyclic peptides with different amino acid residue numbers were mixed to prepare two cDNA display libraries: a linear mixed library and a cyclic mixed library. These libraries were then subjected to parallel and separate permeation experiments. Each mixed library was suspended in PBS and diluted with RPMI 1640 medium without fetal bovine serum (FBS). After removing the medium from the culture inserts and wells, 150 μL of each diluted library solution (approximately 100 fmol–300 fmol of cDNA display molecules) was added to separate culture inserts (apical side), and 600 μL of RPMI 1640 medium was added to each well (basolateral side) to begin the permeation experiment. After incubation at 37°C under 5% CO for 1 hour, the basolateral medium was collected in a 1.5 mL tube and stored at -80°C until quantitative analysis of nucleic acids and the next round of display library construction.

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

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

[0076] <Third and fourth permeation tests and purification and amplification of permeation samples> For the third and fourth rounds of permeation testing, new human iPS cell-derived intestinal epithelial cells, F-hiSIECTM, were prepared in the same manner as for the first and second rounds. Using the cDNA display library from the third round, permeation testing and purification and amplification of permeation samples were performed in the same manner as above to generate the cDNA display library for the fourth round. Using the cDNA display library for four rounds, a penetration test and purification and amplification of the penetration sample were carried out in the same manner as described above.

[0077] <Next-generation sequencing (NGS) analysis> Using Illumina Inc.'s published documentation on the Tailed-PCR workflow, samples collected from each penetration test were used to create libraries for next-generation sequencing by adding adapter sequences using standard PCR, and then analyzed using the MiniSeq (Illumina) next-generation sequencer. The resulting paired-end fastq files (two fastqs) were analyzed using Flash2 to determine the amino acid sequences of peptides that penetrate the cell membrane. The sequences with the highest read counts in the fourth penetration test are listed below.

[0078] [Table 2]

[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 Cells were seeded at 1000 cells / well. After 72 hours of incubation at 37°C under 5% CO2, the medium was removed from each well. A cDNA display library equivalent to that in Experimental Example 2 (a total of six cDNA display libraries with different numbers of amino acid residues in the peptides displayed (three linear peptides and three 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) was added to each well. After 1 hour of incubation at 37°C under 5% CO2, the added library was recovered. Then, 300 μL of fresh medium was added, and incubation was continued for 23 hours. The cells were then washed as follows.

[0080] The cells were washed three times with 300 μL of PBS, 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). The resulting paired-end fastq files were analyzed using Flash2 to determine the amino acid sequences of peptides transported into the cells. The sequences with the highest read counts were listed below.

[0082] [Table 3]

[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 Cells were seeded at 1000 cells / well. A total of two selections were performed using the same method 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 NGS analysis were listed as follows:

[0084] [Table 4]

[0085] In Experimental Example 3, a similar experiment was performed using an mRNA display library instead of a cDNA display library. One hour after cell contact, the residual rates of nucleic acids were over 90% for both linear 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. One hour after cell contact, the residual rates of nucleic acids were 23% and 51%, respectively, for linear 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 the cDNA display method enables highly stable screening, even in systems using living cells.

[0086] [Experimental Example 5: Screening of peptides that translocate into neutrophil cells] HL-60 cells (RIKEN BioResource Research Center) were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and plated at 1.0 × 10 cells per well in a 24-well plate. 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 cultured at 37°C under 5% CO2 for 72 hours to differentiate into neutrophils.

[0087] The medium was removed from each well, and a cDNA display library equivalent to that used in Experimental Example 3 was diluted to 310 μL with RPMI 1640 medium without FBS or antibiotics. 300 μL of this solution was added to each well. After 1 hour of incubation at 37°C and 5% CO2, the added library was recovered. 300 μL of PBS was then added, and the cells were collected by pipetting. The cells were then washed with 300 μL of PBS + 0.05% Tween 20 and similarly collected 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 then collected by centrifugation at 1000 g for 3 minutes. This supernatant was purified and amplified in the same manner as in Experimental Example 2 to prepare a 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 files were analyzed using Flash2 to determine the amino acid sequences of peptides transported into the cells. The sequences with the highest read counts were listed below.

[0089] [Table 5]

[0090] [Experimental Example 6: Screening of peptides that translocate into macrophage cells] THP-1 cells (RIKEN BioResource Research Center) were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and plated at 1.0 × 10 5THP-1 cells were seeded at 1000 cells / well. A 10 μg / mL DMSO stock solution of phorbol 12-myristate 13-acetate (PMA, Funakoshi, AG-CN2-0010-M001) was added at 1 / 1000 volume, and the cells were incubated at 37°C under 5% CO2 with 10 ng / mL PMA for 24 hours. The medium was removed, and 300 μL of fresh RPMI 1640 medium + 10% FBS was added per well and incubated for 24 hours. IFN-γ (Cosmo Bio (Proteintech), HZ-1301) and LPS (Cosmo Bio (Santa Cruz Biotechnology), SC-3535) were added to final concentrations of 20 ng / mL and 250 ng / mL, respectively, and the cells were further incubated at 37°C under 5% CO2 for 24 hours to differentiate the THP-1 cells into macrophages.

[0091] The medium was removed from each well, and a cDNA display library equivalent to that used in Experimental Example 3 was diluted to 310 μL with RPMI 1640 medium without FBS or antibiotics, and 300 μL of this solution was added to each well. After incubation at 37°C under 5% CO for 1 hour, the cells were washed as follows.

[0092] The cells were washed three times with 300 μL of PBS, 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 treated with hypotonicity 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 files were analyzed using Flash2 to determine the amino acid sequences of the peptides transported into the cells. The sequences with the highest read counts were listed below.

[0094] [Table 6]

[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. The assay buffer used was DMEM / F-12 medium (Gibco, 11330-032) + 0.5% BSA (Fujifilm Wako Pure Chemical Industries, 017-22231) + 500 nM hydrocortisone. The first day of culture was considered day 1, and the first (round 1) permeation test was performed on day 5, followed by the second (round 2) test on day 7.

[0096] <First penetration test> The variable region peptide sequences in the cDNA display libraries were 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. 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 parallel and separate penetration experiments. Each mixed library was suspended in PBS to obtain two mixed cDNA display library solutions. On the fifth day of incubation, the inserts were transferred to a washing well. The medium 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 diluted with 200 μL of Assay Buffer, and 200 μL of each was added to separate inserts to begin the permeation assay. After 2 hours of incubation at 37°C and 5% CO2, the receptor solutions were collected.

[0097] Purification and amplification of permeated samples 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, and then used as 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 permeation sample purification and amplification were performed in the same manner as above to prepare a cDNA display library for the fourth round. Using the cDNA display library for four rounds, a penetration test and purification and amplification of the penetration sample were carried out in the same manner as described above.

[0100] Next-generation sequencing (NGS) is performed on the samples collected from each permeation test to confirm the degree of convergence of the amino acid sequences. The permeation test is then repeated until sufficient convergence is achieved. Once sufficient convergence is confirmed, the sequence with the highest read count is identified as the amino acid sequence of the membrane-permeable peptide.

[0101] [Experimental Example 8: Screening of peptides that penetrate the skin model] A 3D skin model similar to human skin, EpiDerm Full Thickness 400 (Kurabo, #EFT-400), was cultured using the kit's culture medium according to the manufacturer's instructions. This resulted in the construction of a skin model on the donor side (inside) of a collagen-coated culture cup, the size of which was equivalent to a 24-well plate. Screening was initiated by adding 100 μL of the peptide library to the donor side of the culture cup. After 24 hours of culture, 2.5 mL of culture medium was collected from the receptor side. The mixture was placed in an ultrafiltration centrifugal filter (Amicon Ultra 10 kDa MWCO, Millipore UFC5010) and centrifuged (TOMY TMP-24, 87 mm radius of rotation) at 14,000 rpm for 10 minutes at 4°C to concentrate the mixture 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 collected. The DNA samples were amplified by PCR according to standard methods to prepare a cDNA display library for round 2. The penetration test was repeated in the same manner, for a total of four rounds.

[0102] The samples collected from 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 sequence of the peptide that permeates the skin model. The sequence with the highest read count was identified as "RWNCVSCAFL" (SEQ ID NO: 40).

[0103] [Experimental Example 9: Skin permeability test of peptide] As in Experimental Example 8, a three-dimensional skin model, EpiDerm Full Thickness 400, was prepared according to the manufacturer's instructions. 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). The permeability test was initiated by adding 100 μL of 1% DMSO-containing PBS solution of each FAM-modified peptide to the center of the donor side of the cup containing the skin model (n=3). After 24 hours of incubation at 37°C under 5% CO2, 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 TAT sequence FAM-modified peptide that permeated outside the cup relative to the amount added, i.e., the permeability, was 21.4±3.3% (mean value of three cases ± standard deviation).The permeability of the FAM-modified peptide of sequence number 40 was 49.8±10.0% (mean value of three cases ± standard deviation), which was significantly higher than that of the TAT sequence (p<0.01).

[0104] [Experimental Example 10: Preparation of eGFP-peptide fusion protein] We commissioned Eurofins to synthesize template DNA necessary for the expression of eGFP or eGFP-peptide fusion proteins, including DNA necessary for protein expression in a cell-free system. The synthesized template DNAs are as follows, and their DNA sequences are shown in Figures 5 to 9. [Table 7]

[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: N100 (Taiyo Nippon Sanso SI Division).

[0106] The template DNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (approximately 100 μg / mL) and further diluted with purified water to the desired concentration according to the manufacturer's instructions. This was then mixed with the kit's premixed reaction mixture to a final volume of 100 μL. Subsequently, 1 mL of the kit's dialysate was added to the outside of the dialysis unit, and the reaction mixture was then placed in the dialysis unit. After shaking at 30°C for 16 hours (amplitude 25 mm, 80 rpm, Bioshaker BR-53FP, Taitec Corporation), the reaction mixture was collected in a new tube and placed on ice for 5 minutes to terminate the reaction. The reaction mixture was then adjusted to approximately 1000 μL with PBS + 0.05% Tween 20 and diluted with HisPurpose HCl. (R) , Ni-NTA Magnetic Beads (Thermo Scientific (R) Purification was performed using a HPLC-MS / MS antibody (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, 400 μL (4 volumes) of equilibration buffer (PBS + 0.05% Tween 20) was added, and the mixture was suspended by vortexing (weak) for 10 seconds. The beads were collected using a magnetic stand, the supernatant was removed, and 1000 μL (10 volumes) of equilibration buffer was added. The mixture was suspended by vortexing (weak) for 10 seconds. The supernatant was similarly removed, and the beads were recovered and equilibrated.

[0107] The equilibrated beads were added to the reaction solution and suspended. After rotating and mixing at 4°C for 1 hour, 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 buffer, and the yellow-green fraction was collected. That is, elution was performed twice with 20 μL of PBS+250 mM imidazole, twice with 20 μL of PBS+250 mM imidazole+1 mM EDTA, and twice with 20 μL of PBS+250 mM imidazole+10 mM EDTA.

[0108] The recovered eGFP and eGFP-peptide fusion proteins were subjected to ultrafiltration (Amicon Ultra 10 kDa 0.5 mL Millipore UFC501024) with 20 mM HEPES (pH 7.0) + 150 mM NaCl, and then used in the following cell experiments. 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 RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) were seeded at 5000 cells / well in a 96-well plate and cultured at 37°C under 5% CO2 for 24 hours. After removing the medium, the cells were washed once with 200 μL of RPMI1640 medium without FBS or antibiotics. Protein solutions (eGFP, eGFP-TAT, eGFP-L10#1) were diluted with the same RPMI1640 medium and added to the wells (final concentration 5 μM, 30 μL / well). After 30 minutes of culture at 37°C under 5% CO2, the cells were incubated with 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 15 minutes of incubation, 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 the plate was observed under a fluorescence microscope. Images taken with the 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, a fusion protein of eGFP and a peptide containing the amino acid sequence of SEQ ID NO: 7, green fluorescence from eGFP was observed surrounding blue fluorescence from the nucleus, indicating that GFP-L10#1 was efficiently translocated into the cytoplasm. Here, the mass of eGFP is 27 kDa, and the mass of the peptide attached to eGFP is 1.2 kDa. In contrast, in the sample using eGFP and the sample using eGFP-TAT, a fusion protein of eGFP and the known membrane-permeable peptide TAT, only slight fluorescence from eGFP was observed around the nucleus. As a result, translocation of eGFP or the eGFP fusion protein into the cytoplasm was hardly confirmed.

[0111] [Experimental Example 12: THP-1 macrophage intracellular internalization test of eGFP-peptide fusion protein] 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 and washing once with 200 μL of RPMI 1640 medium without FBS or antibiotics, a protein solution (eGFP, eGFP-L10#2) was diluted in 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 Fig. 11, in the sample using eGFP-L10#2, which is a fusion protein of a peptide containing the amino acid sequence of SEQ ID NO: 37 and eGFP, green fluorescence derived from eGFP was observed together with blue fluorescence derived from the nucleus, indicating that eGFP-L10#2 had migrated into macrophage cells. Here, the mass of eGFP is 27 kDa, and the mass of the peptide added to eGFP is 1.5 kDa. On the other hand, in the sample using GFP, fluorescence derived from eGFP was not observed, and as a result, the migration of eGFP into macrophage cells was not confirmed.

[0113] [Experimental Example 13: Intracellular Migration Test of Alexa647-Labeled IgG in Macrophage Cells] <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) so that its final concentration was 5 mg / mL, and AlexaFluor647 NHS Ester was added at a 10-fold molar ratio to hIgG, and the mixture was inverted and stirred at 25 °C for 30 minutes. The reaction solution was applied to a Zeba spin desalting column (7K MWCO) (Thermo, #89882) equilibrated with PBS, and Alexa647-labeled IgG was recovered after removing unreacted reagents.From the measurement of absorbance values at 280 nm and 650 nm as described in the manufacturer's manual, it was calculated that approximately 3 molecules of Alexa647 were labeled per molecule of the recovered IgG.

[0114] <Peptide Addition to Alexa647-Labeled IgG> Approximately 1 mg of Alexa647-labeled IgG (in PBS) was mixed with a 20-fold molar equivalent of a 10 mM DMSO solution of maleimide-PEG2-NHS ester (Tokyo Chemical Industry, product code M3339) or a 10 mM PBS solution of sulfo-SMCC crosslinker, which contains a hydrophilic linker. The mixture was then mixed by end-over-end stirring at 25°C for 1 hour. The reaction mixture was then applied to a Zeba spin desalting column (7K MWCO) equilibrated with PBS to remove unreacted crosslinker and recover the IgG fraction. A peptide containing the amino acid sequence obtained in the screening above and containing a C at the N-terminus (synthesized by Eurofins Genomics, Inc.; the amino acid sequence of the peptide conjugated to IgG is shown in the table below) was dissolved in DMSO to a 10 mM DMSO solution. The DMSO solution of the peptide was added to the recovered IgG fraction in a 5-fold molar equivalent to the IgG, and EDTA was added to a final concentration of 1 mM. The mixture was then mixed by end-over-end stirring at 25°C for 1 hour. A five-fold molar excess of cysteine was then added, and the mixture was stirred end-over-end at 25°C for 30 minutes. The reaction mixture was applied to a Zeba spin desalting column (7K MWCO) equilibrated with PBS to remove unreacted peptide, cysteine, and EDTA, and the IgG-peptide conjugate was recovered. For example, in the case of peptide T001, measurements of absorbance at 280 nm and 650 nm and fluorescence intensity derived from tryptophan (excitation wavelength: 280 nm, emission wavelength: 350 nm) indicated that three to four peptide molecules were conjugated per IgG molecule.

[0115] [Table 8]

[0116] <Peptide-mediated IgG delivery into THP-1 macrophages> The experiment 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 them once with 200 μL of FBS- and antibiotic-free RPMI 1640 medium, the protein solution (IgG or IgG-peptide conjugate) was diluted with the same RPMI 1640 medium and added to the wells (final concentration of 5 μM, 30 μL / well). Cells were observed 1 hour after addition, using the same procedure as in Experiment 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 rate after 1 hour was compared based on the ratio of red intensity (integrated) to blue intensity (integrated). The results are shown in Figure 13 (in the figure, bar heights indicate the mean value of n = 3, and error bars indicate the standard deviation).

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

[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 intensity (integrated) to blue intensity (integrated). The results are shown in Figure 14 (in the figure, the bar height indicates the average value for n = 2–3, and the error bars indicate the standard deviation).

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

[0120] [Experimental Example 15: Evaluation of cell specificity of cell membrane permeability 2] The same procedure as in Experimental Example 14 was used, except that A549 cells cultured as in Experimental Example 11 were used. 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 over one hour was compared based on the ratio of red intensity (integrated) to blue intensity (integrated). The results are shown in Figure 15 (in the figure, the bar height indicates the average value for n = 2–3, and the error bars indicate the standard deviation).

[0121] As shown in Figure 15, IgG bound to peptides (R001, R005) containing amino acid sequences obtained by screening using A549 cells showed a greater increase in the amount of translocation into A549 cells than IgG bound to peptides (T001, T006) containing 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 bar height indicates the average value of n = 2 to 3, and the error bars indicate the standard deviation). As shown in Figure 16, peptides R001 and R005 containing amino acid sequences obtained by screening using A549 cells have selective cell membrane permeability for A549 cells, and peptides T001 and T006 containing amino acid sequences obtained by screening using macrophage cells have selective cell membrane permeability for macrophage cells.

[0123] [Experimental Example 17: Preparation of siRNA-encapsulating lipid nanoparticles (LNPs) and peptide modification] siRNA-encapsulating LNPs were prepared using the Lipid Nanoparticle (LNP-102) Exploration Kit (Cayman Chemical) according to the manufacturer's instructions. However, 1,2-Distearoyl-rac-glycero-3-(2'-maleimidoethyl)polyoxyethylene (DSG-PEG(2000)-Maleimide) (SUNBRIGHT GS-020MA, Nippon Oil & Fats Co., Ltd.) was added as a lipid having a maleimide group for peptide modification. The siRNA was prepared by outsourcing the synthesis of the following sequence against luciferase (siRNA(LUC)) 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) were dissolved in ethanol. DSG-PEG(2000)-Maleimide was dissolved in DMF. Meanwhile, siRNA (LUC) was dissolved in 50 mM sodium acetate buffer (pH 5.0) (autoclaved). Based on the lipid molar ratio recommended by the manufacturer (ionic lipid SM-102:neutral phospholipid DSPC:cholesterol:PEGylated lipid DMG-PEG(2000) = 50:10:38.5:1.5), the ratio of PEGylated lipid DMG-PEG(2000) was reduced by 0.5 and replaced with 0.5 DSG-PEG(2000)-Maleimide. Stock solutions of 100 mg / mL SM-102, 25 mg / mL DSPC, 5 mg / mL cholesterol, 1 mg / mL DMG-PEG(2000), and 5 mg / mL DSG-PEG(2000)-Maleimide were used. Each lipid stock solution was added to a 50 mM sodium acetate (pH 5.0) solution of siRNA to achieve the molar ratio of the components and the siRNA content recommended in the manual, and the mixture was mixed by quick pipetting for 15 seconds. 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 min, 3 times), and the recovered sample was used as maleimide-containing LNP. Next, a 10 mM DMSO solution of Cys-added membrane-permeable peptide 1 (CTVTRGERYKH (SEQ ID NO: 54)) with an N-terminal cysteine was added in a 5-fold molar amount 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 an N-terminal cysteine. Subsequently, a 5-fold molar amount of cysteine (10 mM PBS solution) was added and incubated at room temperature for 15 minutes. After dialysis as described 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-attached membrane-permeable peptide 1 (abbreviation: A001), peptide-modified LNP modified with Cys-attached membrane-permeable peptide 2 (abbreviation: B002), and control LNP (Cys (control)) to which cysteine was attached.

[0128] [Experimental Example 18: Inhibitory effect of siRNA (LUC)-encapsulated LNP on luciferase activity in luciferase-expressing cells] A549-Luc cells and BxPC-3-Luc#2 cells (both obtained from the RIKEN BioResource Research Center) were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and seeded at 2000 cells / 0.1 mL / well into Corning 96-well white plates (product number 3610). After 24 hours of incubation at 37°C and 5% CO2, 10 μL of siRNA (LUC)-encapsulated LNP (specifically, Cys (control), A001, or B002) was added to each well to achieve a final siRNA concentration of 100 nM followed by 3-fold dilutions. The cells were then incubated at 37°C and 5% CO2 for 24 hours, 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. Figure 17 shows the results for A549-Luc cells, and Figure 18 shows the results for BxPC-3-Luc#2 cells (in the figures, points represent the mean values of n = 3, and error bars represent the standard deviation. However, if there are no error bars, the standard deviation is the size of the points).

[0129] As shown in Figures 17 and 18, luciferase activity was suppressed in both cells depending on the concentration of siRNA added. When the siRNA concentration was the same, the peptide-modified LNPs (A001 and B002) suppressed luciferase activity more strongly than LNPs (Cys (control)) in 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 (LNPs) and peptide modification] As in Experimental Example 17, DSG-PEG(2000)-Maleimide was added to the Lipid Nanoparticle (LNP-102) Exploration Kit to prepare LNPs encapsulating mRNA. The mRNA used was CleanCap FLuc mRNA (TriLink BioTechnologies, L-7602-100) (mRNA(LUC)) for luciferase.

[0131] To achieve the mRNA content recommended in the manual, the lipid stock solution was added to the mRNA solution in 50 mM sodium acetate (pH 5.0) and mixed for 15 seconds by rapid pipetting. The prepared sample was dialyzed as in Experimental Example 17, and the recovered sample was used as maleimide group-containing LNP. Next, a 10 mM DMSO solution of Cys-tagged 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. A 5-fold molar amount of cysteine (10 mM PBS solution) was then added, and the mixture was incubated at room temperature for 15 minutes. After similar dialysis, the recovered LNP was designated 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)), which has 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-attached membrane-permeable peptide 1 (abbreviation: AM01), peptide-modified LNP modified with Cys-attached membrane-permeable peptide 2 (abbreviation: BM02), peptide-modified LNP modified with Cys-attached membrane-permeable peptide 3 (abbreviation: PM02), and control LNP (Cys (control)) to which cysteine was attached.

[0136] [Experimental Example 20: Effect of peptide modification on luciferase expression effect of mRNA (LUC)-encapsulated LNP] 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 in a Corning 96-well white plate. After 24 hours of incubation at 37°C under 5% CO2, 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. The cells were then incubated at 37°C under 5% CO2 for 24 hours, and the luciferase activity of each well was assessed using the ONE-Glo EX Luciferase Assay System in the same manner as in Experimental Example 18. The luminescence intensity measurements for 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, cells treated with peptide-modified LNPs (AM01, BM02, and PM02) emitted stronger luminescence than 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. [Industrial Applicability]

[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) Contains 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) Contains 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) Contains 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 comprises a substitution, insertion, or deletion of one amino acid residue in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 40, and has a sequence identity of 90% or more with the amino acid sequence; A peptide or a salt thereof, wherein, when both ends of the amino acid sequence X are cysteine residues, the amino acid sequence X has a cyclic structure formed by disulfide bonds between the cysteine residues at both ends, and is linear in other cases.

2. The peptide or salt thereof according to claim 1 , wherein the amino acid residue substitution is a conservative substitution.

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

4. The peptide or salt thereof according to claim 1, which has 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.

5. 2. The peptide or salt thereof according to claim 1, wherein the number of amino acid residues is 9 to 30.

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

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

8. The drug-peptide conjugate of claim 7, wherein the drug is a protein or a nucleic acid.

9. A method for increasing the membrane permeability of a drug or a drug delivery carrier, comprising binding the peptide or its salt described in claim 1 to the drug or drug delivery carrier or a component thereof.

10. Step A: generating a cDNA display library containing cDNA display molecules; Step B: incubating the cells in contact with the cDNA display library; and Step C recovering the cDNA display molecule from the contents of the cell; Including, A method for screening a peptide having cytoplasmic transportability, wherein the incubation time in step B is 20 to 72 hours.

11. The screening method according to claim 10, further comprising generating a sub-cDNA display library using the recovered cDNA display molecules.

12. The screening method according to claim 10, wherein the selection comprising steps A, B, and C in this order is repeated two or more times.

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