Peptide, peptide complex, composition for cell culture, composition for medical, diagnostic, or research use, and method for producing peptide complex

Abstract

This peptide includes an amino acid sequence represented by formula A1 or an amino acid sequence in which one or more amino acid residues in the amino acid sequence represented by formula A1 have been substituted, deleted, added, or inserted. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15

Description

Peptides, peptide complexes, cell culture compositions, compositions used for medical, diagnostic or research purposes, and methods for producing peptide complexes.

[0001] The present invention relates to peptides, peptide complexes, cell culture compositions, compositions used for medical, diagnostic or research purposes, and methods for producing peptide complexes. This application claims priority to Japanese Patent Application No. 2024-169014, filed in Japan on September 27, 2024, the contents of which are incorporated herein by reference.

[0002] Basic fibroblast growth factors (FGFs) are expressed in various tissues and regulate cell proliferation, survival, migration, and differentiation. FGFs are also known to be important components in pluripotent stem cell culture. FGF2, a type of FGF, transmits signals within cells by binding to receptor tyrosine kinases (Fibroblast growth factor receptors: GFRFs), thereby exerting its diverse biological activities.

[0003] Non-patent document 1 discloses that there are multiple subtypes of FGFR, and among them, FGFR1c and FGFR3c are the main receptors for FGF2.

[0004] Non-patent document 2 discloses that in iPSCs (Induced Plurippotent Stem Cells), which are pluripotent stem cells, FGF2 transmits signals within the cell by binding to FGFR1, thereby exhibiting diverse biological activities.

[0005] Various artificial FGFR1 agonists have been reported with the aim of activating FGFR1, the receptor for FGF2, and thereby exerting FGF2-like biological activity. For example, Patent Document 1 discloses an FGFR1 agonist antibody that uses an antibody that binds to FGFR1.

[0006] Non-patent document 3 discloses a homodimeric VHH FGFR1c agonist using an alpaca-derived antibody (Variable domain of Heavy chain of Heavy chain antibody: VHH antibody) that binds to FGFR1c.

[0007] Non-patent document 4 discloses an FGFR1 agonist using a DNA aptamer that binds to FGFR1.

[0008] Non-patent document 5 discloses an FGFR agonist using a peptide derived from the sequence of a protein that binds to FGFR, such as FGF2. Non-patent document 5 also discloses an FGFR1 agonist using a linear peptide that binds to FGFR1, which was discovered by phage display.

[0009] Japanese Patent Publication No. 2016-29084

[0010] Xiuqin Zhang,et al.Receptor Specificity of the Fibroblast Growth Factor Family,The Journal of Biological Chemistry 2006, 281, 15694.Yoshiki Nakashima and Takeshi Omasa,What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?,BioResearch Open Access 2016, 5.1, 84.Ryo Yonehara,et al.A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand,J. Biol. Chem. 2023, 299, 102804.Ryosuke Ueki,et al.DNA aptamer assemblies as fibroblast growth factor mimics and their application in stem cell culture,Chem. Commun, 2019, 55, 2672.Emily Atkinson,Rachael Dickman,Growth factors and their peptide mimetics for treatment of traumatic brain injury,Bioorg. Med. Chem. 2023, 90, 117368.

[0011] The object of the present invention is to create a novel peptide having the ability to bind to FGFR1, which is a receptor for FGF2; a novel peptide complex having the ability to activate FGFR1 agonist, which is a receptor for FGF2; a peptide capable of forming a peptide complex; a cell culture composition containing a peptide or peptide complex; a composition for use in medical, diagnostic, or research applications; and a method for producing a peptide or peptide complex.

[0012] To achieve the above objectives, the present invention employs the following configuration.

[0013] [1] A peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which one or more amino acid residues are substituted, deleted, added, or inserted in the amino acid sequence represented by formula A1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 where, X1 is an amino acid residue having an optionally substituted aryl group in its side chain, X2 is any amino acid residue, X3 is V, X4 is an amino acid residue having an aliphatic hydrocarbon group in its side chain, X5 is an optionally substituted 4-6 membered cyclic secondary amino acid residue, or an amino acid residue having an optionally substituted aliphatic hydrocarbon group in its side chain, X6 is any amino acid residue, X7 is any amino acid residue, X8 is an optionally N-alkylated glycine residue (where the alkyl group constituting the N-alkylation may have substituents), or any D-amino acid residue, X9 is any amino acid residue, X10 is an amino acid residue having an aliphatic hydrocarbon group in its side chain. X11 is an amino acid residue having an optionally substituted aryl group in its side chain, X12 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in its side chain, X13 is I, X14 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in its side chain, or any N-alkyl amino acid residue, and X15 is C. In formula A1, the amino acid sequence is described from the N-terminus to the C-terminus.[2] In formula A1, X1 is F, F2F, or F3C, X2 is W, Cit, or W7N, X4 is V or alI, X5 is P, Hyp, or T, X6 is D, 3Py6NH2, E, F4COO, Hyp, K, N, Nmm, or S, X7 is I, Chg, E, Gthp, K, R, T, V, or W7N, X8 is G, ApG, de, dp, or MeG, X9 is D, E, H, K, or N, X10 is I or Chg, X11 is Y, F4aao, 3Py6Ph, or 4Py, X12 is S, Q, Cit, Har, K, or R, [1] The peptide according to [1], wherein X14 is R, E, MeG, or K. [3] The peptide according to [1] or [2], comprising an amino acid sequence represented by formula A2, or an amino acid sequence represented by formula A2 in which 1 to 12 amino acid residues arbitrarily selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 14th amino acid residues from the N-terminus are substituted or deleted. A2: F-W-V-V-P-D-I-de-D-Chg-F4aao-Q-I-R-C (SEQ ID NO: 13) [4] The peptide according to any one of [1] to [3], comprising any one selected from the peptides of Peptide SEQ ID NOs: 1 to 65. [5] The peptide according to any one of [1] to [4], which is a cyclic peptide. [6] The peptide according to any one of claims [1] to [5], wherein one of the amino acid residues in the amino acid sequence represented by formula A1 is chloroacetylated, and the chloroacetylated amino acid residue and a cysteine ​​residue in the same peptide are linked intramolecularly to form a cyclic structure. [7] The peptide according to any one of claims [1] to [6], further comprising a glycine residue at the C-terminus. [8] The peptide according to any one of claims [1] to [7], further comprising an additional amino acid residue. [9] A peptide complex comprising two or more of the peptides according to any one of claims [1] to [8].

[10] The peptide complex according to [9], having FGFR1 agonist activity.

[11] A peptide complex according to any one of [9] to

[10] , comprising two or more peptides described in any one of [1] to [8] and a linker connecting the peptides, wherein the amino acid sequences of the peptides may be the same or different from each other.

[12] A peptide complex according to any one of [9] to

[11] , wherein the homology of the amino acid sequences of the peptides is 90% or more and 100% or less.

[13] A peptide complex according to any one of [9] to

[12] , wherein the two or more peptides are each cyclic peptides.

[14] A peptide complex according to any one of [9] to

[13] , wherein one of the amino acid residues contained in each of the peptides is chloroacetylated, and the chloroacetylated amino acid residue and a cysteine ​​residue contained in the same peptide are linked intramolecularly to form a cyclic structure.

[15] A peptide complex according to any one of [9] to

[14] , further comprising at least one of the two or more peptides an additional amino acid residue.

[16] The peptide complex according to any one of

[11] to

[15] , wherein the linker is a PEG linker or a linker consisting of PEG and an amino acid residue.

[17] The peptide complex according to any one of

[11] to

[16] , wherein the linker is one arbitrarily selected from linkers represented by linker structure numbers 1 to 23.

[18] The peptide complex according to any one of

[11] to

[17] , wherein the C-terminuses of the peptides or the side chains of the amino acid residues are linked via the linker.

[19] A cell culture composition used for cell culture, comprising the peptide according to any one of [1] to [8], or the peptide complex according to any one of [9] to

[18] .

[20] A composition used for medical, diagnostic, or research purposes, comprising the peptide according to any one of [1] to [8], or the peptide complex according to any one of [9] to

[18] .

[21] A method for producing a peptide complex, comprising the steps of producing a peptide according to any one of [1] to [8] and linking two or more of the peptides together with a linker.

[0014] According to the present invention, it is possible to provide a peptide having the ability to bind FGFR1, which is a receptor for FGF2, and preferably having the ability to bind FGFR1c; a peptide complex having the ability to activate an FGFR1 agonist, which is a receptor for FGF2, and preferably having the ability to activate an FGFR1c agonist; a peptide capable of forming a peptide complex; a cell culture composition containing a peptide or peptide complex; a composition used for medical, diagnostic, or research purposes; and a method for producing a peptide or peptide complex.

[0015] The following describes in detail embodiments of the peptides, peptide complexes, cell culture compositions, and compositions used for medical, diagnostic, or research purposes of the present invention. However, the present invention is not limited to the embodiments described below, and various modifications are possible as long as they do not depart from the spirit of the invention.

[0016] <Abbreviations> Å as angstrom (unit); BSA as bovine serum albumin; Boc as tert-butoxycarbonyl group; ClAc as chloroacetyl group; ClAcOSu as (2,5-dioxopyrrolidine-1-yl)2-chloroacetate; DCM as dichloromethane or methylene chloride; DIPCI as N,N'-diisopropylcarbodiimide; DIPEA or DIEA as N,N-diisopropylethylamine; DMSO as dimethyl sulfoxide; DMF as N,N-dimethylformamide; DODT as 3,6-dioxa-1,8-octanedithiol; DMEM as Dulbecco's modified Eagle medium; EC50 as 50% effective concentration; FBS as fetal bovine serum; Fmoc as 9-fluorenylmethyloxycarbonyl; Fmoc-Lys(Fmoc)-OH as N2,N6-bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine; g as grams; HATU as O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HPLC as high-performance liquid chromatography; LC-MS or LC / MS as liquid chromatography-mass spectrometry; M as molar; MeCN as acetonitrile; mg as milligrams; min as minutes; mL as milliliters; mM as millimolars; mm as millimeters; Mpe group as O-3-methylpento-3-yl group; NHS as N-hydroxysuccinimide; nm as nanometers; μL as microliters; Oxysuccinimide (OSu): 2, 2, 4, 6Pbf as 7-pentamethyldihydrobenzofuran-5-sulfonyl group; PEG as polyethylene glycol; rpm as revolutions per minute; Sub as dibenzosberyl group; tBu as tert-butyl group; TEAA as triethylamine acetate; TFA as trifluoroacetic acid; TIS as triisopropylsilane; Trt or Tr as trityl group; F2F: (S)-2-amino-3-(2-fluorophenyl)propanoic acid (CAS number: 19883-78-4) F3C: (S)-2-amino-3-(3-chlorophenyl)propanoic acid (CAS number: 80126-51-8) Cit: (S)-2-amino-5-ureidopentanoic acid (CAS number: 372-75-8) W7N: (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 49758-35-2) alI: L-alloisoleucine (CAS number: 1509-34-8) Hyp: (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (CAS No.: 51-35-4) 3Py6NH2: (S)-2-amino-3-(6-aminopyridine-3-yl)propanoic acid (CAS No.: 1269968-61-7) F4COO: (S)-4-(2-amino-2-carboxyethyl)benzoic acid (CAS No.: 126109-42-0) Nmm: N4-methyl-L-asparagine (CAS No.: 7175-34-0) Chg: (S)-2-amino-2-cyclohexylacetic acid (CAS No.: 14328-51-9) Gthp: (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (CAS No.: 811842-25-8) ApG: (3-aminopropyl)glycine (CAS number: 2875-41-4) de: D-glutamic acid (CAS number: 6893-26-1) dp: D-proline (CAS number: 344-25-2) MeG: methylglycine (CAS number: 107-97-1) F4aao: (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (CAS number: 24558-63-2) 4Py: (S)-2-amino-3-(pyridine-4-yl)propanoic acid (CAS number: 37535-49-2) 3Py6Ph: (S)-2-amino-3-(6-phenylpyridine-3-yl)propanoic acid (CAS number: 1335566-07-8) Har: N6-carbamimidoyl-L-lysine (CAS number: 156-86-5) cPEG9c: 4,7,10,13,16,19,22,25,28-nononaoxahentricontandedioic acid (CAS number: 1268488-70-5) cPEG13c: 4,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxatritetracontandedioic acid (CAS number: 2225903-66-0) cPEG17c: 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecaoxapentapentacontanediocate (CAS number: 2226897-74-9) NHS-cPEG9c-NHS: bis(2,5-dioxopyrrolidine-1-yl)4,7,10,13,16,19,22,25,28-nononaoxahentricontandioate (CAS number: 1008402-79-6) NHS-cPEG13c-NHS:bis(2,5-dioxopyrrolidine-1-yl)4,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxatritetracontandioate (Bis-PEG13-NHS ester) (CAS number: 2221949-00-2) NHS-cPEG17c-NHS: Bis(2,5-dioxopyrrolidine-1-yl)4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecaoxapentapentacontanediate (Bis-PEG17-NHS ester) (CAS number: 2221948-93-0) NHS-OCOPEG9OCO-NHS: Bis(2,5-dioxopyrrolidine-1-yl)(3,6,9,12,15,18,21-heptaoxatricosane-1,23-diyl)biscarbonate (NOF Corporation) NHS-OCOPEG13OCO-NHS: Bis(2,5-dioxopyrrolidine-1-yl)(3,6,9,12,15,18,21,24,27,30,33-undadecaoxaheptatetracontane-1,35-diyl) biscarbonate (TS-L12-TS) (NOF Corporation) NHS-OCOPEG17OCO-NHS: Bis(2,5-dioxopyrrolidine-1-yl)(3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-pentadecaoxaheptatetracontane-1,47-diyl) biscarbonate (TS-L16-TS) (NOF Corporation) OCOPEG1 OCO: Oxybis(ethane-2,1-diyl)bis(hydrogen carbonate) (CAS number: 57557-13-8) OCOPEG9 OCO: 3,6,9,12,15,18,21-heptaoxatricosan-1,23-diylbis(hydrogen carbonate) (NOF Corporation) OCOPEG13 OCO: 3,6,9,12,15,18,21,24,27,30,33-undedecaxpentatriacontane-1,35-diylbis(hydrogen carbonate) (NOF Corporation) OCOPEG17 OCO: 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-Pentadecaoxaheptatetracontane-1,47-diirbis(hydrogen carbonate) (NOF Corporation) PEG8c: 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-euic acid (CAS number: 756526-04-2) PEG12c: 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontane-39-euic acid (CAS number: 1415408-69-3)

[0017] In the following specification, the range of a numerical value indicated by "~" may be written as "-". The number of carbon atoms may be written using "C". For example, C1 represents 1 carbon atom, and C5 represents 5 carbon atoms.

[0018] <FGFs> FGFs (Fibroblast growth factors) are mitogenic factors that regulate various cellular functions, including migration, proliferation, differentiation, and survival. FGF signaling plays a crucial role in development, metabolism, and tissue homeostasis. Dysfunction of the FGF / FFFR signaling pathway is associated with various diseases, including congenital craniosynostosis, dwarfism, chronic kidney disease, obesity, insulin resistance, and cancer.

[0019] The FGF family is one of the most diverse growth factors in vertebrates, and 23 FGF ligands have been found in mice and humans. Based on sequence homology and phylogeny, 18 classical mammalian FGFs are classified into six subfamilies, including five paracrine subfamilies and one endocrine subfamily. The five paracrine subfamilies are the FGF1 subfamily (FGF1, FGF2), the FGF4 subfamily (FGF4, FGF5, FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, FGF22), the FGF8 subfamily (FGF8, FGF17, FGF18), and the FGF19 subfamily (FGF9, FGF16, FGF20). In addition, the FGF19 subfamily (FGF19, FGF21, FGF23) is endocrine and transmits signals (Signal Transduction and Targeted Therapy 2020, 5, 181.).

[0020] <FGFRs> FGFs bind to and activate high-affinity tyrosine kinase receptors encoded by four genes (FGFR1, FGFR2, FGFR3, FGFR4) and FGFR-L1 in mammals, thereby exerting various effects.

[0021] FGFRs (Fibroblast growth factor receptors) are single-pass transmembrane proteins containing an extracellular domain, a transmembrane domain (TMD), and an intracellular tyrosine kinase domain. Among the above domains, the extracellular domain is composed of three immunoglobulin-like domains (D1-D3), an acidic region, a heparin-binding motif of FGFs, and a heparin cofactor, etc. The TMD supports the receptor in the cell membrane and promotes dimerization of the receptor. In the cytoplasmic matrix, the region near the membrane of FGFRs is involved in receptor dimerization, and the kinase domain is necessary for FGF-related signal transduction (Signal Transduction and Targeted Therapy 2020, 5, 181.).

[0022] <FGF / FFFR Signaling Pathway> When FGFs bind to inactive monomeric FFFRs, a structural change occurs in the FFFRs, leading to dimerization. This phosphorylation of tyrosine residues in the cytoplasmic tail of the FFFRs activates cytoplasmic tyrosine kinases. Subsequently, the phosphorylated tyrosine residues act as docking sites for downstream signaling molecules such as 2α, a substrate of FFFRs located on the cell membrane. Furthermore, FFFRs form an allosteric 2:1 FFFR complex with phospholipase Cγ (PLCγ), recruiting and phosphorylating PLCγ, a substrate containing the SH2 domain.

[0023] As described above, FGF dimerization plays an essential role in substrate phosphorylation. Depending on the cellular contents in different cells and tissues, the classical FGF / FFFR downstream signaling pathway includes Ras / Raf-MEK-MAPKs (mitogenic factor-activated protein kinases), phosphatidylinositol-3-kinase / protein kinase B (PI3K / AKT), PLCγ, and signaling and transcriptional activators (STAT). Furthermore, the precise regulation of these signaling pathways can also be achieved by feedback inhibitors such as Sprouty (Spry), XFLRT3, SEF, and MKP3. These proteins themselves are regulated by FGF signaling and are co-expressed with FGFs. Many of these proteins inhibit the FGF / FFFR signaling pathway through a negative feedback mechanism ((a) Signal Transaction and Targeted Therapy 2020, 5, 181. (b) Developmental Biology 2005, 287, 390.).

[0024] <Peptide> The peptide of the first aspect of the present invention is a peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which one or more amino acid residues in the amino acid sequence represented by formula A1 are substituted, deleted, added or inserted. In formula A1, the amino acid sequence is described from the N-terminal side to the C-terminal side.Formula A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15

[0025] In the above formula A1, X1 is an amino acid residue having an optionally substituted aryl group in the side chain, X2 is an arbitrary amino acid residue, X3 is a Val residue, X4 is an amino acid residue having an aliphatic hydrocarbon group in the side chain, X5 is an optionally substituted 4-6 membered cyclic secondary amino acid residue, or an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, X6 is an arbitrary amino acid residue, X7 is an arbitrary amino acid residue, X8 is a glycine residue which may be N-alkylated (where the alkyl group constituting the N-alkylation may have a substituent), or an arbitrary D-amino acid residue, X9 is an arbitrary amino acid residue, X10 is an amino acid residue having an aliphatic hydrocarbon group in the side chain, X11 is an amino acid residue having an optionally substituted aryl group in the side chain, X12 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, X13 is an Ile residue, X14 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or an arbitrary N-alkyl amino acid residue, X15 is a Cys residue.

[0026] In the present specification and claims, "optionally substituted" for a part such as the side chain of an amino acid means that one or more of any hydrogen atoms possessed by that part may be substituted by any substituent or halogen atom. Examples of arbitrary substituents include an alkyl group having 1 to 4 carbon atoms, a carboxy group, a hydroxy group, an amino group, and the like.

[0027] The peptide in this embodiment is a peptide that can constitute a peptide complex, as described later. In other words, the peptide in this embodiment can also be said to be a material for producing a peptide complex. Furthermore, the peptide in this embodiment is a peptide that may have binding activity to FFFR1, which is preferable because it can achieve the effects of the present invention.

[0028] <Amino Acids> The amino acids mentioned above include not only natural amino acids but also unnatural amino acids. Examples of unnatural amino acids include N-alkylated amino acids, i.e., amino acids modified with a lower alkyl group (e.g., C1-C5, preferably C1-C3, more preferably C1) in which the nitrogen forming the peptide bond is branched or unbranched. As N-alkyl amino acids, N-ethyl amino acids and N-butyl amino acids are preferred, and N-methyl amino acids are more preferred.

[0029] Examples of unnatural amino acids include chemically modified amino acids such as D-type amino acids (also written as D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, and amino acid derivatives, as well as amino acids that do not serve as building blocks for proteins in the body, such as norleucine and ornithine.

[0030] Non-natural amino acids include amino acids in which functional groups have been further added to the side chain of a natural amino acid, or in which a different functional group has been substituted. Specifically, examples include amino acids with substitutions or additions to the arylene group or alkylene group of the side chain, amino acids with an increased number of carbon atoms in the arylene group, alkylene group or alkyl group of the side chain, amino acids with substitutions in the aromatic ring of the side chain, and heterocyclic or fused amino acids.

[0031] Examples of non-natural amino acids include N-methylamino acids, 4Py, alI, Cit, SMe, Atp, Hgl, NaL1, W6N, and W7N.

[0032] By adding or substituting functional groups or other structures to the side chains of natural amino acids, properties different from those of natural amino acids can be conferred. For example, A4p ((S)-2-amino-3-(piperidin-4-yl)propanoic acid (CAS: 342036-77-5)) is alanine with a piperidyl group added to its side chain. Due to the addition of this piperidyl group, it exhibits basic polarity, unlike alanine which belongs to the nonpolar amino acid group.

[0033] <Conservative Amino Acid Substitutions> When one, two, or three amino acid residues are substituted, deleted, added, or inserted from a specific sequence, it is preferable that conservative amino acid substitutions be made. A conservative amino acid substitution means a substitution with functionally equivalent or similar amino acids.

[0034] Conservative amino acid substitutions in peptides result in static changes to the peptide's amino acid sequence. For example, one or more amino acids with similar polarity act functionally equivalently, resulting in a static change to the amino acid sequence of such a peptide. In general, substitutions within a given group can be considered structurally and functionally conserved.

[0035] However, as is obvious to those skilled in the art, the role of a particular amino acid residue can be determined by its significance in the three-dimensional structure of the molecule containing that amino acid. For example, a cysteine ​​residue can take the less polar oxidized (disulfide) form compared to the reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute structurally and functionally important features. Also, side chains containing aromatic rings (such as tryptophan, tyrosine, and phenylalanine) can contribute to ion-aromatic or cation-pi interactions. In such cases, substituting amino acids with these side chains for amino acids belonging to the acidic or nonpolar group may be structurally and functionally conserved. Residues such as proline, glycine, and cysteine ​​(disulfide form) can have a direct effect on the three-dimensional structure of the main chain and often cannot be substituted without structural distortion.

[0036] Conservative amino acid substitutions include specific substitutions based on side chain similarity (Lehninger, Biochemistry, 2nd edition, 1975, pp. 73-75; Worth Publisher, New York (1975)) and typical substitutions, as shown below.

[0037] Furthermore, for conservative amino acid substitutions, it is preferable to substitute an amino acid with an amino acid belonging to the same group to which a given amino acid belongs, in a group of natural amino acids divided based on the properties of their common side chains, as shown below.

[0038] Hydrophobic (also called nonpolar) amino acids are amino acids that exhibit hydrophobicity (nonpolarity) and include alanine (also written as "Ala" or simply "A"), glycine (also written as "Gly" or simply "G"), valine (also written as "Val" or simply "V"), leucine (also written as "Leu" or simply "L"), isoleucine (also written as "Ile" or simply "I"), proline (also written as "Pro" or simply "P"), phenylalanine (also written as "Phe" or simply "F"), tryptophan (also written as "Trp" or simply "W"), tyrosine (also written as "Tyr" or simply "Y"), and methionine (also written as "Met" or simply "M").

[0039] Hydrophobic amino acids can be further divided into the following groups: Aliphatic amino acids are amino acids having an aliphatic hydrocarbon group or hydrogen in their side chain, and include Al, Gly, Val, Ile, and Leu. Aliphatic, branched-chain amino acids are amino acids having a branched aliphatic hydrocarbon group in their side chain, and include Val, Ile, and Leu. Aromatic amino acids are amino acids having an aromatic ring in their side chain, and include Trp, Tyr, and Phe.

[0040] Hydrophilic (also called polar) amino acids are amino acids that exhibit hydrophilicity (polarity), and include serine ("Ser" or simply "S"), threonine ("Thr" or simply "T"), cysteine ​​("Cys" or simply "C"), asparagine ("Asn" or simply "N"), glutamine ("Gln" or simply "Q"), aspartic acid ("Asp" or simply "D"), glutamic acid ("Glu" or simply "E"), lysine (also written as lysine; "Lys" or simply "K"), arginine ("Arg" or simply "R"), and histidine ("His" or simply "H").

[0041] Furthermore, hydrophilic amino acids can be further divided into the following groups: Acidic amino acids are those whose side chains are acidic and include Asp and Glu. Basic amino acids are those whose side chains are basic and include Lys, Arg and His. Neutral amino acids are those whose side chains are neutral and include Ser, Thr, Asn, Glun and Cys.

[0042] Furthermore, Gly and Pro can be classified as "amino acids that affect the orientation of the main chain," and Cys and Met, which are amino acids that contain sulfur molecules in their side chains, can be classified as "sulfur-containing amino acids."

[0043] The aforementioned groups, which classify natural amino acids based on their common side-chain properties, can include non-natural amino acids that possess similar side-chain properties. For example, N-methylarginine (MeR), the N-methylated amino acid of arginine, which belongs to the basic amino acids, is a non-natural amino acid, but because it exhibits basicity, it can be classified as a basic amino acid. In this way, non-natural amino acids that exhibit similar side-chain properties to a given amino acid can also be included as targets for conservative amino acid substitution.

[0044] <Peptides and their pharmaceutically acceptable salts, esters, or solvates> Examples of pharmaceutically acceptable salts include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, acidic and basic amino acid salts, etc.

[0045] Examples of inorganic salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate.

[0046] Examples of organic salts include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.

[0047] Examples of inorganic base salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, and ammonium salts.

[0048] Examples of organic base salts include diethylamine salt, diethanolamine salt, meglumine salt, and N,N'-dibenzylethylenediamine salt.

[0049] Examples of acidic amino acid salts include aspartate and glutamate. Examples of basic amino acid salts include arginine salt, lysine salt, and ornithine salt.

[0050] The peptide and the peptide complex containing the peptide may be pharmaceutically acceptable salts or solvates. Examples of solvates include hydrates.

[0051] In the peptide, X1 is an amino acid residue having an aryl group in its side chain, which may be substituted, and is preferably a Phe residue, an F2F residue, or an F3C residue. F2F and F3C have the structures of formula (1) and formula (2), respectively. In the following chemical formulas, "abs" refers to the stereocenter carbon atom, and indicates a carbon atom (chiral carbon atom) to which four different atoms or substituents are covalently bonded. For example, in formula (1), the carbon atom represented by abs is a carbon atom, a carboxyl group (-CO ２ H), amino group (-NH ２ ), and the chiral carbon atom bonded to the hydrogen atom (details omitted), which is the stereocenter.

[0052]

[0053] In the peptide, X2 is preferably a Trp residue, a Cit residue, or a W7N residue. Cit and W7N have the structures of formula (3) and formula (4), respectively.

[0054]

[0055] In the peptide described above, X3 is a Val residue. Preferably, the Val residue in X3 is not substituted or deleted.

[0056] In the peptide, X4 is an amino acid residue having an aliphatic hydrocarbon group in its side chain. The aliphatic hydrocarbon group in the amino acid represented by X4 may be linear or branched, but it is preferably branched. X4 is preferably a Val residue or an alI residue. alI has the structure of formula (5).

[0057]

[0058] In the peptide, X5 is a 4- to 6-membered cyclic secondary amino acid residue which may be substituted, or an amino acid residue which has an aliphatic hydrocarbon group which may be substituted in its side chain. The aliphatic hydrocarbon group may be an aliphatic cyclic group, and the number of carbon atoms constituting the aliphatic cyclic group is preferably, for example, 5 to 7. X5 is preferably, for example, a Pro residue, a Hyp residue, or a Thr residue. Hyp has the structure of formula (6).

[0059]

[0060] In the peptide, X6 is preferably an Asp residue, a 3Py6NH2 residue, a Glu residue, an F4COO residue, a Hyp residue, a Lys residue, an Asn residue, an Nmm residue, or a Ser residue. 3Py6NH2, F4COO, and Nmm have the structures of formulas (7), (8), and (9), respectively.

[0061]

[0062] In the peptide, X7 is preferably an Ile residue, a Chg residue, a Glu residue, a Gthp residue, a Lys residue, an Arg residue, a Thr residue, a Val residue, or a W7N residue. Chg and Gthp have the structures of formula (10) and formula (11), respectively.

[0063]

[0064] In the peptide, X8 is an N-alkylated glycine residue (wherein the alkyl group constituting the N-alkylation may have any substituent such as an amino group, a carboxyl group, or a hydroxyl group), or any D-amino acid residue. X8 is preferably, for example, a Gly residue, an ApG residue, a de residue, a dp residue, or a MeG residue. ApG, de, dp, and MeG have the structures of formulas (12), (13), (14), and (15), respectively.

[0065]

[0066] In the peptide, X9 is preferably an Asp residue, a Glu residue, a His residue, a Lys residue, or an Asn residue.

[0067] In the peptide, X10 is an amino acid residue having an aliphatic hydrocarbon group in its side chain. The aliphatic hydrocarbon group in the amino acid represented by X10 may be linear or branched, but it is preferably branched. The aliphatic hydrocarbon group may also be an aliphatic cyclic group, and the number of carbon atoms constituting the aliphatic cyclic group is preferably, for example, 5 to 7. X10 is preferably an Ile residue or a Chg residue.

[0068] In the peptide, X11 is an amino acid residue having an aryl group in its side chain, which may be substituted, and is preferably a Tyr residue, an F4aao residue, a 3Py6Ph residue, or a 4Py residue. F4aao, 3Py6Ph, and 4Py have the structures of formulas (16), (17), and (18), respectively.

[0069]

[0070] In the peptide, X12 is an amino acid residue having an optional aliphatic hydrocarbon group in its side chain. The aliphatic hydrocarbon group in the amino acid represented by X12 may be linear or branched, but is preferably linear. X12 is preferably a Ser residue, a Gln residue, a Cit residue, a Har residue, a Lys residue, or an Arg residue. Har has the structure of formula (19).

[0071]

[0072] In the peptide, X13 is an ile residue. Preferably, the ile residue in X13 is not substituted or deleted.

[0073] In the peptide, X14 is an amino acid residue having an optional aliphatic hydrocarbon group in its side chain, or any N-alkyl amino acid residue. The aliphatic hydrocarbon group in the amino acid represented by X14 may be linear or branched, but is preferably linear. X14 is preferably an Arg residue, a Glu residue, a MeG residue, or a Lys residue.

[0074] In the peptide, X15 is a Cys residue. Preferably, the Cys residue in X15 is not substituted or deleted.

[0075] Specific examples of peptides include the peptides with SEQ codes 1 to 65, listed in Tables 1 to 4. In Tables 1 to 4, the numbers shown below SEQ represent the order (amino acid number) counted from the N-terminus of each peptide. The "ClAc" indicated at the N-terminus of each peptide indicates that the amino group of the N-terminal amino acid is chloroacetylated.

[0076]

[0077]

[0078]

[0079]

[0080] The peptide in this embodiment may be a peptide containing the amino acid sequence shown in formula A2. In formula A2, the amino acid sequence is described from the N-terminus to the C-terminus. Furthermore, in the amino acid sequence shown in formula A2, at least one amino acid residue may be substituted or deleted in one to twelve amino acid residues selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 14th amino acid residues from the N-terminus. Formula A2: F-W-V-V-P-D-I-de-D-Chg-F4aao-Q-I-R-C (Sequence ID 13)

[0081] There are no particular limitations on the three-dimensional structure of the peptide, but it is preferable that it be a cyclic peptide. Advantages of a cyclic peptide include, for example, improved metabolic stability due to enhanced protease resistance, increased rigidity due to restrictions on conformational changes, improved membrane permeability, and improved affinity with target proteins.

[0082] If the peptide is a cyclic peptide, it is preferable that one of the amino acid residues in the peptide before cyclization is chloroacetylated, and that the chloroacetylated amino acid residue and a cysteine ​​residue in the same peptide before cyclization are intramolecularly linked to form a cyclic structure. If the peptide is a cyclic peptide, it is preferable that the chloroacetylated amino acid residue in the peptide before cyclization is the N-terminal amino acid residue. In other words, it is preferable that the N-terminal amino acid residue of the peptide before cyclization is a chloroacetylated amino acid residue, that there is a cysteine ​​residue in the same peptide before cyclization, and that the N-terminal amino acid residue and the cysteine ​​residue are linked to form a cyclic structure. The above advantages can be obtained by linking the chloroacetylated amino acid residue and the cysteine ​​residue to form a cyclic structure.

[0083] In this specification, a cyclic peptide includes peptides in which two amino acids are linked together to form a ring, in which amino acids in the peptide form a cross-linking structure, in which a cyclic structure is formed by lactam ring formation or macrocyclization reaction, and in which a lasso-peptide-like structure is present. In other words, in this specification, a cyclic peptide is one in which a part of it forms a cyclic structure, and it may also have a linear portion.

[0084] In this specification, some amino acids may be modified for the purpose of cyclization of peptides. Peptides as used herein also include peptides containing such modified amino acids. An example of modification for cyclization is the addition of a chloroacetyl group to the N-terminal amino acid, which then binds to a cysteine ​​residue in the peptide for cyclization. Peptides containing various (natural or unnatural) amino acids to which chloroacetyl groups have been added are also included in the definition of peptides as used herein.

[0085] There are no particular limitations on the method of peptide cyclization, and it can be carried out according to known methods. For example, by designing the peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed by disulfide bonds after translation. Alternatively, cyclization can be performed by synthesizing a peptide having a chloroacetyl group at the N-terminus using genetic code reprogramming techniques according to the method of Goto et al. (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)), and then placing cysteine ​​residues within the peptide. In this case, after translation, the mercapto group spontaneously nucleophilically attacks the chloroacetyl group, and the peptide is cyclized by thioether bonds. Cyclization may also be performed by placing other combinations of amino acids that bind to form a cyclic structure within the peptide using genetic code reprogramming techniques. Alternatively, cyclization may be performed by synthesizing a peptide having a cycloamide at the N-terminus, placing L-2-aminoadipic acid residues within the peptide, and then binding them together.

[0086] The amino acids that make up a cyclic peptide are those located between the amino acid residue indicated by X1 and the amino acid residue indicated by X15. One or more amino acid residues may be inserted between X1 and X15. In addition, one or more of the amino acid residues X2 to X14 may be deleted.

[0087] There is no particular upper limit to the number of amino acid residues constituting the cyclic peptide, but it is preferably 20 residues or less, more preferably 19 residues or less, or 18 residues or less, even more preferably 16 residues or less, and most preferably 15 residues. There is no particular lower limit to the number of amino acid residues constituting the cyclic peptide, but it is preferably 6 residues or more, 7 residues or more, 8 residues or more, 9 residues or more, 10 residues or more, or 11 residues or more, more preferably 13 residues or more, particularly preferably 14 residues or more, and most preferably 15 residues. The preferred ranges of the upper and lower limits can be combined as appropriate. The number of amino acid residues constituting the cyclic peptide is, for example, preferably 6 to 20 residues, more preferably 7 to 20 residues, more preferably 8 to 19 residues, more preferably 9 to 19 residues, more preferably 10 to 18 residues, more preferably 11 to 18 residues, more preferably 13 to 16 residues, more preferably 14 to 16 residues, and most preferably 15 residues.

[0088] The number of amino acid residues constituting the peptide before cyclization is preferably 6 to 20 residues, more preferably 7 to 20 residues, more preferably 8 to 19 residues, more preferably 9 to 19 residues, more preferably 10 to 18 residues, more preferably 11 to 18 residues, more preferably 13 to 16 residues, more preferably 14 to 16 residues, and most preferably 15 residues.

[0089] As a modification of the first aspect of the present invention, the peptide before cyclization may contain additional amino acid residues that are added to the N-terminus of X1 or the C-terminus of X15.

[0090] The number of amino acid residues added to the peptide can be appropriately selected depending on the type and purpose of the added amino acids, and is not particularly limited.

[0091] In the peptide before cyclization, it is preferable to have an additional Gly residue at the C-terminus of X15. There is no particular limit to the number of Gly residues, but 1 to 6 are preferred, and 1 to 2 are more preferred. When the peptide has the above preferred number of Gly residues at its C-terminus, the Gly residues become linkers or part of the linker structure when forming the peptide complex described later, allowing the peptide to be linked to another peptide. In this specification, such modifications may be referred to as peptide compounds or monomers.

[0092] <Method for producing peptides> A second aspect of the present invention is a method for producing peptides, comprising the steps of synthesizing the peptide of the first aspect and cyclizing the peptide.

[0093] In the step of synthesizing the peptide according to the first embodiment, the peptide is first synthesized by chemical synthesis. There are no particular limitations on the chemical synthesis method, and examples include solid-phase synthesis using a solid-phase synthesizer (e.g., Syro II, Biotage) and liquid-phase synthesis.

[0094] There are no particular limitations on the type of amino acid used as a raw material for peptides. Examples of amino acids that can be used as raw materials for peptides include amino acids protected with a benzyloxycarbonyl group (Cbz group) (Cbz-amino acids), amino acids protected with a tert-butoxycarbonyl group (Boc group) (Boc-amino acids), and amino acids protected with a 9-fluorenylmethoxy group (Fmoc group) (Fmoc-amino acids), with Fmoc-amino acids being preferred. By using Fmoc amino acids, they can be used in solid-phase synthesis.

[0095] The peptides obtained by the above synthesis method are preferably purified. There are no particular limitations on the method of purifying the peptides, and examples include High Performance Liquid Chromatography (HPLC) and crystallization.

[0096] There are no particular limitations on the linker's binding position in the cyclic peptide, but it is preferably on the C-terminal side of X15 of the peptide before cyclization. Specific examples of linkers are described later.

[0097] The temperature, time, and other conditions for peptide synthesis, peptide cyclization, and linker attachment can be appropriately selected to suit the peptide, cyclizing agent, and linker used.

[0098] <Peptide Complex> The peptide complex according to the third aspect of the present invention comprises two or more peptides of the first aspect. That is, the peptide complex according to the third aspect is formed in which two or more peptides of the first aspect are covalently bonded to each other. The form in which two or more peptides of the first aspect are covalently bonded to each other may be such that each of the two or more peptides of the first aspect is directly bonded to each other, or it may be such that they are covalently bonded to a linker, as described later, and bonded via the linker.

[0099] Preferably, the peptide complex has at least one of the following: FFFR1 agonist activity, MSC cell proliferation activity, and bovine cell proliferation activity.

[0100] The peptide complex is preferably used as a substitute peptide for FGF2.

[0101] The peptide complex preferably comprises two or more peptides according to the first embodiment and a linker connecting the peptides.

[0102] The peptide complex may be a homomultimer in which all the constituent peptides are the same peptide. In a homomultimer, the amino acid sequences of each peptide may be the same or different, as long as they are peptides of the first embodiment. Alternatively, the complex may be a heteromultimer having another peptide different from the peptide of the first embodiment.

[0103] There are no particular limitations on the amino acid sequence homology between the peptides contained in the peptide complex, but 90% to 100% is preferred, and 93% to 100% is more preferred. A person skilled in the art can appropriately select the combination of peptides constituting the peptide complex based on the activity of the peptides, etc., depending on the target peptide complex.

[0104] In this specification, amino acid sequence homology between peptides refers to the proportion of identical amino acid residues when two amino acid sequences are placed side by side. Amino acid homology between peptides can be calculated using known mathematical algorithms, for example, by the method described in paragraph

[0056] of Japanese Patent Application Publication No. 2024-054181. Specifically, for example, the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used to calculate under the following conditions (expected value = 10; gap allowed; matrix = BLOSUM62; filtering = OFF).

[0105] The number of peptides contained in the peptide complex is not particularly limited. Those skilled in the art can design and use the number of peptides contained in the peptide complex and the structure of the peptide complex as appropriate, depending on the solubility of the peptide complex in a solvent and the intended use. Examples of such numbers include 2 to 10, 2 to 8, 2 to 6, 2 to 4, 2 to 3, 3 to 5, or 4 to 6.

[0106] The peptides contained in the peptide complex may include peptides having an amino acid sequence different from that of the peptide represented by formula A1, peptide-containing compounds, and pharmaceutically acceptable salts thereof. The peptide complex may contain two or more peptides according to the first embodiment.

[0107] There are no particular limitations on the three-dimensional structure of the peptides included in the peptide complex, but it is preferable that each of the peptides is a cyclic peptide. Advantages of the cyclic structure of the peptides include, for example, improved metabolic stability due to increased protease resistance, increased rigidity due to restrictions on conformational changes, improved membrane permeability, and improved affinity with the target protein.

[0108] In the peptide complex described above, the peptide of the first embodiment is as described above under <Peptide>, so its explanation is omitted.

[0109] <Linker> In this specification, "linker" refers to a structure that binds two or more peptides of the first embodiment, or a structure that can bind two or more peptides of the first embodiment. If an additional amino acid is bound to a peptide that forms a cyclic structure, and two or more peptides are bound via the additional amino acid, the additional amino acid can also be considered a linker. Furthermore, if a linker described later is bound to the additional amino acid, the additional amino acid and the linker bound to the additional amino acid can together be considered a linker.

[0110] Examples of the linkers include amino acid linkers, chemical linkers, fatty acid linkers, nucleic acid linkers, and glycan linkers.

[0111] An amino acid linker is a linker containing at least one amino acid, and in this specification, a peptide linker is a type of amino acid linker. Examples of amino acid linkers that can be used include glycine-rich peptides such as peptides having the sequence [Gly-Gly-Gly-Gly-Ser]n (wherein n is 1, 2, 3, 4, 5, or 6) as described in U.S. Patent No. 7,271,149, and serine-rich peptide linkers as described in U.S. Patent No. 5,525,491. Adding an amino acid linker may change the physical properties of the peptide, such as its solubility.

[0112] Examples of chemical linkers include polyethylene glycol (PEG) linkers. There are no particular limitations on the number of ethylene glycol units (repeating units derived from ethylene glycol) contained in the PEG linker, but 1 to 50 are preferred, and 1 to 40 are more preferred.

[0113] Examples of fatty acid linkers include fatty acid linkers containing a divalent chemical moiety (a divalent organic group) derived from fatty acids.

[0114] As for the linker, a linker combining the above-mentioned linkers is preferred, and a linker combining a PEG linker and an amino acid linker is more preferred.

[0115] Examples of linkers combining PEG linkers and amino acid linkers include linker complexes in which PEG linkers and amino acid linkers are alternately bonded, linkers in which an amino acid linker is bonded to one end of a PEG linker, and linkers in which amino acid linkers are bonded to both ends of a PEG linker.

[0116] Examples of linkers in this embodiment include the linkers with linker structure numbers 1 to 23 shown in Tables 5 to 9. In the table, the dashed lines at the ends of the linker structures indicate that they are bound to a peptide. The linkers shown in Tables 5 to 6, with linker structure numbers 1 to 7, are linkers in which one end, indicated by the dashed line, is bound to a peptide, and the other parts can be bound to other peptides. The "binding to a peptide" indicated by the dashed line includes not only cases where the dashed portion is directly bound to the peptide, but also cases where the dashed portion is bound to the peptide via another linker.

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] There are no particular limitations on the linker binding site in peptides. For example, it may be bound to a Cys residue located at the C-terminus of a peptide, which binds to the first amino acid to form a cyclic structure, or to an amino acid such as glycine attached to the C-terminus of a Cys residue, or it may be bound to the side chain of an amino acid residue other than a Cys residue included in a cyclic peptide.

[0123] In peptide sequence number 12, it is preferable that the linker is bound to the side chain of the 8th ApG residue.

[0124] In peptide sequence number 26, it is preferable that the linker is bound to the side chain of the 12th Lys residue.

[0125] In peptide sequence number 30, it is preferable that the linker is bound to the side chain of the 14th Lys residue.

[0126] In peptide sequence number 34, it is preferable that the linker is bound to the side chain of the 9th Lys residue.

[0127] In peptide SEQ ID NO: 39, it is preferable that the linker is bound to the side chain of the seventh Lys residue.

[0128] In peptide sequence number 48, it is preferable that the linker is bound to the side chain of the sixth Lys residue.

[0129] <Composition> The peptide of the first embodiment and the peptide complex of the third embodiment may have the function of binding to FGFR1. In this case, by forming a composition in which a desired substance to be delivered to FGFR1 is bound to the peptide complex, it is possible to deliver the substance to FGFR1.

[0130] The substance is not particularly limited as long as it is a substance intended to be delivered to FGFR1. Examples of the substance include compounds, peptides different from those in the first embodiment described above, proteins, compounds containing Radioisotopes (RI), and nucleic acids.

[0131] The aforementioned compound may be a low-molecular-weight compound or a medium-molecular-weight compound, and examples include known low-molecular-weight drugs.

[0132] A peptide different from the first embodiment may be a peptide that binds to a target in the body and exerts some effect.

[0133] The aforementioned proteins are not particularly limited as long as they are proteins that exhibit useful functions in the body, such as antibodies and enzymes. Examples include enzymes used in enzyme replacement therapy.

[0134] The aforementioned RI-containing compounds are not particularly limited as long as they are compounds labeled with radioisotopes, and examples include low-molecular-weight and medium-molecular-weight compounds and antibodies labeled with radioisotopes. Examples of compounds labeled with radioisotopes include compounds used for PET scans.

[0135] The types of nucleic acids mentioned above are not particularly limited and include DNA, RNA, and DNA-RNA chimeras.

[0136] The substance may be a molecule used in a drug delivery system (DDS). The molecule used in the DDS may be a known molecule used in DDS, such as a liposome or micelle, and the DDS molecule may further contain compounds such as pharmaceuticals.

[0137] The substance intended to be delivered to FGFR1 may be a complex of the substances listed above. As a method for binding the desired substance to be delivered to FGFR1 to the peptide complex, in addition to the linker binding described above, known methods may be appropriately selected and used.

[0138] The composition comprising the peptide complex of the third embodiment may also contain a carrier. The carrier may be, for example, water such as sterile water, pure water, or distilled water; physiological saline solution; glucose solution; alcohol such as ethanol; polyalcohol such as glycerol, propylene glycol, or polyethylene glycol; sterile organic solvent; or aqueous starch; or a mixture of two or more of the following.

[0139] <Cell Culture Composition> A fourth aspect of the present invention, a cell culture composition used for cell culture, comprises the peptide of the first aspect or the peptide complex of the third aspect.

[0140] The peptide complex of the third embodiment has FGFR1 agonist activity and can therefore be used as a reagent or additive for cell culture media. The cells using the cell culture composition of this embodiment are preferably cells whose cell proliferation is activated by FGFR1 agonist activity. For example, mammalian cells are preferred, and human cells are more preferred.

[0141] The cell culture composition according to this embodiment may be used, for example, as a culture medium reagent or culture medium additive for culturing cells for the production of cultured meat.

[0142] The cell culture composition of this embodiment may be used, for example, as a culture medium reagent or additive for induced pluripotent stem cells or mesenchymal stem cells. FGF2 having FGFR1 agonist activity plays an important role in the proliferation of induced pluripotent stem cells while maintaining pluripotency (Non-Patent Literature 2), and the same is true for mesenchymal stem cells (Stem Cell Research & Therapy 2021, 12, 165). Therefore, the peptide complex of the third embodiment having FGFR1 agonist activity can be used, but is not limited to, for the purpose of maintaining pluripotency and promoting the proliferation of induced pluripotent stem cells or mesenchymal stem cells during culture.

[0143] The culture medium is not particularly limited as long as it is a culture medium for culturing cells or tissues. The culture medium is preferably a serum medium, more preferably a serum-free medium or a low-serum medium.

[0144] The cell culture composition may be in the form of a solution or a dry solid (e.g., solid or powder). If it is in the form of a solution, it may be used as is as a culture medium, or it may be diluted with a solvent and the above-mentioned additives added as necessary before being used as a culture medium. Examples of solvents used for dilution include water, buffer solutions, physiological saline, and various culture media used for cell and tissue culture, which may be used individually or in combination of two or more.

[0145] If the cell culture composition is in the form of a dry solid, it may be dissolved in a solvent such as water, buffer solution, physiological saline, and various cell and tissue culture media, and the above-mentioned additives may be added as necessary before being used as a culture medium.

[0146] The content of the peptide or peptide complex in the culture medium for culturing the cells or tissues, or in the culture medium for the cells obtained thereby, is preferably, for example, about 0.01 to about 10,000 nmol / L, more preferably about 0.1 to about 1,000 nmol / L, even more preferably about 0.5 to about 1,000 nmol / L, and particularly preferably about 1 to about 100 nmol / L, relative to the total amount of the composition or the total amount of the culture medium.

[0147] <Compositions for medical, diagnostic, or research use> A fifth aspect of the present invention, a composition for medical, diagnostic, or research use, comprises the peptide of the first aspect or the peptide complex of the third aspect. [Medical composition] The content of the peptide or the peptide complex in the medical composition which is the first embodiment of this aspect is not particularly limited.

[0148] The amount of the peptide or peptide complex contained in the medical composition when it is actually used is not particularly limited as long as it is an effective amount. The effective amount varies depending on the target organism, symptoms, patient's age, sex, weight, sensitivity differences, administration method, administration interval, type of active ingredient, and type of formulation.

[0149] When the aforementioned medical composition is used in mammals, it can be used in, for example, humans, cattle, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, and sheep. When the aforementioned medical composition is administered to humans, the dosage varies depending on the symptoms, the patient's age, sex, weight, sensitivity differences, method of administration, administration interval, type of active ingredient, and type of formulation, and is not particularly limited. For example, the dosage can be 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg in one dose or in several divided doses. In the case of injection administration, depending on the patient's weight, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg may be administered in one dose or in several divided doses.

[0150] The diseases targeted by the aforementioned medical compositions include, for example, diseases caused by an increase or decrease in the expression of FGFR1 or the activity of FGFR1, diseases that are exacerbated thereby, and all other diseases related thereto. Furthermore, examples include diseases caused by an increase or decrease in FGF / FGFR signaling or any other intracellular signaling cascade activated via FGFR1, diseases that are exacerbated thereby, and all other diseases related thereto. Specific examples of diseases targeted by the pharmaceutical compositions include, but are not limited to, cancers such as breast cancer and lung cancer (British Journal of Cancer 2021, 124, 880), wounds and skin ulcers, and ischemic diseases.

[0151] The administration method of the medical composition is not particularly limited and may be oral or parenteral. Parenteral administration methods include, for example, intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0152] The peptides in the aforementioned medical composition can be modified in various ways, taking into account their easily metabolized and excreted properties. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood retention time and reduce its antigenicity. Alternatively, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, and nanospheres may be used as sustained-release agents, and polypeptides may be encapsulated within them. When administering transdermally, iontophoresis, which involves passing a weak electric current through the skin surface to penetrate the stratum corneum, may be applied.

[0153] The aforementioned medical composition may use the active ingredient as is, or it may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.

[0154] The aforementioned formulation can be carried out by conventional methods, for example, by appropriately using excipients, binders, disintegrants, lubricants, solvents, solubilizers, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc.

[0155] Examples of ingredients used in the aforementioned formulation include, but are not limited to, purified water, saline solution, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicic acid, aluminum magnesium silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia gum, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, trehalose, and polysorbate.

[0156] The absorption enhancer can be used to improve the absorption of poorly absorbed drugs. The absorption enhancer may include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0157] The aforementioned pills or tablets may also be coated with a sugar coating, gastric-soluble, or enteric-coated substance. The aforementioned injectable preparation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, or alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solvents, solubilizers, or preservatives, etc., may be added.

[0158] The aforementioned medical composition may be administered in combination with other pharmaceuticals or treatments useful for the aforementioned disease.

[0159] [Diagnostic Composition] The diagnostic composition according to the second embodiment of this aspect can also be used as a diagnostic agent for detecting FGFR1 using the peptide or the peptide complex. The diagnostic agent may be a detection agent for detecting the expression level of FGFR1, and when used as a detection agent, the peptide or the peptide complex may be labeled in a detectable manner.

[0160] The content of the peptide or peptide complex in the diagnostic composition is not particularly limited as long as the expression level of FGFR1 can be detected.

[0161] The content of the peptide or peptide complex in the diagnostic composition when actually used is not particularly limited as long as it is in an effective amount. The effective amount varies depending on the type of sample being tested.

[0162] [Research Composition] The research composition, which is the third embodiment of this aspect, can be used for solubility in solvents, binding ability to FGFR1, toxicity tests on cells and tissues, and toxicity tests on experimental animals, etc.

[0163] When measuring solubility in a solvent, the solvent is not limited and can be freely selected according to the purpose. Furthermore, regarding the method of measuring solubility, a known method can be appropriately selected depending on the type of solvent.

[0164] The method for measuring the binding ability to FGFR1 is not particularly limited, as long as the binding ability of the peptide or peptide complex to FGFR1 can be measured. As the measurement method, known methods such as surface plasmon resonance (SPR) assay, scachard analysis, radioimmunoassay (RIA), enzyme immunoassay (EIA), and competitive binding assay can be preferably used.

[0165] Toxicity testing of cells and tissues is not limited to any known toxicity assessment test using cells and tissues, and may be performed in vitro, for example.

[0166] The cells and tissues subject to the aforementioned toxicity evaluation test may be any cells and tissues that are normally subjected to toxicity evaluation tests for pharmaceuticals, and there are no particular limitations.

[0167] There are no particular limitations on the methods for testing toxicity in experimental animals, as long as they are known toxicity evaluation tests using experimental animals. Examples include general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames test / chromosomal aberration test / in vitro micronucleus test), carcinogenicity tests, reproductive and developmental toxicity tests (ICH-I, II, III), local irritation tests (eye irritation test, skin irritation test, etc.), other toxicity tests (skin sensitization test, phototoxicity test, antigenicity test), and chemical analysis / biological analysis (TK / PK).

[0168] The experimental animals used in the aforementioned toxicity evaluation tests are not particularly limited as long as they are commonly used, and include, for example, mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cattle, birds (e.g., chickens, quail, etc.), monkeys, and primates other than humans (e.g., crab-eating macaques, marmosets, rhesus macaques, etc.).

[0169] <Method for producing a peptide complex> A sixth aspect of the present invention is a method for producing a peptide complex, comprising the steps of producing the peptide according to the first aspect and linking two or more of the peptides with a linker.

[0170] The peptide according to the first embodiment can be obtained by the method for producing the peptide according to the second embodiment.

[0171] Next, two or more peptides obtained by the peptide production method of the second embodiment are linked together with a linker. In the step of linking two or more peptides with a linker, the two or more peptides and the linker are mixed.

[0172] When mixing two or more peptides with a linker, the temperature, time, and other conditions can be appropriately selected to suit the peptides and linker being used.

[0173] One aspect of the present invention is the use of the peptide of the first embodiment or the peptide complex of the third embodiment for producing the composition. Another aspect of the present invention is the use of the peptide of the first embodiment or the peptide complex of the third embodiment for activating cell proliferation in cell culture.

[0174] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify or change the present invention based on the description herein, and such modifications are within the technical scope of the present invention.

[0175] In the following examples, for convenience, a compound in which one linker molecule is bonded to one peptide molecule will be referred to as a "monomer." A peptide complex in which two peptide molecules are bonded via a linker may be referred to as a "dimer." A dimer contains at least one linker. Examples of dimers include a complex in which the two monomer molecules are further bonded by another linker molecule, and a complex in which two peptide molecules are bonded by one linker molecule. A peptide complex in which three peptide molecules are bonded via a linker may be referred to as a "trimer." A trimer contains at least one linker. Examples of trimers include a complex in which the three monomer molecules are further bonded by another linker molecule, and a complex in which three peptide molecules are bonded by one linker molecule. A peptide complex in which four peptide molecules are bonded via a linker may be referred to as a "tetramer." A tetramer contains at least one linker. Examples of tetramers include a complex in which four monomer molecules are further linked by another linker molecule, and a complex in which four peptide molecules are linked by one linker molecule.

[0176] <Chemical Synthesis> In the following examples, all raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis were either commercially available or synthesized by those skilled in the art using organic chemical methods. Unless otherwise specified, amino acids containing protecting groups were commercially available.

[0177] Peptide chain elongation in solid-phase resins was carried out using the resins described in each example as starting materials, under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were performed using the automated peptide synthesizers Biotage Syro I, Biotage Syro II, CEM Liberty Blue, CEM Liberty Blue HT12, or CEM Liberty Prime, following the manufacturer's manual.

[0178] The resins used were NovaPEG Rink Amide resin or Sieber Amide resin, and the amount used ranged from 5 mg to 2 g depending on the peptide.

[0179] The reagent cocktail used for deprotecting the side chains and cleaving them from the solid resin was 4 mL to 50 mL depending on the peptide, and the following compositions were used: A: TFA / H ２ O / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) B:TFA / H ２ Tables 10-11 list the common Fmoc amino acids used in O / TIS / DODT (90 / 2.5 / 2.5 / 5), with side-chain protecting groups indicated in parentheses.

[0180]

[0181]

[0182] Unless otherwise specified, the crude peptides obtained were purified using one of the following reverse-phase preparative purifying apparatuses: A) Shimadzu prep-HPLC system (LC-20AP, SPD-M20A, CTO-20AC, and CBM-20A) B) Waters AutoPurification System C) Waters AutoPurification System with SQD D) Waters Preparative HPLC System E) YMC Conticrom® CUBE

[0183] Unless otherwise specified, the columns used were one of the following a)-l). a) Kinetex EVO C18 30x150mm b) XBridge C18 5μm 19x150mm c) XBridge C18 5μm 30x150mm d) XBridge C18 5μm 50x150mm e) XBridge C18 5μm 50x250mm f) XSelect C18 5μm 19x150mm g) XSelect C18 5μm 30x150mm h) 50x250mm i)XSelect Fluoro-Phenyl 5μm 10x150mm j) YMC-Actus Triart Prep C18-S 10μm 20x250mm k) YMC-Triart Prep C18-S 10μm 10x250mm l) Jeanious One-Column 20mmx150mm

[0184] The structure of chemically synthesized peptides was determined by calculating the molecular weight, considering the amino acids used according to the target sequence and the building blocks used as needed, and then confirming this by ESI-MS(+) in mass spectrometry. ESI-MS(+) refers to electrospray ionization mass spectrometry performed in positive ion mode. The detected mass was reported in "m / z" units. Compounds with molecular weights greater than approximately 1000 were frequently detected as polyvalent ions.

[0185] <Basic Analytical Instruments and Conditions> Unless otherwise specified, the following basic analytical instruments and conditions were used for the mass spectral analysis of the peptides synthesized in the following examples. The gradient B (%) was analyzed using one of the following conditions: w / x / y / z.

[0186] [Basic analysis equipment] Equipment: Waters AutoPurification System-SQD2 single quadruple mass spectrometer

[0187] [Basic Conditions] Column: Kinetex EVO C18 1.7 μm 2.1 x 50 mm, 100 Å Column temperature: 60°C Mobile phase A: 0.025% TFA in H ２ O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.6 mL / min Wavelength: 220 nm Gradient B (%): w) 5-95% / 2.10 min, 95-95% / 0.75 min

[0188] [Basic analysis equipment] Equipment: Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020)

[0189] [Basic Conditions] Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Column temperature: 60°C Mobile phase A: 0.025% TFA in H ２ O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.5 mL / min Wavelength: 225 nm PDA Gradient B (%): x) 20-60% / 7.15 min, 60-95% / 0.3 min, 95-95% / 1.55 ​​min y) 40-80% / 7.15min, 80-95% / 0.3min, 95-95% / 1.55min z) 5-45% / 7.15min, 45-95% / 0.3min, 95-95% / 1.55min

[0190] <Example 1> Synthesis of dimer structure number 11. Dimer structure number 11 has the structure shown in formula (20).

[0191]

[0192] Using Sieber amide TG-resin, the synthesis began with the removal of the Fmoc group using the general method described above. For the 18th residue, Fmoc-K(Fmoc)-OH (CAS number: 78081-87-5) was used to synthesize the target peptide complex (dimer structure number 11). Biotage's Syro II was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / HATU / DIEA (6.3 equivalents / 6 equivalents / 12.6 equivalents) was used per equivalent of resin, and the reaction was carried out twice for 30 minutes at 75°C in DMF. However, for the 6th, 9th, 12th, 13th, 14th, and 15th residues, the reaction was carried out twice for 30 minutes at 50°C. For the 16th and 18th residues, the reaction was carried out twice for 20 minutes at 75°C. The 17th residue was reacted once at 25°C for 60 minutes.

[0193] To remove the Fmoc group, the mixture was reacted with a 20% piperidine DMF solution at room temperature for 5 minutes, then the solution was removed, and the mixture was reacted again with a 20% piperidine DMF solution at room temperature for 15 minutes.

[0194] The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group in the solid resin holding the Fmoc-protected peptide obtained in the previous step using the method described above, and then adding a DMF solution of ClAcOSu (10 equivalents) to the solid resin and shaking at room temperature for 60 minutes.

[0195] Deprotection of the side chains and cleavage from the solid phase resin are performed by adding the reagent cocktail A (TFA / H) to the reaction vessel containing the solid phase resin. ２ A mixture of O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5 was added and shaken well, then shaken at room temperature for 90 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to chilled excess diisopropyl ether, a turbid precipitate formed, and the mixture was centrifuged and the solution decanted. The obtained solid was washed with a chilled diethyl ether / hexane (1 / 1) mixed solvent and dried for 60 minutes. The obtained solid was used in the next cyclization reaction.

[0196] The peptide cyclization reaction is carried out using DMSO / H2O, where the final peptide concentration is 2.5 mM based on the number of moles of the solid phase resin. ２After dissolving in O(7 / 3), triethylamine (40 equivalents) was added and stirred, and then left standing at room temperature overnight. The resulting reaction solution was concentrated under reduced pressure using GeneVac EZ-2 Elite to a concentration of 12.5 mM.

[0197] The obtained crude product was subjected to solid-phase extraction using a Gilson column in the following steps (column: Gilson ASPEC C18 500 mg 3 mL): (1) The column was washed with extraction solution A (0.1% TFA in 95% MeCN / H ２ O, 3 mL). (2) The column was equilibrated with extraction solution B (0.1% TFA in 5% MeCN / H ２ O, 3 mL). (3) 0.2 mL of the above solution was loaded onto the column. (4) The column was washed with extraction solution B (4 mL). (5) Extraction was performed with extraction solution A (4 mL). The obtained extract was concentrated under reduced pressure using EZ-2 Elite. Thereby, a cyclic peptide complex in which the peptide of peptide sequence number 1 was cyclized was synthesized.

[0198] One of the main peaks of the peptide complex represented by dimer structure number 11, which is the target product, had a purity calculated from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analysis conditions and was 33%. [Analysis conditions] Analysis conditions: retention time = 1.53 minutes Column: Kinetex EVO C18 1.7 μm 2.1x50 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60 °C Gradient (% B conc): 5 - 95% over 2.10 minutes, then 95 - 95% over 0.75 minutes Flow rate: 0.6 mL / min ESI-MS(+) observed value m / z = 1243 (M + 4H)4+

[0199] <Example 2> Synthesis of monomer structure number 1-1. Monomer structure number 1-1 has the structure shown in formula (21).

[0200]

[0201] Using Sieber amide resin, the target peptide (monomer structure number 1-1) was synthesized starting with the removal of the Fmoc group using the general method described above. A CEM Liberty BLUE HT was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (5.25 equivalents / 10 equivalents / 5 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. However, the 4th residue was reacted twice for 30 minutes at 75°C. The 5th residue was reacted twice for 3 minutes at 90°C. The 14th residue was reacted twice for 15 minutes at 50°C. The 15th residue was reacted once for 15 minutes at 50°C.

[0202] The Fmoc group was removed by reacting the residue with a 20% piperidine DMF solution at room temperature for 5 minutes twice. However, residues 10, 11, 12, 13, 14, and 15 were reacted at 75°C for 3 minutes.

[0203] The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group in the solid resin holding the Fmoc-protected peptide obtained in the previous step using the method described above, and then shaking it in DMF at room temperature for 30 minutes with ClAcOH / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents).

[0204] Deprotection of the side chains and cleavage from the solid phase resin are performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, drying under reduced pressure, and then adding the reagent cocktail A (TFA / H) to the reaction vessel containing the solid phase resin. ２ A mixture of O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5 was added and shaken at room temperature for 50 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to a cooled excess diisopropyl ether / hexane (1 / 1) mixed solvent, a turbid precipitate formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed with cooled diethyl ether and dried under reduced pressure. The resulting solid was used in the next cyclization reaction.

[0205] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the number of moles of the solid phase resin, adding triethylamine (10 equivalents), shaking at room temperature for 15 hours, and then adding acetic acid. The resulting reaction solution was concentrated using EZ-2 Elite. The resulting crude product was purified under the following conditions. This resulted in the synthesis of a cyclic peptide in which the peptide with monomer structure number 1-1 was cyclized.

[0206] [Purification Conditions] Column: Jeanious One-Column 20mm x 150mm Mobile Phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: Room temperature Gradient (%B conc): 25.1%–45.1% over 9 minutes, then 45.1–90% over 0.01 minutes Flow rate: 20 mL / min

[0207] The purity of the target peptide, represented by monomer structure number 1-1, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 90.42%.

[0208] [Analysis Conditions] Retention time: 4.40 min Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min; ESI-MS(+) observed value m / z = 941(M+2H)²+

[0209] <Example 3> Synthesis of monomer structure number 13-2. Monomer structure number 13-2 has the structure shown in formula (22).

[0210]

[0211] Using Sieber amide resin, the target peptide (monomer structure number 13-2) was synthesized starting with the removal of the Fmoc group using the general method described above. A Biotage Syro I was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) was used per equivalent of resin, and the reaction was carried out once for 10 minutes at 75°C in DMF. However, the 14th residue was reacted twice at room temperature for 20 minutes each time. The 15th residue was reacted once at room temperature for 20 minutes each time. Furthermore, when introducing Fmoc-F4aao(tBu)-OH and Fmoc-Chg-OH, amino acids dissolved in NMP were used.

[0212] The Fmoc group was removed by reacting it with a 10% pyrrolidine DMF solution at room temperature for 3 minutes.

[0213] The chloroacetyl group was introduced by adding a DMF solution of ClAcOSu (10 equivalents) to the solid resin and shaking it at room temperature for 60 minutes.

[0214] Deprotection of the side chains and cleavage from the solid phase resin are performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, drying under reduced pressure, and then adding the reagent cocktail A (TFA / H) to the reaction vessel containing the solid phase resin. ２ A mixture of O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5 was added and shaken at room temperature for 60 minutes. The reaction mixture was collected by filtration through the frit. When this filtrate was added to a cooled excess diethyl ether / hexane (1 / 1) mixed solvent, a turbid precipitate formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed with cooled diethyl ether again and dried under reduced pressure. The resulting solid was used in the next cyclization reaction.

[0215] The peptide cyclization reaction is carried out using DMSO / H2O, where the final peptide concentration is 5 mM based on the number of moles of the solid resin. ２After dissolving in O(9 / 1), triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 1 hour, after which acetic acid was added. The resulting reaction solution was concentrated using EZ-2 Elite. The resulting crude product was purified under the following conditions. This synthesized a cyclic peptide in which the peptide with monomer structure number 13-2 was cyclized.

[0216] [Purification conditions] Column: Waters XSelect CSH Prep C18 5μm OBD 50x250mm Mobile phase: A = 20mM TEAA in H ２ O, B = 20mM TEAA in MeCN, C = 0.2M TEAA in H ２ O, D = MeCN Temperature: 50°C Gradient (%A conc): 0.1% over 5.0 minutes, then 0.1% over 0.1 minutes, then (100% - %B) after 5.1 minutes; (%B conc): 0% over 5.1 minutes, then 0-4.2% over 1.9 minutes, then 4.2-24.6% over 3 minutes, then 24.6-29.7% over 15.5 minutes, then 29.7-60% over 1.5 minutes, then 60-90% over 4 minutes; (%C conc): 99.9% over 5.0 minutes, then 99.9-0% over 0.1 minutes, then 0% after 5.1 minutes. Flow rate: 18 mL / min over 5.1 minutes, then (18 mL / min - 118 mL / min) over 1.9 minutes, then 118 mL / min. After freeze-drying, 0.1% TFA in MeCN / H ２ O(1 / 1) was added.

[0217] The purity of the target peptide, represented by monomer structure number 13-2, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 96.39%.

[0218] [Analysis Conditions] Retention time: 3.44 mins Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min ESI-MS(+) observed value m / z = 1103(M+2H)²+

[0219] <Example 4> Synthesis of dimer structure number 27. Dimer structure number 27 has the structure shown in formula (23).

[0220]

[0221] The peptide complex (dimer structure number 27) was synthesized by dissolving monomer structure number 13-2 (24 mg) in DMSO, then adding NHS-OCOPEG13OCO-NHS (0.45 equivalents) and DIEA (10 equivalents), stirring at room temperature for 3 hours, and finally adding acetic acid. The resulting crude product was purified under the following conditions.

[0222] [Purification Conditions] Column: Waters XSelect C18 5μm 19x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN. Temperature: 60°C. Gradient (%B conc): 10-35% over 3.0 minutes, then 35-40% over 8 minutes, then 40-60% over 1 minute. Flow rate: 17 mL / min.

[0223] The purity of the target peptide complex, represented by dimer structure number 27, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 98.31%.

[0224] [Analysis Conditions] Retention time: 4.77 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H ２O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min ESI-MS(+) observed value m / z = 1253(M+4H)4+

[0225] <Example 5> Synthesis of monomer structure number 13-5. Monomer structure number 13-5 has the structure shown in formula (24).

[0226]

[0227] Using Sieber amide resin, the target peptide (monomer structure number 13-5) was synthesized starting with the removal of the Fmoc group using the general method described above. A Biotage Syro I was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) was used per equivalent of resin, and the reaction was carried out once for 10 minutes at 75°C in DMF. However, the 14th residue was reacted twice at room temperature for 20 minutes each time. The 15th residue was reacted once at room temperature for 20 minutes each time. Furthermore, when introducing Fmoc-F4aao(tBu)-OH and Fmoc-Chg-OH, amino acids dissolved in NMP were used.

[0228] The Fmoc group was removed by reacting it with a 10% pyrrolidine DMF solution at room temperature for 3 minutes.

[0229] The chloroacetyl group was introduced by adding a DMF solution of ClAcOSu (10 equivalents) to the solid resin and shaking it at room temperature for 60 minutes.

[0230] Deprotection of the side chains and cleavage from the solid phase resin are performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, drying under reduced pressure, and then adding the reagent cocktail A (TFA / H) to the reaction vessel containing the solid phase resin. ２A mixture of O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5 was added and shaken at room temperature for 60 minutes. The reaction mixture was collected by filtration through the frit. When this filtrate was added to a cooled excess diethyl ether / hexane (1 / 1) mixed solvent, a turbid precipitate formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed with cooled diethyl ether again and dried under reduced pressure. The resulting solid was used in the next cyclization reaction.

[0231] The peptide cyclization reaction is carried out using DMSO / H2O, where the final peptide concentration is 5 mM based on the number of moles of the solid resin. ２ After dissolving in O(9 / 1), triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 1 hour, after which acetic acid was added. The resulting reaction solution was concentrated using EZ-2 Elite. The resulting crude product was purified under the following conditions. This synthesized a cyclic peptide in which the peptide with monomer structural numbers 13-5 was cyclized.

[0232] [Purification Conditions] Column: Waters XBridge C18 5μm 50x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc): 5% over 2.0 minutes, then 5-29% over 1 minute, then 29-34% over 8 minutes, then 29-60% over 1 minute Flow rate: 20 mL / min over 1 minute, then (20 mL / min - 120 mL / min) over 1 minute, then 120 mL / min.

[0233] The purity of the target peptide, represented by monomer structure number 13-5, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 92.10%.

[0234] [Analysis Conditions] Retention time: 3.55 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H ２O, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes; Flow rate: 0.5 mL / min; ESI-MS(+) observed value m / z = 877(M+3H)3+

[0235] <Example 6> Synthesis of trimmer structure number 1. Trimmer structure number 1 has the structure shown in formula (25).

[0236]

[0237] The peptide complex (trimer structure number 1) was synthesized by dissolving monomer structure number 13-5 (22 mg) in DMSO, then adding Atcemm_OCOPEG8Me-NHS3 (0.28 equivalents) and DIEA (15 equivalents), stirring at room temperature for 2 hours, and finally adding acetic acid. The resulting crude product was purified under the following conditions.

[0238] [Purification Conditions] Column: Waters XBridge C18 5μm 30x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN. Temperature: 50°C. Gradient (%B conc): 11-36% over 3.0 minutes, then 36-41% over 8 minutes, then 41-60% over 1 minute. Flow rate: 45 mL / min.

[0239] The purity of the target peptide complex, represented by trimer structure number 1, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 97.49%.

[0240] [Analysis Conditions] Retention time: 5.07 mins; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H ２O, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes; Flow rate: 0.5 mL / min; ESI-MS(+) observed value m / z = 1431(M+6H)6+

[0241] <Example 7> Synthesis of tetramer structure number 6. Tetramer structure number 6 has the structure shown in formula (26).

[0242] Monomer structure number 13-5 (20 mg) was dissolved in a DMSO solution of DIPEA (40 mM, 8 equivalents), and then a 10 mM DMSO solution of Boc-PEG8cCebA(NHS)-NHS (0.46 equivalents) was added. After stirring at room temperature for 4 hours, the solvent was removed by distillation, and the resulting crude product was treated with TFA / TIS / H ２ The peptide was dissolved in an O / DODT (92.5 / 2.5 / 2.5 / 2.5) mixed solvent under ice cooling. After stirring for 10 minutes, the reaction solution was added dropwise to ice-cooled isopropyl ether, and the precipitated solid was precipitated by centrifugation, and the supernatant was removed. The obtained solid was washed again with isopropyl ether, and the peptide dimer was obtained by the same procedure as above. The obtained crude product was dissolved in a DMSO solution of DIPEA (40 mM, 4 equivalents), and then a DMSO solution of NHS-OCOPEG1OCO-NHS (5 mM, 0.2 equivalents) was added, and the mixture was stirred at room temperature for 16 hours.

[0243] The obtained crude product was purified using the following conditions.

[0244] [Purification Conditions] Column: Waters XBridge C18 5μm 30x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN. Temperature: 50°C. Gradient (%B conc): 12-37% over 3.0 minutes, then 37-42% over 8 minutes, then 42-60% over 1 minute. Flow rate: 45 mL / min.

[0245] The purity of the target peptide complex, represented by tetramer structure number 6, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 97.30%.

[0246] [Analysis Conditions] Retention time: 5.34 mins Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min SI-MS(+) observed value m / z = 1682(M+7H)7+

[0247] <Example 8> Synthesis of monomer structure number 21-5. Monomer structure number 21-5 has the structure shown in formula (27).

[0248]

[0249] Using Sieber amide resin, the target peptide (monomer structure number 21-5) was synthesized starting with the removal of the Fmoc group using the general method described above. A CEM Liberty BLUE HT was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. However, for the 3rd and 4th residues, the reaction was carried out twice for 10 minutes at 90°C. For the 13th and 17th residues, the reaction was carried out once for 10 minutes at 90°C. For the 14th residue, the reaction was carried out twice for 15 minutes at 50°C. For the 15th residue, the reaction was carried out once for 15 minutes at 50°C.

[0250] The Fmoc group was removed by reacting the residue with a 10% pyrrolidine DMF solution at 50°C for 90 seconds. However, residues 9, 10, 13, 14, 15, 16, 17, and 18 were reacted at 90°C for 1 minute.

[0251] The chloroacetyl group was introduced by adding a DMF solution of ClAcOSu (5 equivalents) to the solid resin and shaking it at room temperature for 60 minutes.

[0252] Deprotection of the side chains and cleavage from the solid phase resin are performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, drying under reduced pressure, and then adding the reagent cocktail A (TFA / H) to the reaction vessel containing the solid phase resin. ２ A mixture of O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5 was added and shaken at room temperature for 60 minutes. The reaction mixture was collected by filtration through the frit. When this filtrate was added to a cooled excess diisopropyl ether / hexane (1 / 1) mixed solvent, a turbid precipitate formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed with cooled diethyl ether and dried under reduced pressure. The resulting solid was used in the next cyclization reaction.

[0253] The peptide cyclization reaction is carried out using DMSO / H2O, where the final peptide concentration is 1.4 mM based on the number of moles of the solid resin. ２ After dissolving in O(9 / 1), triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 1 hour, after which acetic acid was added. The resulting reaction solution was concentrated using HT-12. This synthesized a cyclic peptide in which the peptide with monomer structure number 21-5 was cyclized. The resulting crude product was purified under the following conditions.

[0254] [Purification Conditions] Column: Waters XBridge C18 5μm 50x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc): 5% over 2.0 minutes, then 5-30% over 1 minute, then 30-35% over 8 minutes, then 35-60% over 1 minute Flow rate: 20 mL / min over 1 minute, then (20 mL / min - 120 mL / min) over 1 minute, then 120 mL / min.

[0255] The purity of the target peptide, represented by monomer structure number 21-5, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 91.11%.

[0256] [Analysis Conditions] Retention time: 3.89 min Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min ESI-MS(+) observed value m / z = 844(M+3H)3+

[0257] <Example 9> Synthesis of dimer structure number 42. Dimer structure number 42 has the structure shown in formula (28).

[0258]

[0259] The peptide complex (dimer structure number 42) was synthesized by dissolving monomer structure numbers 21-5 (20 mg) in DMSO, then adding OCOPEG8Me-CebA-NHS2 (0.5 equivalents) and DIEA (4 equivalents), and stirring at room temperature for 20 hours. The resulting crude product was purified under the following conditions.

[0260] [Purification Conditions] Column: Waters XBridge C18 5μm 19x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN. Temperature: 50°C. Gradient (%B conc): 14-39% over 3.0 minutes, then 39-44% over 8 minutes, then 44-60% over 1 minute. Flow rate: 17 mL / min.

[0261] The purity of the target peptide complex, represented by dimer structure number 42, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 99.01%.

[0262] [Analysis Conditions] Retention time: 5.36 mins Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min ESI-MS(+) observed value m / z = 1400(M+4H)4+

[0263] <Example 10> Synthesis of dimer structure number 8. Dimer structure number 8 has the structure shown in formula (29).

[0264] Using Sieber amide resin, the process began with the removal of the Fmoc group using the general method described above. For the 18th residue, Fmoc-K(Fmoc)-OH (CAS number: 78081-87-5) was used to synthesize the target peptide complex (dimer structure number 8). CEM's Liberty BLUE was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (8.4 equivalents / 16 equivalents / 8 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. However, for the 14th residue, the reaction was carried out twice for 15 minutes at 50°C. For the 15th residue, the reaction was carried out once for 15 minutes at 50°C.

[0265] The Fmoc group was removed by reacting the residue with a 10% pyrrolidine DMF solution at room temperature for 1 minute twice. However, the 9th residue was reacted once at 50°C for 90 seconds. The 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, and 20th residues were reacted at 90°C for 1 minute.

[0266] The chloroacetyl group was introduced by adding a DMF solution of ClAcOSu (10 equivalents) to the solid resin and shaking it at room temperature for 30 minutes.

[0267] Deprotection of the side chains and cleavage from the solid phase resin are performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, drying under reduced pressure, and then adding the reagent cocktail A (TFA / H) to the reaction vessel containing the solid phase resin. ２ A mixture of O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5 was added and shaken at room temperature for 90 minutes. The reaction mixture was collected by filtration through the frit. When this filtrate was added to a cooled excess diethyl ether / hexane (1 / 1) mixed solvent, a turbid precipitate formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed with cooled diethyl ether again and dried under reduced pressure. The resulting solid was used in the next cyclization reaction.

[0268] The peptide cyclization reaction is carried out using DMSO / MeCN / H2O, where the final peptide concentration is 2.5 mM based on the number of moles of the solid resin. ２ After dissolving in O(1 / 1 / 1), triethylamine (20 equivalents) was added, and the mixture was shaken at room temperature for 15 hours, after which acetic acid was added. The resulting reaction solution was concentrated using EZ-II elite. This synthesized a cyclic peptide complex in which the peptide of peptide SEQ ID NO: 1 was cyclized. The resulting crude product was purified under the following conditions.

[0269] [Purification Conditions] Column: Waters XBridge C18 5μm 50x250mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: 50°C Gradient (%B conc): 10.3–6.7% over 0.1 minutes, then 6.7% over 4.9 minutes, then 6.7–10.3% over 2 minutes, then 10.3–35.6% over 3 minutes, then 35.8–40.8% over 15 minutes, then 40.8–60% over 3 minutes. Flow rate: 118–18 mL / min over 0.1 minutes, then 18 mL / min over 4.9 minutes, then 18–118 mL / min over 2 minutes, then 118 mL / min.

[0270] The purity of the target peptide complex, represented by dimer structure number 8, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 85.56%.

[0271] [Analysis Conditions] Retention time: 4.80 min Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min ESI-MS(+) observed value m / z = 1318(M+4H)4+

[0272] <Example 11> Synthesis of tetramer structure number 3. Tetramer structure number 3 has the structure shown in formula (30).

[0273]

[0274] In Table 20, described later, the monomers represented by monomer structures 1-11 in the peptide complex represented by tetramer structure number 3 represent a 2:1 bond between the peptide of peptide sequence number 1 and the linker of linker structure number 11.

[0275] The peptide complex was synthesized by dissolving dimer structure number 8 (6 mg) shown in formula (29) in DMSO, then adding NHS-OCOPEG9OCO-NHS (0.4 equivalents) and DIEA (15 equivalents), stirring at room temperature for 6 hours, and finally adding acetic acid. The resulting crude product was purified under the following conditions.

[0276] [Purification Conditions] Column: Waters XBridge C18 5μm 19x150mm Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN. Temperature: 50°C. Gradient (%B conc): 15% over 3.0 minutes, then 15-65% over 9 minutes. Flow rate: 17 mL / min.

[0277] The purity of the target peptide complex, represented by tetramer structure number 3, was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 95.02%.

[0278] [Analysis Conditions] Retention time = 5.52 mins Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å Mobile phase: A = 0.025% TFA in H ２ O, B = 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc): 20-60% over 7.15 minutes, then 60-95% over 0.30 minutes, then 95-95% over 1.55 minutes Flow rate: 0.5 mL / min ESI-MS(+) observed value m / z = 1371(M+8H)8+

[0279] <Example 12> Synthesis of Atcemm_OCOPEG8Me-NHS3 used in Example 6. Atcemm_OCOPEG8Me-NHS3 has the structure shown in formula (31).

[0280] To a 5 mL solution of di-tert-butyl 3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropionate (CAS No.: 175724-30-8) (1.41 g, 2.79 mmol) in DCM (5 mL), TFA (5.37 mL) was added under ice cooling. After stirring at room temperature for 5 hours, the solution was concentrated and azeotropic dilution with toluene was performed.

[0281] The crude product obtained was dissolved in acetonitrile (10 mL), and then a solution of 2,5-Dioxo-1-pyrrolidinyl 2,5,8,11,14,17,20,23,26-nonaoxaheptacosanoate (CAS No.: 1372860-04-2) (1.41 g, 2.79 mmol) in acetonitrile (10 mL) and DIPEA (3.00 mL, 17.17 mmol) were added. After stirring for 24 hours, DCM and H were added. ２ After adding oxygen and concentrating the resulting aqueous layer, it was purified using reverse-phase column chromatography.

[0282] The recovered fraction was freeze-dried and dissolved in DCM (15 mL). WSCI (1.41 g, 2.79 mmol) and HOSu (CAS No.: 6066-82-6) (1.41 g, 2.79 mmol) were added. After stirring at room temperature for 16 hours, water was added, and the mixture was extracted three times with DCM. The resulting organic layer was combined, dried over sodium sulfate, filtered off the solid, and concentrated. The crude product was purified by column chromatography to obtain the target product (599 mg, 0.577 mmol). ESI-MS(+) observed value m / z = 1040(M+H)+

[0283] <Example 13> Synthesis of Boc-PEG8cCebA(NHS)-NHS used in Example 7. Boc-PEG8cCebA(NHS)-NHS has the structure shown in formula (32).

[0284] To a DMF solution (7.39 mL) of Boc-PEG8c-OH (CAS No.: 1334169-93-5) (4 g, 7.39 mmol), WSCI (1.487 g, 7.75 mol) and HOSu (CAS No.: 6066-82-6) (0.892 g, 7.75 mmol) were added and the mixture was stirred at room temperature for 17 hours.

[0285] After dilution with water, the resulting organic layer was extracted three times with ethyl acetate, dried with magnesium sulfate, filtered off the solid, and concentrated.

[0286] The crude product obtained was dissolved in DMF (11.69 mL), and then DIPEA (4.18 mL, 24.55 mmol) and 3,3'-azanediyldipropionic acid (CAS number: 505-47-5) (989 mg, 6.14 mmol) were added. After stirring at 40°C for 16 hours, the mixture was allowed to cool to room temperature, and then 1 M potassium dihydrogen phosphate aqueous solution was added to adjust the pH to 5. Finally, liquid-liquid extraction was performed using a DCM / MeOH (9 / 1) mixed solvent.

[0287] The resulting aqueous layer was again diluted with potassium dihydrogen phosphate aqueous solution to adjust the pH to 3, and then extracted three times with a DCM / MeOH (9 / 1) mixed solvent. The resulting organic layer was dried over magnesium sulfate and concentrated. Next, it was diluted with DMF (8.5 mL) and WSCI (1.41 g, 2.79 mmol) and HOSu (CAS No.: 6066-82-6) (1.41 g, 2.79 mmol) were added. After stirring at room temperature for 5 hours, water was added and extracted three times with DCM. The resulting organic layers were combined, dried over sodium sulfate, filtered off the solid, and concentrated. The resulting crude product was purified by column chromatography to obtain the target product (994 mg, 1.052 mmol). ESI-MS(+) observed value m / z = 880(M+H)+

[0288] <Example 14> Synthesis of OCOPEG8Me-CebA-NHS2 used in Example 9. OCOPEG8Me-CebA-NHS2 has the structure shown in formula (33).

[0289] To an acetonitrile solution of 2,5-dioxopyrrolidin-1-yl (2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)carbonate (CAS No.: 1372860-04-2) (1.381 g, 2.63 mmol) and triethylamine (1.8 mL, 13.14 mmol), 3,3'-azanediyldipropionic acid (CAS No.: 505-47-5) (0.466 g, 2.89 mmol) was added and the mixture was stirred for 16 hours.

[0290] The reaction mixture was concentrated and then diluted with a 10% potassium dihydrogen phosphate aqueous solution and purified using reverse-phase column chromatography. The fractions containing the target product were freeze-dried together to obtain 28-(2-carboxethyl)-27-oxo-2,5,8,11,14,17,20,23,26-nonaoxa-28-azahentriacontan-31-oic acid (767 mg, 1.342 mmol).

[0291] To a DMF solution of the obtained compound (270 mg, 0.472 mmol), WSCI (226 mg, 1.18 mmol) and HOSu (CAS number: 6066-82-6) (136 mg, 1.18 mmol) were added. After stirring at room temperature for 3.5 hours, water was added and the mixture was extracted three times using DCM.

[0292] The obtained organic layer was combined, dried over sodium sulfate, filtered off the solid, and concentrated. The resulting crude product was purified by column chromatography to obtain the target product (229.5 mg, 0.285 mmol). ESI-MS(+) observed value m / z = 766(M+H)+

[0293] <Example 15> [Synthesis of various peptides] In this example, monomers shown in Tables 12-13 were chemically synthesized in the same manner as in Examples 2, 3, 5, or 8. Dimers with linker structure numbers 8-11 from among the dimers shown in Tables 14-18 were synthesized in the same manner as in Example 1. Dimers with linker structure numbers 12-19 from among the dimers shown in Tables 14-18 were synthesized in the same manner as in Example 4. Dimers with linker structure number 20 from among the dimers shown in Tables 14-18 were synthesized in the same manner as in Example 9. Trimers shown in Table 19 were synthesized in the same manner as in Example 6. Tetramers with tetramer structure numbers 2-4 from among the tetramers shown in Table 20 were synthesized in the same manner as in Example 11. Tetramers with tetramer structure numbers 1, 5-17 from among the tetramers shown in Table 20 were synthesized in the same manner as in Example 7. The amino acid sequences of the peptides contained in the synthesized monomers, dimers, trimers, and tetramers are shown in Tables 1-4 above, and the linker structures are shown in Tables 5-9 above.

[0294] In Tables 12-13, the peptide sequence number and linker structure number indicate the structure of the peptide and linker contained in the monomer. The monomer was also analyzed under one of the analytical conditions described in Examples 2, 3, 5, or 8, and its structure was confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observations, retention time, valency, and concentration gradient (%) of mobile phase B used in the analysis are shown in Tables 12-13.

[0295]

[0296]

[0297] In Tables 14-18, the peptide sequence number, monomer structure number, and linker structure number indicate the structures of the peptide, monomer, and linker contained in the dimer. The dimer was also analyzed under any of the analytical conditions described in Examples 1, 4, 9, or 10, and its structure was confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observations (indicated as ESI(m / z) in the table), retention time (indicated as Ret.Time in the table), valency (indicated as [M+XH]X+ in the table), and the concentration gradient (%) of mobile phase B used for analysis (indicated as concentration gradient B(%) in the table) are shown in Tables 14-18.

[0298] In Tables 14 to 20 below, monomer structure numbers are represented by two consecutive numbers: the first number (to the left of the "-") is the peptide sequence number, and the second number (to the right of the "-") is the linker structure number. For example, in the peptide complex represented by dimer structure number 1, the linkers represented by linker structure number 2 in the peptides represented by monomer structure numbers 1-2 are connected by the linker represented by linker structure number 12. For example, in the peptide complex represented by dimer structure number 8, the peptides represented by peptide sequence number 1 are connected by the linker represented by linker structure number 11.

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] In Table 19, the monomer structure number and linker structure number indicate the structures of the monomer and linker contained in the trimer. The trimer was also analyzed under the analytical conditions described in Example 6, and its structure was confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observations (indicated as ESI(m / z) in the table), retention time (indicated as Ret.Time in the table), valence (indicated as [M+XH]X+ in the table), and the concentration gradient (%) of mobile phase B used for analysis (indicated as concentration gradient B(%) in the table) are shown in Table 19. In Table 19, for example, in the peptide complex represented by trimer structure number 1, the linkers represented by linker structure number 5 in monomer structures 13-5 are connected by the linker represented by linker structure number 21.

[0305]

[0306] In Table 20, the monomer structure number and linker structure number indicate the structures of the monomer and linker contained in the tetramer. The tetramer was also analyzed under either the analytical conditions described in Example 7 or 11, and its structure was confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observations (indicated as ESI(m / z) in the table), retention time (indicated as Ret.Time in the table), valence (indicated as [M+XH]X+ in the table), and the concentration gradient (%) of mobile phase B used for analysis (indicated as concentration gradient B(%) in the table) are shown in Table 20. In Table 20, for example, in the peptide complex represented by tetramer structure number 1, the linkers represented by linker structure number 5 in monomer structures 1-5 are connected by the linker represented by linker structure number 23.

[0307]

[0308] <Example 16> [Evaluation of FGFR1 agonist activity by phosphorylation of FGFR1] To evaluate the FGFR1 agonist activity of the peptide complex of the present invention, phosphorylation of FGFR1 was investigated.

[0309] To evaluate the phosphorylation of FGFR1, mouse proB cell line Ba / F3 cells expressing human FGFR1c were generated using the following method.

[0310] Ba / F3 cells were transfected with a human FGFR1c expression vector by electroporation, and then cultured for 7 days in RPMI-1640 (Nacalai Tesque) growth medium containing 10% FBS (Thermo Fisher Scientific), 10 ng / ml mouse IL-3 (R&D), and 50 ug / ml gentamicin (Nacalai Tesque), with 800 ng / ml Geneticin (Thermo Fisher Scientific) added, for drug selection.

[0311] Next, the cells were cultured for 10 days in a growth medium from which mouse IL-3 had been removed, supplemented with 100 ng / mL Recombinant Human FGF2 protein (R&D) (hereinafter referred to as FGF2), 1 IU Heparin (Sigma), and 50 nM β-mercaptoethanol (Thermo Fisher Scientific). After that, the cells were expanded in growth medium supplemented with 800 ng / mL Geneticin, and single clones were obtained by the limiting dilution method.

[0312] The above human FGFR1c-expressing Ba / F3 cells were cultured in growth medium supplemented with 800 ng / mL Geneticin. After harvesting, they were washed twice with HBSS buffer (Thermo Fisher Scientific) containing 0.1% BSA (Sigma) and 1 IU Heparin, resuspended, and seeded into 384-well culture plates at a density of 45,000 cells per well. ２ The samples were incubated at 37°C for 3 hours in an incubator.

[0313] Then, FGF2 or peptide is added, CO ２ The plates were incubated in an incubator at 37°C for 15 minutes while permeating, and the cells were lysed with Lysis buffer supplemented with the blocking buffer included with the HTRF Human Phosph-FGFR1 (Tyr653 / 654) Detection Kit (Revvity).

[0314] The experiment was conducted according to the kit protocol, and Envision HTS (Revvity) was used to detect the signal. The obtained signals were analyzed with GraphPad Prism, and % activity was calculated with the maximum signal induced by FGF2 set to 100% and the unstimulated state to 0%. FGF2 was evaluated at three or four 10-fold dilutions (0.06 nM to 60 nM) starting from 60 nM, and Emax (100% activity) was calculated.

[0315] For dimer structure numbers 1-7, 9-17, 21, 23-33, 35-44, 48-51, 54, 56-58, 60, 63-68, 70-72, 81-90, 96, 97, and 99, the test was conducted with a dimer addition concentration of 10 nM. Dimers with a % activity of 50% or more were evaluated as A-1, and those with an activity of 10% or more but less than 50% were evaluated as A-2. The results are shown in Tables 21-22.

[0316]

[0317]

[0318] For dimer structure numbers 18-20, 22, 34, 45-47, 52, 53, 55, 59, 61, 62, 69, 73-80, 91-95, 98, 100, and 102, the test was conducted with a dimer addition concentration of 10 nM. Dimers with a % activity of 50% or more were evaluated as B-1, and those with an activity of 10% or more but less than 50% were evaluated as B-2. The results are shown in Table 23.

[0319] For dimer structure number 101, the test was conducted with a dimer addition concentration of 100 nM. Dimers with a % activity of 10% or more were evaluated as B-3, and the results are shown in Table 23.

[0320]

[0321] For trimmer structures 1 to 13, tests were conducted with a trimmer additive concentration of 10 nM. Trimmers with a % activity of 50% or more were evaluated as A-1, and those with an activity of 10% or more but less than 50% were evaluated as A-2. The results are shown in Table 24.

[0322]

[0323] For tetramer structures 1 to 17, tests were conducted with a tetramer addition concentration of 10 nM. Tetramers with a % activity of 50% or more were evaluated as A-1, and those with an activity of 10% or more but less than 50% were evaluated as A-2. The results are shown in Table 25.

[0324]

[0325] <Example 17> [Proliferation assay of bone marrow-derived MSCs] To evaluate the FGFR1 agonist activity of the peptide complex of the present invention, the proliferation of bone marrow-derived MSCs was examined.

[0326] MSCs (PromoCell) were cultured in Cellartis MSC Xeno-Free Culture Medium (TAKARA, registered trademark). After detaching the cells with Triple (Thermo), they were washed with Cellartis MSC Xeno-Free Basal Medium w / o Phenol Red (TAKARA) containing 10% FBS, suspended, and seeded at 3000 cells per well in 96-well cell adhesion plates with white sides and a transparent bottom, and cultured overnight.

[0327] The next day, add FGF2 or peptide, CO ２ The cells were cultured in an incubator at 37°C for two days. After two days of culture, FGF2 or a peptide was added, and the cells were cultured for another three days. A white seal was attached to the bottom of the plate, and the number of cells was detected using the CellTiter-Glo (Promega) kit, with the experiment performed according to the attached protocol.

[0328] A SpectraMax Paradigm multimode microplate reader (Molecular Devices) was used for signal detection.

[0329] The obtained signals were analyzed using GraphPad Prism, and % activity was calculated by setting the maximum signal induced by FGF2 as 100% and the unstimulated state as 0%. FGF2 was evaluated at 6 nM and 10-fold dilutions at 3 or 5 points (0.0006 to 6 nM), and Emax (100% activity) was calculated.

[0330] For dimer structures 1-7, 9-13, 15-17, 21, 23, 24, 27-32, 35-44, 48, 51, 54, 60, 66-68, 81, 84-90, 96, 97, 99, trimer structures 1, 5-7, and tetramer structures 1-6, 10, 11, the peptide complex was added at a concentration of 6 nM for the tests. Peptide complexes with a % activity of 50% or higher were evaluated as C-1. The results for dimers are shown in Tables 26-27, the results for trimers in Table 28, and the results for tetramers in Table 29.

[0331]

[0332]

[0333]

[0334]

[0335] <Example 18> [Bovine muscle satellite cell proliferation assay] To evaluate the FGFR1 agonist activity of the peptide complex of the present invention, the proliferation of bovine muscle satellite cells was verified.

[0336] Bovine muscle satellite cells (donated from an organoid farm) were cultured in DMEM containing 20% ​​FBS, 10 mM HEPES (Thermo Fisher Scientific), an antibiotic-antifungal mixture (Nacalai Tesque), 2 ng / mL Gentamicin, and 5 ng / mL bFGF (Reprocell), as well as high glucose, GlutaMAX™ Supplement, and pyruvate (Thermo Fisher Scientific).

[0337] After detaching the cells using Triple, they were washed with DMEM / F12 (Thermo Fisher Scientific) containing a mixture of antibiotics and antifungals, 2 mM L-alanyl-L-Glutamine (Wako), 0.0005% BSA (Sigma), 75 ng / mL Recombinant IL-6 (R&D), 100 ng / mL Recombinant IGF-1 (R&D), 5 ng / mL Recombinant HGF (R&D), and an antibiotic-antifungal agent mixture. After suspension, the cells were seeded into 96-well cell adhesion plates with white sides and a transparent bottom, at a density of 6000 cells per well, and cultured overnight.

[0338] The next day, add FGF2 or peptide, CO ２ The cells were cultured in an incubator at 37°C for 3 days. A white seal was attached to the bottom of the plate, and cell counts were detected according to the CellTiter-Glo (Promega) kit protocol.

[0339] A SpectraMax Paradigm multimode microplate reader (Molecular Devices) was used for signal detection.

[0340] The obtained signals were analyzed using GraphPad Prism, and % activity was calculated by setting the maximum signal induced by FGF2 as 100% and the unstimulated state as 0%. FGF2 was evaluated at five 10-fold dilutions starting from 6 nM (0.0006 to 6 nM) or three 10-fold dilutions starting from 0.6 nM (0.006 to 0.6 nM), and Emax (100% activity) was calculated.

[0341] For dimer structures 1, 2, 5, 6, 11, 12, 21, 23, 29-31, 35, 36, 38-40, 43, 44, 51, 66, 68, 88, 90, 96, and 97, the tests were conducted with a dimer addition concentration of 1 nM. Dimers with a % activity of 50% or higher were evaluated as D-1, and the results are shown in Table 30.

[0342] For dimer structures 27, 32, 84-87, and 89, the tests were conducted with a dimer addition concentration of 6 nM. Dimers with a % activity of 50% or higher were evaluated as E-1, and the results are shown in Table 30.

[0343]

[0344] For trimmer structure number 6, the test was conducted with a trimmer addition concentration of 1 nM. Trimmers with a % activity of 50% or more were evaluated as D-1, and the results are shown in Table 31.

[0345] For trimmer structures 1 and 4, the test was conducted with a trimmer addition concentration of 6 nM. Trimmers with a % activity of 50% or more were evaluated as E-1, and the results are shown in Table 31.

[0346]

[0347] For tetramer structure number 10, the test was conducted with a tetramer addition concentration of 1 nM. Tetramers with a % activity of 50% or more were evaluated as D-1, and the results are shown in Table 32.

[0348] For tetramer structures 1 and 2, the tests were conducted with a tetramer addition concentration of 10 nM. Tetramers with a % activity of 50% or higher were evaluated as D-2, and the results are shown in Table 32.

[0349] For tetramer structures 6 and 9, the tests were conducted with a tetramer addition concentration of 6 nM. Tetramers with a % activity of 50% or higher were evaluated as E-1, and the results are shown in Table 32.

[0350]

[0351] <Example 19> [Evaluation test of intermolecular interaction between FFFR1 and monomer] To evaluate the binding ability of the peptide complex of the present invention to FFFR1, the intermolecular interaction of the peptide with Recombinant human FFFR1 beta(IIIc)Fc chimera protein (R&D) (hereinafter referred to as FFFR1c-Fc) was tested by surface plasmon resonance (SPR) using the method described below.

[0352] A CM3 sensor chip (Cytiva) was inserted into a BiacoreT200 (Cytiva), and several priming operations were performed with running buffer: HBS-P+, pH 7.4 (Cytiva), and equilibration was achieved at a flow rate of 30 μL / min.

[0353] The FGFR1c protein used in this study is an Fc fusion protein, so the Human Antibody Capture Kit, type 2 (Cytiva) was used. The capture molecule (0.5 mg / ml Anti-human IgG (Fc) antibody (hereinafter referred to as Fc antibody) contained in the above capture kit) was immobilized at a flow rate of 10 μL / min.

[0354] 100 μL each of 60 mM EDC solution (Cytiva) and 650 mM NHS solution (Cytiva) were mixed, and then the mixture was reacted at a flow rate of 10 μL / min for 420 seconds to activate the carboxyl groups on the sensor chip.

[0355] A 0.2 μM 0.02 mg / ml Fc antibody solution was prepared by diluting with 10 mM acetic acid solution (pH 5.0), and 200 μL of this solution was reacted at a flow rate of 10 μL / min for 420 seconds to immobilize the Fc antibody onto the CM3 sensor chip.

[0356] After immobilization, capping was performed by reacting with a 1.0 M ethanolamine aqueous solution (Cytiva) at a flow rate of 10 μL / min for 420 seconds. Next, a capture molecule (Fc antibody) of 0.1 μM FGFR1c-Fc was reacted at a flow rate of 5 μL / min for 120 seconds to capture approximately 1,200 RU.

[0357] A peptide lysis solution prepared to 10 mM in DMSO solution was diluted with running buffer to a final concentration of 10 μM. Further peptide dilutions of 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM were prepared using the running buffer. The above peptide samples were reacted at a flow rate of 30 μL / min for 120 seconds, followed by dissociation for 600 seconds, and the peptide kinetics against FGFR1c-Fc were obtained by SPR measurement.

[0358] The kinetics evaluation model was Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Cytiva). Curve fitting was performed on the obtained sensorgrams using the least squares method, and the binding of the peptide to FGFR1c-Fc was evaluated by determining the KD value (equilibrium dissociation constant).

[0359] Table 33 shows the calculated KD values ​​for monomer structure numbers 1-1, 1-2, and 13-2.

[0360]

[0361] <Discussion> In Example 19, the KD values ​​resulting from the binding of human FGFR1c-Fc to monomers were 7.80E-10 for monomer structure number 1-1, 5.80E-10 for monomer structure number 1-2, and 4.00E-10 for monomer structure number 13-2. These results indicate that the above monomers have binding activity to FGFR1.

[0362] The monomers described above are peptide compounds in which a gy residue, or a gy residue and a lys residue, are further bonded to the cyclic peptide of the present invention. Furthermore, the results from Examples 16 to 18 showed that the peptide complex of the present invention has agonist activity against FGFR1. Therefore, it is considered that the peptide of the present invention, in particular the cyclic peptide, has binding activity against FGFR1 even without forming a peptide complex.

Claims

1. A peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which one or more amino acid residues are substituted, deleted, added, or inserted in the amino acid sequence represented by formula A1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 where, X1 is an amino acid residue having an optionally substituted aryl group in its side chain, X2 is any amino acid residue, X3 is V, X4 is an amino acid residue having an aliphatic hydrocarbon group in its side chain, X5 is an optionally substituted 4-6 membered cyclic secondary amino acid residue, or an amino acid residue having an optionally substituted aliphatic hydrocarbon group in its side chain, X6 is any amino acid residue, X7 is any amino acid residue, X8 is an optionally N-alkylated glycine residue (where the alkyl group constituting the N-alkylation may have substituents), or any D-amino acid residue, X9 is any amino acid residue, X10 is an amino acid residue having an aliphatic hydrocarbon group in its side chain. X11 is an amino acid residue having an optionally substituted aryl group in its side chain, X12 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in its side chain, X13 is I, X14 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in its side chain, or any N-alkyl amino acid residue, and X15 is C. In formula A1, the amino acid sequence is described from the N-terminus to the C-terminus.

2. In formula A1, X1 is F, F2F, or F3C, X2 is W, Cit, or W7N, X4 is V or alI, X5 is P, Hyp, or T, X6 is D, 3Py6NH2, E, F4COO, Hyp, K, N, Nmm, or S, X7 is I, Chg, E, Gthp, K, R, T, V, or W7N, X8 is G, ApG, de, dp, or MeG, X9 is D, E, H, K, or N, X10 is I or Chg, X11 is Y, F4aao, 3Py6Ph, or 4Py, X12 is S, Q, Cit, Har, K, or R, The peptide according to claim 1, wherein X14 is R, E, MeG, or K.

3. The peptide according to claim 1, comprising an amino acid sequence represented by formula A2, or an amino acid sequence in which 1 to 12 amino acid residues, arbitrarily selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 14th amino acid residues from the N-terminus, are substituted or deleted in the amino acid sequence represented by formula A2. A2: F-W-V-V-P-D-I-de-D-Chg-F4aao-Q-I-R-C (SEQ ID NO: 13) 4. The peptide according to any one of claims 1 to 3, which consists of any one selected from the peptides of peptide sequence numbers 1 to 65.

5. The peptide according to any one of claims 1 to 3, which is a cyclic peptide.

6. The peptide according to any one of claims 1 to 3, wherein one of the amino acid residues before cyclization is chloroacetylated, and the chloroacetylated amino acid residue and a cysteine ​​residue contained in the same peptide before cyclization are linked intramolecularly to form a cyclic structure.

7. The peptide according to any one of claims 1 to 3, further comprising a glycine residue at the C-terminus.

8. The peptide according to claim 5, further comprising additional amino acid residues.

9. A peptide complex comprising two or more of the peptides described in claim 1.

10. The peptide complex according to claim 9, having FGFR1 agonist activity.

11. The peptide complex according to claim 9, comprising two or more peptides according to claim 1 and a linker connecting the peptides, wherein the amino acid sequences of the peptides may be the same or different from each other.

12. The peptide complex according to claim 9, wherein the amino acid sequence homology between the peptides is 90% or more and 100% or less.

13. The peptide complex according to claim 9, wherein each of the two or more peptides is a cyclic peptide.

14. The peptide complex according to claim 9, wherein one of the amino acid residues contained in each of the peptides is chloroacetylated, and the chloroacetylated amino acid residue and a cysteine ​​residue contained in the same peptide are linked intramolecularly to form a cyclic structure.

15. The peptide complex according to claim 9, wherein at least one of the two or more peptides comprises an additional amino acid residue.

16. The peptide complex according to claim 11, wherein the linker is a PEG linker, or a linker consisting of PEG and amino acid residues.

17. The peptide complex according to claim 11, wherein the linker is one arbitrarily selected from the linkers represented by linker structure numbers 1 to 23.

18. The peptide complex according to claim 11, wherein the C-terminuses of the peptides or the side chains of the amino acid residues are linked to each other via the linker.

19. A cell culture composition comprising the peptide described in claim 1 or the peptide complex described in claim 9, used for cell culture.

20. A composition comprising the peptide described in claim 1 or the peptide complex described in claim 9, for use in medical, diagnostic, or research purposes.

21. A method for producing a peptide complex, comprising the steps of producing a peptide according to any one of claims 1 to 3, and linking two or more of the peptides together with a linker.

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