Intracellular Delivery Peptides

Abstract

The present disclosure provides a peptide complex that includes a segment X having a prescribed amino acid sequence and a membrane-damaging segment Z.

Description

Technical Field

[0001] The present disclosure relates to a peptide complex for delivering a target substance into cells and an intracellular delivery agent.

Background Art

[0002] To date, various intracellular delivery reagents for proteins and nucleic acids (including siRNA, plasmids, etc.) (including liposomes, peptides, etc.) have been developed, but there is still room for further improvement in terms of efficiency and cytotoxicity. For example, it is known that nucleic acids can be introduced into cells by forming conjugates with peptides such as LK15 peptide and bee venom melittin, but it has high cytotoxicity due to high cell membrane damage and is not a practical intracellular introduction method.

[0003] In intracellular introduction via endocytosis, for a substance to exert its function inside the cell, it needs to be taken up into the endosome and then released from the endosome into the cytoplasm. Patent Document 1 and Patent Document 2 disclose peptides that can promote uptake into the endosome and release from the endosome. However, the conditions and situations required for intracellular delivery reagents are diverse, and an increase in the types of intracellular delivery reagents is still strongly desired so that the intracellular delivery reagents can be appropriately selected according to the circumstances.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0006] The disclosures of all prior art documents cited in this specification are incorporated herein by reference.

Summary of the Invention

Problems to be Solved by the Invention

[0007] An example of the problem of the present disclosure is to provide a means for effectively delivering a target substance such as a nucleic acid, a protein, or a medicine into cells.

Means for Solving the Problems

[0008] SDF1 (stromal cell-derived factor 1), also known as CXCL12 (C-X-C motif chemokine 12), is a chemokine protein and is a ligand of the chemokine receptor CXCR4. SDF1 is encoded by the CXCL12 gene on chromosome 10 in humans and is produced in two forms, SDF1α (CXCL12a) and SDF1β (CXCL12b), by alternative splicing of the gene.

[0009] It is known that human SDF1α induces macropinocytosis through the activation of CXCR4 (Non-Patent Document 1), and it is also known that the amino acid residues in the N-terminal region of human SDF1α are important for the activation of CXCR4 (Non-Patent Documents 2 and 3).

Table 1

[0010] The inventors have found that peptides having the N-terminal 8, 17, and 21 amino acid residues of SDF1α (SDF1αN8, SDF1αN17, and SDF1αN21 below, respectively) promote the introduction of a target substance into cells, and in particular, have revealed that SDF1αN21 has a high ability to introduce substances. Furthermore, the inventors have revealed that SDF1αN21 induces endocytosis (macropinocytosis and clathrin-related endocytosis), and that a conjugate of SDF1αN21 and a target substance (for example, an apoptosis-promoting PAD peptide or an apoptosis-promoting BH3 peptide) is effectively introduced into cells.

[0011] When the inventors conjugated a membrane-damaging peptide to a peptide derived from the SDF1α N-terminus, they surprisingly found that the resulting peptide conjugate exhibited a much higher intracellular introduction effect compared to the SDF1α N-terminus-derived peptide alone and the membrane-damaging peptide alone. Even more surprisingly, they found that the cytotoxicity of the peptide conjugate was much lower than that of the membrane-damaging peptide alone, leading to the technology disclosed in the present application. In addition, it has been found that by substituting valine (V) at the 18th position or arginine (R) at the 20th position of the amino acid sequence of SEQ ID NO: 1 with alanine (A), the intracellular introduction effect of the SDF1α N-terminus-derived peptide alone is significantly improved. Exemplary embodiments of the present disclosure are described in detail below.

[0012] [1] A peptide conjugate comprising segment X and a membrane-damaging segment Z, wherein segment X (a) the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1; or (b) an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a), and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both. having (or consisting of) The integer n ranges from 14 to 30, the membrane-damaging segment Z has (or consists of) an amino acid sequence derived from a membrane-damaging peptide, a peptide complex. [2] The peptide complex according to [1], wherein the C-terminus of the segment X and the terminus of the segment Z are conjugated directly or via a linker. [3] The peptide complex according to [2], Formula (I): R 1 -U-X-J 1 -Z-R 2 (I) [wherein, X represents the segment X, Z represents the segment Z, J 1 represents a divalent linker, the C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J 1 and, R 1 is a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U is absent or is an amino acid sequence consisting of 1 to 7 amino acids, R 2 is a hydroxyl group (OH), an amino group (NH2), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group] a peptide complex represented by. [4] The peptide complex according to [2], Formula (Ia): R 1 -(U-X-J 1 -Z-J 2 ) m1 -R 2 (Ia) [wherein, X each independently represents the segment X, Z, independently of one another, represents the segment Z, J 1 , independently of one another, represents a divalent linker, The C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J 1 . R 1 , independently of one another, is a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U, independently of one another, is either absent or an amino acid sequence consisting of 1 to 7 amino acids, R 2 , independently of one another, is a hydroxyl group (OH), an amino group (NH2), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group, J 2 , independently of one another, is a single bond or a divalent linker, The integer m1, independently of one another, is in the range of 2 to 10 A peptide complex represented by The peptide complex according to [5][2], The following formula (Ib): J 3 (-R a ) m2 (Ib) [In the formula, J 3 is a branched polyvalent linker, R a is the same as or different from R 1 -U-X-J 1 -Z-J 2 - or -J 2 -U-X-J 1 -Z-R 2 (where J 2 is independently bonded to J 3 ), X, independently of one another, represents the segment X, Z independently represents the segment Z, J 1 each independently represent a divalent linker; The C-terminus of the segment X and the N-terminus of the segment Z are J 1 It is conjugated via R 1 each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, each U is independently absent or an amino acid sequence of 1 to 7 amino acids; R 2 are each independently a hydroxyl group (OH), an amino group (NH), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group, and J 2 are each independently a single bond or a divalent linker; The integer m2 ranges from 2 to 8. The peptide complex represented by [6] The peptide complex according to any one of [1] to [5], wherein the segment X is (a) the amino acid sequence from position 1 to position n of the amino acid sequence of SEQ ID NO: 1; or (b) an amino acid sequence in which one or more amino acids other than those at positions 9 and 11 in the amino acid sequence of (a) have been deleted and / or substituted, and / or one or more amino acids have been added, wherein the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; wherein the integer n is in the range of 18 to 30; Peptide complexes. [7] The segment X is (a) the amino acid sequence from position 1 to position n of the amino acid sequence of SEQ ID NO: 1; or (b) An amino acid sequence in which one or several amino acids other than the 9th and 11th amino acids in the amino acid sequence of (a) are deleted and / or substituted and / or one or several amino acids are added, and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; having (or consisting of), where the integer n ranges from 20 to 30, The peptide complex according to any one of [1] to [6]. [8] The segment X is (a) The amino acid sequence from the 1st to the nth amino acids in the amino acid sequence of SEQ ID NO: 1; or (b) An amino acid sequence in which one or several amino acids other than the 9th and 11th amino acids in the amino acid sequence of (a) are deleted and / or substituted and / or one or several amino acids are added, and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; having (or consisting of), where the integer n ranges from 20 to 27, The peptide complex according to any one of [1] to [7]. [9] The segment X is (a) The amino acid sequence from the 1st to the nth amino acids in the amino acid sequence of SEQ ID NO: 1; or (b) An amino acid sequence in which one or several amino acids other than the 9th and 11th amino acids in the amino acid sequence of (a) are deleted and / or substituted and / or one or several amino acids are added, and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; having (or consisting of), where the integer n ranges from 20 to 25, The peptide complex according to any one of [1] to [8].

[10] The segment X is (b) In the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1, one or several consecutive amino acids (e.g., 2 to 6 amino acids) at the 1st to 6th positions are deleted, and one or several (e.g., 1 to 3) amino acids other than the 9th and 11th positions are arbitrarily substituted, and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; having (or consisting of), and the integer n is in the range of 14 to 30 (e.g., 14 to 25), the peptide complex according to any one of [1] to [5].

[11] The peptide complex according to any one of [1] to

[10] , wherein the segment X has (or consists of) an amino acid sequence selected from SEQ ID NOs: 2 to 27.

[12] When the segment X contains two or more cysteines, the segment X contains one or more disulfide bonds formed by any two cysteines, the peptide complex according to any one of [1] to

[11] .

[13] The peptide complex according to any one of [1] to

[12] , wherein the membrane-damaging segment Z has (or consists of) an amino acid sequence of a basic amphiphilic membrane-damaging peptide, and the number of amino acid residues of the amino acid sequence is 5 to 50.

[14] The peptide complex according to any one of [1] to

[13] , wherein the membrane-damaging segment Z is an amino acid sequence derived from a membrane-damaging peptide selected from the group consisting of LK15, M-lycotoxin, Chrysophsin, Cryptonin, Osmin, Grammistin, Brevinin, Mastoparan, Melectin, Ponericin, and Melittin.

[15] The peptide complex according to any one of [3] to

[14] , wherein the divalent linker is each independently a divalent linker selected from the group consisting of an ester bond (-CO-O-, -O-CO-), an ether bond (-O-), an amide bond (-NHCO-, -CONH-), a sugar chain linker, a polyethylene glycol linker, and a peptide linker.

[16] The peptide conjugate according to any one of [1] to

[15] , which is conjugated with a molecule that enhances affinity for target cells.

[17] A target substance-peptide complex comprising the peptide complex according to any one of [1] to

[16] and a target substance conjugated to the peptide complex.

[18] A cytoplasmic delivery agent comprising the peptide complex according to any one of [1] to

[16] .

[19] An intracellular introduction agent comprising the peptide complex according to any one of [1] to

[16] .

[20] The intracellular introduction agent described in

[19] , further containing a target substance.

[21] An intracellular introduction agent comprising the target substance-peptide complex described in

[17] .

[22] An intracellular introduction agent comprising a vector, wherein the vector comprises the peptide complex according to any one of [1] to

[16] .

[23] The intracellular introduction agent described in

[22] , wherein the vector further contains a target substance.

[24] An intracellular introduction agent comprising a vector, the vector comprising the target substance-peptide complex described in

[17] .

[25] The intracellular introduction agent according to any one of

[22] to

[24] , wherein the peptide complex or the target substance-peptide complex is conjugated to a component of the vector.

[26] The intracellular introduction agent according to any one of

[22] to

[25] , wherein the vector is a liposome, lipid microsphere, polymeric micelle, polymeric hollow carrier, nanogel, high-density lipoprotein (HDL), synthetic polymer, self-assembling nucleic acid-derived vector, viral coat protein-derived vector, or nanoparticle.

[27] The intracellular introduction agent according to

[25] , wherein the constituent component is cholesterol or a phospholipid.

[28] An intracellular introduction agent described in any one of

[20] ,

[23] ,

[26] , and

[27] , wherein the peptide complex and the target substance are non-covalently associated either directly or via another molecule that interacts with the target substance. A method for introducing a target substance into the cytoplasm, comprising contacting a cell with the peptide complex according to any one of [1] to

[16] .

[30] Conjugating a target substance with the peptide complex according to any one of [1] to

[16] to form a target substance-peptide complex, and contacting the target substance-peptide complex with a cell, a method for introducing a target substance into the cytoplasm.

[31] A peptide having (or consisting of) the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1, wherein the valine (V) at the 18th position and / or the arginine (R) at the 20th position is substituted, and the integer n is in the range of 18 to 30.

[32] The peptide according to

[31] , wherein the valine (V) at the 18th position and / or the arginine (R) at the 20th position is independently substituted with an amino acid selected from the group consisting of alanine and glycine.

[33] A peptide having (or consisting of) an amino acid sequence in which one or several consecutive amino acids (e.g., 2 to 6) at the 1st to 6th positions are deleted, and one or several (e.g., 1 to 3) amino acids other than the 9th and 11th positions are arbitrarily substituted in the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1, and the integer n is in the range of 14 to 30 (e.g., 14 to 25).

[34] The peptide according to

[33] , having (or consisting of) an amino acid sequence in which one or several consecutive amino acids (e.g., 2 to 6) at the 1st to 6th positions are deleted, and the proline (P) at the 10th position is substituted with alanine (A), and the integer n is in the range of 14 to 30 (e.g., 14 to 25).

[35] The peptide according to any one of

[31] to

[34] , having (or consisting of) an amino acid sequence selected from SEQ ID NOs: 23 to 27.

[36] The peptide according to any one of

[31] to

[35] , wherein the peptide is acetylated at the N-terminus, amidated at the C-terminus, or acetylated at the N-terminus and amidated at the C-terminus.

[37] The peptide according to any one of

[31] to

[36] , which exhibits the ability to promote intracellular introduction.

[38] The peptide according to any one of

[31] to

[37] , conjugated with a molecule that enhances the affinity for a target cell.

[39] A target substance-peptide complex comprising the peptide according to any one of

[31] to

[38] and a target substance conjugated to the peptide.

[40] A cytoplasmic delivery agent comprising the peptide according to any one of

[31] to

[38] .

[41] An intracellular introduction agent comprising the peptide according to any one of

[31] to

[38] .

[42] The intracellular introduction agent according to

[41] , further comprising a target substance.

[43] An intracellular introduction agent comprising the target substance-peptide complex according to

[39] .

[44] An intracellular introduction agent comprising a vector, wherein the vector comprises the peptide complex according to any one of

[31] to

[38] .

[45] The intracellular introduction agent according to

[44] , wherein the vector further comprises a target substance.

[46] An intracellular introduction agent comprising a vector, wherein the vector comprises the target substance-peptide complex according to

[39] .

[47] The intracellular introduction agent according to any one of

[44] to

[46] , wherein the peptide or the target substance-peptide complex is conjugated to a constituent component of the vector.

[48] The intracellular introduction agent according to any one of

[44] to

[47] , wherein the vector is a liposome, a lipid microsphere, a polymer micelle, a polymer hollow carrier, a nanogel, a high-density lipoprotein (HDL), a synthetic polymer, a self-assembling nucleic acid-derived vector, a virus outer shell protein-derived vector, or a nanoparticle.

[49] The intracellular introduction agent according to

[47] , wherein the constituent component is cholesterol or phospholipid.

[50] The intracellular introduction agent according to any one of

[42] ,

[45] ,

[48] , and

[49] , wherein the peptide and the target substance are non-covalently associated directly or via another molecule that interacts with the target substance.

[51] A method for introducing a target substance into the cytoplasm of a cell, comprising contacting the cell with the peptide according to any one of

[31] to

[38] .

[52] Conjugating the target substance with the peptide according to any one of

[31] to

[38] to form a target substance-peptide complex, and contacting the target substance-peptide complex with a cell, A method for introducing a target substance into the cytoplasm of a cell. [Advantages of the Invention]

[0013] In one embodiment, the peptide complex and peptide of the present disclosure can effectively deliver a target substance into a cell, for example, and can promote the introduction of the target substance into the cell. [Brief Description of the Drawings]

[0014]

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Figure 21

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Figure 23

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Figure 25

Mode for Carrying Out the Invention

[0015] In one aspect, the present disclosure provides a peptide complex comprising segment X and a membrane-damaging segment Z, wherein segment X (a) the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1; or (b) an amino acid sequence in which one or several (e.g., 1 to 6, 1 to 5, 1 to 3, or 1 to 2) amino acids are deleted, substituted, and / or added in the amino acid sequence of (a), and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both, and (or, consisting of) the integer n is any one of 14 to 30, the membrane-damaging segment Z has (or, consisting of) an amino acid sequence derived from a membrane-damaging peptide, and provides a peptide complex.

[0016] In the present disclosure, the "amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1" means the amino acid sequence of the amino acid residues up to the nth position counted with the N-terminal amino acid residue (K) of SEQ ID NO: 1 as the 1st position. The integer n is in the range of, for example, any of 8 to 30, 9 to 30, 11 to 30, 14 to 30, 15 to 30, 17 to 30, 18 to 30, 20 to 30, 20 to 27, 20 to 25. Specific examples of the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1 include the amino acid sequences in the following table. Peptides of these amino acid sequences may exhibit the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both. [Table 2]

[0017] Each amino acid included in the "amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1" may be an L-amino acid, a D-amino acid, or L-amino acids and D-amino acids may be mixed in one amino acid sequence. In one embodiment, all amino acids included in the amino acid sequence are L-amino acids. In one embodiment, all amino acids included in the amino acid sequence are D-amino acids.

[0018] In the present disclosure, since glycine (G, Gly) does not have an asymmetric carbon, it is treated as an L-amino acid in this specification, but glycine is treated as a D-amino acid when all amino acids included in the amino acid sequence are D-amino acids.

[0019] In the present disclosure, in the "amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a), and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both", "Showing the ability to promote intracellular introduction" means that a peptide (alone) having an amino acid sequence in which one or several amino acids are deleted, substituted and / or added has the ability to promote intracellular introduction. The method for evaluating the ability to promote intracellular introduction is not particularly limited, and for example, it can be carried out according to Test Example 3 (70 kDa dextran uptake assay) of the present application; "Showing the activity of reducing the cytotoxicity of the membrane-damaging segment Z" means that when an amino acid sequence in which one or several amino acids are deleted, substituted and / or added is bound to the membrane-damaging segment Z via linker-GG, the cytotoxicity of the resulting peptide complex is reduced as compared with the cytotoxicity of the membrane-damaging segment Z alone. Alternatively, it may mean that when an amino acid sequence in which one or several amino acids are deleted, substituted and / or added is bound to LK15 via linker-GG, the cytotoxicity of the resulting peptide complex is reduced as compared with the cytotoxicity of LK15 alone. The method for evaluating cytotoxicity is not particularly limited, and for example, it can be carried out by the method of Test Example 22 (cell viability assay) of the present application.

[0020] In the "amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence of (a)" in the present disclosure, the total number of amino acids to be deleted, substituted and / or added is not limited as long as the amino acid sequence shows the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the amino acid sequence derived from the membrane-damaging peptide, or both of them. For example, 1 to 6, 1 to 5, 1 to 3, and 1 to 2 can be mentioned. The amino acids used for substitution or addition may be standard amino acids or non-standard amino acids.

[0021] In the present disclosure, "standard amino acid" means alanine, leucine, arginine, lysine, asparagine, methionine, aspartic acid, phenylalanine, cysteine, proline, glutamine, serine, glutamic acid, threonine, glycine, tryptophan, histidine, tyrosine, isoleucine, and valine. In the present disclosure, the standard amino acid may be an L-type amino acid or a D-type amino acid.

[0022] In the present disclosure, "non-standard amino acid" means an amino acid other than a standard amino acid. Examples of non-standard amino acids include 2-aminoadipic acid, 3-aminoadipic acid, 2-aminobutyric acid, 4-aminobutyric acid, 2,4-diaminobutyric acid, 2-aminohexanoic acid, 6-aminohexanoic acid, β-alanine, 2-aminopentanoic acid, 2,3-diaminopropanoic acid, 2-aminopimelic acid, 2,6-diaminopimelic acid, cysteic acid, 2,4-diaminobutyric acid, 2,6-diaminopimelic acid, 2,3-diaminopropanoic acid, 4-carboxyglutamic acid, 5-oxoproline (pyroglutamic acid), homocysteine, homoserine, homoserine lactone, homoserine lactone, 5-hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, alloisoleucine, norleucine, norvaline, ornithine, sarcosine, allothreonine, thyroxine.

[0023] In one embodiment, in the "amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a)", the positions of the deletion and / or substitution are other than the 9th and 11th positions of the amino acid sequence of SEQ ID NO: 1.

[0024] In one embodiment, in the "amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a)", the positions of the deletion and / or substitution are other than the 7th to 9th and 11th to 14th positions of the amino acid sequence of SEQ ID NO: 1.

[0025] In one embodiment, in the "amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a)", the positions of the deletion and / or substitution include the 18th and / or 20th positions of the amino acid sequence of SEQ ID NO: 1 (for example, substituted with an amino acid selected from the group consisting of alanine and glycine).

[0026] In one embodiment, in the "amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a)", the position of substitution is the 10th position of the amino acid sequence of SEQ ID NO: 1. In one embodiment, in the "amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a)", proline (P) at the 10th position of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (A).

[0027] In one embodiment, the segment X is in the amino acid sequence from the 1st position to the nth position of the amino acid sequence of SEQ ID NO: 1, where one or several consecutive amino acids (e.g., 2 - 6 amino acids) at the 1st to 6th positions are arbitrarily deleted, and one or several (e.g., 1 - 3 amino acids) amino acids other than the 9th and 11th positions (e.g., other than the 7th - 9th and 11th - 14th positions) are substituted, and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; has (or consists of), and the integer n is in the range of 14 - 30 (e.g., 14 - 25).

[0028] In one embodiment, the segment X is in the amino acid sequence from the 1st position to the nth position of the amino acid sequence of SEQ ID NO: 1, where one or several consecutive amino acids (e.g., 2 - 6 amino acids) at the 1st to 6th positions are deleted, and one or several (e.g., 1 - 3 amino acids) amino acids other than the 9th and 11th positions (e.g., other than the 7th - 9th and 11th - 14th positions) are arbitrarily substituted, and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; has (or consists of), and the integer n is in the range of 14 - 30 (e.g., 14 - 25).

[0029] In one embodiment, in the amino acid sequence from the 1st to the nth position of the amino acid sequence of SEQ ID NO: 1, one or several consecutive amino acids (for example, 2 to 6 amino acids) at the 1st to 6th positions are deleted, and the proline (P) at the 10th position is substituted with alanine (A), and the amino acid sequence exhibits the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both; has (or consists of), and the integer n is in the range of 14 to 30 (for example, 14 to 25).

[0030] Examples of "amino acid sequences in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of (a)" include the following amino acid sequences. APVSLSYRCPCRFFESHVARA (SEQ ID NO: 15) KPASLSYRCPCRFFESHVARA (SEQ ID NO: 16) KPVSASYRCPCRFFESHVARA (SEQ ID NO: 17) KPVSLSARCPCRFFESHVARA (SEQ ID NO: 18) KPVSLSYACPCRFFESHVARA (SEQ ID NO: 19) KPVSLSYRCPCAFFESHVARA (SEQ ID NO: 20) KPVSLSYRCPCRAFESHVARA (SEQ ID NO: 21) KPVSLSYRCPCRFAESHVARA (SEQ ID NO: 22) KPVSLSYRCPCRFFESHAARA (SEQ ID NO: 23) KPVSLSYRCPCRFFESHVAAA (SEQ ID NO: 24) YRCPCRFF (SEQ ID NO: 25) YRCACRFF (SEQ ID NO: 26) YRCGCRFF (SEQ ID NO: 27). Peptides of these amino acid sequences may exhibit the ability to promote intracellular introduction, the activity of reducing the cytotoxicity of the membrane-damaging segment Z, or both.

[0031] In one embodiment, when segment X contains two or more cysteines, segment X has one or more (e.g., 1 to 2, or 1) disulfide bonds formed by any two cysteines. In another embodiment, in the peptide complex of the present disclosure, the cysteines at positions 9 and 11 in the amino acid sequence of SEQ ID NO: 1 are disulfide-bonded.

[0032] In the present disclosure, a membrane-damaging peptide means a peptide with an IC 50 of < 20 μM in a WST-1 assay or a WST-8 assay (e.g., a method according to Test Example 22 of the present application). Examples of membrane-damaging peptides include, but are not limited to, LK15, M-lycotoxin, Chrysophsin, Cryptonin, Osmin, Grammistin, Brevinin, Mastoparan, Melectin, Ponericin, and Melittin.

[0033] In the present disclosure, an amino acid sequence derived from a membrane-damaging peptide means an amino acid sequence having (or consisting of) the amino acid residues of a membrane-damaging peptide. The number of amino acid residues in the amino acid sequence is not particularly limited, and examples include 5 to 60 residues, 6 to 50 residues, 7 to 40 residues, and 8 to 35 residues. Each amino acid contained in the amino acid sequence may be a standard amino acid or a non-standard amino acid. Each amino acid contained in the amino acid sequence may be an L-amino acid or a D-amino acid, and an L-amino acid and a D-amino acid may be mixed in one amino acid sequence. In one embodiment, all amino acids contained in the amino acid sequence are L-amino acids. In one embodiment, all amino acids contained in the amino acid sequence are D-amino acids.

[0034] In one embodiment, the sequence derived from a membrane-damaging peptide is the amino acid sequence of LK15 (KLLKLLLKLLLKLLK: SEQ ID NO: 71).

[0035] In one embodiment, the sequence derived from the membrane-damaging peptide is the amino acid sequence of Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-amide: SEQ ID NO: 72).

[0036] In one embodiment, the C-terminus of segment X and the terminus of segment Z of the peptide complex are conjugated.

[0037] The "C-terminus of segment X" means the amino acid residue at the rightmost of segment X when the N-terminal amino acid residue (K) of SEQ ID NO: 1 is at the leftmost. The C-terminus of segment X may be conjugated to the N-terminus of segment Z or to the C-terminus of segment Z. In one embodiment, the C-terminus of segment X is conjugated to the N-terminus of segment Z.

[0038] In the present disclosure, "conjugated" or "conjugation" means that two or more substances (entities, such as peptides) are covalently bonded directly or via a linker to form one substance (entity). The method of conjugation in the present disclosure is not particularly limited and can be carried out using methods well known in the art.

[0039] In one embodiment, the peptide complex is Formula (I): R 1 -U-X-J 1 -Z-R 2 (I) [wherein, X represents the segment X, Z represents the segment Z, J 1 represents a divalent linker, the C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J 1 and, R 1is a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U is either absent or an amino acid sequence consisting of 1 to 7 amino acids, R 2 is a hydroxyl group (OH), an amino group (NH2), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group is represented by

[0040] In one embodiment, the peptide complex is in the form of a dimer or multimer. For example, a peptide complex represented by the following formula (Ia) can be mentioned. Formula (Ia): R 1 -(U-X-J 1 -Z-J 2 ) m1 -R 2 (Ia) [wherein, X each independently represents the segment X, Z each independently represents the segment Z, J 1 each independently represents a divalent linker, the C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J 1 and R 1 each independently is a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U each independently is either absent or an amino acid sequence consisting of 1 to 7 amino acids, R 2is, independently of each other, a hydroxyl group (OH), an amino group (NH2), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group, J 2 is, independently of each other, a single bond or a divalent linker, the integer m1 is, independently of each other, in the range of 2 to 10.

[0041] In one embodiment, the peptide complex may be linked by a branched polyvalent linker such as a dendrimer, or a metal complex or the like. The dendrimer is represented by, for example, the following formula (Ib). J 3 (-R a ) m2 (Ib) [wherein, J 3 is a branched polyvalent linker, R a is the same as or different from R 1 -U-X-J 1 -Z-J 2 - or -J 2 -U-X-J 1 -Z-R 2 (where J 2 is independently bonded to each J 3 ), X independently represents the segment X, Z independently represents the segment Z, J 1 independently represents a divalent linker, the C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J 1 ), R 1 is, independently of each other, a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U is, independently of each other, absent or an amino acid sequence consisting of 1 to 7 amino acids, R 2 is, independently of each other, a hydroxyl group (OH), an amino group (NH2), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group, J 2 is, independently of each other, a single bond or a divalent linker, The integer m2 ranges from 2 to 8.

[0042] R 1 When R is a hydrogen atom, the N-terminus of "U-X" is an amino group (NH2). R 1 When R is an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, or an aryloxycarbonyl group, its N-terminus is a monoalkylamino, acylamino, alkoxycarbonylamino, aralkyloxycarbonylamino, or aryloxycarbonylamino group, respectively.

[0043] In one embodiment, R 1 is a hydrogen atom (H), an alkyl group, an alkoxycarbonyl group, or an acyl group. In another embodiment, R 1 is a hydrogen atom.

[0044] R 2 When R is a hydroxyl group (OH), the C-terminus is a carboxyl group (COOH), and when R 2 is an amino group (NH2), the C-terminus becomes an amide group (CONH2), and when R 2 is an alkoxy group, an aralkyloxy group, or an aryloxy group, the C-terminus becomes the corresponding ester. When R 2 is a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, or a dialkylamino group, the C-terminus becomes the corresponding amide.

[0045] In one embodiment, R 2 is a hydroxyl group (OH), an alkoxy group, or an amino group (NH2).

[0046] An alkoxycarbonyl group, an aralkyloxycarbonyl group, and an aryloxycarbonyl group may each be directly bonded to the peptide complex of the present disclosure, but may also be bonded to the peptide complex of the present disclosure via a suitable linker such as PEG (polyethylene glycol), an amide group (-CONH-, -NHCO-), an ester group (-COO-, -O-CO-), an ether group (-O-), an amino group (-NH-), an alkylene (methylene, ethylene, propylene, butylene, pentylene, hexylene, etc.), an amino acid (for example, the 20 natural amino acids), etc. For example, a cholesteryl group or a phospholipid (for example, phosphatidylethanolamine) may be bonded to the peptide complex of the present disclosure via a linker, and the conjugate may constitute a component of a vector such as a liposome.

[0047] In one embodiment, the peptide complex of the present disclosure comprises (or consists of) SEQ ID NO: 56.

[0048] In one embodiment, the peptide complex of the present disclosure comprises (or consists of) SEQ ID NO: 57.

[0049] In one embodiment, the peptide complex of the present disclosure comprises (or consists of) SEQ ID NO: 58.

[0050] In one embodiment, the peptide complex of the present disclosure comprises (or consists of) SEQ ID NO: 59.

[0051] In one embodiment, the peptide complex of the present disclosure SDF1αN21-Melittin: comprises (or consists of) TIFF0007716101000003.tif55168.

[0052] In one embodiment, the peptide complex of the present disclosure comprises (or consists of) SEQ ID NO: 70.

[0053] Examples of the alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0054] Examples of the acyl group include linear or branched acyl groups having 2 to 22 carbon atoms, preferably 2 to 18 carbon atoms, such as acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, lauroyl, myristoyl, palmitoyl, stearoyl, isostearoyl, oleoyl, and linoleoyl. Further, an acyl group containing an aromatic group such as 1-pyreneacetyl or 1-pyrenebutyryl may also be used.

[0055] Examples of the alkoxycarbonyl group include cholesteryloxycarbonyl group, tert-butyloxycarbonyl group, phytosteryloxycarbonyl group, stearyloxycarbonyl group, palmityloxycarbonyl group, 2-octyldodecyloxycarbonyl group, behenyloxycarbonyl group, and the like.

[0056] Examples of the aralkyloxycarbonyl group include benzyloxycarbonyl group, phenethyloxycarbonyl group, fluorenylmethyloxycarbonyl group, anthrylmethyloxycarbonyl group, biphenylylmethyloxycarbonyl group, tetrahydronaphthylmethyloxycarbonyl group, chromanylmethyloxycarbonyl group, 2,3-dihydro-1,4-dioxanaphthalenylmethyloxycarbonyl group, indanylmethyloxycarbonyl group, and phenanthrylmethyloxycarbonyl group.

[0057] Examples of the aryloxycarbonyl group include a fluorenyloxycarbonyl group, a phenyloxycarbonyl group, a naphthyloxycarbonyl group, an anthryloxycarbonyl group, a biphenylyloxycarbonyl group, a tetrahydronaphthyloxycarbonyl group, a chromanyloxycarbonyl group, a 2,3-dihydro-1,4-dioxanaphthalenyloxycarbonyl group, an indanyloxycarbonyl group, and a phenanthryloxycarbonyl group.

[0058] Examples of the alkoxy group include a cholesteryloxy group, a phytosteryloxy group, a stearyloxy group, a palmitinyloxy group, a 2-octyldodecyloxy group, and a behenyloxy group.

[0059] Examples of the aryloxy group include a fluorenyloxy group, a phenyloxy group, a naphthyloxy group, an anthryloxy group, a biphenylyloxy group, a tetrahydronaphthyloxy group, a chromanyloxy group, a 2,3-dihydro-1,4-dioxanaphthalenyloxy group, an indanyloxy group, and a phenanthryloxy group.

[0060] Examples of the aralkyloxy group include a benzyloxy group, a phenethyloxy group, a fluorenylmethyloxy group, an anthrylmethyloxy group, a biphenylylmethyloxy group, a tetrahydronaphthylmethyloxy group, a chromanylmethyloxy group, a 2,3-dihydro-1,4-dioxanaphthalenylmethyloxy group, an indanylmethyloxy group, and a phenanthrylmethyloxy group.

[0061] Examples of the monoalkylamino group include amino groups substituted with linear or branched alkyl groups having 1 to 6 carbon atoms, such as methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, and hexylamino.

[0062] Examples of the monoaryl amino group include phenylamino, naphthylamino, anthrylamino, biphenylylamino, tetrahydronaphthylamino, chromanylamino, fluorenylamino, 2,3-dihydro-1,4-dioxanaphthalenylamino, indanylamino, and phenanthrylamino.

[0063] Examples of the monocycloalkyl amino group include cycloalkyl aminos having 3 to 8 carbon atoms such as cyclopropylamino, cyclobutylamino, cyclopentylamino, cyclohexylamino, cycloheptylamino, and cyclooctylamino.

[0064] Examples of the dialkyl amino group include amino groups disubstituted with linear or branched alkyl groups having 1 to 6 carbon atoms such as dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-sec-butylamino, di-tert-butylamino, dipentylamino, and dihexylamino.

[0065] In the present disclosure, the "divalent linker" includes divalent linkers commonly used in chemical conjugation methods of peptides. Examples of chemical conjugation methods include, but are not limited to, carbodiimide chemistry, reductive animation, cyanilation chemistry (e.g., CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroxysuccinimide chemistry, click chemistry, etc. Examples of the divalent linker include, but are not limited to, ester bonds (-CO-O-, -O-CO-), ether bonds (-O-), amide bonds (-NHCO-, -CONH-), sugar chain linkers, polyethylene glycol linkers, peptide linkers, etc. Also, as crosslinking reagents, BS3 (bis(sulfosuccinimidyl)suberate), BMOE (bismaleimidoethane), GMBS (N-γ-maleimidobutyryl-oxysuccinimide ester), DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate), DSP (dithiobis(succinimidyl propionate)), DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), DST (disuccinimidyl tartrate), EGS (ethylene glycol bis(succinimidyl succinate)), Sulfo-EGS (ethylene glycol bis(sulfosuccinimidyl succinate)), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), DFDNB (1,5-difluoro-2,4-dinitrobenzene), BMB (1,4-bismaleimidobutane), BMH (bismaleimidohexane), TMEA (tris(2-maleimidoethyl)amine), BM(PEG)2 (1,8-bismaleimido-diethyleneglycol), BM(PEG)3 (1,11 - bismaleimido - triethyleneglycol), DTME (dithiobismaleimidoethane), AMAS (N - α - maleimidoacet - oxysuccinimide ester), BMPS (N - β - maleimidopropyl - oxysuccinimide ester), Sulfo - GMBS (N - γ - maleimidobutyryl - oxysulfosuccinimide ester), MBS (m - maleimidobenzoyl - N - hydroxysuccinimide ester), Sulfo - MBS (m - maleimidobenzoyl - N - hydroxysulfosuccinimide ester), SMCC (succinimidyl 4 - (N - maleimidomethyl)cyclohexane - 1 - carboxylate), Sulfo - SMCC (sulfosuccinimidyl 4 - (N - maleimidomethyl)cyclohexane - 1 - carboxylate), EMCS (N - ε - maleimidocaproyl - oxysuccinimide ester), Sulfo - EMCS (N - ε - maleimidocaproyl - oxysulfosuccinimide ester), SMPB (succinimidyl 4 - (p - maleimidophenyl)butyrate), Sulfo - SMPB (sulfosuccinimidyl 4 - (N - maleimidophenyl)butyrate), SMPH (Succinimidyl 6 - ((beta - maleimidopropionamido)hexanoate)), LC - SMCC (succinimidyl 4 - (N - maleimidomethyl)cyclohexane - 1 - carboxy - (6 - amidocaproate)), Sulfo - KMUS (N - κ - maleimidoundecanoyl - oxysulfosuccinimide ester), SM(PEG)2 (PEGylated SMCC crosslinker),SM(PEG)4 (PEGylated SMCC crosslinker), SM(PEG)6 (PEGylated, long-chain SMCC crosslinker), SM(PEG)8 (PEGylated, long-chain SMCC crosslinker), SM(PEG)12 (PEGylated, long-chain SMCC crosslinker), SM(PEG)24 (PEGylated, long-chain SMCC crosslinker), SPDP (succinimidyl 3-(2-pyridyldithio)propionate), LC-SPDP (succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate), Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene), SDA (NHS-Diazirine) (succinimidyl 4,4'-azipentanoate), Sulfo-SDA (Sulfo-NHS-Diazirine) (sulfosuccinimidyl 4,4'-azipentanoate), LC-SDA (NHS-LC-Diazirine) (succinimidyl 6-(4,4'-azipentanamido)hexanoate), Sulfo-SDAD (Sulfo-NHS-SS-Diazirine) (sulfosuccinimidyl 2-((4,4'-azipentanamido)ethyl)-1,3'-dithiopropionate), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), EDAC (1-Ethyl-3-(3-Dimethylaminopropyl)carbodiimide,Hydrochloride), NHS (N-hydroxysuccinimide), Sulfo-NHS (N-hydroxysulfosuccinimide), BMPH (N-β-maleimidopropionic acid hydrazide), EMCH (N-ε-maleimidocaproic acid hydrazide), MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide), KMUH (N-κ-maleimidoundecanoic acid hydrazide), PDPH (3-(2-pyridyldithio)propionyl hydrazide), PMPI (p-maleimidophenyl isocyanate), SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate), etc. may also be used.,

[0066] Examples of the peptide linker include a linker containing at least one standard amino acid (e.g., L-type standard amino acid). Two or more amino acids contained in the peptide linker may be linked by a chemical conjugation method (e.g., maleimide chemistry, click chemistry), for example, using the above cross-linking reagents. The number of amino acids contained in the peptide linker may be 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4, but is not limited thereto. Examples of the peptide linker include arginine dimer, arginine trimer, arginine tetramer, lysine dimer, lysine trimer, lysine tetramer, glycine dimer, glycine trimer, glycine tetramer, glycine pentamer, glycine hexamer, alanine-alanine-tyrosine, isoleucine-leucine-alanine, arginine-valine-lysine-arginine, etc.

[0067] The amino acid that may be contained in U of formula (I), (Ia), or (Ib) may be a standard amino acid, a non-standard amino acid, or a mixture of a standard amino acid and a non-standard amino acid. U may be bound to the N-terminus of segment X. The amino acids that may be included in U are either absent or are 1 to 7 (for example, 1 to 5, 1 to 3, 1) in number, and are independently selected from standard amino acids and non-standard amino acids. In one embodiment, U is absent or is 1 4-aminobutanoic acid. In one embodiment, U is absent.

[0068] Examples of branched polyvalent linkers include diethylenetriamine, spermine, spermidine, triethanolamine, ethylenediaminetetraacetate (EDTA), pentaerythritol, azido-propyl(alkyl)amine, lysine, ornithine, aspartic acid, glutamic acid, polyfunctional peptides (dipeptides, tripeptides or tetrapeptides containing lysine, ornithine, aspartic acid or glutamic acid), organic polyvalent amino compounds (for example, poly(amidoamine) (PAMAM), tris(ethyleneamine)ammonia, poly(propyleneimine) (Astramol (商標) ) etc.).

[0069] The integer m1 is in any range of 2 to 10, 2 to 8, 2 to 6, or 2 to 4, or is 2 or 3.

[0070] The integer m2 is in any range of 2 to 8, 2 to 6, 2 to 4, or is 2 or 3.

[0071] In formula (Ib), for example, a dimer in which the N-termini of the amino acid sequences represented by U-X are linked together is included in the dendrimers of the present disclosure. Furthermore, a tetramer in which the dimer in which the N-termini of the amino acid sequences represented by U-X are linked together and the dimer in which the C-termini of the amino acid sequences represented by Z are linked together are further linked is also included in the dendrimers of the present disclosure.

[0072] In one aspect, the present disclosure provides a peptide having (or consisting of) the amino acid sequence from position 1 to position n of the amino acid sequence of SEQ ID NO: 1, wherein valine (V) at position 18 and / or arginine (R) at position 20 are substituted, and the integer n is in the range of, for example, 14 to 30 (sometimes referred to as "the peptide of the present disclosure").

[0073] In the peptide of the present disclosure, the amino acid that substitutes valine (V) at position 18 and / or arginine (R) at position 20 is not particularly limited as long as the resulting peptide exhibits the ability to promote intracellular introduction. For example, each is independently substituted with an amino acid selected from the group consisting of alanine and glycine.

[0074] Examples of the peptide of the present disclosure include KPVSLSYRCPCRFFESHAARA (SEQ ID NO: 23) KPVSLSYRCPCRFFESHVAAA (SEQ ID NO: 24) peptides consisting of these, or peptides containing these.

[0075] Furthermore, in one aspect, the present disclosure provides YRCPCRFF (SEQ ID NO: 25), YRCACRFF (SEQ ID NO: 26), and YRCGCRFF (SEQ ID NO: 27) peptides having (or consisting of) an amino acid sequence selected from the group consisting of these (sometimes referred to as "the peptide of the present disclosure").

[0076] The peptide of the present disclosure may be in the form of a dimer or multimer linked by a linker or the like.

[0077] In the present disclosure, the peptide complex or peptide may be chemically modified to such an extent that these structures and properties are not significantly changed. The chemical modification can be carried out using ordinary methods known to those skilled in the art. The chemical modification can be performed on each amino acid unit in the peptide, and the sites of chemical modification include, for example, hydroxyl groups, amino groups, and carboxyl groups contained in amino acids, as well as amino groups located at the N-terminus of the peptide, hydroxyl groups or carboxyl groups located at the C-terminus of the peptide, and the like.

[0078] As used herein, "protein", "peptide", or "polypeptide" can be used interchangeably. A protein may be a polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a composite polypeptide containing a structure other than the polypeptide.

[0079] In the present disclosure, target substances include bioactive substances, such as proteins, peptides, nucleic acids, pharmaceuticals, sugars, or labeled substances thereof, as well as synthetic polymers, liposomes, organic / inorganic / semiconductor microparticles, and the like. Bioactive substances such as proteins, peptides, nucleic acids, pharmaceuticals, sugars, or labeled substances thereof, synthetic polymers, liposomes, organic / inorganic / semiconductor microparticles, etc. may be bound to the peptide complex or peptide (e.g., at the end) directly or via an appropriate linker.

[0080] In the present disclosure, the "cytoplasmic delivery agent" means an agent that does not itself contain the target substance and, when used in combination with the target substance, promotes the introduction of the target substance into the cell. The peptide complex / peptide of the present disclosure can be used as a cytoplasmic delivery agent.

[0081] In the present disclosure, the "intracellular introduction agent" is an agent used to introduce the target substance into the cell, and the intracellular introduction agent may include a plurality of components such as the peptide complex / peptide of the present disclosure, the target substance, the target substance-peptide complex, and the vector.

[0082] The peptide complex of the present disclosure / the peptide of the present disclosure can be used to enable intracellular localization, behavior, measurement, and control of the intracellular environment of target substances through the delivery of target substances such as proteins and sensor molecules that have been chemically modified, including fluorescent labeling, into the cytoplasm.

[0083] Examples of proteins introduced into cells include antibodies, enzymes, cell signaling factors, transcription factors, DNA or RNA binding proteins, intracellular organelle constituent proteins such as those of the nucleus, mitochondria, and cytoskeleton, ubiquitin-proteasome system-related proteins such as ubiquitin and heat shock proteins, apoptosis-related proteins such as caspases, cell cycle regulatory proteins such as p53, lectins, etc. Proteins having gene cleavage and recombination ability such as Cre recombinase, TALEN, and Cas9 are also included. Regarding antibodies, in addition to immunoglobulins, their fragment proteins and single-chain antibodies derived from camelids are also included. Examples of targets for these antibodies include kinases, transcription factors such as HIF-1, and cytoskeletal proteins such as microtubules. Examples of peptides include helix peptides and cyclic peptides that regulate intracellular protein interactions, fragment peptides of intracellular proteins, DNA / RNA binding peptides, various enzyme substrates and inhibitors, and antigen peptides for cancer vaccine production. Examples of pharmaceuticals include antitumor agents, antiviral agents, etc. Examples of sugars include dextran, sialic acid, etc. Examples of nucleic acids include DNA, RNA (preferably siRNA, miRNA, shRNA, rRNA, ribozymes, antisense RNA, etc.), DNA / RNA aptamers, and chemically modified forms thereof. Protein-nucleic acid complexes such as Cas9 / sgRNA are also included in bioactive substances. Derivatives obtained by chemically modifying the above bioactive substances as necessary for improving bioactivity and functions in cells can also be introduced into cells as target substances.

[0084] For intracellular visualization, measurement, and interaction analysis, physiologically active substances such as the above-mentioned proteins and nucleic acids are modified with fluorophores, quantum dots, radioisotopes, fluorescent proteins, luciferase, photocrosslinking groups, etc., and stable isotope-labeled proteins for intracellular NMR measurement, etc. are also listed as target substances, but are not limited thereto. As the fluorophore, it is also possible to modify with a fluorophore whose fluorescence characteristics change according to the intracellular environment as needed.

[0085] The target substance introduced into the cell does not particularly need to be limited to the above, and it is also possible to use it in combination with other cell-introducing agents such as peptides having membrane permeability and various transfection reagents, and to use the present invention to further improve the cytoplasmic translocation efficiency of these.

[0086] The target substance does not particularly need to be limited to the above, and it may be used in combination with other intracellular introducing agents such as peptides having membrane permeability and various transfection reagents, and this method may be used to further improve the cytoplasmic translocation efficiency of these.

[0087] The peptide complex / peptide of the present disclosure may be administered by mixing with the target substance, or may be covalently bound directly to the target substance or via an appropriate linker, or may form a non-covalent complex directly or via other molecules that interact with the target substance. Furthermore, it may be included in a vector that can be encapsulated in an endosome. For example, the peptide complex / peptide of the present disclosure may be contained inside the vector, or may be bound directly or via a linker to a component of the vector. Alternatively, the peptide complex / peptide of the present disclosure may be included on the surface of the vector by forming a non-covalent complex via other molecules that interact with the components of the vector. Furthermore, by encapsulating the peptide complex / peptide of the present disclosure together with the target substance inside the vector, after the target substance is taken up into the endosome, the transfer from the endosome to the cytoplasm can be promoted.

[0088] In formula (I), formula (Ia) and formula (Ib), R 1and / or R 2 Alternatively, the target substance may be optionally bound via a linker.

[0089] R 1 and R 2 Examples of the peptide complex of the present disclosure in which one or both of R (target substance)-U-X-J 1 -Z-(target substance); (target substance)-(linker)-U-X-J 1 -Z-(linker)-(target substance); (target substance)-(linker)-U-X-J 1 -Z-(target substance); (target substance)-U-X-J 1 -Z-(linker)-(target substance); (target substance)-(linker)-U-X-J 1 -Z-R 2 ; R 1 -U-X-J 1 -Z-(linker)-(target substance); (target substance)-U-X-J 1 -Z-R 2 ; R 1 -U-X-J 1 -Z-(target substance).

[0090] The peptide complex of the present disclosure / the peptide of the present disclosure can supply a target substance to target cells by DDS, or introduce the target substance into the cytoplasm of specific cells by combining a cell-specific vector with the peptide complex of the present disclosure / the peptide of the present disclosure. Examples of the cell-specific vector include vectors having a cell-specific antibody, ligand, etc. introduced on the surface. Examples of the vector include liposomes (cationic liposomes, anionic liposomes), lipid microspheres, lipofectamine, polymeric micelles composed of block copolymers containing a hydrophilic segment and a core-forming segment (hydrophobic segment, cationic segment, metal complex-forming segment, etc.), polymeric hollow carriers (drug carriers containing polysarcosine derivatives described in Patent No. 5142313, electrostatically bound polymeric micelles composed of block copolymers having a non-charged segment and a charged segment described in WO2004 / 105799, etc.), nanogels, high-density lipoproteins (HDL), synthetic polymers, nanoparticles, etc. It may also be a self-assembled nucleic acid-derived vector or a virus outer shell protein-derived vector. For example, when the vector is a liposome, R 1Either an acyl group having 10 or more carbon atoms is used, or a group with high lipophilicity such as a cholesteryloxycarbonyl group at the N-terminus or a cholesteryl group at the C-terminus, or a phospholipid such as phosphatidylethanolamine is bonded to the peptide complex / disclosed peptide of the present disclosure via an appropriate linker by an ester bond or an amide bond as needed, whereby liposomes containing the peptide complex / disclosed peptide of the present disclosure can be obtained. Examples of the linker include PEG (polyethylene glycol), amide groups (-CONH-, -NHCO-), ester groups (-COO-, -O-CO-), ether groups (-O-), amino groups (-NH-), alkylene (methylene, ethylene, propylene, butylene, pentylene, hexylene, etc.), amino acids, etc. The amino acids may be bonded via the COOH or NH2 group in the side chain. Examples of virus (outer shell protein)-derived vectors include retrovirus vectors, adenovirus vectors, Sendai virus vectors, etc. Examples of self-assembling nucleic acid-derived vectors include those described in WO 2012144560 A1 and WO2016 / 006628.

[0091] In the present disclosure, the "molecule that enhances the affinity of the peptide complex / disclosed peptide of the present disclosure for target cells" refers to a molecule that can transfer and accumulate the peptide complex of the present disclosure more efficiently / effectively into the endosome of cells and enhance the destabilizing effect of the endosome membrane and the delivery effect of the target substance to the cytoplasm by conjugation with the peptide complex of the present disclosure. Examples of molecules that enhance the affinity of the peptide complex of the present disclosure for target cells include receptors presented on the surface of cells (folate receptor, transferrin receptor, receptors for sugars such as glucose, growth factor receptors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), etc.), ligands that bind to receptors of the extracellular matrix such as integrin, or fatty acids, hydrophobic peptides, basic peptides such as polyarginine, sugar chains, etc. In addition, the membrane-damaging peptides described in the present disclosure are also mentioned as examples.

[0092] The peptide complex of the present disclosure / the peptide of the present disclosure and the target substance may form a non-covalent complex directly or via another molecule that interacts with the target substance. Such non-covalent complexes include (a) A complex of [a peptide showing affinity for an antibody (selected using a phage display system, etc.) and a conjugate of the peptide complex of the present disclosure / the peptide of the present disclosure] and an antibody (b) A non-covalent complex of a nucleic acid and the peptide complex of the present disclosure / the peptide of the present disclosure (c) A non-covalent complex of [a conjugate of the peptide complex of the present disclosure / the peptide of the present disclosure and a nucleic acid interacting molecule] and a nucleic acid, etc.

[0093] The species to which the target substance is to be delivered to cells is a vertebrate such as a mammal, for example. Examples of mammals include, but are not limited to, humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc.

Examples

[0094] Hereinafter, the content of the present disclosure will be described with reference to test examples, but the present invention is not limited to these test examples.

[0095] Materials and Reagents All salts, reagents, inhibitors, and media were purchased from Wako (Japan), Sigma-Aldrich, or Thermo Fischer unless otherwise specified. In the following examples section, capital letters in the amino acid sequence indicate L-amino acids, and small letters indicate D-amino acids.

[0096] Test Example 1 Peptide Synthesis The following peptides were synthesized by Fmoc solid-phase peptide synthesis using Rink amide resin described by Ikuhiko Nakase et al. (Interaction of Arginine-Rich Peptides with Membrane-Associated Proteoglycans Is Crucial for Induction of Actin Organization and Macropinocytosis. (2006). doi:10.1021 / BI0612824). Deprotection and cleavage of the peptide from the resin were performed using trifluoroacetic acid (TFA) / ethanedithiol (95:5) (room temperature, 2 hours). The peptides were purified by reverse-phase high-performance liquid chromatography using a C18 column. The mass was confirmed by MALDI-TOF mass spectrometry. In the preparation of OxN21 and OxN21-LK15, the intramolecular cysteine of SN21 was oxidized according to the protocol described by Taguchi et al. (Taguchi, A. et al. 3-Nitro-2-pyridinesulfenates as Efficient Solution- and Solid-Phase Disulfide Bond Forming Agents. Chem. - A Eur. J. 23, 8262-8267 (2017)).

[0097]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0098] SDF1αN21-Melittin: JPEG0007716101000008.jpg56168[M+H+] calc.:6213.42 [M+H+] obs. :6214.22 KPVSLSYRCPCRFFESHVARA-G: SEQ ID NO: 63 GGIGAVLKVLTTGLPALISWIKRKRQQ-amide: SEQ ID NO: 64

[0099] P4A-LK15: YRCACRFF-GG-KLLKLLLKLLLKLLK-amide: SEQ ID NO: 70

[0100] Test Example 2 Cell Culture HeLa cells were maintained in minimum essential medium alpha modified containing 10% bovine serum (BS) [α-MEM(+)] at 37 °C in a humidified 5% CO2 environment. HeLa-TBS-Luci cells were maintained under the same conditions in the presence of 0.8 μg / mL puromycin.

[0101] Test Example 3 70 kDa Dextran Uptake Assay-1 HeLa cells (70,000 cells / well) were cultured overnight in a 24-well plate and serum-starved for 16 hours before the assay. The starved cells were washed twice with phosphate-buffered saline (PBS) and incubated for 30 minutes in α-MEM(-) in the presence of 1 mg / mL FITC-dextran (70 kDa), 5 μM peptide, and 100 nM SDF1α (BioLegend), 10 μM phorbol-12-myristate-13-acetate (PMA) or 50 ng / mL PDGF-BB. After incubation, the cells were washed three times with ice-cold PBS(-), detached with 0.1% trypsin, and transferred to a microcentrifuge tube. The samples were pelleted by centrifugation (800 g, 5 minutes, 4 °C) and washed once more with PBS(-). Flow cytometry analysis (FCM) was performed on 10,000 events. The data are shown as relative data normalized to the set without peptide (blank subtracted). The results are shown in Figure 1.

[0102] Test Example 4 70 kDa Dextran Uptake Assay-2 (Comparison with known cell-penetrating peptides) Using octaarginine (R8), dodecaarginine (R12), and the basic peptide (TAT) derived from the Tat protein of HIV-1, the uptake of 70 kDa dextran was evaluated in the same manner as in Test Example 3. The results are shown in Figure 2. As shown in Figure 2, SDF1αN21 showed an effect equal to or greater than that of known inducers.

[0103] Test Example 5 Delivery of luciferase siRNA - 1 HeLa - GL3 cells were maintained in α - MEM(+) (containing 500 μg / mL G418). The cells were seeded in a 12 - well plate (70,000 cells / well) and allowed to adhere for 48 hours. The peptide was combined with luciferase siRNA in OptiMEM (200 μL) at charge ratios of 1, 3, 5, 7, 10, and 20 shown by the following formula and complexed for 30 minutes. HeLa - GL3 cells were washed twice with PBS, and the culture medium was changed to 800 μL of α - MEM(-). To this, the peptide - siRNA complex was added (final siRNA concentration = 50 nM), and the mixture was incubated for 6 hours. The culture medium was changed to fresh α - MEM(+), and incubation was continued for another 24 hours. TIFF0007716101000009.tif23153 Regarding the test using Lipofectamine 2000 (LF2000), LF2000 (1 μL) was dissolved in OptiMEM (Invitrogen, 49 μL), and this was added to 500 nM siRNA in OptiMEM (100 μL) and allowed to stand at room temperature (~23°C) for 20 minutes. αMEM(-) was added to this so that the final siRNA concentration was 50 nM, and HeLa - GL3 cells were incubated for 6 hours in the same manner as above. The culture medium was changed to fresh α - MEM(+), and incubation was continued for another 24 hours.

[0104] For the luciferase activity assay, cells were washed three times with PBS and lysed using GloLysis Buffer (Promega). The lysates were collected and transferred to a 96-well plate partitioned with a white wall at 50 μL lysate / well. SteadyGlo (Promega) reagent was added to the lysates at a 1:1 ratio, and then incubated at room temperature for 5 minutes, after which luminescence was measured. The total protein concentration of each lysate was measured by Pierce BCA assay (Thermo). Luciferase activity was calculated as relative light units per 1 mg of protein. The results are shown in Figure 3. From the results, it was shown that SDF1αN21-LK15 is useful for siRNA delivery.

[0105] Test Example 6 Delivery of EGFP-N1 Plasmid DNA - 1 HeLa cells were seeded in a 35-mm glass-bottom dish (IWAKI) and allowed to adhere overnight. EGFP-N1 plasmid (1 μg) and the peptide were combined in OptiMEM at a charge ratio of 5. Complex formation was carried out at room temperature for 30 minutes. The cells were washed three times with PBS, and then α-MEM(-) was added. To this, the pDNA-peptide solution was added. The cells were incubated for 6 hours and further incubated in α-MEM(+) for 24 hours. Observation was carried out using an FV1000 CLSM system (Olympus). The results are shown in Figure 4. As shown in Figure 4, in the transfection using SDF1αN21-LK15, FGFP expression was observed in more than 50% of the cells, whereas when LK15 was used, the cells showing the EGFP signal were 20% or less. From this, it was shown that SDF1αN21-LK15 is useful for plasmid DNA delivery.

[0106] Test Example 7 Delivery of EGFP-N1 Plasmid DNA - 2 (Quantification of EGFP-Expressing Cells) HeLa cells were seeded in a 12-well plate, and then the same procedure as in Test Example 6 was performed. The EGFP-N1 plasmid (1 μg) and the peptide were combined in OptiMEM at a charge ratio of 5. Complex formation was carried out at room temperature for 30 minutes. In the study using LF2000, LF2000 (1 μL) was dissolved in 49 μL of OptiMEM, and this was mixed with OptiMEM (50 μL) of the EGFP-N1 plasmid (1 μg), and after standing at room temperature (~23 °C) for 20 minutes, it was added to the cells. As a control, only the EGFP-N1 plasmid (1 μg) was added. After the 24-hour incubation, the cells were trypsinized, washed with PBS, and analyzed using Attune NxT Flow Cytometry (Thermo) (10,000 events). The results are shown in Fig. 5. From the results, it was shown that SDF1αN21-LK15 is useful for the delivery of plasmid DNA.

[0107] Test Example 8 IgG-Alexa488 Delivery-1 Before performing the assay, human serum immunoglobulin G (IgG) was labeled with Alexa488-sdp-ester (Thermo) according to the description by Akishiba et al. (Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide. Nat. Chem. 9, 751-761 (2017)). HeLa cells (150,000 cells) were seeded in a 35-mm glass-bottom dish (Iwaki) and allowed to adhere for 48 hours. The obtained cells were washed 3 times with PBS, and then incubated in -MEM(-) with 5 μM SDF1αN21, 2 μM LK15, or SDF1αN21-LK15, and 100 μg / mL IgG-Alexa488 for 60 minutes. After incubation, the cells were washed 3 times with PBS. The non-internalized signal was quenched with 5% trypan blue in PBS for 1 minute, followed by washing 3 times with PBS. Observation was carried out using an FV1000 CLSM system (Olympus). The results are shown in Fig. 6. As shown in Fig. 6, no fluorescent signal was observed for SDF1αN21 alone, LK-15 alone, and the mixture of SDF1αN21 and LK-15, whereas a fluorescent signal spreading throughout the cells was observed for SDF1αN21-LK15, indicating that SDF1αN21-LK15 is useful for the delivery of IgG.

[0108] Test Example 9 Uptake of SDF1αN21 and Its Invasion Pathway Adherent cells were pretreated with 100 μM EIPA for 30 minutes, and the cells were pretreated with a predetermined concentration of inhibitor diluted in α-MEM(-) for 30 minutes before treatment. Without washing, the medium was replaced with fresh α-MEM(-) containing 5 μM SDF1αN21-FITC (KPVSLSYRCPCRFFESHVARA-K-[fluorescein isothiocyanate (FITC)]-NH2 (SEQ ID NO: 65: KPVSLSYRCPCRFFESHVARA-K)) and each inhibitor, and the cells were further incubated at 37°C for 30 minutes. After washing with PBS(-), the cells were collected and analyzed by FCM. The results are shown in Fig. 7. From the results, it was shown that the uptake of SDF1αN21 is mediated via macropinocytosis and clathrin-mediated endocytosis.

[0109] Test Example 10 CXCR4 Knockdown Test Using Lipofectamine 2000 (Invitrogen), HeLa cells were transfected with three different anti-CXCR4 siRNAs or control siRNA (siCTR) according to the manufacturer's protocol. After 6 hours of incubation, the medium was changed to fresh α-MEM(+), and after further incubation for 48 hours, the intracellular uptake of SDF1αN21-FITC was evaluated as in Test Example 9. The results are shown in Fig. 8. From the results, the involvement of CXCR4 in the intracellular uptake of SDF1αN21 was shown.

[0110] Test Example 11 Delivery of Cre Recombinase HeLa cells (150,000 cells) were seeded in a 35 mm glass-bottom dish and allowed to adhere overnight. The LoxP-DsRed-LoxP-EGFP-N1 plasmid was transfected using Lipofectamine LTX with Plus reagent according to the manufacturer's protocol. After 24 hours, the cells were washed and incubated for 1 hour with 3 μM peptide or (40 μM L17E) together with 5 μM Cre recombinase (in αMEM(-)). Non-internalized proteins were washed with PBS, fresh culture medium (αMEM(+)) was added, and the cells were incubated for an additional 24 hours. Observation was carried out using an FV1000 CLSM system. The assay scheme is shown in Fig. 9-1. The results are shown in Fig. 9-2. As shown in Fig. 9-2, the expression of EGFP was <15% in the presence of SDF1αN21 and LK15. In contrast, in the presence of L17E and SDF1αN21-LK15, they were 40% and 60% respectively, indicating that SDF1αN21-LK15 is useful for the delivery of Cre recombinase.

[0111] Test Example 12 Delivery of dHax3 (TALE)-VP64 - 1

[0112] (1) Construction of the dHax3-TBS-luciferase plasmid To construct a TBS-dHax3 plasmid (TALE reporter plasmid) with Puro, a DNA fragment encoding puromycin, the EF1 promoter, and an insulator sequence was amplified from GFP-Puro (#HR700PA-1, SBI system) using the following primer pair; 5’- GGT AAA ATC GAT AAG CCG CGT ACG CGT GTG AAG ACT C -3’ (SEQ ID NO: 66) / 5’- TCG CCA CCA TGA CCG AGT ACA AGC CCA CGG-3’ (SEQ ID NO: 67); 5’-CGG TCA TGG TGG CGA ATT CGT AGG CGC C-3’ (SEQ ID NO: 68) / 5’-GGG CAT CGG TCG ACG CGA CGG CCA GTG AAA CTA GTG GT-3’ (SEQ ID NO: 69). These two fragments were assembled with the TALE luciferase reporter vector digested with BamHI using the Gibson assembly System.

[0113] (2) Establishment of stable expression strains HeLa cells were transfected with the above plasmid, and cells surviving puromycin were selected.

[0114] (3) Preparation of TALE protein To construct the E. coli expression vector dHax3-VP64-pET42b(+) plasmid, the DNA fragment encoding the VP64 sequence was digested from dHax3-VP64_pCMV using XmaI and XbaI, and the DNA fragment was inserted into His-dHax3-pET42b(+).

[0115] HeLa-dHax3 reporter cells were incubated with the indicated concentration of peptide and 1 μM dHax3-VP64 in αMEM(-) for 1 hour, then the medium was changed to fresh αMEM(+), and further incubated for 6 hours to measure luciferase activity. The assay scheme is shown in Figure 10-1. The results are shown in Figure 10-2. The results showed that SDF1αN21-LK15 is useful for the delivery of dHax3 (TALE)-VP64 (artificial transcription factor protein).

[0116] Test Example 13 70 kDa Dextran Uptake Assay - 3 Using SDF1αN21 and OxN21, tests were conducted in the same manner as in Test Example 3. The results are shown in Figure 11. As shown in Fig. 11, the amount of 70 kDa dextran uptake by OxN21 was higher than that by SDF1αN21.

[0117] Test Example 14 70 kDa Dextran Uptake Assay - 4 Using OxN21, the test was conducted in the same manner as in Test Example 4. The results are shown in Fig. 12. As shown in Fig. 12, OxN21 showed an effect equal to or better than that of known inducers.

[0118] Test Example 15 Delivery of Luciferase siRNA - 2 Using SDF1αN21-LK15 (designated as N21-LK15 in the figure), OxN21-LK15, and SDF1αN21-Melittin (designated as N21-Mel in the figure), the test was conducted in the same manner as in Test Example 5. The results are shown in Fig. 13. As shown in Fig. 13, SDF1αN21-LK15, OxN21-LK15, and SDF1αN21-Melittin showed excellent siRNA delivery effects.

[0119] Test Example 16 IgG-Alexa488 Delivery - 2 Using OxN21 and OxN21-LK15, the test was conducted in the same manner as in Test Example 8. The results are shown in Fig. 14. As shown in Fig. 14, no significant fluorescence signal was observed for OxN21, whereas a fluorescence signal spreading throughout the cells was observed for OxN21-LK15, indicating that OxN21-LK15 is useful for IgG delivery.

[0120] Test Example 17 70 kDa Dextran Uptake Assay - 5 Using SN8, SN17, SN21, SN23, SN25, and SN27, the test was conducted in the same manner as in Test Example 3. The results are shown in Fig. 15.

[0121] Test Example 18 70 kDa Dextran Uptake Assay - 6 (Alanine Scanning) Using K1A, V3A, L5A, Y7A, R8A, R12A, F13A, F14A, V18A, R20A in which the hydrophobic amino acids or basic amino acids in SN21 were replaced with alanine, tests were conducted in the same manner as in Test Example 3. The results are shown in Fig. 16. As shown in Fig. 16, the uptake effects of V18A and R20A were significantly improved compared to SN21.

[0122] Test Example 19 IgG-Alexa488 Delivery - 3 Using SN23, SN25, SN21-LK15, SN23-LK15, and SN25-LK15, tests were conducted in the same manner as in Test Example 8. The results are shown in Fig. 17. As shown in Fig. 17, no significant fluorescence signal was observed for SN23 and SN25, while fluorescence signals spreading throughout the cells were observed for SN21-LK15, SN23-LK15, and SN25-LK15, indicating that they are useful for IgG delivery. The spread of the fluorescence signal was SN21-LK15 = SN23-LK15 > SN25-LK15.

[0123] Test Example 20 Delivery of dHax3(TALE)-VP64 - 2

[0124] An assay for the delivery of dHax3(TALE)-VP64 was conducted in the same manner as in Test Example 12. The results are shown in Fig. 18.

[0125] Test Example 21 Delivery of Luciferase siRNA - 3 Tests were conducted in the same manner as in Test Example 5. The results are shown in Fig. 19. As shown in Fig. 19, SN21-LK15, SN23-LK15, and SN25-LK15 showed excellent siRNA delivery effects.

[0126] In previous studies, the present inventors have clarified that the SDF1αN-terminal-derived peptide SDF1αN21 activates endocytosis (macropinocytosis and clathrin-related endocytosis). In recent years, it has been found that the lipids of the cell membrane (the membrane on the cell surface) are almost neutral, while the endosome membrane is rich in acidic lipids such as Bis(monoacylglycero)phosphate (BMP). Since membrane-damaging peptides such as LK15 and melittin have a positive charge, it is expected that due to their electrostatic interaction, they will show higher damage to the endosome membrane rich in acidic lipids compared to the cell membrane rich in neutral lipids. The peptide complex of the present disclosure enhances the uptake amount of the target substance by inducing endocytosis (especially macropinocytosis, which is known to enable large-capacity uptake) by the SDF1αN-terminal-derived segment, and further efficiently releases the target substance due to the electrostatic interaction between the membrane-damaging segment and the endosome membrane. It is presumed that the high intracellular substance-introducing ability of the peptide complex is exhibited. Note that the speculation of this mechanism does not limit the present invention in any way.

[0127] Test Example 22 Cell Viability Assay (WST-8 Assay) The cell viability assay was performed using Cell Counting Kit-8 (Dojinjo). Briefly, cells were seeded at a density of 5,000 cells / well in a 96-well plate and cultured for 48 hours. After washing the cells with α-MEM(-), the cells were treated with 10 μM peptide (in α-MEM(+)) at 37 °C for 24 hours. The cells were washed twice with α-MEM(-). Then, 100 μL of α-MEM(-) and 10 μL of WST-8 reagent were added to each well. The plate was then incubated for an additional 2 hours. The absorbance at 450 nm was measured to analyze the cell viability. The results are shown in the table below.

Table 4

[0128] Test Example 23 70 kDa Dextran Uptake Assay-7 Using SN21, sSN21, sP4A, and sP4G, tests were conducted in the same manner as in Test Example 3. The results of this assay are shown in Figure 20. The vertical axis in Figure 20 indicates the relative dextran uptake (%). As shown in Figure 20, the uptake effects of sP4A and sP4G were significantly improved compared to SN21 and sSN21.

[0129] Test Example 24 Delivery of AlexaFluor594-labeled anti-nuclear pore complex IgG HeLa cells (200,000 cells / dish) were seeded in 35-mm glass-bottom dishes (Iwaki) and incubated overnight. The obtained cells were washed three times with PBS(-), and then incubated for 60 minutes in αMEM(-) with 2 μM SN21-LK15, 5 μM SN21, or 25 μM LK15, and 50 μg / mL AlexaFluor594-labeled anti-nuclear pore complex IgG (anti-NPC IgG(Alexa594), Biolegends). After incubation, the cells were washed three times with PBS(-). Nuclear staining was performed by using 10 μg / mL Hoechst 33342 in αMEM(-) for 10 minutes and then washing with PBS(-). The cells were visualized using an FV3000 confocal laser scanning microscope system. The results are shown in Figure 21. In cells treated with anti-NPC IgG(Alexa594) in the presence of SN21-LK15, a significant signal of anti-NPC IgG(Alexa594) was observed inside the cells, confirming intracellular introduction by SN21-LK15. Furthermore, accumulation of anti-nuclear pore complex IgG (staining the cytoplasmic side of the nuclear envelope in the right figure (2)) was observed around the nucleus stained with Hoechst 33342 (right figure (1)), indicating that the introduced antibody was correctly bound to the nuclear pore complex inside the cell. On the other hand, no signal of anti-NPC IgG(Alexa594) was observed inside the cells in the presence of SN21 and LK15.

[0130] Test Example 25 Intracellular introduction of Cas9 / RNP Lipofectamine (TM) CRISPRMAX (TM) (Thermo Fisher Scientific)'s recommended protocol for RNP (Cas9 (TrueCut (TM) Cas9 Protein v2, Thermo Fisher Scientific) / RNA (TrueGuide (TM) sgRNA Positive Control, HPRT1 (human), Thermo Fisher Scientific) complex) treatment resulted in knockout of the HPRT gene with a gene modification efficiency of 57%. Knockout occurred with a gene modification efficiency of 26% when the same amount of RNP and N21-LK15 (3 μM) as above were administered to the cells. It was confirmed that N21-LK15 can also be used in methods using the CRISPR-Cas9 system. (N21-LK15 means "SDF1αN-LK15".)

[0131] Test Example 26 Delivery of KRas siRNA HeLa cells were seeded in a 12-well plate (2×10 5 cells / well) and allowed to adhere overnight. The cells were transfected with KRAS Silencer (商標) siRNA (Ambion) using Lipofectame 2000 (LF2000) or SN21-LK15 (charge ratio 5). After 6 hours, the transfection medium was removed, fresh α-MEM(+) was added, and the cells were incubated for an additional 24 hours. The transfected cells were trypsinized and seeded in a 96-well plate (5,000 cells / well) for cell viability or (1.5×10 5 cells / well), or in a 12-well plate for Western blot. Regarding the cell viability assay, the cells seeded in a 96-well plate were incubated to allow cell attachment. After 3 hours, WST-8 reagent (Dojindo) was added to the designated wells, and the cells were incubated for an additional 2 hours, followed by measuring the absorbance at 450 nm. This process was repeated at 24 hours, 48 hours, and 72 hours later. Western blot was performed to confirm the protein expression of KRAS after siRNA transfection. The transfected cells were washed three times with ice-cold PBS and lysed using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, pH 7.6) in combination with 1×cOmplete (商標) Protease Inhibitor Cocktail (Roche). The cells were scraped and collected, transferred to a pre-cooled 1.5 mL microcentrifuge tube, incubated on ice for 15 minutes, and centrifuged (16,000×g, 20 minutes, 4°C). The clarified supernatant was transferred to a new pre-cooled microcentrifuge tube. The total protein concentration was determined using the Pierce BCA assay. SDS-PAGE loading buffer was added to the samples, boiled for 5 minutes, and loaded onto a 5-20% SuperSep (商標) Ace precast SDS-PAGE gel (FIJIFILM Wako) (8 μg / well). Subsequently, the gel was electrophoresed (25 mA, 60 minutes, 25°C), and Transblot (商標) Turbo (商標)Blotted onto a Mini PVDF membrane (BioRad). Blocking was performed with 5% non-fat milk (in Tris-buffered saline containing 0.1% tween 20 (TBST, pH 7.6)) at 25°C for 1 hour. The blocked membrane was incubated with the diluted primary antibody with constant stirring (4°C, overnight). The primary antibodies used were as follows: Anti-KRAS antibody (1:2000, abcam); β-actin: Monoclonal anti-β-actin mouse IgG, Clone AC-74 (1:10000, Sigma). The membrane was washed 3 times with TBST (10 minutes for each wash), then incubated with secondary antibodies [Anti-rabbit IgG, HRP-labeled antibody (1:2000, Cell Signaling Technologies); Amersham ECL anti-mouse IgG, horseradish peroxidase-labeled species-specific Whole antibody (1:5000, GE Healthcare)] (25°C, 1 hour), washed again in rounds with TBST, detected using ECL Prime Western Blotting Detection reagent (Amersham), and visualized with a LAS3000 CCD camera (GE Lifescience). After detecting KRAS, the membrane was stripped using Restore (商標) PLUS Western Blot Stripping buffer and re-detected for actin. The results are shown in Figure 23. LF2000 is a commercially available reagent with very high siRNA transfection. On the other hand, LF2000 consisting of cationic lipids is known to be difficult to use in vivo due to non-specific adsorption and aggregation to in vivo tissues. Western blot analysis revealed that transfection of KRAS Silencer siRNA using SN21-LK15 showed a decrease in intracellular KRAS protein similar to that when using LF2000 (A). Also, SN21-LK15 (C) obtained a cell growth inhibitory effect by KRAS Silencer siRNA similar to that when using LF2000 (B).

[0132] Test Example 27 Delivery of Nuclear Localization Signal (EGFP-NLS) -Fused Enhanced Green Fluorescent Protein HeLa cells (2×10 5 cells) were seeded in a 35 mm glass well plate and incubated overnight. The cells were washed three times with PBS and incubated for 1 hour with 40 μM EGFP-NLS in the presence of 40 μM L17E or 5 μM P4A-LK15. After incubation, the cells were washed three times and incubated with Hoechst 33342. Observation was performed using an Olympus FV3000 confocal laser microscope (60×). The results are shown in Figure 24. HeLa cells incubated with EGFP-NLS and 40 μM L17E or 5 μM P4A-LK15 showed nuclear co-localization of EGFP and Hoescht 33342 signals.

[0133] Test Example 28 Delivery of TALE-VP64 HeLa-TBS-Luci(1×10 4Cells were seeded in a 96-well assay plate (Nunc) partitioned by white walls and allowed to adhere for 48 hours in the presence of antibiotics. Before performing the assay, ultrafiltration was carried out using Amicon Ultra-0.5 mL (30,000 MWCO, Merck), and the buffer of purified dHax3-VP64 was exchanged with PBS. The concentration was measured using NanoDrop. The cells were washed with PBS, and 1 μM dHax3-VP64 was added in α-MEM(-) (50 μL) in the presence of 2 μM SN21-LK15, 5 μM P4A, or 5 μM P4A-LK15. The untreated set received only α-MEM(-) (50 μL), and the peptide-free set received only 1 μM TALE-VP64 (in α-MEM(-) (50 μL)). The cells were incubated for 1 hour, then the medium was replaced with 50 μL α-MEM(+) (50 μL) and incubated for an additional 8 hours. An equal volume of Steady-Glo Luciferase assay reagent (Promega) was added to each well, and luminescence was measured using a GloMax-Multi Detection System (Promega) to measure luciferase activity. The results are shown in Fig. 25. Luciferase expression (expressed as luminescence) was enhanced in HeLa-TBS-Luci cells incubated for 1 hour with TALE-VP64 and 2 μM SN21-LK15, 5 μM sP4A (designated as P4A in the figure), or 5 μM P4A-LK15.

Industrial Applicability

[0134] The technology of the present disclosure is used, for example, in the following applications, but is not limited thereto: 1. Protein / biologically active substance introduction reagents and introduction kits for living cells 2. Basic research in the fields of molecular cell biology and medicine (intracellular visualization, measurement, interaction analysis, cell activity control, etc.) 3. Basic research for intracellular activity evaluation of antibody pharmaceuticals, biopharmaceuticals, etc. in the field of drug discovery (biopharmaceutical design support methods) 4. Ex vivo and in vivo intracellular delivery of antibody drugs, nucleic acid drugs, and biopharmaceuticals

Claims

1. A peptide complex comprising segment X and a membrane-damaging segment Z, wherein segment X has an amino acid sequence selected from SEQ ID NOs: 5 to 27, and the membrane-damaging segment Z has an amino acid sequence selected from SEQ ID NOs: 71 to 72, peptide complex.

2. The peptide complex according to claim 1, wherein the C-terminus of segment X and the terminus of segment Z are conjugated directly or via a linker.

3. The peptide complex according to claim 2, Formula (I): R １ -U-X-J １ -Z-R ２ (I) [wherein, X represents segment X, Z represents segment Z, J １ represents a divalent linker, The C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J １ and R １ is a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U is absent or an amino acid sequence consisting of 1 to 7 amino acids, R ２ is a hydroxyl group (OH), an amino group (NH ２ ), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group] peptide complex represented by.

4. The peptide complex according to claim 2, Formula (Ia): R １ -(U - X - J １ - Z - J ２ ) ｍ１ - R ２ (Ia) [wherein, X each independently represents segment X, Z each independently represents segment Z, J １ each independently represents a divalent linker, The C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J １ and R １ each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U each independently is absent or an amino acid sequence consisting of 1 to 7 amino acids, R ２ is, independently of each other, a hydroxyl group (OH), an amino group (NH ２ ), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group, J ２ each independently is a single bond or a divalent linker, the integer m1 each independently ranges from 2 to 10] peptide complex represented by.

5. The peptide complex according to claim 2, the following formula (Ib): J ３ (-R ａ ) ｍ２ (Ib) [wherein, J ３ is a branched polyvalent linker, R ａ is, independently of one another, R １ -U-X-J １ -Z-J ２ -or -J ２ -U-X-J １ -Z-R ２ (where J ２ is each independently J ３ bonded to), X each independently represents segment X, Z each independently represents segment Z, J １ each independently represents a divalent linker, The C-terminus of the segment X and the N-terminus of the segment Z are conjugated via J １ and R １ each independently represents a hydrogen atom, an alkyl group, an acyl group, an alkoxycarbonyl group, an aralkyloxycarbonyl group, an aryloxycarbonyl group, an alkoxy group, an aralkyloxy group, or an aryloxy group, U each independently is absent or an amino acid sequence consisting of 1 to 7 amino acids, R ２ is, independently of one another, a hydroxyl group (OH), an amino group (NH ２ ), a monoalkylamino group, a monoarylamino group, a monocycloalkylamino group, a dialkylamino group, an alkoxy group, an aralkyloxy group, or an aryloxy group, and J ２ each independently is a single bond or a divalent linker, the integer m2 ranges from 2 to 8] peptide complex represented by.

6. The peptide complex according to any one of claims 1 to 5, wherein segment X contains a disulfide bond formed by two cysteines.

7. The peptide complex according to any one of claims 1 to 6, conjugated with a molecule that enhances affinity for target cells.

8. A target substance-peptide complex comprising the peptide complex according to any one of claims 1 to 7 and a target substance conjugated to the peptide complex.

9. A cell-introducing agent comprising the peptide complex according to any one of claims 1 to 7.

10. The cell-introducing agent according to claim 9, further comprising a target substance.

11. A cell-introducing agent comprising the target substance-peptide complex according to claim 8.

12. A peptide having an amino acid sequence selected from SEQ ID NOs: 23 to 27.

13. An object substance-peptide complex comprising the peptide according to claim 12 and an object substance conjugated to the peptide.

Images