Cyclic peptide or salt thereof, and use thereof

Abstract

The present invention addresses the problem of providing a cyclic peptide, a salt thereof, and a cyclic peptide complex or a salt thereof, which have excellent bonding properties with an FGFR protein and excellent stability, and of providing a culture medium composition, a purification material, a labeling material, a cell control material, and an integration material using the cyclic peptide, the salt thereof, and the cyclic peptide complex or the salt thereof. According to the present invention, provided is a cyclic peptide or a salt thereof, which comprises an amino acid sequence represented by X1-X2-X3-X4 and comprises a cyclization part that is cyclized by a covalent bond, the cyclization part comprising a structure represented by formula (2), x1 represents an alanine residue, a leucine residue, an isoleucine residue, a valine residue, a phenylalanine residue, a tyrosine residue, or a threonine residue, X2 represents an amino acid residue having an aromatic residue in a side chain, X3 represents an arbitrary amino acid residue, and X3 represents an arbitrary amino acid residue. X4 represents an alanine residue, a glycine residue, a phenylalanine residue, a tyrosine residue, a tryptophan residue, a serine residue or a histidine residue, Z1 and Z2 each independently represent a linking group, and m represents an integer from 1 to 10.

Description

Technical Field

[0001] This invention relates to a cyclic peptide or its salt that exhibits excellent binding and stability to FGFR (Fibroblast Growth Factor Receptor) proteins. The invention also relates to a cyclic peptide complex or its salt formed by linking two or more molecules of the above-mentioned cyclic peptide or its salt via linkers. Furthermore, the invention relates to the utilization of the above-mentioned cyclic peptide or its salt, and the cyclic peptide complex or its salt. Background Technology

[0002] As a treatment for diseases that cannot be treated with traditional low-molecular-weight and antibody drugs, cell therapy products are being developed that introduce cells with healing capabilities into the body. Furthermore, in recent years, as a measure to address the food crisis and greenhouse gases, research and development are underway toward the practical application of cell products such as cultured meat (cell agriculture) or cells used to support drug development. In the research and manufacturing of these cell therapy products and cell products, it is crucial to enable cells to proliferate effectively while maintaining qualities such as undifferentiated capacity. Among the components of the culture medium used for cell proliferation, the proteome of proteomes called growth factors (also known as cytokines) is a crucial component that significantly influences quality and cell proliferation rate. However, growth factors are generally very expensive and have low stability, contributing to the high manufacturing cost of cell therapy products and cell products. In particular, the production of cultured meat is costly and difficult to commercialize; a significant cost factor is the need for large quantities of specialized cell culture media, especially suitable growth factors that tend to be the most expensive component of cell culture media. Currently, the majority (up to 96%) of production costs are influenced by the cost of growth factors, making production economically unfeasible.

[0003] Among growth factors, fibroblast growth factor (FGF) is known to play a crucial role in maintaining the undifferentiated capacity of cells and promoting cell proliferation across a wide range of applications. Basic fibroblast growth factor (bFGF) is widely used for the proliferation of mesenchymal stem cells, which constitute the majority of cell therapy products, and is also a critical component for maintaining the undifferentiated capacity and promoting the proliferation of induced pluripotent stem cells (iPSCs) and adult stem cells.

[0004] bFGF proliferation is promoted, for example, by the binding of the bFGF FGFR protein to its extracellular region. Therefore, substances that bind to the extracellular region of the FGFR protein have long been investigated in order to reduce the cost of bFGF and improve its stability.

[0005] Patent Document 1 describes a disulfide-type cyclic peptide that bonds to FGFR proteins. Patent Document 2 describes a thioether-type cyclic peptide as a substance that balances bonding and stability in proteins other than FGFR.

[0006] Existing technical documents

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. WO2000 / 003245

[0009] Patent Document 2: Japanese Patent Application Publication No. 2017-95443 Summary of the Invention

[0010] The technical problem to be solved by the invention

[0011] The peptide described in Patent Document 1 exhibits significantly low binding affinity to FGFR proteins, rendering it impractical. Furthermore, the disulfide bonds contained in disulfide-bonded cyclic peptides are known to be unstable and easily decomposed under cell culture conditions. Moreover, combining the FGFR-bound amino sequence of Patent Document 1 with the stable thioether bond of Patent Document 2 results in significantly low binding affinity to FGFR proteins, rendering it impractical.

[0012] The present invention aims to provide a cyclic peptide or its salt, and a cyclic peptide complex or its salt, that exhibit excellent binding affinity and stability to FGFR proteins. A further objective of the present invention is to provide a culture medium composition, culture medium additive, purification material, labeling material, cell control material, and integration material utilizing the aforementioned cyclic peptide or its salt, and the cyclic peptide complex or its salt.

[0013] means for solving technical problems

[0014] The inventors, through in-depth research to solve the aforementioned problems, discovered that cyclic peptides with specific amino acid residues at specific positions in the amino acid sequence exhibit excellent binding affinity and stability to FGFR proteins. This invention is based on the above insights. According to this invention, the following invention is provided.

[0015] <1> A cyclic peptide or a salt thereof, comprising an amino acid sequence represented by X1-X2-X3-X4, and comprising a cyclized portion cyclized by covalent bonds, wherein the cyclized portion comprises a structure represented by formula (2).

[0016] [Chemical Formula 1]

[0017]

[0018] In the formula,

[0019] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0020] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0021] X3 represents any amino acid residue.

[0022] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0023] Z1 and Z2 represent the linking groups independently.

[0024] m represents an integer from 1 to 10.

[0025] <2> The cyclic peptide or its salt as described in <1> is represented by formula (1).

[0026] [Chemical Formula 2]

[0027]

[0028] In the formula,

[0029] V represents -NH-Y4 or R,

[0030] When V is -NH-Y4, L0 represents L; when V represents R, L0 represents L1.

[0031] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0032] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0033] X3 represents any amino acid residue.

[0034] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0035] Y1 represents a single bond or an amino acid residue or peptide residue containing 1 to 13 arbitrary amino acid residues.

[0036] Y3 represents an amino acid residue or peptide residue containing 1 to 14 arbitrary amino acid residues.

[0037] Y4 represents a hydrogen atom, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0038] Y5 represents OH, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0039] The amino terminus of X1 is bonded to the carboxyl terminus of Y1, and the carboxyl terminus of X1 is bonded to the amino terminus of X2.

[0040] L indicates that...

[0041] [Chemical Formula 3]

[0042]

[0043] or

[0044] [Chemical Formula 4]

[0045]

[0046] The cyclization part is indicated by Z11 and Z12, which independently represent linking groups, m represents an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0047] L1 indicates that by

[0048] [Chemical Formula 5]

[0049]

[0050] The cyclization part is indicated by Z12, the linking group is indicated by m, an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0051] R represents the side chain structure of an amino acid.

[0052] <3> The cyclic peptide or its salt according to <1> is represented by formula (1A1) or formula (1A2).

[0053] [Chemical Formula 6]

[0054]

[0055] In the formula,

[0056] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0057] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0058] X3 represents any amino acid residue.

[0059] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0060] Y1 represents a single bond or an amino acid residue or peptide residue containing 1 to 13 arbitrary amino acid residues.

[0061] Y3 represents an amino acid residue or peptide residue containing 1 to 14 arbitrary amino acid residues.

[0062] Y4 represents a hydrogen atom, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0063] Y5 represents OH, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0064] The amino terminus of X1 is bonded to the carboxyl terminus of Y1, and the carboxyl terminus of X1 is bonded to the amino terminus of X2.

[0065] L indicates that...

[0066] [Chemical Formula 7]

[0067]

[0068] or

[0069] [Chemical Formula 8]

[0070]

[0071] The cyclization part is indicated by Z11 and Z12, which independently represent linking groups, m represents an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0072] L1 indicates that by

[0073] [Chemical Formula 9]

[0074]

[0075] The cyclization part is indicated by Z12, the linking group is indicated by m, an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0076] R represents the side chain structure of an amino acid.

[0077] <4> The cyclic peptide or its salt according to <1> comprises an amino acid sequence represented by X1-X2-X3-X4-X5.

[0078] In the formula,

[0079] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0080] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0081] X3 represents any amino acid residue.

[0082] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0083] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

[0084] <5> The cyclic peptide or its salt according to <1> comprises an amino acid sequence represented by X1-X2-X3-X4-X5.

[0085] In the formula,

[0086] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0087] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0088] X3 represents any amino acid residue.

[0089] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0090] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0091] <6> The cyclic peptide or its salt according to <1> comprises an amino acid sequence represented by X1-X2-X3-X4-X5-Xn-X6.

[0092] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0093] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0094] X3 represents any amino acid residue.

[0095] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0096] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

[0097] Xn represents a peptide residue containing 1 to 3 arbitrary amino acid residues.

[0098] X6 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

[0099] <7> The cyclic peptide or its salt according to <1> comprises an amino acid sequence represented by X1-X2-X3-X4-X5-Xn-X6.

[0100] In the formula,

[0101] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0102] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0103] X3 represents any amino acid residue.

[0104] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0105] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0106] Xn represents a peptide residue containing 1 to 3 arbitrary amino acid residues.

[0107] X6 represents an alanine residue, glycine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or histidine residue.

[0108] <8> The cyclic peptide or its salt according to <1>, wherein,

[0109] The number of amino acid residues that make up the ring is 10 to 22.

[0110] <9> The cyclic peptide or its salt according to <1>, wherein,

[0111] The cyclized portion contains homocysteine ​​residues.

[0112] <10> The cyclic peptide or its salt according to <1> comprises any one of the amino acid sequences of sequence numbers 38-335 and 337-342 listed in Tables 4-6, or

[0113] The amino acid sequence containing 1 to 4 amino acids that have been substituted, deleted or inserted in any of the amino acid sequences of sequence numbers 38 to 335 and sequence numbers 337 to 342 listed in Tables 4 to 6, and which has the ability to bind to FGFR proteins.

[0114] <11> The cyclic peptide or its salt according to <1> comprises the sequences 38-54, 56-57, 63-64, 69-70, 76-77, 87, 89-90, 97-98, 100-105, 108-109, 111-117, 124, 131-136, 140-147, 155-159, 162, 165-168, 171-172, 175-181, and 183 listed in Tables 4-6. Any one of the amino acid sequences of serial numbers 185–192, 199–206, 219–220, 223–225, 227, 230, 232, 235–237, 239–240, 242, 244–245, 250, 252–253, 256, 258, 265–267, 269, 274–275, 278, 280, 283, 290–335, and 340, or

[0115] Includes the serial numbers 38-54, 56-57, 63-64, 69-70, 76-77, 87, 89-90, 97-98, 100-105, 108-109, 111-117, 124, 131-136, 140-147, 155-159, 162, 165-168, 171-172, 175-181, 183, 185-192, 199-206, and 219 recorded in Tables 4-6. The amino acid sequence of sequence number 220, 223-225, 227, 230, 232, 235-237, 239-240, 242, 244-245, 250, 252-253, 256, 258, 265-267, 269, 274-275, 278, 280, 283, 290-335, and 340 contains 1-4 amino acid substitutions, deletions, or insertions, and has the ability to bind to FGFR proteins.

[0116] <12> The cyclic peptide or its salt according to <1> comprises the sequence numbers 38-45, 47-54, 57, 63, 70, 87, 97-98, 100-101, 103-105, 111-117, 131-134, 136, 140-146, 155-159, 162, 165-168, 176-178, 185-190, and 192 as listed in Tables 4-6. Any one of the amino acid sequences of serial numbers 199–206, 219–220, 223–224, 227, 230, 232, 235–237, 239–240, 244–245, 252, 256, 258, 266–267, 269, 274–275, 278, 280, 290–325, 327–328, 330, and 332–335, or

[0117] Includes the serial numbers 38-45, 47-54, 57, 63, 70, 87, 97-98, 100-101, 103-105, 111-117, 131-134, 136, 140-146, 155-159, 162, 165-168, 176-178, 185-190, 192, 199-206, 219-220, and 223-206 recorded in Tables 4-6. 24. An amino acid sequence in any one of the following sequences: 227, 230, 232, 235–237, 239–240, 244–245, 252, 256, 258, 266–267, 269, 274–275, 278, 280, 290–325, 327–328, 330, and 332–335, wherein 1–4 amino acids are substituted, deleted, or inserted, and the sequence possesses the ability to bind to FGFR proteins.

[0118] <13> The cyclic peptide or its salt as described in <1> is modified by other substances.

[0119] <14> A cyclic peptide complex or a salt thereof, which is formed by linking two or more molecules of any one of <1> to <13> cyclic peptides or salts thereof by linkers.

[0120] <15> A cyclic peptide complex or a salt thereof, which is formed by linking two molecules of any one of <1> to <13> cyclic peptides or salts thereof by a linker.

[0121] <16> The cyclic peptide complex or its salt as described in <14> is modified by other substances.

[0122] <17> A culture medium composition or culture medium additive comprising any one of <1> to <13> of a cyclic peptide or a salt thereof.

[0123] <18> A purification material comprising any one of <1> to <13> a cyclic peptide or a salt thereof.

[0124] <19> A labeling material comprising any one of <1> to <13> a cyclic peptide or a salt thereof.

[0125] <20> A material for cell control comprising any one of <1> to <13> a cyclic peptide or a salt thereof.

[0126] <21> An integrated material comprising any one of <1> to <13> a cyclic peptide or a salt thereof.

[0127] <22> A culture medium composition or culture medium additive comprising the cyclic peptide complex described in <14> or a salt thereof.

[0128] <23> A purification material comprising the cyclic peptide complex described in <14> or a salt thereof.

[0129] <24> A labeling material comprising the cyclic peptide complex described in <14> or a salt thereof.

[0130] <25> A material for cell control comprising the cyclic peptide complex described in <14> or a salt thereof.

[0131] <26> An integrated material comprising the cyclic peptide complex described in <14> or a salt thereof.

[0132] Invention Effects

[0133] The cyclic peptides or their salts of the present invention exhibit excellent binding affinity and stability to FGFR proteins. Attached Figure Description

[0134] Figure 1 The structures of the compounds used in the examples are shown.

[0135] Figure 2 This indicates the cell proliferation-promoting effect of the cyclic peptide complex (serial number 345).

[0136] Figure 3 This indicates the stem cell proliferation-promoting effects of the cyclic peptide complex (serial number 346) and bFGF (basic fibroblast growth factor).

[0137] Figure 4 This indicates the role of the cyclic peptide complex (serial number 346) and bFGF (basic fibroblast growth factor) in maintaining the undifferentiated capacity of stem cells.

[0138] Figure 5 This indicates the bovine cell proliferation activity of the cyclic peptide complex (serial number 346). Detailed Implementation

[0139] The present invention will now be described in detail. These descriptions and embodiments are illustrative and do not limit the scope of the embodiments. The mechanisms of action described in the present invention include speculation, and their correctness does not limit the scope of the embodiments.

[0140] In this invention, the term "process" or the term representing a process includes not only processes that are independent of other processes, but also processes that can achieve their purpose, even if they cannot be clearly distinguished from other processes.

[0141] In this invention, the numerical range represented by “~” indicates the range included by taking the values ​​recorded before and after “~” as the minimum and maximum values, respectively.

[0142] In the numerical ranges described in stages in this invention, the upper or lower limit value described in one numerical range can be replaced with the upper or lower limit value of other numerical ranges described in stages. Furthermore, the upper or lower limit value of the numerical range described in this invention can also be replaced with the value shown in the embodiments.

[0143] <Amino acids, amino acid residues, and peptides>

[0144] Amino acids are, in principle, named using names and abbreviations adopted by the Joint Commission on Biochemical Nomenclature (IUPAC-IUB) of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology (JCBN). Furthermore, amino acid residues are represented using the abbreviations of the amino acids derived from them. Additionally, amino acid residues can be N-terminal amino acids (N-terminal residues) or C-terminal amino acids (C-terminal residues).

[0145] Unless otherwise specified, the amino acid sequence (also known as the "primary structure") of a peptide or protein is represented by arranging amino acid residues from left to right in a single column from the N-terminus to the C-terminus.

[0146] Examples of amino acids include α-amino acids, β-amino acids, γ-amino acids, and amino acids in which the amino or carboxyl groups are similarly substituted with reactive groups (e.g., substituted by a secondary or tertiary amine of a primary amine or substituted by an ester of a carboxyl group), but are not particularly limited thereto. An α-amino acid is a molecule containing both an amino group and a carboxyl group bonded to a carbon atom designated as α-carbon. A β-amino acid is a molecule containing both an amino group and a carboxyl group in a β configuration. A γ-amino acid is a molecule containing both an amino group and a carboxyl group in a γ configuration. α-amino acids are preferred as amino acids.

[0147] As an amino acid, it can be any of the natural or non-natural amino acids, and can be either a D- or L-isomer. When an amino acid is represented by its name, and enantiomers (L- and D-isoforms) exist, the L-isoform is generally used unless explicitly stated otherwise. For example, "isoleucine" represents "L-isoleucine," and the enantiomer of "isoleucine" is "D-isoleucine." The same applies to amino acid residues.

[0148] Table 1 lists the officially recognized one-character and three-character abbreviations of amino acids (one-character and three-character abbreviations).

[0149] [Table 1]

[0150]

[0151] Amino acids are not limited to those listed in Table 1; amino acids referred to as non-natural amino acids may also be used. Examples of non-natural amino acids are given in Table 2 below, but are not limited to these. Furthermore, amino acids can be N-alkyl amino acids in which the hydrogen atom on the N-terminal amino group is replaced by an alkyl group (methyl, propyl, etc.). As an example of an N-alkyl amino acid, N-methylleucine (hereinafter referred to as...) can be given. me L).

[0152] [Table 2]

[0153]

[0154] Examples of amino acids containing aromatic residues include phenylalanine, tryptophan, tyrosine, and histidine, as well as non-natural amino acids containing a benzene ring, imidazole ring, or pyridine ring in their side chain structure, with phenylalanine, tryptophan, tyrosine, and histidine being preferred.

[0155] Methionine, cysteine, and lysine can be used as amino acids, but from the viewpoint of oxidation resistance, it is preferable not to use methionine and cysteine. From the viewpoint of peptide chemical synthesis cost, it is preferable not to include cysteine ​​and lysine.

[0156] Peptides are generally structures consisting of 3 to 100 amino acids, but they can also contain structures other than amino acids at their ends and / or inside. All substances containing 3 to 100 amino acids are peptides.

[0157] Cyclic peptides are peptides that contain a closed ring structure with three or more amino acid residues.

[0158] <Cyclic Peptide>

[0159] The cyclic peptide of the present invention comprises a peptide with an amino acid sequence represented by X1-X2-X3-X4 and includes a cyclized portion cyclized by covalent bonds, wherein the cyclized portion comprises a structure represented by formula (2).

[0160] [Chemical Formula 10]

[0161]

[0162] In the formula,

[0163] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0164] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0165] X3 represents any amino acid residue.

[0166] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0167] Z1 and Z2 represent the linking groups independently.

[0168] m represents an integer from 1 to 10.

[0169] Preferably, the cyclic peptide of the present invention is represented by formula (1).

[0170] [Chemical Formula 11]

[0171]

[0172] In the formula,

[0173] V represents -NH-Y4 or R,

[0174] When V is -NH-Y4, L0 represents L; when V represents R, L0 represents L1.

[0175] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0176] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0177] X3 represents any amino acid residue.

[0178] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0179] Y1 represents a single bond or an amino acid residue or peptide residue containing 1 to 13 arbitrary amino acid residues.

[0180] Y3 represents an amino acid residue or peptide residue containing 1 to 14 arbitrary amino acid residues.

[0181] Y4 represents a hydrogen atom, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0182] Y5 represents OH, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0183] The amino terminus of X1 is bonded to the carboxyl terminus of Y1, and the carboxyl terminus of X1 is bonded to the amino terminus of X2.

[0184] L indicates that...

[0185] [Chemical Formula 12]

[0186]

[0187] or

[0188] [Chemical Formula 13]

[0189]

[0190] The cyclization part is indicated by Z11 and Z12, which independently represent linking groups, m represents an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0191] L1 indicates that by

[0192] [Chemical Formula 14]

[0193]

[0194] The cyclization part is indicated by Z12, the linking group is indicated by m, an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0195] R represents the side chain structure of an amino acid.

[0196] Preferably, the cyclic peptide of the present invention is represented by formula (1A1) or formula (1A2).

[0197] [Chemical Formula 15]

[0198]

[0199] In the formula,

[0200] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0201] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0202] X3 represents any amino acid residue.

[0203] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0204] Y1 represents a single bond or an amino acid residue or peptide residue containing 1 to 13 arbitrary amino acid residues.

[0205] Y3 represents an amino acid residue or peptide residue containing 1 to 14 arbitrary amino acid residues.

[0206] Y4 represents a hydrogen atom, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0207] Y5 represents OH, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues.

[0208] The amino terminus of X1 is bonded to the carboxyl terminus of Y1, and the carboxyl terminus of X1 is bonded to the amino terminus of X2.

[0209] L indicates that...

[0210] [Chemical Formula 16]

[0211]

[0212] or

[0213] [Chemical Formula 17]

[0214]

[0215] The cyclization part is indicated by Z11 and Z12, which independently represent linking groups, m represents an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0216] L1 indicates that by

[0217] [Chemical Formula 18]

[0218]

[0219] The cyclization part is indicated by Z12, the linking group is indicated by m, an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone.

[0220] R represents the side chain structure of an amino acid.

[0221] In a more preferred embodiment, the cyclic peptide comprises an amino acid sequence represented by X1-X2-X3-X4-X5.

[0222] In the formula,

[0223] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0224] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0225] X3 represents any amino acid residue.

[0226] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0227] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

[0228] In a more preferred embodiment, the cyclic peptide comprises an amino acid sequence represented by X1-X2-X3-X4-X5.

[0229] In the formula,

[0230] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0231] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0232] X3 represents any amino acid residue.

[0233] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0234] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0235] In a further preferred embodiment, the cyclic peptide comprises an amino acid sequence represented by X1-X2-X3-X4-X5-Xn-X6.

[0236] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0237] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0238] X3 represents any amino acid residue.

[0239] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0240] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

[0241] Xn represents a peptide residue containing 1 to 3 arbitrary amino acid residues.

[0242] X6 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

[0243] In a further preferred embodiment, the cyclic peptide comprises an amino acid sequence represented by X1-X2-X3-X4-X5-Xn-X6.

[0244] In the formula,

[0245] X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue.

[0246] X2 represents an amino acid residue whose side chain contains an aromatic residue.

[0247] X3 represents any amino acid residue.

[0248] X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue.

[0249] X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0250] Xn represents a peptide residue containing 1 to 3 arbitrary amino acid residues.

[0251] X6 represents an alanine residue, glycine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or histidine residue.

[0252] X1 preferably represents an alanine residue, a leucine residue, an isoleucine residue, or a valine residue. X1 more preferably represents an isoleucine residue or a valine residue.

[0253] X2 preferably represents a phenylalanine residue, a tyrosine residue, a tryptophan residue, or a histidine residue. X2 more preferably represents a tyrosine residue or a histidine residue.

[0254] X3 preferably represents an alanine residue, glycine residue, leucine residue, isoleucine residue, valine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or histidine residue. X3 more preferably represents an alanine residue, leucine residue, glutamine residue, serine residue, or threonine residue. X3 is particularly preferably a serine residue or threonine residue.

[0255] X4 preferably represents a phenylalanine residue, a tyrosine residue, a tryptophan residue, or a histidine residue. X4 more preferably represents a phenylalanine residue, a tyrosine residue, or a tryptophan residue. X4 is particularly preferably a phenylalanine residue.

[0256] X5 preferably represents a glycine residue, leucine residue, isoleucine residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. X5 more preferably represents a glycine residue, isoleucine residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, arginine residue, or histidine residue. X5 is particularly preferably an asparagine residue or aspartic acid residue.

[0257] Xn preferably represents a peptide residue containing an amino acid residue represented by Xn1 or two amino acid residues represented by Xn1-Xn2. Xn more preferably represents a peptide residue containing two amino acid residues represented by Xn1-Xn2.

[0258] Xn1 preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, histidine residue, an amino acid residue containing a thiol group forming a cyclization portion, or an amino acid residue containing a halocarboxyl group forming a cyclization portion. Xn1 more preferably represents an alanine residue, glycine residue, proline residue, isoleucine residue, valine residue, tyrosine residue, tryptophan residue, glutamine residue, serine residue, glutamic acid residue, arginine residue, histidine residue, an amino acid residue containing a thiol group forming a cyclization portion, or an amino acid residue containing a halocarboxyl group forming a cyclization portion. Xn1 further preferably represents an alanine residue, proline residue, valine residue, tryptophan residue, glutamine residue, serine residue, glutamic acid residue, arginine residue, or histidine residue. Xn1 is particularly preferably represented by an alanine residue, a glutamine residue, a serine residue, a glutamic acid residue, or an arginine residue.

[0259] Xn2 preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, an amino acid residue containing a thiol group forming the cyclization part, or an amino acid residue containing a halogenated carboxyl group forming the cyclization part. Xn2 more preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, an amino acid residue containing a thiol group forming the cyclization part, or an amino acid residue containing a halogenated carboxyl group forming the cyclization part. Xn2 is further preferably represented by an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, an amino acid residue containing a thiol group forming a cyclization portion, or an amino acid residue containing a halocarboxyl group forming a cyclization portion. Xn2 is particularly preferably represented by an alanine residue, glycine residue, leucine residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0260] X6 preferably represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or lysine residue. X6 more preferably represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, serine residue, threonine residue, arginine residue, or lysine residue. X6 is particularly preferably alanine residue.

[0261] Y1 preferably represents a single bond.

[0262] Y3 preferably represents a peptide residue consisting of 8 amino acid residues represented by Y31-Y32-Y33-Y34-Y35-Y36-Y37-Y38.

[0263] Y31 preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. Y31 more preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. Y31 particularly preferably represents an alanine residue, glycine residue, proline residue, leucine residue, valine residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0264] Y32 preferably represents an alanine residue, glycine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamate residue, arginine residue, or histidine residue. Y32 more preferably represents an alanine residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, or glutamate residue. Y32 is particularly preferably an asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, or glutamate residue.

[0265] Y33 preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or histidine residue. Y33 more preferably represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, serine residue, threonine residue, or histidine residue. Y33 is particularly preferably represented by an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, or tyrosine residue.

[0266] Y34 preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. Y34 more preferably represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, tyrosine residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. Y34 particularly preferably represents an alanine residue, glycine residue, proline residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, or histidine residue.

[0267] Y35 preferably represents an alanine residue, glycine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. Y35 more preferably represents an alanine residue, glycine residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

[0268] Y36 preferably represents an alanine residue, an isoleucine residue, a valine residue, or a serine residue. Y36 more preferably represents an alanine residue.

[0269] Y37 preferably represents a leucine residue, an isoleucine residue, a valine residue, a phenylalanine residue, or a threonine residue. Y37 more preferably represents a leucine residue.

[0270] Y38 preferably represents an alanine residue, glycine residue, proline residue, valine residue, phenylalanine residue, asparagine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or histidine residue. Y38 more preferably represents an asparagine residue, glutamine residue, threonine residue, arginine residue, or histidine residue.

[0271] Y4 preferably represents a hydrogen atom, or an arbitrary amino acid residue.

[0272] Y5 preferably represents OH, or represents one arbitrary amino acid residue. Y5 more preferably represents one arbitrary amino acid residue.

[0273] Z1 and Z2 each independently represent a linking group, preferably an alkylene group having 1 to 10 carbon atoms, more preferably an alkylene group having 1 to 5 carbon atoms, and especially preferably an alkylene group having 2 to 4 carbon atoms.

[0274] m preferably represents an integer from 1 to 5, more preferably an integer from 1 to 3, and especially preferably an integer representing 1.

[0275] Z11 and Z12 each independently represent a linking group, preferably representing an alkylene group having 1 to 10 carbon atoms, more preferably representing an alkylene group having 1 to 5 carbon atoms, and especially preferably representing an alkylene group having 2 to 4 carbon atoms.

[0276] Cyclic peptides are closed ring structures formed by multiple amino acid residues in a polypeptide chain bonded together by covalent bonds other than the peptide bonds in the main chain. In a cyclic peptide, the portion containing these bonded amino acid residues is called the cyclization region.

[0277] From a stability perspective, the cyclization region of a cyclic peptide should not contain disulfide bonds. Examples of cyclization regions without disulfide bonds include structures containing thioether bonds.

[0278] In the case of forming a thioether bond, for example, a thiol bond can be formed by reacting a side-chain thiol group derived from an amino acid residue having a thiol group in its side chain with a side-chain chloroacetyl group derived from an amino acid residue having a chloroacetyl group in its side chain. Specifically, for example, a linear polypeptide can be cyclized by forming a thiol bond between a side-chain thiol group of an L-homocysteine ​​residue and a side-chain chloroacetyl group of an N-ε-chloroacetyl-L-lysine residue.

[0279] Alternatively, amino acids with a higher number of methylene units (such as chloropropionyl groups) in their side chains than those with more halogenated acetyl groups can be used instead of amino acids with halogenated acetyl groups in their side chains. However, the fewer the number of methylene units, the higher the cyclization efficiency, and therefore this is preferred. For example, compared to propionyl (-C(=O)-(CH2)2-X; X is a halogen atom, number of methylene units 2), acetyl (-C(=O)-CH2-X; X is a halogen atom, number of methylene units 1) has a higher cyclization efficiency and is therefore preferred.

[0280] When forming thioethers, from the viewpoint of FGFR bonding, it is more preferable to use amino acids with halogenated carbonyl groups in the side chains than to use amino acids with halogenated carbonyl groups in the main chain.

[0281] From the perspectives of stability and suppressing the formation of racemic compounds during peptide synthesis, the amino acid with a thiol group is preferably homocysteine ​​rather than cysteine ​​when forming a thioether bond. That is, the cyclization portion of a cyclic peptide preferably contains a homocysteine ​​residue.

[0282] From the viewpoint of FGFR protein bonding, the number of amino acid residues constituting the ring of the cyclic peptide is preferably 10 to 22, more preferably 13 to 20, even more preferably 17 or 18, and especially preferably 18.

[0283] From the perspective of FGFR protein bonding, the amino acids used in cyclic peptides can be of similar structures (generally considered similar structures). Similar structures include non-natural amino acids. Non-natural amino acids can be amino acids with elongated carbon chains (such as homoserine), amino acids endowed with methyl groups (such as N-methylalanine), and amino acids that have undergone one or more modifications (such as phosphorylated tyrosine).

[0284] In cyclic peptides, the amino acids at sites not specifically defined can be natural amino acids, non-natural amino acids, or amino acid analogs (peptide mimics).

[0285] As specific examples of cyclic peptides, the following cyclic peptides can be cited:

[0286] Includes any one of the amino acid sequences of sequence numbers 38-335 and 337-342 listed in Tables 4-6, or

[0287] The amino acid sequence containing 1 to 4 amino acids that have been substituted, deleted or inserted in any of the amino acid sequences of sequence numbers 38 to 335 and sequence numbers 337 to 342 listed in Tables 4 to 6, and which has the ability to bind to FGFR proteins.

[0288] As a preferred example of a cyclic peptide, the following cyclic peptides can be cited:

[0289] Includes the serial numbers 38-54, 56-57, 63-64, 69-70, 76-77, 87, 89-90, 97-98, 100-105, 108-109, 111-117, 124, 131-136, 140-147, 155-159, 162, 165-168, 171-172, 175-181, 183, and 185-19 recorded in Tables 4-6. 2. Any one of the amino acid sequences of serial numbers 199–206, 219–220, 223–225, 227, 230, 232, 235–237, 239–240, 242, 244–245, 250, 252–253, 256, 258, 265–267, 269, 274–275, 278, 280, 283, 290–335, and 340, or

[0290] Includes the serial numbers 38-54, 56-57, 63-64, 69-70, 76-77, 87, 89-90, 97-98, 100-105, 108-109, 111-117, 124, 131-136, 140-147, 155-159, 162, 165-168, 171-172, 175-181, 183, 185-192, 199-206, and 219 recorded in Tables 4-6. The amino acid sequence of sequence number 220, 223-225, 227, 230, 232, 235-237, 239-240, 242, 244-245, 250, 252-253, 256, 258, 265-267, 269, 274-275, 278, 280, 283, 290-335, and 340 contains 1-4 amino acid substitutions, deletions, or insertions, and has the ability to bind to FGFR proteins.

[0291] As a more preferred specific example of a cyclic peptide, the following cyclic peptides can be cited:

[0292] This includes serial numbers 38-45, 47-54, 57, 63, 70, 87, 97-98, 100-101, 103-105, 111-117, 131-134, 136, 140-146, 155-159, 162, 165-168, 176-178, 185-190, 192, and 199-206 as recorded in Tables 4-6. Any one of the amino acid sequences of serial numbers 219–220, 223–224, 227, 230, 232, 235–237, 239–240, 244–245, 252, 256, 258, 266–267, 269, 274–275, 278, 280, 290–325, 327–328, 330, and 332–335, or

[0293] Includes the serial numbers 38-45, 47-54, 57, 63, 70, 87, 97-98, 100-101, 103-105, 111-117, 131-134, 136, 140-146, 155-159, 162, 165-168, 176-178, 185-190, 192, 199-206, 219-220, and 223-206 recorded in Tables 4-6. 24. An amino acid sequence in any one of the following sequences: 227, 230, 232, 235–237, 239–240, 244–245, 252, 256, 258, 266–267, 269, 274–275, 278, 280, 290–325, 327–328, 330, and 332–335, wherein 1–4 amino acids are substituted, deleted, or inserted, and the sequence possesses the ability to bind to FGFR proteins.

[0294] The number of amino acids that are substituted, deleted, or inserted can be 1 to 4, preferably 1, 2, or 3, and more preferably 1 or 2.

[0295] The binding affinity to FGFR proteins is expressed as the amount of cyclic peptide that binds to the FGFR protein when the cyclic peptide is applied at a specific concentration to a certain amount of FGFR protein. In this invention, ELISA and SPR methods were used for determination, but other methods (ITC, alphascreen, etc.) can also be used. FGFR proteins can be full-length, local domains, or mutants.

[0296] Regarding the molecular stability of cyclic peptides, this invention uses resistance to reduction and alkali as indicators for measurement, but it also plays a role in resistance to other stimuli (e.g., X-ray resistance, gamma-ray resistance, ultraviolet resistance, heat resistance, and chemical resistance). This is because molecular stability essentially indicates that the molecule is more stable in terms of its free energy.

[0297] Cyclic peptides can be modified by other substances as exemplified below.

[0298] Cyclic peptides can be endowed with 1 to 20 amino acids and / or modified structures. From the viewpoint of cost of peptide chemical synthesis, it is preferable not to endow with amino acids. From the viewpoint of stability, it is preferable to endow with modified structures. Examples of modified structures include N-terminal acetylation and similar structures, and C-terminal amidation and similar structures.

[0299] Cyclic peptides can be modified by phosphorylation, methylation, adenosylation, ADP (adenosine diphosphate) ribosylation, glycan addition, etc., depending on their intended use.

[0300] Furthermore, cyclic peptides can be endowed with functional structures from the viewpoints of water solubility, stability, ease of separation, and / or traceability. Examples of functional structures include water-soluble polymers such as polyethylene glycol (PEG), IgG and other proteins, various affinity tags (e.g., histidine (His) tags, FLAG tags), immobilized carriers (e.g., cellulose beads, magnetic beads, various gels), fluorescent dyes, and radioactive isotopes.

[0301] Cyclic peptides may lack amino acids and / or have structural modifications. From the viewpoint of FGFR protein binding, the absence of amino acids should be no more than four, preferably no more than two, and especially preferably none.

[0302] The cyclic peptides of the present invention can be salts. Salts are preferably salts of physiologically permissible inorganic and organic acids and bases. Examples of suitable acid salts include the following: acetates, adipates, benzoates, benzenesulfonates, butyrates, citrates, digluconates, dodecyl sulfates, formates, fumarates, glycolates, hemisulfates, heptanoates, hexanoates, hydrobromide, hydroiodates, lactates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, pamoates, phosphates, picrates, neopentanoates, propionates, salicylates, succinates, sulfates, tartrates, toluenesulfonates, trifluoroacetates, and undecanoates. Salts derived from suitable bases include alkali metal (e.g., sodium) salts, alkaline earth metal (e.g., magnesium) salts, ammonium salts, and N-(alkyl)4 salts. + Salt.

[0303] <Methods for manufacturing cyclic peptides>

[0304] The method of manufacturing cyclic peptides is not particularly limited and can be achieved through chemical synthesis, genetic engineering synthesis, or enzymatic synthesis based on cell-free translation systems. From the viewpoint of purity, chemical synthesis is preferred.

[0305] Peptide synthesis based on chemical synthesis can be carried out using either solid-phase synthesis or liquid-phase synthesis. For small-scale synthesis, solid-phase synthesis is preferred due to its simplicity when using automated peptide synthesis equipment. For large-scale synthesis, liquid-phase synthesis is preferred from an operational point of view. Examples of large-scale synthesis include 1g or more, but it is not limited to this.

[0306] Solid-phase synthesis of peptides is well known to those skilled in the art, for example, by esterifying the hydroxyl group of a resin containing hydroxyl groups with the carboxyl group of a first amino acid (typically the C-terminal amino acid of the target peptide) protected by a protecting group of its α-amino group. Known dehydrating condensing agents such as 1-mesinesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used as esterification catalysts. Next, the protecting group of the α-amino group of the first amino acid is deprotected, and a second amino acid with all functional groups protected except for the carboxyl group of the main chain is added, activating the carboxyl group to bond the first and second amino acids. Then, the α-amino group of the second amino acid is deprotected, and a third amino acid with all functional groups protected except for the carboxyl group of the main chain is added, activating the carboxyl group to bond the second and third amino acids. This process is repeated until a peptide of the target length is synthesized, after which all functional groups are deprotected. Examples of resins synthesized in the solid phase include Merrifield resin, MBHAresin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGAresin (Merck), and HMPA-PEGAresin (Merck). These resins can be used after washing with solvents such as dimethylformamide (DMF), 2-propanol, and dichloromethane. Examples of protecting groups for the α-amino group include benzyloxycarbonyl (Cbz or Z), tert-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), benzyl, allyl, and allyloxycarbonyl (Alloc). The Cbz group can be deprotected by hydrofluoric acid or hydrogenation, the Boc group by trifluoroacetic acid (TFA), and the Fmoc group by piperidine treatment. α-Carboxyl groups can be protected using methyl esters, ethyl esters, benzyl esters, tert-butyl esters, and cyclohexyl esters. Other functional groups of amino acids include the hydroxyl groups of serine or threonine, which can be protected with benzyl or tert-butyl groups, and the hydroxyl groups of tyrosine, which can be protected with 2-bromobenzyloxycarbonyl or tert-butyl groups. The amino group of lysine side chains and the carboxyl group of glutamic acid or aspartic acid can be protected in the same way as the α-amino and α-carboxyl groups. Activation of the carboxyl group can be achieved using condensing agents. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonate ammonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU). Peptide chain cleavage from the resin can be achieved by treatment with acids such as TFA or hydrofluoric acid (HF).

[0307] Genetic engineering synthesis refers to the method of introducing genes into cells and synthesizing peptides. Cells that can be used include bacteria, yeast, nematode cells, insect cells, and animal cells (mammalian cells, etc.).

[0308] For example, non-natural amino acids can be introduced using a 4-base codon method for synthesis. Furthermore, it can be synthesized by synthesizing chain peptides and reacting the cross-linking functional groups of the side chains of the amino acid residues introduced into the cyclic portion to achieve cyclization.

[0309] Cell-free translation systems, also known as cell-free protein synthesis systems, are translation systems that utilize components present within cells such as E. coli without directly using cells. There are systems that use cell extracts and systems that use reaction solutions that are purified and reconstructed from the components of cell extracts (reconstructed cell-free translation systems).

[0310] Examples of systems that use cell extracts include those using Escherichia coli extract, wheat germ extract, rabbit erythrocyte extract, and insect cell extract.

[0311] The reconstructed cell-free translation system can be constructed from separately purified ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, GTP, ATP, translation initiation factor (IF), elongation factor (EF), termination factor (RF), ribosome regeneration factor, and other factors required for translation.

[0312] In translation systems, dialysis can be used to continuously supply energy. RNA polymerase can be added to facilitate transcription from DNA.

[0313] As for commercially available cell-free translation systems, among systems derived from E. coli, Roche Diagnostics' RTS-100 (registered trademark) can be cited; among systems derived from wheat germ extract, products from Zyagen or CellFree Sciences Co., Ltd. can be cited; and among reconstructive translation systems, PGI's PURESYSTEM (registered trademark), Gene Frontier Co., Ltd.'s Purefrex (registered trademark), and New England BioLabs' PURExpress In Vitro Protein Synthesis Kit can be cited.

[0314] <Cyclic Peptide Complex>

[0315] According to the present invention, a cyclic peptide complex or salt thereof formed by linking two or more molecules of the cyclic peptide or its salt thereof through linkers is provided. The number of molecules of the cyclic peptide or its salt linked by linkers is not particularly limited as long as there are two or more molecules, for example, 2 to 10 molecules, preferably 2 to 5 molecules, more preferably 2 to 4 molecules, further preferably 2 or 3 molecules, and especially preferably 2 molecules.

[0316] FGFR is a single-membrane transmembrane receptor tyrosine kinase. Starting with the binding of FGF, FGFR dimerizes, and the intracellular kinase regions of FGFR become close together, leading to cross-phosphorylation and promoting cell proliferation induction and / or differentiation control. Therefore, it is believed that FGFR phosphorylation can be achieved by polymerizing cyclic peptides with FGFR protein-binding affinity through linker structures (hereinafter, the polymerized cyclic peptides are referred to as cyclic peptide complexes) and applying them to cells expressing FGFR, thereby inducing FGFR polymerization through the cyclic peptide complexes. Given the proximity of the intracellular kinase regions of FGFR leading to phosphorylation, it is considered preferable to polymerize FGFR at an appropriate distance and orientation.

[0317] Furthermore, FGFR phosphorylation intended to maintain the proliferative or differentiation state of cells expressing FGFR sometimes requires the inclusion of multiple cyclic peptides. The multiple cyclic peptides contained in a cyclic peptide complex can be covalently bonded or non-covalently bonded (e.g., complex formation, affinity bonds, nucleic acid hybridization). From the viewpoint of synthetic cost, the aforementioned bonds can be non-covalent, but from the viewpoint of complex stability, covalent bonds (e.g., amide bonds, various click chemistry) are preferred.

[0318] Multiple cyclic peptides can be complexed through direct bonding between cyclic peptides or through other molecules. The multiple cyclic peptides contained in a cyclic peptide complex can have the same structure or different structures. From the viewpoint of promoting cell proliferation and / or controlling cell differentiation state, different structures are acceptable, but from the viewpoint of synthetic cost, having the same structure is preferred.

[0319] The length of the linker is not particularly limited, but from the viewpoint of FGFR2 phosphorylation, it is preferred to have a length that is not particularly limited. More preferably

[0320] When calculating the length of the linker, the bending of the molecular chain is taken into account, and the length of each single bond is calculated. (Each amino acid residue is) The unit distance is used to calculate the length of connectors based on molecular structure.

[0321] In cyclic peptide complexes, from the viewpoint of FGFR phosphorylation, the linker can be used by binding to either the N-terminus or the C-terminus of the cyclic peptide, but binding to the N-terminus is preferred.

[0322] In the cyclic peptide complex, from the viewpoint of FGFR phosphorylation, the linker structure can be PEG (polyethylene glycol), alkyl chain (polyethylene), polypeptide, polyester, polyacrylamide, polycarbonate, polypropylene, polystyrene, and / or polyurethane. Furthermore, the linker structure can form a salt. The salt in which the linker structure forms a salt is preferably one of the aforementioned salts. From the viewpoint of the water solubility of the cyclic peptide complex, PEG (polyethylene glycol), alkyl chain (polyethylene), polypeptide, polyester, and / or polyacrylamide are more preferred.

[0323] The fabrication of cyclic peptide complexes can be carried out, for example, by linking two cyclic peptides having one amino group using a linker molecule having two NHS-activated carboxyl groups (hereinafter referred to as Bis-NHS linkers). Bis-NHS linkers with various linker lengths are commercially available from various companies; examples include BS(PEG)5 (PEGylated bis(sulfosuccinimidyl) suberate) (Thermo, 21581) or Bis(NHS)PEG9 (Tokyo Chemical Industry Co., Ltd., B4688). The linking of cyclic peptides using Bis-NHS linkers can be carried out by mixing the cyclic peptides and linkers in water at a pH near 7.

[0324] Furthermore, in both solid-phase and liquid-phase peptide synthesis, cyclic peptide complexes can be fabricated using branched-chain amino acids with multiple amino groups. Examples of branched-chain amino acids include lysine and 2,4-diaminobutyric acid. In this case, after linking the branched-chain amino acid to a solid-phase resin or a label for liquid-phase synthesis, the multiple amino groups are deprotected, and peptide chains are elongated on both, thereby enabling the synthesis of cyclic peptide complexes on the solid-phase resin or the label for liquid-phase synthesis. Peptide chain elongation can be performed simultaneously on all amino groups or sequentially.

[0325] The cyclic peptide complex of the present invention can be modified with other substances. These other substances are the same as those used for the modification of the cyclic peptides described above.

[0326] <Utilization of Cyclic Peptides and Cyclic Peptide Complexes>

[0327] The cyclic peptides or their salts of the present invention, as well as the cyclic peptide complexes or their salts of the present invention, can be used for functional analysis, functional control, labeling, proliferation promotion, differentiation control, or purification of living substances (proteins, cells, tissues, etc.) containing FGFR proteins, etc. In particular, the proliferation-promoting effect of the cyclic peptide complexes or their salts of the present invention helps reduce the manufacturing costs of various cell therapies and cultured meat, thus having high industrial value. Especially in cultured meat, commercial production can be achieved by replacing most (up to 96%) of the growth factors in production costs with the cyclic peptide complexes or their salts of the present invention.

[0328] From the viewpoints of binding affinity, functional control, labeling efficiency, purification, proliferation promotion, and / or differentiation state control, the object for which the cyclic peptide or its salt, or the cyclic peptide complex or its salt, functions can be an FGFR protein, or other proteins, compounds, and / or cells, but is preferably an FGFR protein and / or cells expressing FGFR, cell secretions and / or cell lysates, or cells known to have proliferative or undifferentiated maintenance effects based on bFGF. More preferably, it is an FGFR1, FGFR3, or FGFR4 protein and / or cells expressing FGFR1, FGFR3, or FGFR4, cell secretions and / or cell lysates. Particularly preferred are FGFR1 or FGFR4 proteins and / or cells expressing FGFR1 or FGFR4, cell secretions and / or cell lysates.

[0329] The substance that enables the cyclic peptide or its salt, or a cyclic peptide complex or its salt, to function can be derived from a human, or from a mouse, cow, or other animal. From the viewpoints of binding affinity, functional controllability, labeling efficiency, purification, proliferation promotion, and / or differentiation state control, substances derived from animals with high homology to the amino acid sequence constituting the human FGFR are preferred. Examples of animals with high homology to the amino acid sequence constituting the human FGFR include cattle, pigs, chickens, and tuna.

[0330] Cyclic peptides or their salts, or cyclic peptide complexes or their salts, can be used dissolved in aqueous solutions or organic solvents, or bonded to an immobilized support (e.g., plates, beads). When used in conjunction with an immobilized support, from the viewpoint of bonding efficiency and / or the manifestation of target performance, it is preferable to introduce reactive groups (e.g., amino, thiol, biotin) for bonding with the cyclic peptide and / or cyclic peptide complex.

[0331] Specifically, the cyclic peptides or their salts of the present invention, as well as the cyclic peptide complexes or their salts of the present invention, can be used as culture medium compositions, culture medium additives, purification materials, labeling materials, cell control materials, or integration materials.

[0332] When using cyclic peptides or their salts or cyclic peptide complexes or their salts as culture medium compositions, they can be prepared using conventional methods with the constituent components. Their form is not particularly limited as long as the desired effects such as cell proliferation promotion and / or cell control are achieved; for example, they can be prepared as liquid, semi-fluid, or solid culture media. Furthermore, the culture medium compositions of the present invention can be prepared in powder form. Preparing them in powder form makes them extremely easy to transport and store. Moreover, by adding sterile water and / or agar during use, liquid, semi-liquid, or solid culture media can be easily prepared. When preparing liquid, semi-liquid, or solid culture media, from the viewpoint of FGFR phosphorylation, the concentration of the cyclic peptide or its salt or cyclic peptide complex or its salt is preferably 0.001 to 100 nmol / L, more preferably 0.01 to 10 nmol / L, and particularly preferably 0.1 to 3 nmol / L.

[0333] Furthermore, the culture medium composition of the present invention can also be used in any of the following culture methods: adhesion culture, suspension culture, embedding culture, tissue culture, etc.

[0334] When using cyclic peptides or their salts or cyclic peptide complexes or their salts as a culture medium composition, in addition to the cyclic peptides or their salts or cyclic peptide complexes or their salts of the present invention, components commonly used for cell culture, such as amino acids, vitamins, buffer materials, inorganic salts, carbon sources, serum and serum substitutes, can be appropriately used.

[0335] When using cyclic peptides or their salts, or cyclic peptide complexes or their salts, as a culture medium composition, they can be dissolved in the culture medium for use. From the viewpoint of FGFR phosphorylation, the concentration when dissolved in the culture medium is preferably 0.001–100 nmol / L, more preferably 0.01–10 nmol / L, and particularly preferably 0.1–3 nmol / L.

[0336] When using cyclic peptides or their salts or cyclic peptide complexes or their salts dissolved in a culture medium, the culture medium may be any medium commonly used for cell culture, but from the viewpoint of promoting cell proliferation and / or controlling cell growth, a culture medium prepared with components suitable for the cells used is preferred.

[0337] Culture media may contain serum, but from a safety perspective, serum-free culture media are preferred when used in the manufacture of cell products such as cell therapy products or cultured meat. Serum-free culture media do not contain serum, but may contain purified components derived from serum, or recombinant proteins derived from serum. Serum-free culture media may contain serum substitutes; for example, suitable culture media containing serum albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, or 3'-thiol glycerol, or their equivalents, can be cited. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. The cyclic peptides of the present invention, or their salts, or cyclic peptide complexes, or their salts, can also be cited as serum substitutes. Commercially available serum substitutes can be used. Knockout is an example of such a commercially available serum substitute. TM Serum Replacement (Life Technologies Corporation), Chemically-defined Lipid Concentrated (Life Technologies Corporation), Glutamax TM (Life Technologies Corporation), B27 (Life Technologies Corporation), N2 (Life Technologies Corporation), etc., but not limited to these.

[0338] When using cyclic peptides or their salts or cyclic peptide complexes or their salts as culture medium additives, they can be used when adding cyclic peptides or their salts or cyclic peptide complexes or their salts to the culture medium. The culture medium additives of the present invention may contain appropriate amounts of any additives, such as stabilizers, isotonic agents, pH adjusters, etc., in addition to the cyclic peptides or their salts or cyclic peptide complexes or their salts of the present invention, provided that they do not impair the desired effects such as cell proliferation promotion and / or cell control.

[0339] The culture medium additive of the present invention can be in any dosage form, such as solution, solid, or powder, as long as the desired effect can be obtained. From the viewpoint of reducing transportation costs, solid or powder form is preferred. In the case of solid or powder form, it can be dissolved to the desired concentration using a suitable buffer solution or solvent, and can also be used in solid or powder form. When the culture medium additive is a solution, it is preferable that the solution undergoes sterilization treatment such as filtration sterilization using a membrane filter.

[0340] The number of times and the timing of adding the culture medium additive of the present invention to the culture medium are not particularly limited. It can be done before, during, or after cell culture. However, from the viewpoint of promoting proliferation and / or controlling differentiation status, it is preferred to do so before and / or during cell culture.

[0341] When cyclic peptides or their salts, or cyclic peptide complexes or their salts, are used as culture medium compositions or additives, the culture medium compositions or additives can be used in the manufacture of cell therapeutics or cultured meat. Methods for manufacturing cell therapeutics or cultured meat may, for example, include the step of culturing cells using a culture medium containing cyclic peptides or their salts, or cyclic peptide complexes or their salts.

[0342] Cultured meat refers to edible meat produced through cell culture. It is an innovative technology that uses cell engineering to produce animal meat from cultured tissues, replacing traditional methods of raising livestock. The production process involves culturing animal cells to grow into muscle tissue or fat. This technology allows for the production of protein-rich foods that are materially and nutritionally equivalent to traditional meat or fish. Regarding livestock farming, in addition to obvious animal welfare concerns, the increasing use of water and land for cultivating feed crops creates significant resource demands. Furthermore, due to increased awareness of greenhouse gas emissions, deforestation, pollution, antibiotic resistance, the ecological impacts of meat production, and growing concerns about animal welfare, the concept of "cultured meat" or "clean meat" has emerged to replace traditional meat consumption with "laboratory-cultured meat."

[0343] Generally, edible meat refers to an aggregate of muscle fibers, connective tissue, and fat. Cultured meat preferably mimics the structure of edible meat, but it may not contain all the structures of so-called edible meat, as long as it contains cultured cells selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells. More preferably, it is a culture containing multiple cell types.

[0344] In addition to cultured cells selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells, cultured meat may also contain an extracellular matrix. Methods for manufacturing cultured meat may include, for example, a step of culturing cultured cells selected from the group consisting of fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells, and a step of recovering and integrating the cultured cells. Methods for manufacturing cultured meat may also include a differentiation induction step or a post-integration culture step.

[0345] When cyclic peptides or their salts or cyclic peptide complexes or their salts are used as culture medium compositions or additives, cell culture can be carried out by seeding cells into a culture medium containing cyclic peptides or their salts or cyclic peptide complexes or their salts. Culture is performed under conditions known in the art, such as in a 37°C CO2 incubator. Culture can be plate culture or suspension culture. Proliferating cells can be recovered as a culture by trypsin treatment or the like, and can then be further passaged. Cell culture can also be performed by seeding cells into a detachable construct. The construct with attached proliferating cells can be recovered as a culture. This construct can be constructed from extracellular matrix components such as collagen, elastin, fibronectin, laminin, and glandular adhesion proteins, and the constructed with attached cells can be integrated to form cultured meat.

[0346] The integration process includes the step of shaping the culture of one or more recovered cells. The culture shaped in the integration process can be a piece of meat such as a steak, branched meat, or minced meat. The integration process includes the step of integrating the cell culture with at least one substance selected from the group consisting of other cells, blood, and tissues. The other cells can be cultured cells or cells collected from animals. More specifically, it can be shaped together with other cells cultured in a medium containing the cyclic peptide of the present invention or its salt or cyclic peptide complex or its salt. As an example, muscle tissue-derived cells cultured in a medium containing the cyclic peptide of the present invention or its salt or cyclic peptide complex or its salt can be integrated with adipose tissue-derived cells and / or fibroblasts cultured in a medium containing the cyclic peptide of the present invention or its salt or cyclic peptide complex or its salt. Co-culture can also be further performed after integration. As an example, the cultures of one or more recovered cells can be mixed and seeded into an extracellular matrix for co-culture. Collagen, elastin, fibronectin, laminin, glandular adhesion globulin, etc., can be used as the extracellular matrix. Regarding the culture medium at this time, a culture medium containing the cyclic peptide of the present invention or its salt or cyclic peptide complex or its salt can also be used.

[0347] In the integration process, cultures of one or more recovered cell types can be integrated with blood and / or tissues. The tissues can be obtained from animals or can be cultured tissues. For example, cultured meat can be manufactured by integrating blood, adipose tissue, or muscle tissue separated during the processing of edible meat with a culture.

[0348] Differentiation induction can be performed after cell culture, or before, during, or after integration. Through differentiation induction, mononuclear muscle satellite cells and myoblasts can differentiate into multinucleated myotube cells, which then mature into muscle fibers. Differentiation induction can be performed using methods known in the art. For example, methods involving culture at high carbon dioxide concentrations are known; for instance, culture at a CO2 atmosphere of 5–10% (v / v) can promote differentiation into myotube cells.

[0349] Culture media containing cyclic peptides or their salts, or cyclic peptide complexes or their salts, can culture any animal cells. From the viewpoint of producing cultured meat, cells derived from livestock such as cattle, pigs, goats, sheep, rabbits, chickens, ostriches, and ducks can be used. In particular, when using bovine cells, any type of cell from Holstein, Jersey, Black-haired, Brown-haired, Shorthorn, Polled, and their hybrids can be used; however, from the viewpoint of producing edible meat, cells from Black-haired, Brown-haired, Shorthorn, and Polled varieties are preferred as meat-type varieties.

[0350] Culture media containing cyclic peptides or their salts, or cyclic peptide complexes or their salts, can also culture tissues formed by cell aggregation. Animal cells can be primary cells obtained from animals, passaged cells derived from primary cells, or lined cells or genetically modified cells with missing or inserted genes. Primary cells can be obtained by processing animal tissues in the culture medium. Cells can also be differentiated from stem cells, such as stem cells. From the viewpoint of manufacturing cell therapy products, it is preferable to culture adult stem cells or artificial pluripotent stem cells containing mesenchymal stem cells. From the viewpoint of manufacturing cultured meat, it is preferable to culture cells selected from the group including fibroblasts, adipose tissue-derived cells, and muscle tissue-derived cells.

[0351] Fibroblasts are cells that make up connective tissue, producing extracellular matrix components such as collagen and elastin. Fibroblasts found in muscle are specifically called myofibroblasts. Myofibroblasts form connective tissue surrounding muscle fiber bundles in skeletal muscle. Myofibroblasts express α-SMA, produce extracellular matrix, and can accumulate fat, contributing to chewiness and texture.

[0352] Adipose tissue-derived cells refer to the cells that make up adipose tissue; they are cells isolated and cultured from adipose tissue. As adipose tissue-derived cells, they are selected from at least one cell type chosen from the group consisting of adipose stem cells, multicystic adipocytes, and monocystic adipocytes. Adipose stem cells are mesenchymal stem cells capable of differentiating into various cell types, including muscle cells, adipocytes, and connective tissue cells. Multicystic adipocytes, also known as brown adipocytes, contribute to fat burning in vivo. Monocystic adipocytes, also known as white adipocytes, store fat droplets within their cells. The presence of fat in adipose tissue-derived cells contributes to the texture and flavor of edible meat.

[0353] Muscle tissue-derived cells refer to the cells that make up muscle tissue; they are cells isolated and cultured from muscle tissue. Examples of muscle tissue-derived cells include myoblasts, muscle satellite cells, and myotube cells. However, myotube cells are not proliferative; therefore, from a proliferative perspective, myoblasts and / or muscle satellite cells are preferred. Muscle satellite cells are adult stem cells contained in muscle, capable of proliferating and differentiating into myoblasts. Myoblasts are the cells that become the source of muscle fibers; they are proliferative mononuclear cells. If myoblasts differentiate, they fuse together to form multinucleated myotube cells, which then mature into muscle fibers. Muscle fibers are composed of myofibrils, which are the proteins that make up muscle, namely actin fibers and myosin fibers. They are classified into red muscle fibers (type I, type IIA) and white muscle fibers (type IIB) based on the isotype of myosin, contributing to the differences in the texture of edible meat.

[0354] When using cyclic peptides or their salts, or cyclic peptide complexes or their salts, as purification materials, the cyclic peptides can be used either bonded to a solid-phase support or not. As a method of using them without bonding, there are methods for endowing the cyclic peptide with a separation structure (e.g., a biotin tag, an antibody epitope, a polar residue for electrostatic separation) or methods for endowing the cyclic peptide with a toxin to kill cells other than the target.

[0355] When using cyclic peptides or their salts or cyclic peptide complexes or their salts as labeling materials, fluorescent dyes, radioisotopes, nuclear polar molecules, affinity tags (e.g., biotin, FLAG tags) or enzymes (e.g., luciferase) can be used as labeling groups.

[0356] When using cyclic peptides or their salts, or cyclic peptide complexes or their salts, as materials for cell control, the cyclic peptides can be used either bonded to a solid support (e.g., a flask, plate) or unbonded. Examples of cell control include promoting FGFR phosphorylation, inhibiting FGFR phosphorylation, promoting cell proliferation, inhibiting cell proliferation, promoting cell differentiation, and inhibiting cell differentiation. Furthermore, by linking cyclic peptides to other cell stimulants, the effects of selectively linking cell stimulants to cells possessing FGFR can be observed.

[0357] When using cyclic peptides or their salts, or cyclic peptide complexes or their salts, as materials for integrating specific molecules near the FGFR, it is necessary to link the molecule to be integrated with the cyclic peptide. The link can be one molecule to one molecule or one molecule to multiple molecules, and the link can be a reversible bond or an irreversible bond.

[0358] Example

[0359] The following specific examples will provide a more detailed description of the cyclic peptides and the like involved in this invention. The materials, processing steps, etc., shown in the following specific examples can be appropriately modified without departing from the spirit of this invention. The scope of the cyclic peptides and the like involved in this invention should not be interpreted as limiting based on the specific examples shown below. In the following description, unless otherwise specified, synthesis, processing, manufacturing, etc., are carried out at room temperature (25°C ± 3°C). The percentage of substance concentration is based on mass.

[0360] <Example 1: Obtaining FGFR-bonded candidate peptides using mRNA display>

[0361] Candidate sequences of cyclic peptides bound to FGFR were obtained using mRNA display.

[0362] The library used for mRNA display employed the sequence number 1. Translation of this library began from the 86th–88th start codon (ATG) at the 5' end, and proceeded to the next set of bases (NNNTGT(NNN)) after the 107th base. 6～18TAGNNN (NNN stands for trimeric oligonucleotide, where N independently represents A, T, G, or C) is a random sequence. The base set located at the 3' end of the random sequence is the pyrimidine linker binding site and the stop codon. The base set from the 5' end up to the 85th base is the sequence required for transcription and translation initiation, such as the T7 promoter sequence and the Shine-Dalgarno sequence. The triad TAG in this random sequence corresponds to the codon for chloroacetylated lysine. Therefore, in the peptide encoded by this random sequence, the thiol group of cysteine ​​spontaneously forms a thioether bond with the chloroacetyl group of chloroacetylated lysine, thus becoming a cyclic peptide. In nucleic acids with sequence number 1, the trimeric oligonucleotide represented by NNN is an equal mixture of trimeric oligonucleotides corresponding to the 18 types of codons shown in Table 3, where one amino acid is assigned one codon. Regarding (NNN)... 6～18 In one section, 13 different libraries with 6 to 18 repeats were prepared separately, and mRNA display was performed on each.

[0363] [Table 3]

[0364] Amino acids (one-character marker) Assign codons Amino acids (one-character marker) Assign codons A GCT N AAC I ATC Q CAG L CTG R CGT V GTT H CAT F TTC K AAA W TGG D GAC Y TAC E GAA S TCT G GGT T ACT P CCG

[0365] Serial Number 1:

[0366] GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGTTAAGAAAACAAAAACANNNTGT(NNN) 6～18 TAGNNNGGTGGCTCTGGCGGTAGCAGGACGGGGGGCGGCGGGGGGTAAATAAATAAGCTTGAGTAT

[0367] (NNN is a trimeric oligonucleotide, where N independently represents A, T, G, or C.)

[0368] The library for sequence number 1 was prepared using overlap-amplification PCR. Specifically, DNA from sequence number 2, sequence number 3, and any one of sequences 4–16 was mixed to concentrations of 3 μmol / L, 1 μmol / L, and 1 μmol / L, respectively, and then processed in a Platinum PCR system. TMIn the presence of SuperFi II DNA Polymerase (Thermo, 12361010), after seven cycles of 98°C / 30 seconds followed by 98°C / 10 seconds, 60°C / 10 seconds, and 72°C / 10 seconds, and finally 72°C / 5 minutes, three DNA molecules were ligated to create the target library. The prepared library was purified and diluted to 10 ng / μL. In the DNA sequences 4–17, the trimeric oligonucleotides, denoted as NNN, are equal-volume mixtures of trimeric oligonucleotides corresponding to the 18 types of codons shown in Table 1, with one codon assigned to each amino acid.

[0369] Serial Number 2:

[0370] GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGTTAAAAAAACAAAAAC

[0371] Serial Number 3:

[0372] ATACTCAAGCTTATTTATTTATTACCCCCCGCCGCCCCCCGTCCTGCTACCGCCAGAACCACC

[0373] Serial Number 4:

[0374] AAAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN)6TAGNNNGGCGGTTCTGGCGGTAGC

[0375] Serial Number 5:

[0376] AAAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN)7TAGNNNGGCGGTTCTGGCGGTAGC

[0377] Serial Number 6:

[0378] AAAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN)8TAGNNNGGCGGTTCTGGCGGTAGC

[0379] Serial Number 7:

[0380] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN)9TAGNNNGGCGGTTCTGGCGGTAGC

[0381] Sequence number 8:

[0382] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 10 TAGNNNGGCGGTTCTGGCGGTAGC

[0383] Sequence number 9:

[0384] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 11 TAGNNNGGCGGTTCTGGCGGTAGC

[0385] Sequence number 10:

[0386] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 12 TAGNNNGGCGGTTCTGGCGGTAGC

[0387] Sequence number 11:

[0388] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 13 TAGNNNGGCGGTTCTGGCGGTAGC

[0389] Sequence number 12:

[0390] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 14 TAGNNNGGCGGTTCTGGCGGTAGC

[0391] Sequence number 13:

[0392] AAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 15 TAGNNNGGCGGTTCTGGCGGTAGC

[0393] Sequence number 14:

[0394] AAAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 16 TAGNNNGGCGGTTCTGGCGGTAGC

[0395] Serial Number 15:

[0396] AAAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 17 TAGNNNGGCGGTTCTGGCGGTAGC

[0397] Serial Number 16:

[0398] AAAGAAGGAGATATACATATGGTTAAAAAAACAAAAACANNNTGT(NNN) 18 TAGNNNGGCGGTTCTGGCGGTAGC

[0399] As the FGFR, magnetic beads (NHS MagSepharose, Cytiva, 28951380) were immobilized using Recombinant Human FGFR1 alpha(IIIc)Fc Chimera Protein, CF (R&D systems, 658-FR-050) according to the manufacturer's (Cytiva) specified protocol (FGFR concentration at immobilization: 1 μg / μL). Furthermore, to remove the cyclic peptides bonded to the magnetic beads, IgG1 Fc, Human, recombinant (FUJIFILM Wako Pure Chemical Corporation, 098-07141) was immobilized into magnetic beads in the same manner as described above.

[0400] After repeating the incubation process for a total of 8 rounds by contacting the library with magnetic beads to immobilize FGFR, the bases of the peptides bonded to FGFR were identified using a sequencer.

[0401] The specific steps for each round are as follows.

[0402] First, the prepared library (Sequence No. 1) was reacted at 37°C for 30 minutes in the presence of T7 RNA Polymerase (TaKaRa, 2540A) to produce the library transcript. The DNA fragment was then purified and diluted to 10 μmol / L.

[0403] Next, the library transcript (final concentration 5 μmol / L) and pyrimidine linker 17 (final concentration 10 μmol / L) were mixed in TBS buffer (1.25 mmol / L Tris, 25 mmol / L NaCl, pH 7.5), heated at 95 °C for 5 minutes, and then irradiated with UV (365 nm) on ice to prepare the complex of the library transcript and pyrimidine linker.

[0404] Serial number 17: (PsoralenC6) - UACCCCCCGCCGCCCCCCGUCCU -(Sp18)-(Sp18)-(Sp18)-(Sp18)-CC-(Puro)

[0405] (Structural references for PsoralenC6, Sp18, and Puro) Figure 1 The underlined nucleotides are those where the 2'OH of RNA forms the 2'OMe body; ununderlined nucleotides represent unmodified DNA.

[0406] To translate chloroacetylated lysine, a non-natural amino acid, tRNA with the anticodon CUA paired with the UAG codon of mRNA was prepared by transcribing DNA at sequence number 18. This tRNA was aminoacylated using an ester of N-chloroacetylated lysine pdCpA (phospho 2'deoxyribocytidylylriboadenosine). This aminoacylated tRNA was designated as aminoacylated tRNA (1).

[0407] Serial Number 18:

[0408] GTTGTAAAACGGACGGCCAGTGCCAAGCTTGGGCTAATACGACTCACTATAGGGAGAGTAGTTCAATGGTAGAACGTCGGTCTCTAAAACCGAGCGTTGAGGGTTCGATTCCTTTCTCTCCCAC

[0409] The library transcript and pyrimidine linker complex were translated in a translation buffer containing PUREfrex2.0 (Gene Frontier, PF201-0.25-5) and aminoacyl-tRNA. The mRNA-cyclic peptide linker was prepared by mixing 5.25 μL of the complex, 7.5 μL of PUREfrex2.0 Solution I, 0.75 μL of Solution II, 1.5 μL of Solution III, and a dry volume of aminoacyl-tRNA (1) (final concentration 0.5 μg / μL), and reacting at 37 °C for 60 min.

[0410] In the presence of ReverTra Ace (TOYOBO, TRT-101), 15 μL of the translation product and DNA of sequence number 19 (final concentration 10 μmol / L) were mixed (reaction volume 37.5 μL) and reacted at 37 °C for 30 minutes to perform reverse transcription and produce cDNA-mRNA-cyclic peptide linkers.

[0411] Serial Number 19: GCTACCGCCAGAACCACC

[0412] Next, 22.5 μL of the reverse transcription product was mixed with 10 μL of magnetically immobilized IgG1 Fc in TBS buffer (20 mmol / L Tris, 150 mmol / L NaCl, 0.1% BSA, 0.05% Tween 20, pH 7.4) (reaction volume 100 μL). After reacting at room temperature for 45 minutes, the supernatant was recovered, thereby removing the cDNA-mRNA-cyclic peptide linker bonded to the magnetic beads.

[0413] Next, the supernatant was mixed with 10 μL of magnetically immobilized FGFR (reaction volume 50 μL), and reacted at room temperature for 45 minutes. The magnetic beads were then washed three times with 100 μL of TBS buffer to extract the cDNA-mRNA-cyclic peptide linker bound to FGFR. The extracted cDNA-mRNA-cyclic peptide linker was amplified by PCR in two stages.

[0414] In the first stage of PCR, 25 μL of the cDNA-mRNA-peptide linker was mixed with DNA from sequence number 20 (final concentration 0.5 μmol / L) and DNA from sequence number 21 (final concentration 0.5 μmol / L) (reaction volume 50 μL), and then precipitated in a Platinum reactor. TM In the presence of SuperFi II DNA Polymerase (Thermo, 12361010), after 6–15 cycles of 98℃ / 10 seconds, 60℃ / 10 seconds, and 72℃ / 10 seconds, followed by a final treatment at 72℃ / 5 minutes, the amplification product was obtained. The amplification product was purified and diluted to 20 nmol / L.

[0415] Serial number 20: GGAGATATACATATGGTTAAGAAAACAAAAAC

[0416] Serial Number 21: CTGCTACCGCCAGAACCACC

[0417] In the second stage of PCR, the amplified product from the first stage PCR (final concentration 10 nmol / L) was mixed with DNA from sequence number 2 (final concentration 0.5 μmol / L) and DNA from sequence number 3 (final concentration 0.5 μmol / L), and then... TM In the presence of SuperFiII DNA Polymerase (Thermo, 12361010), after 98℃ for 30 seconds, six cycles of 98℃ for 10 seconds, 60℃ for 10 seconds, and 72℃ for 10 seconds were repeated, followed by a final treatment at 72℃ for 5 minutes. This yielded DNA with the same sequence as the original library and random sequence. The prepared library was purified, diluted to 2 ng / μL, and used in the next round.

[0418] The base sequences of the stage 1 PCR products from round 8 were identified using MiSeq (manufactured by Illumina, Inc.) and the MiSeq Reagent kit v2 (300 cycles) (Illumina, MS-102-2022) according to Illumina, Inc.'s standard protocol. Analysis of the identified cyclic peptide genome revealed a large number of cyclic peptides containing the following amino acid sequences.

[0419] X1-X2-X3-X4

[0420] X1: Alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue

[0421] X2: phenylalanine residue, tryptophan residue, tyrosine residue, or histidine residue

[0422] X3: Any amino acid residue

[0423] X4: Alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue

[0424] In the bonding evaluation of Example 2, cyclic peptides in which amino acid residues of a portion of the cyclic peptides contained in the identified cyclic peptide group were substituted, deleted, or inserted were evaluated.

[0425] <Example 2: Evaluation of the binding affinity of peptides (enzyme synthesizers)>

[0426] The FGFR binding of the peptides was evaluated using ELISA.

[0427] The peptides used for evaluation were synthesized enzymatically using the following method.

[0428] First, a fusion peptide (Sequence No. 25) was designed, consisting of a translation-promoting sequence (Sequence No. 22), a peptide, a Myc tag (Sequence No. 23), and a HiBiT tag (Sequence No. 24). Next, a template DNA sequence (Sequence No. 26) was designed, endowing the DNA sequence encoding the fusion peptide with the T7 promoter sequence, Shine-Dalgarno sequence, start codon (ATG), and other sequences required for transcription and translation initiation at the 5' end, and with a stop codon set at the 3' end. In the case where the template DNA sequence contains a triplet TAG codon, this codon was mapped to the codon for chloroacetylated lysine. Therefore, the thiol group of cysteine ​​in the peptide encoded by the above DNA sequence spontaneously forms a thioether bond with the chloroacetyl group of chloroacetylated lysine, thus becoming a cyclic peptide.

[0429] Serial Number 22: VKKTKT

[0430] Serial Number 23: EQKLISEEDL

[0431] Serial Number 24: VSGWRLFKKIS

[0432] Serial number 25:

[0433] MVKKTKT[HCSYERLQFHGHEAPFRVXV]GSGSGSEQKLISEEDLGGSVSGWRLFKKIS

[0434] (X represents chloroacetylated lysine, and the [] contains examples of the amino acid sequence of the peptide to be evaluated, which will vary depending on the peptide.)

[0435] Serial number 26:

[0436] GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATGGTTAAAAAAACAAAAACA[CATTGCTCTTACGAACGTCTGCAGTTCCAT GGTCATGAAGCTCCGTTCCGTGTTTAGGTT]GGTTCTGGCAGTGGTTCCGAACAGAAACTGATCAGCGAAGAAGATCTGGGTGGCTCTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCTAATGAATAACTAATCC

[0437] (The brackets [] contain examples of DNA sequences encoding the peptide to be evaluated; the brackets will vary depending on the peptide.)

[0438] The template DNA was prepared by two-stage overlap amplification PCR.

[0439] In the first stage of PCR, DNA from sequence number 27, 28, 29, and 30 was mixed at concentrations of 0.3 μmol / L, 0.3 μmol / L, 0.05 μmol / L, and 0.05 μmol / L, respectively. In the presence of PrimeSTARMax (TaKaRa, R045B), the ligation process was repeated 27 times at 98°C for 10 seconds, 39°C for 5 seconds, and 72°C for 5 seconds, followed by three cycles of 98°C for 10 seconds, 58°C for 5 seconds, and 72°C for 5 seconds. This ligation of sequence number 28 and sequence number 29 resulted in the ligation of DNA from sequence number 28 to 29. The ligated DNA was then purified and diluted to a concentration of 50 ng / μL.

[0440] Serial number 27: GAAATTAATACGACTCACTATAGG

[0441] Serial Number 28: GAACCACTGCCAGAACC

[0442] Serial number 29:

[0443] GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATGGTTAAAAAAACAAAAAC

[0444] Serial number 30:

[0445] GAACCACTGCCAGAACC[AACCTAAACACGGAACGGAGGCTTCATGACCATGGAACTGCAGACGTTCGTAAGAGCAATG]TGTTTTTGTTTTTTTAAC

[0446] (The brackets [] contain examples of DNA sequences encoding the peptide to be evaluated; the brackets will vary depending on the peptide.)

[0447] In the second stage of PCR, the DNA from sequence number 27, sequence number 31, sequence number 32, and the purified product from stage 1 were mixed to concentrations of 0.3 μmol / L, 0.3 μmol / L, 0.0025 μmol / L, and 0.4 ng / μL, respectively. Under PrimeSTAR Max (TaKaRa, R045B), the mixture was repeatedly subjected to 30 cycles of 98℃ / 10 seconds, 58℃ / 5 seconds, and 72℃ / 5 seconds. This ligation of sequence number 32 DNA with the purified product from stage 1 yielded the template DNA (sequence number 25). The template DNA was then purified and diluted to 50 ng / μL.

[0448] Serial number 31: GGATTAGTTATTCATTAGCTAATC

[0449] Serial number 32:

[0450] GGTTCTGGCAGTGGTTCCGAACAGAAACTGATCAGCGAAGAAGATCTGGGTGGCTCTGTAAGTGGATGGCGATTATTCAAGAAGATTAGCTAATGAATAACTAATCC

[0451] Cell-free enzymatic synthesis of peptides was performed in a translation buffer containing template DNA, PUREfrex 2.0 (Genefrontier, PF201-0.25-5), and aminoacyl-tRNA (1). A dry mixture (final concentration 0.5 μg / μL) of template DNA (prepared to 50 ng / μL), 2.5 μL of PUREfrex 2.0 Solution I, 0.25 μL of Solution II, 0.5 μL of Solution III, and aminoacyl-tRNA (1) was mixed and reacted at 37 °C for 1 hour.

[0452] Regarding the concentration of the enzymatically synthesized peptide, the concentration of the chemically synthesized peptide (serial number 33) was determined using a Nano Glo HiBiT Lytic Detection System (Promega, N3040) and a calibration curve of known concentrations, and the peptide diluted with Can Get Signal Immunoreaction Enhancer Solution I (TOYOBO, NKB-101) was measured according to the standard protocol of the Nano Glo HiBiT Lytic Detection System.

[0453] Serial Number 33: EQKLISEEDLGGSVSGWRLFKKIS

[0454] The FGFR binding of enzyme-synthesized peptides was determined by ELISA evaluation of the amount of binding between the peptide and FGFR.

[0455] First, 5 ng of Recombinant Human FGFR1 alpha(IIIc)FcChimera Protein, CF (R&D systems, 658-FR-050) was immobilized in each well of a 96-well plate. After blocking with Pierce Protein-Free (PBS) Blocking Buffer (Thermo, 37572), peptide diluted with CanGet Signal Immunoreaction Enhancer Solution I (TOYOBO, NKB-101) was added, and the plate was incubated at room temperature for 3 hours. After washing with 0.05% Tween 20 PBS (phosphate buffered saline), anti-Myc antibody (Cell Signaling, 14038S) diluted with CanGet Signal Immunoreaction Enhancer Solution II (TOYOBO, NKB-101) was added, and the plate was incubated at room temperature for 2 hours. After washing with PBS containing 0.05% Tween 20, SuperSignal ELISA Femto Substrate (Thermo, 37075) was added, and the luminescence intensity was measured. The dosage-response relationship was calculated based on the difference in luminescence intensity between wells with and without FGFR immobilization and the peptide concentration at the time of reaction. FGFR binding was evaluated according to the following evaluation criteria, preferably criteria A, B, or C.

[0456] (Evaluation criteria for FGFR bonding)

[0457] The luminescence signal emitted by the cyclic peptide with sequence number 239 at a reaction concentration of 10 nmol / L was set to 1, while the luminescence signal emitted by the aforementioned peptide at a reaction concentration of 10 nmol / L under normalization was [value missing].

[0458] A... is above 0.7.

[0459] B... is greater than 0.5 and less than 0.7.

[0460] C... is greater than 0.1 and less than 0.5.

[0461] D……less than 0.1.

[0462] The evaluation results of the cyclic peptides are shown in Table 4.

[0463] The amino acid residues listed in parentheses represent amino acid residues that form a cyclized structure based on thioether bonds or disulfide bonds between the side chains. For example, K(acetyl) refers to a structure in which a haloacetyl group is modified on the side chain of a lysine residue, and the cyclic peptide has a cyclized structure in which the halogen atom of the haloacetyl group is replaced by a thiol group of other residues.

[0464] Serial number 34 is a cyclic peptide reported in International Publication No. WO2000 / 003245 (Patent Document 1), and serial number 36 is a cyclic peptide that converts serial number 34 into a thioether-type cyclic structure.

[0465] Serial number 35 is the peptide reported in Japanese Patent Application Publication No. 2021-70652, and serial number 37 is the peptide in which serial number 35 is converted into a thioether cyclized structure.

[0466] Serial numbers 38 and later are cyclic peptides that are the subject of this invention.

[0467] [Table 4]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476] <Example 3: Evaluation of the bonding properties of cyclic peptides (chemically synthesized products)>

[0477] The FGFR binding of cyclic peptides was evaluated using the surface plasmon resonance (SPR) method.

[0478] The cyclic peptides used for evaluation were custom-synthesized by Toray Research Center, Inc.

[0479] The FGFR binding of chemically synthesized cyclic peptides was evaluated using the following steps.

[0480] A CM5 sensor chip (Cytiva, 29149603) was placed in a Biacore-T200 (Cytiva) surface plasmon resonance device. HBS-EP buffer (Cytiva, BR100826) was added at a flow rate of 10 μL / min as a run buffer to equilibrate the flow path. Next, 70 μL of a mixed aqueous solution of 0.2 mol / L EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 0.05 mol / L NHS (N-hydroxysuccinimide) was added to activate the sensor chip. Then, 50 nmol / L Recombinant Human FGFR1 alpha(IIIc)Fc Chimera Protein CF (R&D systems, 658-FR-050) prepared with Acetate 4.5 buffer (Cytiva, BR100350) was added and immobilized to approximately 5000 RU. Subsequently, a sealing process was performed using an ethanolamine solution. Furthermore, the FGFR was not immobilized in another flow path of the sensor chip; instead, a sealing process was performed immediately after activation.

[0481] In each flow path of the FGFR solid-state sensor chip fabricated above, cyclic peptides diluted to 10 nmol / L with HBS-EP were added for 10 minutes at 25°C. Then, HBS-EP was used as the running buffer and flowed for 30 minutes to measure the bonding of the cyclic peptides to the FGFR. Subsequently, a regeneration process to remove the bonded cyclic peptides was performed twice, twice by flowing a regeneration solution containing sodium chloride powder dissolved in glycine 1.5 (Cytiva, BR100354) to a final concentration of 0.15 mol / L in each flow path for 1 minute. The amount of cyclic peptide bonding was evaluated based on the difference between the Biacore-T200 value measured in the FGFR-fixed flow path when the 10 nmol / L cyclic peptide was passed through and the Biacore-T200 value measured in the unfixed FGFR flow path.

[0482] Using the molecular weights of the cyclic peptides and FGFRs, the bonding amount of the cyclic peptides evaluated above was standardized to the cyclic peptide bonding amount per FGFR molecule, and the FGFR bonding was evaluated according to the following evaluation criteria. Evaluation criteria A, B, or C are preferred.

[0483] (Evaluation criteria for FGFR bonding)

[0484] A... The bonding amount is more than 0.7 molecules of cyclic peptide / FGFR1 molecule.

[0485] B... The amount of bonded peptides is greater than 0.5 molecules / FGFR1 molecule and less than 0.7 molecules / FGFR1 molecule.

[0486] The C... bond amount is greater than 0.1 molecules / FGFR1 molecule and less than 0.5 molecules / FGFR1 molecule.

[0487] The amount of D... bonded is less than 0.1 molecules of cyclic peptide / FGFR1 molecule.

[0488] The evaluation results are shown in Table 5. The amino acid residues listed in parentheses indicate amino acid residues that form a cyclization structure based on thioether bonds or disulfide bonds between the side chains. For example, K(acetyl) refers to a structure in which a haloacetyl group is modified on the side chain of a lysine residue, and the cyclic peptide has a cyclization structure in which the halogen atom of the haloacetyl group is replaced by a thiol group of other residues.

[0489] [Table 5]

[0490]

[0491] <Example 4: Evaluation of the FGFR universality of cyclic peptides (chemically synthesized products)>

[0492] The bonding of cyclic peptides to various types of FGFRs was evaluated using the surface plasmon resonance (SPR) method.

[0493] The cyclic peptides used for evaluation were custom-synthesized by Toray Research Center, Inc.

[0494] The FGFR binding of chemically synthesized cyclic peptides was evaluated using the following steps.

[0495] A CM5 sensor chip (Cytiva, 29149603) was placed in a Biacore-T200 (Cytiva) surface plasmon resonance (SPR) device. HBS-EP buffer (Cytiva, BR100826) was added as a run buffer at a flow rate of 10 μL / min to equilibrate the flow path. Next, 70 μL of a mixed aqueous solution of 0.2 mol / L EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 0.05 mol / L NHS (N-hydroxysuccinimide) was added to activate the sensor chip. Then, a cyclic peptide prepared at 2 mg / mL using HBS-EP buffer was added and immobilized to approximately 200 RU. Blocking was then performed using an ethanolamine solution. Furthermore, FGFR was not immobilized in another flow path of the sensor chip; instead, blocking was performed immediately after activation.

[0496] At 25°C, various FGFRs (FGFR1 Protein, Human, Recombinant (His Tag) (Sino Biological, 10616-H08H), FGFR3 Protein, Human, Recombinant (ECD, His Tag) (Sino Biological, 16044-H08H), and FGFR4 Protein, Human, Recombinant (His Tag) (Sino Biological, 10538-H08H)) diluted to 1 μmol / L with HBS-EP were added to each flow path of the solid-state sensor chip fabricated above for 10 minutes. Then, HBS-EP was used as the running buffer and flowed for 30 minutes to determine the binding of various FGFRs to the cyclic peptides. Subsequently, the regeneration process to remove the bonded FGFRs was performed twice, with each flow path flowing for 1 minute in a regeneration solution prepared by dissolving sodium chloride (final concentration 0.4 mol / L) and sodium dodecyl sulfate aqueous solution (mixed concentration 1%, final concentration 0.2%) in glycine 1.5 (Cytiva, BR100354). The amount of cyclic peptide bonded was evaluated based on the difference between the Biacore-T200 value measured in the flow path after cyclic peptide immobilization and the Biacore-T200 value measured in the flow path without immobilized cyclic peptides when 1 μmol / L FGFR was passed through.

[0497] Using the molecular weights of the cyclic peptides and FGFRs, the bonding amount of the FGFRs evaluated above was standardized to the FGFR molecular bonding amount per cyclic peptide molecule, and the FGFR bonding was evaluated according to the following evaluation criteria. Evaluation criteria A, B, or C are preferred.

[0498] (Evaluation criteria for FGFR bonding)

[0499] A... The bonding amount is more than 0.05 molecules of FGFR / 1 molecule of cyclic peptide.

[0500] B... The amount of bonding is greater than or equal to 0.01 molecules of FGFR / 1 cyclic peptide and less than 0.05 molecules of FGFR / 1 cyclic peptide.

[0501] The C... bond amount is greater than or equal to 0.005 molecules of FGFR per 1 cyclic peptide molecule and less than 0.01 molecules of FGFR per 1 cyclic peptide molecule.

[0502] D... The amount of bonding is less than 0.005 molecules of FGFR / 1 molecule of cyclic peptide.

[0503] The evaluation results are shown in Table 6.

[0504] [Table 6]

[0505]

[0506] These results demonstrate that the cyclic peptides of the present invention can bind to a variety of types of FGFRs.

[0507] <Example 5: Stability Evaluation of Cyclic Peptides>

[0508] The molecular stability of cyclic peptides was evaluated by analyzing aqueous solutions of cyclic peptides treated with reducing agents using LC (liquid chromatography).

[0509] The reducing agent treatment was performed based on the glutathione concentration contained in the cells under the following conditions. 25 μL of a 0.2 mg / mL cyclic peptide aqueous solution was prepared, and 58.8 μL of a 12.6 mmol / L DTT aqueous solution was added to this solution. The mixture was incubated at room temperature for 1 hour, thus obtaining the reducing agent-treated cyclic peptide aqueous solution. The DTT aqueous solution was used after diluting a 1 mol / L (+-)-dithiotriol (DTT) solution (FUJIFILM Wako Pure Chemical Corporation, 044-33871) with water. The total area of ​​all peaks in the LC / MS of the cyclic peptide before reducing agent treatment was set to 100%. The cyclic peptide residual rate was calculated by determining the proportion of the total area of ​​all peaks in the LC / MS of the reducing agent-treated cyclic peptide aqueous solution. Molecular stability was evaluated according to the following evaluation criteria. Evaluation criteria A, B, or C are preferred.

[0510] (Evaluation criteria for the residual rate of cyclic peptides)

[0511] A... The residual rate of cyclic peptides is over 90%.

[0512] B... The residual rate of the cyclic peptide is over 70% but less than 90%.

[0513] The residual rate of C...cyclic peptides is greater than 50% and less than 70%.

[0514] The residual rate of D...cyclic peptides is less than 50%.

[0515] In addition, the LC used in the evaluation of molecular stability is set under the following conditions.

[0516] • LC System: Prominence Series (pump, column oven, autosampler, detector) (manufactured by Shimadzu Corporation)

[0517] • Detector: Photodiode array detector (SPD-M20A), measurement wavelength 280nm

[0518] • Column: TSKgel ODS-100V, inner diameter 4.6mm × length 150mm, particle size 5μm (manufactured by Tosoh Corporation)

[0519] • Eluent A: A solution containing 0.1% trifluoroacetic acid as a solvent and 100% water as the solvent.

[0520] • Eluent B: A solution containing 0.1% trifluoroacetic acid as a solvent and 100% acetonitrile as the solvent.

[0521] • Flow rate: 1.0 mL / min

[0522] Injection volume: 25μL

[0523] • Gradient: 20-50%: Eluent B (0-15 minutes), 100%: Eluent B (15 minutes)

[0524] Column temperature: 40℃

[0525] The evaluation results are shown in Table 7. The amino acid residues listed in parentheses indicate amino acid residues that form a cyclization structure based on thioether bonds or disulfide bonds between the side chains. For example, K(acetyl) refers to a structure in which a haloacetyl group is modified on the side chain of a lysine residue, and the cyclic peptide has a cyclization structure in which the halogen atom of the haloacetyl group is replaced by a thiol group of other residues.

[0526] [Table 7]

[0527]

[0528] These results demonstrate that cyclic peptides cyclized via thioether bonds are more stable than those cyclized via disulfide bonds.

[0529] Furthermore, the stability of a cyclic peptide containing homocysteine ​​residues in the cyclization region (Sequence No. 335) and a cyclic peptide containing cysteine ​​residues in the cyclization region (Sequence No. 327) were compared under the following alkaline treatment as a more severe condition.

[0530] Alkali treatment was performed as follows: 25 μL of a 0.2 mg / mL cyclic peptide aqueous solution was prepared, and 58.8 μL of a 0.5 mol / L sodium hydroxide aqueous solution was added to this solution. The mixture was incubated at room temperature for 3 hours, thus obtaining an alkali-treated cyclic peptide aqueous solution. The total area of ​​all peaks in the LC / MS of the cyclic peptide before alkali treatment was set to 100%, and the proportion of the total area of ​​all peaks in the LC / MS of the alkali-treated cyclic peptide aqueous solution was calculated, thereby determining the cyclic peptide residue. The residue of sequence number 335 was shown to be 15%, and the residue of sequence number 327 was 28%. Cyclic peptides containing homocysteine ​​residues in the cyclization region are more stable than those containing cysteine ​​residues in the cyclization region.

[0531] In addition, the LC conditions used for evaluating molecular stability are the same as those used for the determination of cyclic peptides treated with reducing agent described above.

[0532] <Example 6: Evaluation of the cell proliferation-promoting effect of the cyclic peptide complex>

[0533] The FGFR phosphorylation capacity of the cyclic peptide complex was evaluated using the proliferation-promoting effect of BaF3 cells stably expressing FGFR.

[0534] BaF3 cells stably expressing the FGFR gene were established as follows: The FGFR gene-conjugated vectors (human: GeneCopoeia, EX-Y2820-M67, bovine: Accession number A4IFL5 sequences were introduced into pcDNA3.1 / Hygro(+)Mammalian Expression Vector (Thermo, V87020); porcine: Accession number A0A8D1E4H1 sequences were introduced into pcDNA3.1 / Hygro(+)Mammalian Expression Vector (Thermo, V87020); and chicken: Accession number P21804 sequences were introduced into pcDNA3.1 / Hygro(+)Mammalian Expression Vector (Thermo, V87020))) were introduced into BaF3 cells. Hygromycin was used for drug selection of cells containing the FGFR gene.

[0535] The cyclic peptide complexes used for evaluation were custom-synthesized by Toray Research Center, Inc.

[0536] The proliferation-promoting effect of stable FGFR-expressing BaF3 cells was evaluated using the following method. First, RPMI-1640 with L-Glutamine and Phenol Red (FUJIFILM Wako Pure Chemical Corporation, 189-02025) and Fetal Bovine Serum (Thermo, 10270-106) at a volume ratio of 9:1 was mixed, and heparin (STEM CELL, 07980) was added to a final concentration of 8 μg / mL to prepare the evaluation medium (hereinafter referred to as RPMI (10% FBS) medium). Next, stable FGFR-expressing BaF3 cells were washed twice in RPMI (10% FBS) medium, suspended in RPMI (10% FBS) medium with the cyclic peptide complex dissolved to a final concentration of 0.001–10 nmol / L, and seeded into 384-well plates. As a negative control, conditions without the addition of the cyclic peptide complex were prepared. After culturing at 37°C (5% CO2) for two nights, Hoechst 33342, Trihydrochloride, Trihydrate-10 mg / mL Solution in Water (Thermo, H3570), -Cellstain (registered trademark)-Calcein-AM solution (1 mg / mL DMSO solution) (DOJINDO, C396), and -Cellstain (registered trademark)-PI solution (DOJINDO, P378) were each added to RPMI (10% FBS) medium at their final concentrations at 1 / 1000 volume. The mixture was incubated at room temperature for 1 hour for viability staining, and images were taken using a confocal microscope (YOKOGAWA ELECTRICCORPORATION, CQ1). The number of viable cells was calculated based on the imaging data, and the cell proliferation-promoting effect was evaluated according to the following criteria. The number of viable cells in the negative control was approximately 20 cells / well. Evaluation criteria A, B, or C were preferred.

[0537] (Evaluation criteria for cell proliferation promotion)

[0538] The concentration of the cyclic peptide complex with the highest number of live cells was [not specified].

[0539] A... The number of live cells is 400 cells / well or more.

[0540] B... The number of live cells is 200 cells / well or more but less than 400 cells / well.

[0541] C……The number of live cells is more than 100 cells / well and less than 200 cells / well.

[0542] D……The number of live cells is less than 100 cells / well.

[0543] The evaluation results are shown in Table 8 and... Figure 2 The amino acid residues listed in parentheses represent amino acid residues that form a cyclic structure based on thioether bonds or disulfide bonds between side chains. For example, K (acetyl) refers to a structure in which a haloacetyl group is modified on the side chain of a lysine residue, and the cyclic peptide has a cyclic structure in which the halogen atom of the haloacetyl group is replaced by a thiol group of other residues.

[0544] [Table 8]

[0545]

[0546] These results demonstrate that the cyclic peptide complex formed by linking the cyclic peptides of the present invention can phosphorylate FGFRs from a variety of animals.

[0547] The structures of the cyclic peptide complexes with sequence numbers 343-346 are shown below.

[0548] [Chemical Formula 19]

[0549]

[0550] [Chemical Formula 20]

[0551]

[0552] [Chemical Formula 21]

[0553]

[0554] [Chemical Formula 22]

[0555]

[0556] <Example 7: Evaluation of the stem cell proliferation-promoting effect of the cyclic peptide complex>

[0557] The proliferative effect of the cyclic peptide complex on MSC (mesenchymal stem cells) was evaluated.

[0558] For MSCs, hMSC-Human Mesenchymal Stem Cells (Lonza, PT-2501) were used as the culture medium with PRIME-XV MSC Expansion XSFM (FUJIFILM Irvine Scientific, Inc., 91149). Resuscitation and passage were performed according to the protocol recommended by FUJIFILM Irvine Scientific, Inc. MSCs from passages 1 to 4 were used for evaluation.

[0559] The proliferation-promoting effect of MSCs was evaluated using the following method. First, a culture medium (hereinafter referred to as custom medium) was prepared by removing bFGF from PRIME-XV MSC Expansion XSFM (FUJIFILM Irvine Scientific, Inc., 91149). Next, Prime-XV Human Fibronectin (FUJIFILM Irvine Scientific, Inc., 31002) was dissolved in PBS (FUJIFILM Wako Pure Chemical Corporation, 164-23551) and added to 96-well plates for evaluation at a rate of 2 μg / cm². The plates were then incubated at room temperature for 3 hours, thereby spreading Fibronectin onto the 96-well plates. Subsequently, passaged MSCs were dissected using Tryple Express (Thermo, 12604-013), washed once with custom-made medium, and then resuspended in custom-made medium diluted to a final concentration of 0.001–3 nmol / L with either cyclic peptide complex or bFGF (R&D Systems, 3718-GMP). The MSCs were then seeded into 96-well plates pre-coated with Fibronectin. As a negative control, conditions without the addition of cyclic peptide complex and bFGF were prepared. After incubation at 37°C (5% CO2) for two nights, the supernatant was removed, and the same amount of medium prepared to the same composition as the removed medium was added, thus performing medium replacement. After further culturing at 37°C (5% CO2) for 2 nights, the supernatant was removed, and custom-made culture medium containing Hoechst 33342, Trihydrochloride, Trihydrate-10 mg / mL Solution in Water (Thermo, H3570), and Cellstain-Calcein-AM solution (1 mg / mL DMSO solution) (DOJINDO, C396) at final concentrations of 1 / 10000 and 1 / 4000 respectively was added. The culture was then incubated at 37°C (5% CO2) for 30 minutes, and all cells and viable cells were stained and photographed using a confocal microscope (YOKOGAWA ELECTRIC CORPORATION, CQ1). The viable cell count was calculated based on the photographic data. The viable cell count in wells containing 1 nmol / L bFGF was set to 100, and the viable cell count in the negative control wells was set to 0, thus standardizing the viable cell count. The evaluation results are shown below. Figure 3 middle.

[0560] The cyclic peptide complex (serial number 346) exhibited the same degree of MSC proliferation promotion at the same molar concentration as bFGF, demonstrating that the cyclic peptide complex of the present invention can replace bFGF in promoting MSC proliferation.

[0561] <Example 8: Evaluation of the effect of cyclic peptide complex in maintaining the undifferentiated capacity of stem cells>

[0562] The role of the cyclic peptide complex in maintaining the undifferentiated capacity of MSCs (mesenchymal stem cells) was evaluated.

[0563] MSCs were prepared using the same cells as in Example 7.

[0564] The maintenance effect of MSC undifferentiated capacity was evaluated using the following method. First, a culture medium (hereinafter referred to as custom medium) was prepared from PRIME-XVMSC Expansion XSFM (FUJIFILM Irvine Scientific, Inc., 91149) with bFGF removed. Next, Prime-XV Human Fibronectin (FUJIFILM Irvine Scientific, Inc., 31002) was dissolved in PBS (FUJIFILM Wako Pure Chemical Corporation, 164-23551) and added to 12-well plates for proliferation at a rate of 2 μg / cm². The plates were then incubated at room temperature for 3 hours, thereby spreading the Fibronectin onto the 12-well plates. Subsequently, passaged MSCs were dissected using Tryple Express (Thermo, 12604-013). After washing once with custom-made medium, the cyclic peptide complex or bFGF (R&D Systems, 3718-GMP) was resuspended in custom-made medium diluted to final concentrations of 10 nmol / L and 1 nmol / L, respectively, and seeded into 12-well plates pre-coated with Fibronectin. As a negative control, conditions without the addition of the cyclic peptide complex and bFGF were prepared. After incubation at 37°C (5% CO2) for two nights, the supernatant was removed, and the same volume of medium prepared to the same composition as the removed medium was added, thus performing medium replacement. After further incubation at 37°C (5% CO2) for two nights, RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, 74136) according to the manufacturer's protocol. RNA was extracted using PrimeScript according to the manufacturer's protocol. TMThe extracted RNA was reverse transcribed using an RT reagent kit with a gDNA Eraser (Perfect Real Time) (Takara, RR047A), and the expression levels of genes exhibiting undifferentiated ability were quantified using the TaqMan Gene Expression Assay (Thermo, 4331182, GAPDH: Hs02758991g1, Sox2, Hs04234836s1, Oct4, Hs04260367gH, Nanog, Hs02387400g1) according to the manufacturer's specifications. The expression levels of each gene were normalized by setting the wells containing 1 nmol / L bFGF as 1. The evaluation results are shown below. Figure 4 middle.

[0565] Of the three genes (Sox2, Oct4, and Nanog) that showed maintenance of undifferentiated MSC capacity, Sox2 expression changed under conditions without bFGF (negative control) and under conditions with 1 nmol / L bFGF, therefore it was determined to be a suitable indicator of the role of bFGF in maintaining MSC undifferentiated capacity. On the other hand, Oct4 and Nanog expression levels remained almost unchanged under conditions without bFGF (negative control) and under conditions with 1 nmol / L bFGF, therefore they were determined not to be suitable indicators of the role of bFGF in maintaining MSC undifferentiated capacity. In Sox2, the cyclic peptide complex (serial number 346) showed the same level of gene expression as bFGF, demonstrating that the cyclic peptide complex of the present invention can replace bFGF in maintaining undifferentiated capacity.

[0566] <Example 9: Evaluation of the Bovine Cell Proliferation-Promoting Effect of the Cyclic Peptide Complex>

[0567] The proliferative effect of the cyclic peptide complex on bovine cell (myoblast) proliferation was evaluated.

[0568] Bovine myoblasts were isolated from bovine muscle tissue using the following method. First, the muscle tissue of Iberian black cattle was minced in HBSS (-) (phenol red-free) (FUJIFILM Wako Pure Chemical Corporation, 085-09355), and protnase from Streptomyces Griseus (Sigma-Aldrich, 10165921001) was added to a final concentration of 1 mg / mL. The mixture was then incubated at 37°C for 1 hour. After incubation, HBSS containing 10% FBS was added, and cells were obtained by centrifugation. The obtained cells were then suspended in D-MEM (high glucose) (containing L-glutamine and phenol red) (FUJIFILM Wako Pure Chemical Corporation, 044-29765) containing 10% bovine serum and 30 ng / mL bFGF. Suspension cells were cultured in culture flasks coated with iMatrix-511 solution (FUJIFILM WakoPure Chemical Corporation, 381-07363) at a concentration of 0.55 μg / cm². Bovine myoblasts that had been passaged more than twice after the start of culture were used for evaluation.

[0569] The proliferative effect of bovine myoblasts was evaluated using the following method. Passaged bovine myoblasts were dissected using Trypsin-EDTA (0.05%) and phenol red (Thermo, 25300-054), suspended in D-MEM (high glucose) containing 10% FBS (containing L-glutamine and phenol red) diluted to a final concentration of 30 ng / mL for bFGF or 0.03–10 nmol / L for the cyclic peptide complex, and seeded into 96-well plates plated with ImaMatrix-511 at 0.55 μg / cm². As a negative control, conditions without the addition of bFGF and the cyclic peptide complex were prepared. After culturing at 37°C (5% CO2) for 4 days, the supernatant of the culture medium was removed, and Hoechst 33342, Trihydrochloride, Trihydrate-10mg / mL Solution in Water (Thermo, H3570), and -Cellstain (registered trademark)-Calcein-AM solution (1mg / mL DMSO solution) (DOJINDO, C396) were added at final concentrations of 1 / 10000 and 1 / 4000 volume of D-MEM (high glucose) containing 10% FBS (containing L-glutamine and phenol red). The mixture was then incubated at 37°C (5% CO2) for 30 minutes to stain all cells and viable cells. The staining was performed using a confocal microscope (YOKOGAWA ELECTRIC CORPORATION, CQ1). The viable cell count was calculated based on the imaging data. The viable cell count in wells containing 30 ng / mL bFGF was set to 100, and the viable cell count in the negative control wells was set to 0, thus standardizing the viable cell count. The evaluation results are shown below. Figure 5 middle.

[0570] The cyclic peptide complex (serial number 346) exhibits the same or greater degree of bovine myoblast proliferation-promoting effect as bFGF, demonstrating that the cyclic peptide complex of the present invention can replace bFGF in promoting bovine myoblast proliferation.

Claims

1. A cyclic peptide or a salt thereof, comprising an amino acid sequence represented by X1-X2-X3-X4, and comprising a cyclized portion cyclically bound by covalent bonds, said cyclized portion comprising a structure represented by formula (2). [Chemical Formula 1] In the formula, X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. Z1 and Z2 represent the linking groups independently. m represents an integer from 1 to 10.

2. The cyclic peptide or its salt according to claim 1, represented by formula (1), [Chemical Formula 2] In the formula, V represents -NH-Y4 or R, When V is -NH-Y4, L0 represents L; when V represents R, L0 represents L1. X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. Y1 represents a single bond or an amino acid residue or peptide residue containing 1 to 13 arbitrary amino acid residues. Y3 represents an amino acid residue or peptide residue containing 1 to 14 arbitrary amino acid residues. Y4 represents a hydrogen atom, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues. Y5 represents OH, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues. The amino terminus of X1 is bonded to the carboxyl terminus of Y1, and the carboxyl terminus of X1 is bonded to the amino terminus of X2. L indicates that... [Chemical Formula 3] or [Chemical Formula 4] The cyclization part is indicated by Z11 and Z12, which independently represent linking groups, m represents an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone. L1 indicates that by [Chemical Formula 5] The cyclization part is indicated by Z12, the linking group is indicated by m, an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone. R represents the side chain structure of an amino acid.

3. The cyclic peptide or its salt according to claim 1, represented by formula (1A1) or formula (1A2), [Chemical Formula 6] In the formula, X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. Y1 represents a single bond or an amino acid residue or peptide residue containing 1 to 13 arbitrary amino acid residues. Y3 represents an amino acid residue or peptide residue containing 1 to 14 arbitrary amino acid residues. Y4 represents a hydrogen atom, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues. Y5 represents OH, or an amino acid residue or peptide residue containing 1 to 7 arbitrary amino acid residues. The amino terminus of X1 is bonded to the carboxyl terminus of Y1, and the carboxyl terminus of X1 is bonded to the amino terminus of X2. L indicates that... [Chemical Formula 7] or [Chemical Formula 8] The cyclization part is indicated by Z11 and Z12, which independently represent linking groups, m represents an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone. L1 indicates that by [Chemical Formula 9] The cyclization part is indicated by Z12, the linking group is indicated by m, an integer from 1 to 10, and * indicates the position of the α-carbon atom in the amino acid backbone. R represents the side chain structure of an amino acid.

4. The cyclic peptide or a salt thereof according to claim 1, comprising an amino acid sequence represented by X1-X2-X3-X4-X5. In the formula, X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

5. The cyclic peptide or a salt thereof according to claim 1, comprising an amino acid sequence represented by X1-X2-X3-X4-X5. In the formula, X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue.

6. The cyclic peptide or a salt thereof according to claim 1, comprising an amino acid sequence represented by X1-X2-X3-X4-X5-Xn-X6, wherein, X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue. Xn represents a peptide residue containing 1 to 3 arbitrary amino acid residues. X6 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, histidine residue, or lysine residue.

7. The cyclic peptide or a salt thereof according to claim 1, comprising an amino acid sequence represented by X1-X2-X3-X4-X5-Xn-X6, wherein, In the formula, X1 represents an alanine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, or threonine residue. X2 represents an amino acid residue whose side chain contains an aromatic residue. X3 represents any amino acid residue. X4 represents an alanine residue, glycine residue, phenylalanine residue, tyrosine residue, tryptophan residue, serine residue, or histidine residue. X5 represents an alanine residue, glycine residue, proline residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, arginine residue, or histidine residue. Xn represents a peptide residue containing 1 to 3 arbitrary amino acid residues. X6 represents an alanine residue, glycine residue, leucine residue, isoleucine residue, valine residue, phenylalanine residue, tyrosine residue, tryptophan residue, asparagine residue, glutamine residue, serine residue, threonine residue, glutamic acid residue, arginine residue, or histidine residue.

8. The cyclic peptide or its salt according to claim 1, wherein, The number of amino acid residues that make up the ring is 10 to 22.

9. The cyclic peptide or its salt according to claim 1, wherein, The cyclized portion contains homocysteine ​​residues.

10. The cyclic peptide or its salt according to claim 1, comprising any one of the amino acid sequences of sequence numbers 38-335 and 337-342 listed in Tables 4-6, or The amino acid sequence containing 1 to 4 amino acids that have been substituted, deleted or inserted in any of the amino acid sequences of sequence numbers 38 to 335 and sequence numbers 337 to 342 listed in Tables 4 to 6, and which has the ability to bind to FGFR proteins.

11. The cyclic peptide or its salt according to claim 1, comprising the sequences 38-54, 56-57, 63-64, 69-70, 76-77, 87, 89-90, 97-98, 100-105, 108-109, 111-117, 124, 131-136, 140-147, 155-159, 162, 165-168, 171-172, 175-181, and 183 listed in Tables 4-6. Any one of the amino acid sequences of serial numbers 185–192, 199–206, 219–220, 223–225, 227, 230, 232, 235–237, 239–240, 242, 244–245, 250, 252–253, 256, 258, 265–267, 269, 274–275, 278, 280, 283, 290–335, and 340, or Includes the serial numbers 38-54, 56-57, 63-64, 69-70, 76-77, 87, 89-90, 97-98, 100-105, 108-109, 111-117, 124, 131-136, 140-147, 155-159, 162, 165-168, 171-172, 175-181, 183, 185-192, 199-206, and 219 recorded in Tables 4-6. The amino acid sequence of sequence number 220, 223-225, 227, 230, 232, 235-237, 239-240, 242, 244-245, 250, 252-253, 256, 258, 265-267, 269, 274-275, 278, 280, 283, 290-335, and 340 contains 1-4 amino acid substitutions, deletions, or insertions, and has the ability to bind to FGFR proteins.

12. The cyclic peptide or its salt according to claim 1, comprising the sequences 38-45, 47-54, 57, 63, 70, 87, 97-98, 100-101, 103-105, 111-117, 131-134, 136, 140-146, 155-159, 162, 165-168, 176-178, 185-190, and 192 listed in Tables 4-6. Any one of the amino acid sequences of serial numbers 199–206, 219–220, 223–224, 227, 230, 232, 235–237, 239–240, 244–245, 252, 256, 258, 266–267, 269, 274–275, 278, 280, 290–325, 327–328, 330, and 332–335, or Includes the serial numbers 38-45, 47-54, 57, 63, 70, 87, 97-98, 100-101, 103-105, 111-117, 131-134, 136, 140-146, 155-159, 162, 165-168, 176-178, 185-190, 192, 199-206, 219-220, and 223-206 recorded in Tables 4-6.

24. An amino acid sequence in any one of the following sequences: 227, 230, 232, 235–237, 239–240, 244–245, 252, 256, 258, 266–267, 269, 274–275, 278, 280, 290–325, 327–328, 330, and 332–335, wherein 1–4 amino acids are substituted, deleted, or inserted, and the sequence possesses the ability to bind to FGFR proteins.

13. The cyclic peptide or its salt according to claim 1, wherein it is modified by other substances.

14. A cyclic peptide complex or a salt thereof, comprising two or more molecules of the cyclic peptide or a salt thereof as described in any one of claims 1 to 13 linked together by a linker.

15. A cyclic peptide complex or a salt thereof, comprising two molecules of the cyclic peptide or a salt thereof as described in any one of claims 1 to 13 linked together by a linker.

16. The cyclic peptide complex or a salt thereof according to claim 14, wherein it is modified with other substances.

17. A culture medium composition or culture medium additive comprising any one of claims 1 to 13 of a cyclic peptide or a salt thereof.

18. A purification material comprising the cyclic peptide or a salt thereof as described in any one of claims 1 to 13.

19. A labeling material comprising any one of claims 1 to 13, or a salt thereof.

20. A material for cell control comprising the cyclic peptide or a salt thereof as described in any one of claims 1 to 13.

21. An integration material comprising any one of claims 1 to 13, or a salt thereof.

22. A culture medium composition or culture medium additive comprising the cyclic peptide complex of claim 14 or a salt thereof.

23. A purification material comprising the cyclic peptide complex of claim 14 or a salt thereof.

24. A labeling material comprising the cyclic peptide complex of claim 14 or a salt thereof.

25. A material for cell control comprising the cyclic peptide complex of claim 14 or a salt thereof.

26. An integration material comprising the cyclic peptide complex of claim 14 or a salt thereof.

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