WO2025009503A1 - 細胞膜透過性を有するペプチドおよびそのスクリーニング方法 - Google Patents

細胞膜透過性を有するペプチドおよびそのスクリーニング方法 Download PDF

Info

Publication number
WO2025009503A1
WO2025009503A1 PCT/JP2024/023780 JP2024023780W WO2025009503A1 WO 2025009503 A1 WO2025009503 A1 WO 2025009503A1 JP 2024023780 W JP2024023780 W JP 2024023780W WO 2025009503 A1 WO2025009503 A1 WO 2025009503A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
peptide
cells
acid sequence
acid residues
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2024/023780
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
充訓 原田
幸一郎 北村
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mescue Janusys Inc
Original Assignee
Mescue Janusys Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mescue Janusys Inc filed Critical Mescue Janusys Inc
Priority to JP2025515752A priority Critical patent/JP7723457B2/ja
Publication of WO2025009503A1 publication Critical patent/WO2025009503A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Definitions

  • the present invention relates to a peptide having cell membrane permeability and a screening method thereof.
  • a method for delivering drugs into cells a method is known in which a peptide with cell membrane permeability (hereinafter also referred to as a "membrane-permeable peptide") is attached to the drug or its carrier to impart cell membrane permeability (for example, Patent Document 1).
  • a membrane-permeable peptide a peptide with cell membrane permeability
  • membrane-permeable peptides There are few membrane-permeable peptides that have excellent intracellular delivery properties and are actually being used in clinical trials, so there is a demand for new membrane-permeable peptides.
  • existing membrane-permeable peptides can be introduced into many cells, they are thought to lack cell selectivity. Therefore, to reduce side effects, cell-selective membrane-permeable peptides are desired.
  • the primary objective of the present invention is to provide a novel peptide with excellent cell membrane permeability, and a further objective is to provide a novel cell-selective membrane-permeable peptide.
  • a peptide or a salt thereof having cell membrane permeability comprising 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 wherein the total number of these three types of amino acid residues is 1 to 7; (2) comprising at least one amino acid residue selected from Y, W, and F, and wherein the total number of these three types of amino acid residues is 1 to 5; (3) comprising at least one amino acid residue selected from L, V, and M, and wherein the total number of these three types of amino acid residues is 1 to 4; and (4) comprising 6 to 15 amino acid residues.
  • 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.
  • the peptide or salt thereof according to any one of the above [1] to [3] may have cell-selective cell membrane permeability.
  • the peptide or salt thereof according to any one of the above [1] to [4] 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.
  • the peptide or salt thereof according to any one of the above [1] to [5] may have 6 to 30 amino acid residues.
  • the peptide or salt thereof according to any one of the above [1] to [6] may have a GRAVY value of -2.6 to 1.9 and may be linear.
  • 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.
  • 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.
  • the drug may be a protein or a nucleic acid.
  • the peptide or a salt thereof according to any one of the above [1] to [8] in drug delivery.
  • a method for screening for a peptide having cell membrane permeability comprising: step A: preparing a cDNA display library containing cDNA display molecules; step B: contacting and incubating a cell with the cDNA display library; and step C: recovering the cDNA display molecules from the contents or permeate of the cell.
  • the screening method according to [12] above may further comprise preparing a sub-cDNA display library using the recovered cDNA display molecules.
  • the selection comprising the steps A, B, and C in this order may be repeated two or more times.
  • FIG. 1 is a schematic diagram illustrating an example of a method for preparing a cDNA display library.
  • FIG. 1 is a schematic diagram illustrating the structure of an example of a cDNA display molecule.
  • FIG. 1 is a schematic diagram illustrating a screening method according to one embodiment of the present invention.
  • FIG. 1 is a schematic diagram illustrating a screening method according to one embodiment of the present invention.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • FIG. 1 shows template DNA sequences for protein expression in a cell-free system.
  • 1 shows photographs depicting the results of an intracellular translocation test of eGFP-peptide fusion protein.
  • 1 shows photographs depicting the results of an intracellular translocation test of eGFP-peptide fusion protein.
  • 1 shows photographs depicting the results of an intracellular internalization test of IgG-peptide conjugates in macrophage cells.
  • FIG. 1 shows the ratio of the amount of intracellular internalization of IgG-peptide conjugates to the amount of intracellular internalization of IgG in macrophage cells.
  • FIG. 1 shows the ratio of the amount of intracellular internalization of IgG-peptide conjugates to the amount of intracellular internalization of IgG in macrophage cells.
  • FIG. 1 shows the ratio of the amount of IgG-peptide conjugate translocated into cells to the amount of IgG translocated into cells of A549 cells.
  • FIG. 13 also shows the ratio of the amount of cellular internalization of IgG-peptide conjugates to the amount of cellular internalization of IgG in macrophage cells and A549 cells.
  • FIG. 1 shows the luciferase activity inhibitory effect of siRNA (LUC)-encapsulated lipid nanoparticles in A549-Luc cells.
  • FIG. 1 shows the luciferase activity inhibitory effect of siRNA (LUC)-encapsulated lipid nanoparticles in BxPC-3-Luc#2 cells.
  • FIG. 1 shows the luciferase expression effect of mRNA (LUC)-encapsulated lipid nanoparticles in A549 cells.
  • FIG. 1 shows the luciferase expression effect of mRNA (LUC)-encapsulated lipid nanoparticles in Panc-1 cells.
  • a peptide having cell membrane permeability, or a salt thereof which comprises an amino acid sequence X that satisfies the following (1) to (4): (1) It contains at least one amino acid residue selected from R, K, and H, and the total number of these three types of 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.
  • a peptide comprising an amino acid sequence X consisting of 6 to 15 amino acid residues, which contains a certain 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.
  • the total number of the three types of amino acid residues, R, K and H may be 1 to 6.
  • the ratio of the total number of the three types of amino acid residues R, K and H 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 ratio of the total number of the three types of amino acid residues Y, W and F 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 ratio of the total number of the three types of amino acid residues L, V and M to the total number of amino acid residues constituting the amino acid sequence X can be, for example, 7% to 40%.
  • 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 may be, for example, 7-15 or, for example, 8-14.
  • the membrane-permeable peptide may be linear or may have a cyclic structure.
  • the cyclic structure may be formed, for example, by binding between the N-terminal amino acid residue and the C-terminal amino acid residue of the peptide, by binding between either one of the terminal amino acid residues and an amino acid residue in a non-terminal portion, or by binding between amino acid residues in a non-terminal portion.
  • the cyclic structure may be formed, for example, by a disulfide bond between two cysteine residues (C).
  • the amino acid sequence X may be linear or may form a cyclic structure.
  • the amino acid sequence X is linear; and when amino acid residues selected from D, F, L, Q, R, S, V, and Y are present in the amino acid sequence X, the number of consecutive 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.
  • 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 amino acid residues of each of these amino acid residues is 2 or less, the number of consecutive R is 3 or less, and the number of consecutive amino acid residues other than these may be 1.
  • the number of consecutive amino acid residues represents the number of consecutive amino acid residues that can be present in the amino acid sequence X.
  • the amino acid residues do not exist consecutively (in other words, adjacently) in the amino acid sequence X.
  • the amino acid sequence X may contain 25% to 60% of amino acid residues having a tendency to form an ⁇ -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)).
  • the above percentage is the percentage of the total number of amino acid residues having a tendency to form the above-mentioned ⁇ -helix structure to the total number of amino acid residues constituting the amino acid sequence X in a linear peptide.
  • the above percentage is the percentage of the total number of amino acid residues having a tendency to form the above-mentioned ⁇ -helix structure to the total number of amino acid residues constituting the amino acid sequence X excluding the two Cs that form disulfide bonds.
  • the above percentage is preferably 30% to 50%.
  • 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 the six amino acids consisting of V, T, R, E, Y, and K.
  • the amino acid sequence X is linear, contains only R as basic amino acid residues, and satisfies (iii) and/or (iv) below: (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 at the N-terminus is included as two consecutive amino acid residues, RV or RW.
  • amino acid sequence X is linear, contains only K as a basic amino acid residue, and contains at least two or more types of amino acid residues selected from F, V, L, and I, the total number of which is 40% to 50% of the total number of amino acid residues constituting amino acid sequence X.
  • the amino acid sequence X is linear and does not contain H as a basic amino acid residue, but contains one or more residues each of K and R, with the fourth and eighth amino acid residues from the N-terminus to the C-terminus being K or R.
  • the membrane-permeable peptide e.g., amino acid sequence X
  • the GRAVY value is an index of the hydrophilicity or hydrophobicity of the entire peptide or protein calculated from the amino acid composition, and a positive value indicates a tendency toward hydrophobicity.
  • a peptide e.g., a linear peptide that contains 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 has a GRAVY value in the above range may have excellent cell membrane permeability.
  • the GRAVY value of the membrane-permeable peptide e.g., amino acid sequence X
  • the GRAVY value can be calculated according to the method of Kyte et al. (J. Kyte, RF. Doolittle, J. Mol. Biol.
  • the GRAVY value of any consecutive 10 amino acid residues is preferably 1.0 or less, more preferably 0.7 or less, and even more preferably 0.3 or less.
  • the membrane-permeable peptide contains an amino acid residue having a functional group in the side chain, more preferably at the N-terminus and/or C-terminus.
  • a membrane-permeable peptide can be suitably bound to a drug (e.g., a protein, a nucleic acid) and its carrier, etc., by a reaction utilizing the functional group.
  • a drug e.g., a protein, a nucleic acid
  • amino acids having a functional group in the 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.
  • the cells in which the membrane-permeable peptide exhibits cell membrane permeability may be any cells.
  • Examples include cancer cells; immune cells such as macrophage cells, neutrophil cells, T cells, and B cells; mast cells; cells or cell groups that constitute skin tissues such as the epidermis and dermis; gastrointestinal epithelial cells; vascular epithelial cells; cells or cell groups that constitute the blood-brain barrier; etc.
  • the membrane-permeable peptide has cell-selective membrane permeability.
  • the peptide may exhibit higher cell membrane permeability for a specific cell (typically, the cell used in screening) than for other cells.
  • a peptide screened using lung cancer cells may exhibit higher cell membrane permeability for lung cancer cells than for other cells.
  • a peptide screened using macrophage cells may exhibit higher cell membrane permeability for macrophage cells than for other cells.
  • a peptide when a peptide is 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 and eGFP or GFP is prepared and incubated with cells at a concentration of 5 ⁇ M for 1 hour, and fluorescence from the labeled peptide or fusion protein is confirmed within the cells by observation under a fluorescent microscope, preferably when fluorescence of higher intensity than that of a control without the addition of the peptide is confirmed, the peptide can be determined to have cell membrane permeability, but the determination of cell membrane permeability is not limited to this.
  • the membrane-permeable peptide can be bound to a drug to improve the intracellular transport of the drug.
  • one or more amino acid residues may be added to the N-terminus and/or C-terminus of amino acid sequence X.
  • 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 viewpoints of imparting reactivity with a drug or its carrier, imparting a linker, imparting hydrophilicity or hydrophobicity, facilitating purification, etc. may also be included in the membrane-permeable peptide according to an embodiment 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.
  • 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.
  • amino acid sequence X include the amino acid sequences represented by any of SEQ ID NOs: 1 to 40 in Table 1, or sequences homologous thereto.
  • the membrane-permeable peptide is a peptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 40.
  • the membrane-permeable peptide is a peptide consisting of a homologous sequence of 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), the content ratio of basic amino acid residues, aromatic amino acid residues, and/or amino acid residues having a hydrophobic linear side chain, the above-mentioned predetermined number of consecutive amino acid residues, the content ratio of amino acid residues having a tendency to form an ⁇ -helix structure, and the above-mentioned predetermined GRAVY value range, as described for amino acid sequence X).
  • a "homologous sequence” means a sequence having, 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.
  • the homologous sequence may include substitutions, insertions, or deletions of 1, 2, 3, 4, or 5, preferably 1, 2, or 3, more preferably 1 or 2 amino acid residues to the original amino acid sequence.
  • the substitution of the amino acid residues may be, for example, conservative substitution. Conservative substitution means replacing one amino acid residue with another amino acid residue having similar structural and/or chemical properties.
  • non-polar 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
  • acidic amino acids include D and E.
  • the substitution of the amino acid residues may be performed so that the total number of amino acid residues (specifically, E, M, A, L, Q, K, R, and H) that tend to form the ⁇ -helix structure in the peptide sequence after substitution (i.e., homologous sequence) is, for example, 25% to 60%, preferably 30% to 50%.
  • 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 ⁇ -helical structure with an amino acid residue selected from the amino acid residues having a tendency to form an ⁇ -helical structure.
  • the membrane-permeable peptide is a peptide in which one or more amino acid residues have been added to the N-terminus and/or C-terminus of an amino acid sequence represented by any one of SEQ ID NOs: 1 to 40 or a homologous sequence thereof.
  • a peptide in which a desired amino acid residue has been added to the N-terminus and/or C-terminus of an amino acid sequence represented by 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, imparting a linker, imparting hydrophilicity or hydrophobicity, facilitating purification, etc., may also be included in the membrane-permeable peptide according to the embodiment of the present invention, so long as it has cell membrane permeability.
  • the number of added amino acid residues may be, for example, 1, 2, or 3 or more, as described above, and may be, for example, 24 or less, 15 or less, 10 or less, 8 or less, or 6 or less.
  • amino acid sequences represented by SEQ ID NOs: 1 to 40 correspond to the amino acid sequence X according to embodiment a
  • the amino acid sequences represented by SEQ ID NOs: 14, 18, 20, 21, 24, 25, 37, and 40 correspond to the amino acid sequence X according to embodiment b
  • the amino acid sequences represented by SEQ ID NOs: 4, 22, 23, and 27 correspond to the amino acid sequence X according to embodiment c
  • the amino acid sequences represented by SEQ ID NOs: 6 and 10 correspond to the amino acid sequence X according to embodiment d.
  • Specific examples of amino acid sequence X that can favorably exert the effects of the present invention include the amino acid sequences represented by SEQ ID NOs: 8, 15, 19, 23, and 37 and their homologous sequences.
  • 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.
  • the membrane-permeable peptide may be in the form of a derivative or a salt, so long as the effects of the present invention are obtained.
  • the term "peptide” may include a derivative of the peptide or a salt of the peptide or its derivative.
  • 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 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 protective groups. These substituents may be further substituted with halogens such as fluorine. The substitution may also be the introduction of labels such as fluorescent labels and biotin labels.
  • the salt of the peptide is preferably a pharmacologically acceptable salt.
  • Pharmacologically acceptable salts include acid addition salts and base addition salts.
  • acid addition salts include inorganic acid salts and organic acid salts.
  • inorganic acid salts include hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate.
  • organic acid salts include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate.
  • Examples of base addition salts include inorganic base salts and organic base salts.
  • Examples of inorganic base salts include sodium salt, potassium salt, calcium salt, magnesium salt, and ammonium salt.
  • Examples of organic base salts include triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt.
  • a drug-peptide conjugate according to an embodiment of the present invention comprises a drug and a membrane-permeable peptide described in Section A bound to the drug. Due to the cell membrane permeability of the membrane-permeable peptide, a drug-peptide conjugate according to an embodiment of the present invention may also have cell membrane permeability.
  • the drug is not particularly limited, and examples thereof include proteins (e.g., antibodies or functional fragments thereof, hormones, enzymes), nucleic acids (e.g., high molecular weight nucleic acids such as plasmid DNA and mRNA, 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, metabolic antagonists, analgesics, anti-inflammatory agents, antibacterial agents), fluorescent dyes, contrast agents, and the like.
  • the mass (molecular weight) of the drug is not particularly limited.
  • the mass of the drug may be less than 500 Da or 500 Da or more, for example, 1000 Da to 150,000 Da, or for example, 3000 Da to 150,000 Da.
  • the membrane-permeable peptide according to the embodiment of the present invention can also impart membrane permeability to biopolymers such as proteins and nucleic acids.
  • 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.
  • the drug and the membrane-permeable peptide may be directly bonded or may be bonded via a linker.
  • a drug-peptide complex may be formed by reacting a functional group of the drug with a functional group of the membrane-permeable peptide.
  • a drug-peptide complex may be formed by reacting a functional group at one end of the linker with a functional group of the drug and reacting a functional group at the other end of the linker with a functional group of the membrane-permeable peptide.
  • an alkylene group having 2 to 10 carbon atoms that may contain an ether bond, an amide bond, an ester bond, etc.
  • a drug-peptide complex can also be obtained as a fusion protein in which a membrane-permeable peptide is linked to the N-terminus and/or C-terminus of the protein by genetic engineering techniques.
  • Combinations of functional groups in the above reaction include, for example, azide and alkyne, thiol and (meth)acryloyl, thiol and maleimide, thiol and thiol, thiol and carboxyl, (meth)acryloyl and hydroxyl, (meth)acryloyl and amino, carboxyl and amino, carboxyl and hydroxyl, amino and hydroxyl, etc.
  • the membrane-permeable peptide described in Section A can be used in drug delivery.
  • the membrane-permeable peptide can be used as a targeting moiety in drug delivery to a target present in a cell.
  • it is preferable that the membrane-permeable peptide has selective membrane permeability to a specific cell.
  • the membrane-permeable peptide can be used in blood or oral administration of a drug to improve the transfer of the drug from blood vessels to tissues or from the digestive tract to tissues.
  • the membrane-permeable peptide has membrane permeability to vascular epithelial cells or digestive tract epithelial cells, and it is more preferable that the membrane-permeable peptide has selective membrane permeability.
  • the membrane-permeable peptide can be used in drug delivery to the brain to improve the transfer of the drug into the brain. In this case, it is preferable that the membrane-permeable peptide has brain barrier permeability.
  • the membrane-permeable peptide can be used in drug delivery via the skin to improve the transfer from the skin to the blood. In this case, it is preferable that the membrane-permeable peptide has skin permeability.
  • the compound has the ability to migrate into epidermal or dermal cells.
  • the membrane-permeable peptide can be bound to a drug or its carrier (specifically, a component of the carrier).
  • a method for increasing the membrane permeability of a drug or a drug delivery carrier comprising binding the membrane-permeable peptide to a drug or a drug delivery carrier or a component thereof, and a method for producing a drug or a drug delivery carrier with imparted or increased membrane permeability, comprising binding the membrane-permeable peptide to a drug or a drug delivery carrier or a component thereof.
  • the above-mentioned bond may be a covalent bond or a non-covalent bond.
  • the complex (conjugate) in which the drug and the membrane-permeable peptide are covalently bonded is as described in Section B.
  • the carrier to which the membrane-permeable peptide is bonded 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 (polyethylene glycol, etc.).
  • the membrane-permeable peptide can be bonded to the end of these components that constitute the nanoparticle so as to be exposed on the nanoparticle surface.
  • the bonding method can be appropriately selected depending on the purpose.
  • a covalent bond it can be performed in the same manner as the bond between the drug and the membrane-permeable peptide, and a combination of functional groups that react with each other as described above can be used.
  • a non-covalent bond it can be bonded (complexed) by utilizing electrostatic interactions, hydrophobic interactions, hydrogen bonds, van der Waals forces, etc.
  • the screening method for peptides having cell membrane permeability includes the steps of: Step A of generating a cDNA display library comprising cDNA display molecules; B. incubating cells in contact with the cDNA display library; and C. recovering the cDNA display molecules from the contents or permeate of the cells. Includes.
  • the above screening method utilizes a cDNA display method.
  • the cDNA display method is one of the genotype-phenotype matching techniques, and can match the function and/or phenotype of a gene expression product such as a protein with the cDNA encoding the corresponding gene in a 1:1 manner.
  • the cDNA display molecule recovered in step C is subjected to PCR, and the gene encoding the peptide carried by the molecule is amplified and identified, thereby identifying the amino acid sequence of the membrane-permeable peptide. Since the above screening method utilizes a cDNA display method using a highly stable cDNA display molecule, the desired peptide can be obtained efficiently even in a system using living cells. In addition, by selecting the cells to be used for screening, a peptide having membrane permeability for the desired cells can be obtained efficiently.
  • a sub-cDNA display library can be prepared using the recovered cDNA display molecules.
  • the preparation of the sub-cDNA display library can be performed 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, and the next selection is started by performing step A again using the selection product (recovered cDNA display molecules), and 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 are selected out, 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 (for example, until the sequence with the highest number of reads accounts for 0.1% or more, preferably 1% or more of the total number of reads).
  • the number of rounds of selection is, for example, 1 or more, preferably 2 or more, and may be 3 or more or 4 or more, and is, for example, 6 or less, preferably 5 or less.
  • 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 prepared, for example, as shown in FIG.
  • step a preparing a DNA library containing DNA encoding random amino acid sequences
  • step b transcribing the DNA of this DNA library into mRNA to obtain an mRNA library
  • step c linking a puromycin-bound linker X to the 3' end of the mRNA of the mRNA library to obtain an mRNA-linker conjugate
  • step d translating the mRNA of the mRNA-linker conjugate in a cell-free translation system to generate a peptide, which is linked to puromycin, thereby obtaining an mRNA-linker-peptide conjugate
  • step e reverse transcribing the mRNA of the mRNA-linker-peptide conjugate to generate cDNA and obtain a cDNA display molecule
  • the linker X has puromycin P linked to a main backbone m containing single-stranded DNA and/or peptide nucleic acid (PNA), and has a ligation site at the 5' end of the main backbone that can hybridize with mRNA and link to its 3' end, and may have a reverse transcription primer site at the 3' end.
  • PNA peptide nucleic acid
  • the main backbone preferably has a length of 10mer to 60mer, more preferably 10mer to 45mer, and even more preferably 15mer to 30mer.
  • Puromycin is a compound that functions as an analogue of the 3'-terminus of aminoacyl-tRNA and can bind to a peptide chain elongating within a ribosome.
  • Puromycin may be a puromycin-like compound that has a structure similar to the 3'-terminus of aminoacyl-tRNA and has the ability to bind to the C-terminus of a synthesized protein when the protein is synthesized in a translation system.
  • the linker X preferably further has a solid-phase binding site b capable of binding to a solid-phase site in the main backbone m, and a pair of cleavage sites c1, c2 provided at positions sandwiching the solid-phase binding site b.
  • 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 at the pair of cleavage sites c1, c2 after reverse transcription, a cDNA display molecule can be suitably produced.
  • the solid-phase binding site may be any site capable of binding the mRNA-linker-peptide conjugate to the solid phase via a linker.
  • 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
  • biotin-deoxythymine is preferred from the viewpoint of affinity with avidin.
  • the pair of cleavage sites are, for example, enzyme cleavage sites. This allows the mRNA-linker-peptide conjugate bound to the solid phase by the solid phase binding sites to be cleaved with an enzyme or the like that cleaves the cleavage sites, and the cDNA display molecule can 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 an enzyme cleavage site is ribo G (Guanosine).
  • FIG. 2 is a schematic diagram illustrating the structure of an example of a cDNA display molecule.
  • the cDNA display molecule 10 is a cleavage residue of 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.
  • the diversity of the cDNA display library used in the initial selection can be, for example, 1.0 ⁇ 10 10 to 1.0 ⁇ 10 13 , preferably 1.0 ⁇ 10 11 to 1.0 ⁇ 10 13 , and more preferably 1.0 ⁇ 10 11 to 1.0 ⁇ 10 12 .
  • step B cells are contacted with the cDNA display library and incubated.
  • the cells to be contacted can be appropriately selected depending on the purpose, and may be one type or two or more types.
  • the cells may form a tissue. Therefore, the 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 the embodiment of the present invention.
  • the incubation temperature and incubation time can be appropriately set depending on the cells to be contacted and the application of the membrane-permeable peptide.
  • 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.
  • step C the cDNA display molecule is recovered from the contents or permeate of the cells after the incubation.
  • the recovery of the cDNA display molecule can be carried out using a commercially available kit or the like.
  • the recovered cDNA display molecule is subjected to PCR to identify the cDNA base sequence and the encoded amino acid sequence. If the sequence convergence is insufficient, a sub-cDNA display library can be prepared (Step A) using the PCR product and subjected to selection again. If the sequence convergence is sufficient, the selection can be terminated without performing Step A, and a peptide consisting of the identified amino acid sequence can be obtained as a membrane-permeable peptide.
  • 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.
  • FIG. 3 and 4 are schematic diagrams illustrating a screening method according to one embodiment of the present invention.
  • the 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 culture vessel 30 containing cells 32 and a culture solution 34, followed by incubation.
  • the amount of the cDNA display library added (more specifically, the amount of the cDNA display molecules added) may be, for example, 100 fmol to 1000 fmol, preferably 200 fmol to 400 fmol.
  • the amount of the cDNA display library added may be such that the number of cDNA display molecules per cell at the time of display is, for example, 1.0 ⁇ 10 5 to 1.0 ⁇ 10 8 , preferably 1.0 ⁇ 10 6 to 1.0 ⁇ 10 8 , more preferably 1.0 ⁇ 10 7 to 1.0 ⁇ 10 8 .
  • the incubation time is appropriately set depending on the purpose of the test, and may be a relatively short time (e.g., 5 minutes to 4 hours, preferably 30 minutes to 2 hours) or a relatively long time (e.g., 20 hours to 72 hours, preferably 24 hours to 48 hours).
  • the cells 32 are collected and washed as necessary, and the cell contents are collected by hypotonic treatment, homogenization, etc., and then the cDNA display molecules 10 are collected from the cell contents using a nucleic acid purification kit or the like (step C).
  • the collected 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 sub-cDNA display library 20' can be used to carry out the subsequent steps B and C.
  • Any appropriate cell may be used as the above-mentioned cell depending on the purpose.
  • Examples include cancer cells; immune cells such as macrophage cells, neutrophil cells, T cells, and B cells; mast cells; and cells or cell groups that constitute skin tissues such as the epidermis and dermis.
  • immune cells such as macrophage cells, neutrophil cells, T cells, and B cells
  • mast cells such as the epidermis and dermis.
  • FIG. 3 it is possible to suitably screen for peptides that can permeate cell membranes and remain in cells (in other words, peptides that have cytoplasmic migration properties).
  • the 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 that is inserted into a culture well 40 and contains cells 44 and a culture medium 46, followed by incubation.
  • the bottom surface 42a of the cell culture insert 42 is preferably in a confluent state and is preferably completely covered with a single or multiple layer of cells.
  • the amount of the cDNA display library added (more specifically, the amount of the cDNA display molecule added) may be, for example, 100 fmol to 1000 fmol, and preferably 200 fmol to 400 fmol.
  • the incubation time may be, for example, 5 minutes to 24 hours, preferably 15 minutes to 4 hours, and more preferably 30 minutes to 2 hours.
  • the bottom surface 42a of the cell culture insert 42 is formed of a porous membrane, so that the culture medium 46 outside the cell culture insert 42 may contain a permeant that has permeated the cells 44 from the inside to the outside of the cell culture insert. Therefore, the cDNA display molecule 10 that has permeated the cells 44 can be recovered from the culture medium 46 outside the cell culture insert (step C).
  • the recovery of the cDNA display molecule 10 from the culture medium can be performed using a nucleic acid purification kit or the like.
  • 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.
  • the cDNA display library may be added to the culture medium outside the cell culture insert, contacting the cells through the porous membrane on the bottom of the cell culture insert, and the cDNA display molecules that have permeated the cells may be collected from the culture medium inside the cell culture insert.
  • any appropriate cell may be used as the above-mentioned cell 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.
  • FIG. 4 it is possible to suitably screen for peptides that can permeate from one side of a cell layer to the other side (e.g., peptides that can permeate from the gastrointestinal tract to blood, from blood to tissue, or from blood to the brain, or that can be absorbed transdermally).
  • mRNA library was created using the RiboMAX Large Scale RNA Production System-SP6 kit (Promega) based on a DNA library containing DNA encoding random peptides of 8 to 15 amino acid residues.
  • 1 pmol to 2 pmol of cDNA, 4 ⁇ L of rNTP mixture, 2 ⁇ L of transcription enzyme, and 4 ⁇ L of reaction buffer were mixed and then adjusted to 20 ⁇ L with purified water (reagents other than cDNA were included in the kit).
  • the transcription solution was incubated at 37°C for 2.5 hours, after which 1 ⁇ L of RQ1 RNase-Free DNase (1 U/1 ⁇ L) was added and incubated at 37°C for 20 minutes.
  • the mRNA was then purified using the RNeasy Mini kit (QIAGEN) according to the manual included with the kit.
  • linker a conjugate between the mRNA and the puromycin linker (hereinafter, referred to as linker) described in WO2006/041194 was prepared as follows. After mixing 20 pmol of mRNA and 20 ⁇ M of linker, a solution was prepared by adjusting the volume to 20 ⁇ L with purified water (Ambion). The solution was incubated at 90° C. for 30 seconds, cooled to 70° C. at 0.12° C./sec, incubated for 30 seconds, and cooled to 4° C. at 0.06° C./sec.
  • T4 polynucleotide kinase (Takara Bio)
  • 1 ⁇ L of T4 RNA ligase (Takara Bio)
  • 2 ⁇ L of T4 RNA ligase buffer (included with T4 RNA ligase)
  • 1.5 ⁇ L of 0.1% BSA (included with T4 RNA ligase) were added to the cooled solution and incubated at 25° C. for 1 hour to prepare an mRNA-linker conjugate.
  • peptide synthesis was performed by mixing the mRNA-linker conjugate into a cell-free translation system.
  • a translation reaction solution was prepared by mixing 20 ⁇ L of the conjugate, 50 ⁇ L of Wheat Germ Extract (Promega), 8 ⁇ L of amino acid mixture (Promega), 5 ⁇ L of 1M KOAc (Ambion), 5 ⁇ L of RNase inhibitor (Thermo Fisher Scientific), and 12 ⁇ L of purified water, and incubated at 25° C. for 15 minutes. Then, 46 ⁇ L of Salt mix (18.25 ⁇ L of 3M KCl, 4.75 ⁇ L of 1M MgCl 2 ) was added, and the mixture was incubated at 25° C. for 1 hour. Further, 28 ⁇ L of 0.5 M EDTA pH 8.0 (Thermo Fisher Scientific) was added to obtain an mRNA-linker-peptide conjugate (post-translation product).
  • the biotin contained in the linker in the post-translation product was used to purify the product using DynaBeads MyOne Streptavidin C1 (Thermo Fisher Scientific) (hereafter referred to as magnetic beads). After thoroughly suspending 150 ⁇ L of magnetic beads, the product was washed twice with an equal volume of 2x Binding buffer (buffer composition: 20 mM Tris-HCl (pH 7.5), 2 M NaCl, 2 mM EDTA, 0.2% Polyoxyethylene (20) Sorbitan Monolaurate) (after addition and suspension, the supernatant was discarded). Next, 348 ⁇ L of the post-translation product was added, and the product was incubated at 25°C for 30 minutes while rotating on a rotator. The supernatant was then discarded and the cells were washed twice with 400 ⁇ L of 1x Binding Buffer. They were then washed once with 200 ⁇ L of 1x RT Buffer (Promega).
  • 2x Binding buffer
  • the supernatant of the magnetic beads was discarded, and the beads were washed once with 100 ⁇ L of 1x binding buffer, and then washed once with 50 ⁇ L of equilibration buffer (composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM imidazole, 0.05% Polyoxyethylene (20) Sorbitan Monolaurate).
  • equilibration buffer composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM imidazole, 0.05% Polyoxyethylene (20) Sorbitan Monolaurate.
  • 99.5 ⁇ L of equilibration buffer and 0.5 ⁇ L of RNase T1 (Thermo Fisher Scientific) were added, and the beads were incubated at 37°C for 20 minutes while rotating on a rotator, after which the supernatant was recovered.
  • the solution thus recovered is an unpurified cDNA display library containing cDNA display molecules
  • 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 for 60 minutes at 4°C against 60 mL of dialysis solution (PBS), the dialysis solution was replaced and stirred for another 60 minutes at 4°C. The solution recovered by dialysis was used as a cDNA display library for subsequent screening.
  • the cells were cultured at 37°C and 5% CO 2 , and the medium was replaced according to the schedule of medium replacement example 2 described in the manual (F-hiSIECTM Culture Medium (Fujifilm Corporation) was used as the medium during the culture period).
  • F-hiSIECTM Culture Medium (Fujifilm Corporation) was used as the medium during the culture period).
  • the start date of culture was the first day
  • the first (round 1) permeation test was performed on the 12th day and the second (round 2) on the 14th day.
  • each diluted library solution (approximately 100 fmol to 300 fmol as the amount of cDNA display molecule added) was added to a separate culture insert (apical side), and 600 ⁇ L of RPMI1640 medium was added to each well (basolateral side), and the permeation test was started. After incubation at 37° C. under 5% CO 2 for 1 hour, the medium on the receptor (basolateral) side was entirely collected in a 1.5 mL tube. Each collected solution was stored at ⁇ 80° C. until quantitative analysis of nucleic acid and the next round of display library construction.
  • the second permeation test was carried out in the same manner as the first permeation test, except that the cDNA display library for the second round was used, and all of the medium on the receptor (basolateral) side was collected in a 1.5 mL tube.
  • a cDNA display library for the third round was prepared from the collected liquid in the same manner as above.
  • Third and fourth permeation tests and purification and amplification of permeation samples For the third and fourth permeation tests, new human iPS cell-derived intestinal epithelial cells F-hiSIECTM were prepared in the same manner as for the first and second permeation tests.
  • permeation tests and purification and amplification of permeation samples were performed in the same manner as described above to prepare a cDNA display library for the fourth round.
  • a penetration test and purification and amplification of the penetration sample were carried out in the same manner as described above.
  • NGS Next-generation sequencing
  • 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 seeded at 0.5 x 10 5 cells/well on a 24-well plate. After culturing for 72 hours at 37°C and 5% CO 2 , the medium in each well was removed.
  • RPMI1640 medium ATCC modification Thermo-Fisher Scientific product number A10491-01
  • FBS containing penicillin-streptomycin, Thermo-Fisher Scientific, product number "15140-122", 1/100 volume
  • a cDNA display library equivalent to that in Experimental Example 2 (a cDNA display library of a total of six types (linear peptides: 3 types, cyclic peptides: 3 types) with different numbers of amino acid residues in the peptides to be displayed) was prepared. These were mixed and diluted to 310 ⁇ L with RPMI 1640 medium without FBS and antibiotics, and 300 ⁇ L of the diluted solution (approximately 100 fmol to 300 fmol of added cDNA display molecules) was added to the well. After 1 hour of uptake at 37°C and 5% CO2 , the added library was recovered. Then, 300 ⁇ L of fresh medium was added, and the culture was continued for 23 hours, after which the cells were washed as follows.
  • the cells were washed three times with 300 ⁇ L of PBS + 0.05% Tween 20, and once with 300 ⁇ L of 10 mM hydrochloric acid + 150 mM NaCl. Then, 300 ⁇ L 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 that the cells had burst under a microscope, the samples containing the cell contents were all collected in 1.5 mL tubes. The supernatant was obtained by centrifugation at 1000 g for 3 minutes. Nucleic acids were purified and amplified from this supernatant in the same manner as in Experimental Example 2, and a cDNA display library for the next round was prepared.
  • 10 mM HEPES buffer pH 7.0
  • 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 seeded at 0.5 x 105 cells/well on a 24-well plate. A total of two selections were performed in the same manner as in Experimental Example 3 to determine the amino acid sequences of peptides that were transferred into the cells. The sequences with the largest read counts in the NGS analysis were listed as follows.
  • HL-60 cells (RIKEN BioResource Research Center) were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and seeded at 1.0 x 105 cells/well on a 24-well plate.
  • Dimethyl sulfoxide (DMSO, Fujifilm Wako Pure Chemical Industries, 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 the HL-60 cells into neutrophils.
  • the medium in each well was removed, and a cDNA display library equivalent to that in Experimental Example 3 was diluted to 310 ⁇ L with RPMI1640 medium without FBS and antibiotics, and 300 ⁇ L of the diluted library was added to the well. After 1 hour of uptake at 37°C and 5% CO2 , the added library was collected. Then, 300 ⁇ L of PBS was added, the cells were collected by pipetting, and the cells were washed by centrifugation at 200 g for 3 minutes. The cells were further washed with 300 ⁇ L of PBS + 0.05% Tween 20, and similarly collected by centrifugation.
  • the above test procedure was repeated four more times (a total of five selections), and the samples collected as cell contents were subjected to NGS analysis.
  • the resulting paired-end fastq file was analyzed using flash2 to determine the amino acid sequence of the peptide that was transported into the cells. The sequences with the highest read counts were listed below.
  • THP-1 cells (RIKEN BioResource Research Center) were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and seeded at 1.0 x 105 cells/well on a 24-well plate.
  • FBS penicillin-streptomycin
  • the medium was removed, and 300 ⁇ L of fresh RPMI1640 medium + 10% FBS was added per well, followed by culture for 24 hours.
  • Stock solutions of IFN ⁇ (Cosmo Bio (Proteintech), HZ-1301) and LPS (Cosmo Bio (Santa Cruz Biotechnology), SC-3535) were added to give final concentrations of 20 ng/mL and 250 ng/mL, respectively, and the cells were further cultured at 37° C. under 5% CO 2 for 24 hours to differentiate the THP-1 cells into macrophages.
  • the medium in each well was removed, and the cDNA display library equivalent to that in Experimental Example 3 was diluted with RPMI 1640 medium without FBS or antibiotics to a total volume of 310 ⁇ L, and 300 ⁇ L of the diluted solution was added to each well. After incubation at 37° C. and 5% CO 2 for 1 hour, the cells were washed as follows.
  • the cells were washed three times with 300 ⁇ L of PBS + 0.05% Tween 20, and once with 300 ⁇ L of 10 mM hydrochloric acid + 150 mM NaCl. Then, 300 ⁇ L 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 that the cells had burst under a microscope, the samples containing the cell contents were all collected in 1.5 mL tubes. 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.
  • the above test procedure was repeated three more times (a total of four selections), and the samples collected as cell contents were subjected to NGS analysis.
  • the resulting paired-end fastq file was analyzed using flash2 to determine the amino acid sequence of the peptide that was transported into the cells. The sequences with the highest read counts were listed below.
  • the inserts in the BBB culture were transferred to a washing well, the medium in the inserts was carefully removed with an aspirator, and then transferred to a test well to which 900 ⁇ L of Assay Buffer had been added in advance.
  • 20 ⁇ L of each cDNA display library solution was mixed and diluted with 200 ⁇ L of Assay Buffer, and 200 ⁇ L of each was added to separate inserts to start the permeation test. After incubation at 37 ° C. and 5% CO 2 for 2 hours, each receptor solution was collected.
  • Third and fourth permeation tests and purification and amplification of permeation samples For the third and fourth permeation tests, a new monkey-type BBB kit 6 inserts/24 wells (Pharmacocell Co., Ltd.) was prepared in the same manner as for the first and second permeation tests. Using the cDNA display library for the third round, permeation tests and purification and amplification of permeation samples were performed in the same manner as described above to prepare a cDNA display library for the fourth round. Using the cDNA display library for the fourth round, a penetration test and purification and amplification of the penetration sample were carried out in the same manner as described above.
  • the mixture was set in an ultrafiltration centrifugal filter (Amicon Ultra 10 kDa MWCO, Millipore UFC5010) and centrifuged at 4 ° C. and 14,000 rpm for 10 minutes (TOMY TMP-24, rotation radius Rmax 87 mm) to concentrate to about 100 ⁇ L.
  • DNA purification was performed using a DNA purification kit (QIA quick PCR Purification Kit QIAGEN 28104), and 25 ⁇ L of DNA sample was recovered.
  • the DNA sample was PCR amplified according to a conventional method to prepare a cDNA display library for round 2. Similarly, rounds of the penetration test were repeated until a maximum of four rounds were performed in total.
  • 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).
  • TECAN spark emission wavelength: 494 nm
  • 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 SEQ ID NO:40 was 49.8 ⁇ 10.0% (mean value of three cases ⁇ standard deviation), which was significantly improved compared to the TAT sequence (p ⁇ 0.01).
  • eGFP and eGFP-peptide fusion proteins were prepared and purified using "Muceibo -kun® N100" (Taiyo Nippon Sanso SI Division) in the following manner.
  • the template DNA was dissolved in 10 mM Tris-HCl buffer (pH 7.5) (approximately 100 ⁇ g/mL), and further diluted with purified water to a specified concentration according to the manufacturer's manual, and mixed with the reaction solution premix included in the kit to make a total of 100 ⁇ L (reaction solution). Subsequently, 1 mL of the dialysis solution included in the kit was placed outside the dialysis unit, and the reaction solution was placed in the dialysis unit. After shaking at 30°C for 16 hours (amplitude 25 mm, 80 rpm, Bioshaker BR-53FP, Taitec Co., Ltd.), the reaction solution was collected in a new tube and left on ice for 5 minutes to stop the reaction.
  • the solution was then adjusted to approximately 1000 ⁇ L with PBS + 0.05% Tween 20 and purified using HisPur® Ni-NTA Magnetic Beads (Thermo Scientific® , product number: 88831).
  • the beads were equilibrated in advance by the following procedure. 100 ⁇ L of magnetic bead slurry was transferred to a new 1.5 mL tube, 400 ⁇ L (4 volumes) of equilibration buffer (PBS + 0.05% Tween 20) was added, and the mixture was suspended with a vortex (weak) for 10 seconds.
  • the beads were collected with a magnetic stand, the supernatant was removed, and 1000 ⁇ L (10 volumes) of equilibration buffer was added, the mixture was suspended with a vortex (weak) for 10 seconds, the supernatant was similarly removed, and the beads were collected and equilibrated.
  • the equilibrated beads were added with the reaction solution and suspended. After rotating and mixing for 1 hour at 4°C, the beads were collected using a magnetic stand. The beads were washed three times with approximately 1 mL of PBS + 0.05% Tween 20 + 5 mM imidazole. The beads were then eluted with the following buffer, and the yellow-green fraction was collected. That is, elution was performed twice with 20 ⁇ L of PBS+250 mM imidazole, twice with 20 ⁇ L of PBS+250 mM imidazole+1 mM EDTA, and twice with 20 ⁇ L of PBS+250 mM imidazole+10 mM EDTA.
  • the recovered eGFP and eGFP-peptide fusion proteins were used in the following cell experiments after replacing the medium with 20 mM HEPES (pH 7.0) + 150 mM NaCl using an ultrafiltration membrane (Amicon Ultra 10 kDa 0.5 mL Millipore UFC501024). The concentration was also calculated based on the extinction coefficient at 280 nm calculated from the amino acid sequence.
  • 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 for 24 hours at 37°C under 5% CO 2. After removing the medium, the cells were washed once with 200 ⁇ L of RPMI1640 medium without FBS or antibiotics, and the protein solutions (eGFP, eGFP-TAT, eGFP-L10#1) were diluted with the same RPMI1640 medium and added to the wells (final concentration 5 ⁇ M, 30 ⁇ L/well).
  • LYSO-ID (R) ReD detection kit reagent (Cosmo Bio, product number ENZ-51005-0100) was diluted 500 times with RPMI1640 medium, and 10 ⁇ L of the diluted solution was added. After further incubation for 15 minutes, 3 ⁇ L of NucBlue (R) reagent (Thermo-Fisher Scientific) was added. After continued incubation for 15 minutes, the plate was washed three times with 200 ⁇ L of PBS containing 0.5 mg/mL heparin sulfate cooled to 4°C while cooling on ice.
  • eGFP-L10#1 which is a fusion protein of eGFP and a peptide containing the amino acid sequence of SEQ ID NO:7
  • green fluorescence derived from eGFP was observed to surround blue fluorescence derived from the nucleus, which indicates that GFP-L10#1 was efficiently translocated to the cytoplasm.
  • the mass of eGFP is 27 kDa
  • the mass of the peptide added to eGFP is 1.2 kDa.
  • Example 12 Translocation test of eGFP-peptide fusion protein into THP-1 macrophages
  • THP-1 macrophage cells were used, which were seeded at 5000 cells/well in a 96-well plate and activated with PMA, IFN ⁇ , and LPS. After removing the medium from the wells, the wells were washed once with 200 ⁇ L of RPMI1640 medium without FBS or antibiotics, and protein solutions (eGFP, eGFP-L10#2) were diluted with the same RPMI1640 medium and added to the wells (final concentration 3 ⁇ M, 30 ⁇ L/well). The cells were observed 1 hour after addition, using the same procedure as in Experimental Example 11. The results are shown in FIG. 11.
  • eGFP-L10#2 which is a fusion protein of eGFP and a peptide containing the amino acid sequence of SEQ ID NO:37
  • green fluorescence derived from eGFP was observed along with blue fluorescence derived from the nucleus, indicating that eGFP-L10#2 had migrated into the macrophage cells.
  • the mass of eGFP is 27 kDa
  • the mass of the peptide added to eGFP is 1.5 kDa.
  • GFP no fluorescence derived from eGFP was observed, and as a result, migration of eGFP into the macrophage cells was not confirmed.
  • hIgG 1 mg was added to a 1.5 mL tube (total volume 200 ⁇ L) so that the final concentration was 5 mg/mL, and AlexaFluor647 NHS Ester was added to a 10-fold molar concentration relative to hIgG, and the mixture was stirred by inversion 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 the unreacted reagent was removed to recover Alexa 647-labeled IgG. 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 Alexa 647 were labeled per molecule of recovered IgG.
  • the reaction solution was applied to a Zeba spin desalting column (7K MWCO) equilibrated with PBS, and the unreacted crosslinker was removed to recover the IgG fraction.
  • a peptide containing the amino acid sequence obtained in the above screening and having C at the N-terminus was dissolved in DMSO to give a 10 mM DMSO solution.
  • a DMSO solution of peptide was added to the collected IgG fraction in a molar amount 5 times that of IgG, and EDTA was added to a final concentration of 1 mM, followed by end-over-end stirring at 25°C for 1 hour. Then, 5 times the molar amount of cysteine was added, followed by end-over-end stirring at 25°C for 30 minutes.
  • the reaction solution was applied to a Zeba spin desalting column (7K MWCO) equilibrated with PBS, and the unreacted peptide, cysteine, and EDTA were removed to collect the IgG-peptide conjugate.
  • the amount of IgG translocated into cells was increased by binding peptides (T001, T005, and T006) containing the amino acid sequences obtained by screening using macrophage cells.
  • TAT was bound to the IgG.
  • Example 14 Evaluation of cell specificity of cell membrane permeability 1
  • the cells were observed and photographed 1 hour after addition in the same manner as in Experimental Example 13 using an all-in-one fluorescence microscope BZ-X810 (Keyence Corporation) and images were analyzed using the accompanying software BZ-X800 Analyzer, except that the protein solution was added to the wells so that the final concentration of IgG or IgG-peptide conjugate was 1 ⁇ M.
  • the ratio of red brightness (integrated) to blue brightness (integrated) was used to compare the uptake ratios at an uptake time of 1 hour.
  • IgG bound to peptides (T001, T006) containing amino acid sequences obtained in screening using macrophage cells showed a greater increase in migration into macrophage cells than IgG bound to peptides (R001, R005) containing amino acid sequences obtained in screening using A549 cells.
  • IgG bound to peptides (R001, R005) containing amino acid sequences obtained in screening using A549 cells showed a greater increase in translocation into A549 cells than IgG bound to peptides (T001, T006) containing amino acid sequences obtained in screening using macrophage cells.
  • siRNA As the siRNA, the following sequence against luciferase (siRNA (LUC)) was prepared by outsourcing its synthesis to Ajinomoto Bio-Pharma Services Gene Design, Inc. ⁇ siRNA (LUC) Sense strand: 5'-CUUACGCUGAGUACUUCGAdTdT (SEQ ID NO:52) Antisense strand: 5'-UCGAAGUACUCAGCGUAAGdTdT (SEQ ID NO:53)
  • DSPC 1,2-Distearoyl-sn-Glycero-3-Phosphatidylcholine
  • cholesterol 1,2-Distearoyl-sn-Glycero-3-Phosphatidylcholine
  • DMG-PEG(2000) included in the kit were dissolved in ethanol.
  • DSG-PEG(2000)-Maleimide was dissolved in DMF.
  • siRNA (LUC) was dissolved in 50 mM sodium acetate buffer (pH 5.0) (autoclaved).
  • a molar ratio was adopted in which SM-102 was 100 mg / mL, DSPC was 25 mg / mL, cholesterol was 5 mg / mL, DMG-PEG (2000) was 1 mg / mL, and DSG-PEG (2000)-Maleimide was 5 mg / mL.
  • a stock solution of each lipid was added to a 50 mM sodium acetate (pH 5.0) solution of siRNA to achieve the above-mentioned constituent molar ratio and the siRNA content recommended in the manual, and the mixture was quickly mixed by pipetting for 15 seconds.
  • the prepared sample (about 60 ⁇ L) was injected into a microdialysis cartridge Xpress Micro Dialyzer MD100 molecular weight cutoff 140 kDa (Scienova) (Funakoshi (SCI), code: 40931) set in a 2 mL tube.
  • the sample was dialyzed against 1.2 mL of PBS (20 minutes, 3 times), and the recovered sample was used as maleimide group-containing LNP.
  • Cys-added membrane-permeable peptide 1 (CTVTRGERYKH (SEQ ID NO: 54)) having a cysteine at the N-terminus was added in a 5-fold molar amount relative to the maleimide group, and 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 a cysteine added to the N-terminus.
  • 5 times the molar amount of cysteine (10 mM PBS solution) was added and incubated at room temperature for 15 minutes.
  • the recovered LNP was designated as peptide-modified LNP (abbreviation: A001) and stored at 4°C until use.
  • a 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 a cysteine at the N-terminus was used instead of Cys-added membrane-permeable peptide 1. Cys-added membrane-permeable peptide 2 has an amino acid sequence with a cysteine added to the N-terminus of No. 1-8 (SEQ ID NO: 8).
  • LNP in which cysteine was bound to the maleimide group was prepared in the same manner as above, except that a Cys-attached membrane-permeable peptide was not used.
  • peptide-modified LNP modified with Cys-added membrane-permeable peptide 1 (abbreviation: A001)
  • peptide-modified LNP modified with Cys-added membrane-permeable peptide 2 (abbreviation: B002)
  • control LNP with cysteine attached (Cys (control))
  • Example 18 Luciferase activity inhibitory effect of siRNA (LUC)-encapsulated LNPs on luciferase-expressing cells
  • A549-Luc cells and BxPC-3-Luc#2 cells were cultured in RPMI1640 medium + 10% FBS (containing penicillin-streptomycin) and seeded at 2000 cells/0.1 mL/well on a Corning 96-well white plate (product number 3610).
  • siRNA (LUC)-encapsulated LNP (specifically, Cys (control), A001, or B002) was added to each well so that the final concentration of siRNA was 100 nM and then diluted 3-fold.
  • the cells were then incubated at 37°C and 5% CO2 for 24 hours, and the luciferase activity of each well was evaluated using the ONE-Glo EX Luciferase Assay System (Promega, E8110).
  • luciferase activity was suppressed depending on the concentration of siRNA added.
  • concentration of siRNA added was the same, the peptide-modified LNPs (A001 and B002) suppressed luciferase activity more strongly than the LNPs to which cysteine was bound instead of peptide (Cys (control)).
  • siRNA could be delivered efficiently to the cytoplasm by modifying the siRNA (LUC)-encapsulated LNPs with a membrane-permeable peptide.
  • the lipid stock solution was added to the 50 mM sodium acetate (pH 5.0) solution of mRNA to obtain the mRNA content recommended by the manual, and mixed for 15 seconds with rapid pipetting.
  • the prepared sample was dialyzed in the same manner as in Experimental Example 17, and the collected sample was used as maleimide group-containing LNP.
  • a 10 mM DMSO solution of Cys-added membrane-permeable peptide 1 was added in a 5-fold molar amount relative to the maleimide group, and incubated at room temperature for 60 minutes.
  • a 5-fold molar amount of cysteine (10 mM PBS solution) was added, and incubated at room temperature for 15 minutes.
  • the collected LNP was made into peptide-modified LNP (abbreviation: AM01) and stored at 4°C.
  • Peptide-modified LNP (abbreviation: BM02) was prepared using the same procedure as above, except that Cys-tagged membrane-permeable peptide 2 was used instead of Cys-tagged membrane-permeable peptide 1.
  • Cys-added membrane-permeable peptide 3 (CVRVKTYWENH (SEQ ID NO: 56)) having a cysteine at the N-terminus 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.
  • LNP in which cysteine was bound to the maleimide group was prepared in the same manner as above, except that a Cys-attached membrane-permeable peptide was not used.
  • mRNA (LUC)-encapsulating LNPs were obtained: peptide-modified LNP modified with Cys-added membrane-permeable peptide 1 (abbreviation: AM01), peptide-modified LNP modified with Cys-added membrane-permeable peptide 2 (abbreviation: BM02), peptide-modified LNP modified with Cys-added membrane-permeable peptide 3 (abbreviation: PM02), and control LNP with cysteine attached (Cys (control)).
  • AM01 Cys-added membrane-permeable peptide 1
  • BM02 Cys-added membrane-permeable peptide 2
  • PM02 peptide-modified LNP modified with Cys-added membrane-permeable peptide 3
  • control LNP with cysteine attached Cys (control)
  • the cells were incubated at 37°C under 5% CO2 for 24 hours, and the luciferase activity of each well was evaluated in the same manner as in Experimental Example 18 using the ONE-Glo EX Luciferase Assay System.
  • the measurement results of the luminescence intensity of each well were plotted.
  • Membrane-permeable peptides according to embodiments of the present invention can be suitably used, for example, in the manufacture of DDS systems, pharmaceutical compositions, etc.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
PCT/JP2024/023780 2023-07-03 2024-07-01 細胞膜透過性を有するペプチドおよびそのスクリーニング方法 Ceased WO2025009503A1 (ja)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2025515752A JP7723457B2 (ja) 2023-07-03 2024-07-01 細胞膜透過性を有するペプチドおよびそのスクリーニング方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2023109588 2023-07-03
JP2023-109588 2023-07-03

Publications (1)

Publication Number Publication Date
WO2025009503A1 true WO2025009503A1 (ja) 2025-01-09

Family

ID=94171533

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2024/023780 Ceased WO2025009503A1 (ja) 2023-07-03 2024-07-01 細胞膜透過性を有するペプチドおよびそのスクリーニング方法

Country Status (2)

Country Link
JP (1) JP7723457B2 (https=)
WO (1) WO2025009503A1 (https=)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006041194A1 (ja) 2004-10-15 2006-04-20 Japan Science And Technology Agency ｍＲＮＡ－ピューロマイシン－タンパク質連結体作製用リンカー
JP2009527251A (ja) * 2006-02-20 2009-07-30 エファ・ユニバーシティ・インダストリー・コラボレイション・ファウンデイション 細胞膜透過性ペプチド
WO2010134537A1 (ja) * 2009-05-20 2010-11-25 東レ株式会社 細胞膜透過性ペプチド
WO2016186140A1 (ja) * 2015-05-19 2016-11-24 国立大学法人熊本大学 新規な細胞膜透過性ペプチド
WO2017134148A1 (en) * 2016-02-02 2017-08-10 Stichting Katholieke Universiteit Selective cell penetrating peptides
JP2019528693A (ja) * 2016-09-21 2019-10-17 アマル セラピューティクス エスエー 癌を治療するための細胞透過性ペプチド、マルチエピトープ、及びｔｌｒペプチドアゴニストを含む融合体
WO2021039884A1 (ja) * 2019-08-28 2021-03-04 国立研究開発法人理化学研究所 細胞膜透過性ベシクル
JP2021531274A (ja) 2018-07-17 2021-11-18 サントル・ナシオナル・ドゥ・ラ・ルシェルシュ・シアンティフィクCentre National De Larecherche Scientifique 細胞膜透過性ペプチドとして使用するためのペプチド
JP2022540498A (ja) * 2019-07-16 2022-09-15 ジェンセン・カンパニー・リミテッド 自己免疫疾患および炎症性疾患のためのペプチド治療薬
WO2022238530A1 (en) * 2021-05-12 2022-11-17 University Of Copenhagen Psd-95 inhibitors and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005013073A (ja) * 2003-06-25 2005-01-20 Mitsubishi Pharma Corp 輸送ポリペプチドのスクリーニング方法

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006041194A1 (ja) 2004-10-15 2006-04-20 Japan Science And Technology Agency ｍＲＮＡ－ピューロマイシン－タンパク質連結体作製用リンカー
JP2009527251A (ja) * 2006-02-20 2009-07-30 エファ・ユニバーシティ・インダストリー・コラボレイション・ファウンデイション 細胞膜透過性ペプチド
WO2010134537A1 (ja) * 2009-05-20 2010-11-25 東レ株式会社 細胞膜透過性ペプチド
WO2016186140A1 (ja) * 2015-05-19 2016-11-24 国立大学法人熊本大学 新規な細胞膜透過性ペプチド
WO2017134148A1 (en) * 2016-02-02 2017-08-10 Stichting Katholieke Universiteit Selective cell penetrating peptides
JP2019528693A (ja) * 2016-09-21 2019-10-17 アマル セラピューティクス エスエー 癌を治療するための細胞透過性ペプチド、マルチエピトープ、及びｔｌｒペプチドアゴニストを含む融合体
JP2021531274A (ja) 2018-07-17 2021-11-18 サントル・ナシオナル・ドゥ・ラ・ルシェルシュ・シアンティフィクCentre National De Larecherche Scientifique 細胞膜透過性ペプチドとして使用するためのペプチド
JP2022540498A (ja) * 2019-07-16 2022-09-15 ジェンセン・カンパニー・リミテッド 自己免疫疾患および炎症性疾患のためのペプチド治療薬
WO2021039884A1 (ja) * 2019-08-28 2021-03-04 国立研究開発法人理化学研究所 細胞膜透過性ベシクル
WO2022238530A1 (en) * 2021-05-12 2022-11-17 University Of Copenhagen Psd-95 inhibitors and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KITAMURA, KOICHIRO: "Creation of functional peptides using cDNA display technology", JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 18, no. 2, 1 January 2018 (2018-01-01), pages 44, XP009560772, ISSN: 1347-2194 *
KYTE ET AL., J. KYTE, RF. DOOLITTLE, J MOL. BIOL, vol. 152, 1982, pages 105
P. Y. CHOUG. D. FASMANN, ADV. ENZYMOL. RELAT. AREAS MOL. BIOL., vol. 47, 1978, pages 45 - 148

Also Published As

Publication number Publication date
JP7723457B2 (ja) 2025-08-14
JPWO2025009503A1 (https=) 2025-01-09

Similar Documents

Publication Publication Date Title
US20200390700A1 (en) Targeted extracellular vesicles comprising membrane proteins with engineered glycosylation sites
EP3341394B1 (en) Improved cell-permeable (icp)-socs3 recombinant protein and uses thereof
US10538570B2 (en) Targeted and modular exosome loading system
EP2480672B1 (fr) Nouveaux polynucleotides et polypeptides chimeriques permettant la secretion d'un polypeptide d'interet en association avec des exosomes et leurs utilisations
EP3518981A1 (en) Delivery of therapeutic rnas via arrdc1-mediated microvesicles
US20140094404A1 (en) Methods and reagents for efficient and targeted delivery of therapeutic molecules to cxcr4 cells
CN114364704B (zh) 修饰的piggybac转座酶多肽、编码其的多核苷酸、引入载体、试剂盒、将靶序列整合到细胞基因组中的方法以及生产细胞的方法
JP2018064573A (ja) ハイスループットペプチドミメティックを調製するための方法、経口バイオアベイラブルな薬物およびそれを含有する組成物
CN116271102A (zh) 用于药物递送的含修饰蛋白的双层膜囊泡及其制备方法和应用
JP7723457B2 (ja) 細胞膜透過性を有するペプチドおよびそのスクリーニング方法
JP7697656B2 (ja) 人工ウイルスキャプシド
KR20220117091A (ko) 생체내 세포에서 합성 및 분비되고 세포로 투과하는 관심 폴리펩타이드를 코딩하는 발현카세트 및 이의 용도
EP3309178A1 (en) Fusion protein or conjugated protein, intracellular delivery carrier, partial peptide, cell membrane permeation enhancer, dna, and vector
EP1907544B1 (en) Membrane-translocating peptides
JP7716101B2 (ja) 細胞内送達ペプチド
TW202348801A (zh) 基於噬菌體展示之細胞穿透肽發現平台及製備及使用其之方法
CN107635548A (zh) 利用突变型伴侣蛋白复合体的细胞内局部性药物递送系统用纳米胶囊
JP7329750B2 (ja) リポソーム結合ペプチド、リポソーム結合ペプチドの作製用コンストラクト及びリポソーム
JP2022527351A (ja) 結合ペプチドを選択し検出するための方法
JP7477886B2 (ja) 核酸アプタマー
WO2025005142A1 (ja) 細胞外小胞と連結させて使用するための複合体及びその利用
WO2024081641A2 (en) Methods for preparing protein-conjugated nanocarriers
WO2024075767A1 (ja) ミトコンドリアへの核酸の導入方法
CN120248079A (zh) 一种干扰素基因刺激因子突变体及其应用
Alexandrova Tissue-specific expression of the human Glycyl-tRNA synthetase: connection with the Charcot-Marie-Tooth disease

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24836016

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2025515752

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2024836016

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024836016

Country of ref document: EP

Effective date: 20260203