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

Abstract

[Problem] To provide: a novel peptide complex that has agonist activity for FGFR2b, which is a KGF receptor; a peptide that forms the peptide complex; a pharmaceutical composition comprising the peptide complex; a composition for cell culture; and a composition for medical, diagnostic, or research use. [Solution] A peptide comprising the amino acid sequence which is indicated by expression A1 or an amino acid sequence which is obtained by substituting, deleting, adding, or inserting one or more amino acid residues in the amino acid sequence indicated by expression A1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14

Description

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

[0001] The present invention relates to peptides, peptide complexes, pharmaceutical compositions, cell culture compositions, and compositions used for medical, diagnostic, or research purposes.

[0002] Epidermal growth factor (KGF / FGF7) is a paracrine growth factor expressed in fibroblasts that acts on epithelial cells and is known to exhibit various biological activities, including the induction of epithelial cell proliferation, migration, and differentiation.

[0003] KGF transmits signals into cells by binding to the receptor tyrosine kinase FFFR, thereby exerting its diverse biological activities. Among the multiple subtypes of FFFR, KGFR / FFFR2b has been reported to be the main receptor for KGF (Non-Patent Literature 1).

[0004] KGF is used in cell culture for purposes such as cell proliferation and differentiation induction. For example, KGF is used in organoid culture of hepatocytes (Non-Patent Literature 2). Furthermore, KGF has been reported to be important for the differentiation and maturation of induced pluripotent stem cells (iPSCs) into conjunctival epithelium and goblet cells (Non-Patent Literature 3).

[0005] KGF can be a therapeutic target for diseases related to KGF and FFFR2b. For example, KGF is known to promote wound healing and is an important factor involved in liver regeneration by stimulating adult hepatic progenitor cells, suggesting it could be a therapeutic target for liver diseases (Non-Patent Literature 4). In addition, parifermin (Kepivance: Biovitram), a recombinant shortened human KGF expressed by E. coli, has been reported to reduce the duration and severity of oral mucositis after intensive chemotherapy and radiotherapy for hematological malignancies (Non-Patent Literature 5), and is FDA approved.

[0006] Japanese Patent Publication No. 2016-29084

[0007] Receptor Specificity of the Fibroblast Growth Factor Family Ornitz et al. , Journal of Biological Chemistry 1996, 21. 271, 25. Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids Hu et al. , Cell 2018, 175, 1591-1606Generation of functional conjunctive epithelium, including goblet cells, from human iPSCs Nomi et al. , Cell Reports 2021, 34, 108715FGF7 is a functional niche signal required for stimulation of adult liver progenitor cells that support liver regeneration Takase et al. , Genes & Development 2013, 27, 169-181 Palifermin for Oral Mucositis after Intensive Therapy for Hematologic Cancers Spielberger et al. , The New England Journal of Medicine 2024, 351, 2590-8

[0008] The object of the present invention is to create a peptide having binding activity to KGFR / FFFR2b, a receptor for KGF; a peptide complex having KGFR / FFFR2b agonist activity; a peptide that forms the peptide complex; a pharmaceutical composition containing the peptide complex; a cell culture composition; and a composition for use in medical, diagnostic, or research applications.

[0009] To achieve the above objectives, the present invention employs the following configuration.

[0010] [1] A peptide comprising the 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, or a pharmaceutically acceptable salt thereof. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 where, X1 is any amino acid residue, X2 is an amino acid residue having an N-alkylated or substituted aryl group in its side chain, X3 is any amino acid residue, X4 is any amino acid residue, X5 is a N-alkylated secondary amino acid or peptoid, X6 is a secondary amino acid or peptoid, X7 is an amino acid residue having an substituted aryl group in its side chain, X8 is an amino acid residue having an substituted aryl group in its side chain, X9 is glycine or any d-amino acid residue, X10 is an amino acid residue having an substituted aliphatic hydrocarbon group in its side chain, X11 is any amino acid residue, X12 is any amino acid residue, X13 is an acidic amino acid residue that may be of type D, and X14 is C. In formula A1, the amino acid sequence is described from the N-terminus to the C-terminus.

[0011] [2] In formula A1, X1 is MeF, dmor, MeKCOPipzaa, MeE, KCOPipzaa, dp or dhyp, which may be modified with a chloroacetyl group, and X2 is MeF, MeF4COO, MeY, MeF4CON, MeF3T, MeF3C, F, MeF3COO, MeF3CON, Me4Py, Me3Py, MeF4F, MeF3F, MeF4C, MeF4T, MeF3Et, MeF4Et or MeF4OMe, X3 is Tic, P, T, MeT, Hyp, P4Sh, MeKAc, Pipz4Me, R, Mor, alT, dMeS, DapAc, DapMs, DapCOPipzaa, V, Gthp, N, H, A1mor, MeS, MeDapAc, MeE, MeD, MeQ, MeN, MeL, MeKCOPipzaa, MeCit, MeKMor, Aib, P4Cp, P4F2, P4RNHAAc, P4SNHAAc, P4Raao, or P4Saao, and X4 is S, R, H, Q, E, Cit, 4Py, H3Py6NH2, H4Py2NH2, H3Py, or H4Py, X5 is MeG, CeG, CmG, P, P4Sh or Mor, and X6 is P, Hyp, PpG, MsapG, IeG, ImG, 3PypG, 4PypG, 2PyeG, 3PyeG, 4PyeG, 2PymG, 4PymG, P4F2, P4RNHAc, P4RO3Py, P4RO2Py, P4SPh, HypBn, P4Raao, P4RNHCONHmCOO or P4RNCOPipzaa, X7 is Y, F4aao, F4F, F3CON, Ym2Py, F34dOMe, F35dOMe, F4COO, F4OEt, F, F4C, F4OMe, F3H, F3OMe, F3C, Yph, YBn, Ym4Py, Ym3Py, F3OPh, F3OBn, Dopa, F345tOMe, F34diox, F34mdio, W, W5H, W7N, F4CON, F4T, or F3T, X8 is Bph, Bph4COO, Bph4NAc, Bph3COO, Bph4CON, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F44Py, F42Py, 3Py6Ph, Bph3NAc, Bph3F, pBph2F, pBph3F, pBph3C, or Nal2, and X9 is G, da, ds, or dn,X10 is Bph, Bph3COO, 3Py6Ph, Bph4COO, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F43Py, F42Py, Bph2F, 3Py6O4pip1aa, Bph3F, Bph2H, Bph4NAc, Bph3NAc, pBph3C, pBph3F, pBph2C, pBph2F, pBph2Me, or pBph2OMe, and X11 is D, G, A, E, Hgl, R, P, or Hyp, The peptide described in [1] above, or a pharmaceutically acceptable salt thereof, wherein X12 is N, R, Hgl, MeN, MeF, dp, dhpr, Q, Hgn, D, E, Cit, KCOPIPZAa, or dn, and X13 is D, E, or dd.

[0012] [3] The peptide described in [1] above, or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence represented by formula A2, or the amino acid sequence represented by formula A2, in which 1 to 13 amino acid residues arbitrarily selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th amino acid residues from the N-terminus are substituted or deleted. A2: MeE-MeF3T-T-R-MeG-P-F35dOMe-Bph4COO-G-Bph3COO-D-N-D-C (SEQ ID NO: 57)

[0013] [4] A peptide comprising the amino acid sequence shown in SEQ ID NOs: 1 to 261, or a peptide according to any one of the above [1] to [3], having an amino acid sequence in which the N-terminal amino acid residue of the amino acid sequence shown in SEQ ID NOs: 1 to 261 is modified with a chloroacetyl group, or a pharmaceutically acceptable salt thereof.

[0014] [5] A cyclic peptide as described in any one of the above items [1] to [3], or a pharmaceutically acceptable salt thereof.

[0015] [6] A peptide according to any one of the above [1] to [3], wherein one of the amino acid residues 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, or a pharmaceutically acceptable salt thereof.

[0016] [7] The peptide described in any one of the above [1] to [3], further having a glycine residue at its C-terminus, or a pharmaceutically acceptable salt thereof.

[0017] [8] The peptide described in [5] above, or a pharmaceutically acceptable salt thereof, further comprising additional amino acid residues.

[0018] [9] A peptide having the amino acid sequence of Sequence ID No. 57, in which one amino acid residue may be substituted, and having a cyclic structure in which the first amino acid residue and the 14th cysteine ​​residue of the peptide are linked intramolecularly, as described in any one of the above [1] to [3], or a pharmaceutically acceptable salt thereof.

[0019]

[10] A peptide complex comprising two or more peptides described in [1] above, or pharmaceutically acceptable salts thereof.

[0020]

[11] The peptide complex described in

[10] above, having FGFR2b agonist activity.

[0021]

[12] The peptide complex according to

[10] , comprising two or more peptides described in [1] above, or pharmaceutically acceptable salts thereof, and a linker connecting the peptides, wherein the amino acid sequences of the peptides may be the same or different.

[0022]

[13] The peptide complex according to

[10] above, wherein the amino acid sequence homology between the peptides is 90% or more and 100% or less.

[0023]

[14] The peptide complex according to

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

[0024]

[15] The peptide complex according to

[10] , wherein one of the amino acid residues contained in each peptide 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.

[0025]

[16] The peptide complex according to

[10] above, wherein at least one of the two or more peptides further comprises an additional amino acid residue.

[0026]

[17] The peptide complex according to

[12] above, wherein the linker is a PEG linker or a linker composed of PEG and an amino acid residue.

[0027]

[18] The peptide complex according to

[12] above, wherein the linker is any one arbitrarily selected from the linkers represented by linker structure numbers 1 to 11.

[0028]

[19] The peptide complex according to

[12] above, wherein the C-termini of the peptides or the side chains of amino acid residues of the peptides are bound to each other via a linker.

[0029]

[20] A composition for cell culture, which comprises the peptide complex according to

[10] above and is used for cell culture.

[0030]

[21] A composition, which comprises the peptide complex according to

[10] above and is used for medical use, diagnostic use, or research use.

[0031]

[22] A method for producing a peptide complex, comprising: a step of producing the peptide according to any one of [1] to [3] above, or a pharmaceutically acceptable salt thereof; and a step of binding two or more peptides to each other via a linker.

[0032] According to the present invention, there can be provided a peptide complex having KGFR / FGFR2b agonist activity, which is a receptor for KGF, a peptide forming the peptide complex, a pharmaceutical composition comprising the peptide complex, a composition for cell culture, and a composition used for medical use, diagnostic use or research use.

[0033] Hereinafter, embodiments of the peptide, peptide complex, pharmaceutical composition, composition for cell culture, and composition used for medical use, diagnostic use or research use of the present invention will be described in detail. However, the present invention is not limited to the embodiments described below, and various modifications can be made without departing from the gist of the present invention.

[0034] <Abbreviations> Å as angstrom (unit); Boc as tert-butoxycarbonyl group; ClAc as chloroacetyl; DIPEA or DIEA as N,N-diisopropylethylamine; DMSO as dimethyl sulfoxide; DMF as N,N-dimethylformamide; DODT as 3,6-dioxa-1,8-octanedithiol; 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 gram (unit); HATU as O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HPLC as high-performance liquid chromatography; HA tag as hemagglutinin antigen tag; LC-MS or LC / MS as liquid chromatography-mass spectrometry; M as molar; MeCN as acetonitrile; mg as milligram; min as minute; mL as milliliter; mM as millimolar; mm as millimeter; NHS as N-hydroxysuccinimide; nm as nanometer; μL as microliter; Pbf as 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group; PEG as polyethylene glycol; rpm as revolutions per minute; Sub as dibenzosperyl group; tBu as tert-butyl group; TFA as trifluoroacetic acid; HOAt as 1-hydroxy-7-azabenzotriazole; HOBt as 1-hydroxybenzotriazole monohydrate; THPTA as tris[[1-(3-hydroxypropyl)-1H-1,2,3-triazole-4-yl]methyl]amine; TIS as triisopropylsilane; Trt or Tr as trityl group; Oxyma pure as ethyl cyano(hydroxyimino)ethyl acetate; DTT as dithiothreitol; NMP as N-methyl-2-pyrrolidone; Alloc as allyloxycarbonyl group;cPEG-1c: 3,3'-oxydipropanoic acid (CAS number: 5961-83-1);

[0035] The abbreviations and full names of amino acids used in this specification are shown in Table 3 of the Examples.

[0036] In the following specifications, the "~" symbol used to indicate a range of values ​​may be referred to as "-".

[0037] <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.

[0038] The FGF family is one of the most diverse growth factors in vertebrates, with 23 different FGF ligands identified in mice and humans. Based on sequence homology and phylogenetics, the 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), FGF4 subfamily (FGF4, FGF5, FGF6), FGF7 subfamily (FGF3, FGF7, FGF10, FGF22), FGF8 subfamily (FGF8, FGF17, FGF18), and FGF19 subfamily (FGF9, FGF16, FGF20). Furthermore, the FGF19 subfamily (FGF19, FGF21, FGF23) transmits signals in an endocrine-type manner (Signal Transmission and Targeted Therapy 2020, 5, 181).

[0039] <FGFRs> In mammals, FGFs bind to and activate high-affinity tyrosine kinase receptors encoded by four genes (FGFR1, FGFR2, FGFR3, FGFR4) and FGFRL1, thereby exerting various effects.

[0040] Fibroblast growth factor receptors (FFFRs) are single-pass transmembrane proteins containing an extracellular domain, a transmembrane domain (TMD), and an intracellular tyrosine kinase domain. Within these domains, the extracellular domain consists of three immunoglobulin-like domains (D1-D3), an acidic region, the heparin-binding motif of FGFs, and heparan cofactors. The TMD supports the receptor within the cell membrane and promotes receptor dimerization. Within the cytoplasm, the near-membrane region of FFFRs is involved in receptor dimerization, while the kinase domain is necessary for FGF-related signal transduction (Signal Transduction and Targeted Therapy 2020, 5, 181).

[0041] <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.

[0042] 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.).

[0043] <KGF> Epidermal growth factor (KGF / FGF7; Gene ID: 2252), a paracrine growth factor expressed in fibroblasts and acting on epithelial cells, is known to exhibit various biological activities, such as promoting epithelial cell proliferation, migration, and differentiation, by binding to its receptor, FGFR2b.

[0044] <FGFR2b Agonist Activity> FGFR2b (also known as KGFR) is a transmembrane receptor tyrosine kinase and an isoform of the FGFR2 gene (Gene ID: 2263). As described above, KGF binds to FGFR2b and transmits signals into the cell, resulting in various physiological activities. FGFR2 has two variants, iiib and iiiic, due to splicing of the extracellular domain. In this specification, FGFR2b refers to the protein encoded by the base sequence represented by Gene ID: 2263, and is the splicing variant FGFR2(iiib). FGFR2b agonist activity refers to the ability to act on FGFR2b and phosphorylate and activate it. Furthermore, it refers to the ability to produce effects similar to those brought about by naturally occurring KGF, such as activating signaling pathways associated with the activation of FGFR2b, and is also called exhibiting KGF-like activity. FGFR2b agonist activity can be evaluated by known methods, for example, by its binding ability to FGFR2b and its phosphorylation ability to FGFR2b. It can also be evaluated by its ability to activate signaling pathways associated with the activation of FGFR2b and the resulting cell proliferation activity.

[0045] <Peptide> The peptide according to 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 are substituted, deleted, added, or inserted in the amino acid sequence represented by formula A1. In formula A1, the amino acid sequence is described from the N-terminus to the C-terminus. Formula A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14

[0046] In formula A1, X1 is any amino acid residue, X2 is an amino acid residue having an N-alkylated or substituted aryl group in its side chain, X3 is any amino acid residue, X4 is any amino acid residue, X5 is a N-alkylated secondary amino acid or peptoid, X6 is a secondary amino acid or peptoid, X7 is an amino acid residue having an substituted aryl group in its side chain, X8 is an amino acid residue having an substituted aryl group in its side chain, X9 is glycine or any d-amino acid residue, X10 is an amino acid residue having an substituted aryl group in its side chain, X11 is any amino acid residue, X12 is any amino acid residue, X13 is a d-type acidic amino acid residue, and X14 is C.

[0047] In this specification and in the claims, "may be substituted" means that one or more of the side chains of an amino acid may be substituted with any substituent or halogen atom. Examples of substituents include C1-C4 alkyl groups, aryl groups, carboxyl groups, hydroxyl groups, amino groups, and so on.

[0048] The peptide in this embodiment is a peptide that constitutes a peptide complex, which will be described later. In other words, the peptide in this embodiment can be said to be a material for producing a peptide complex.

[0049] <Amino Acids> Amino acids include not only natural amino acids but also unnatural amino acids. Examples of unnatural amino acids include N-alkyl amino acids, in which amino acids have been N-alkylated, and amino acids modified with lower alkyl groups (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.

[0050] Examples of non-natural 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, peptoids, 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.

[0051] 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 arylene groups, alkylene groups, etc., in the side chain; amino acids with an increased number of carbon atoms in the arylene groups, alkylene groups, or alkyl groups in the side chain; amino acids with substitutions in the aromatic ring of the side chain; and heterocyclic or fused amino acids.

[0052] Examples of non-natural amino acids include N-methyl amino acids, peptoids, 4Py, 4PyeG, 4PymG, 4PypG, A1mor, Aib, alT, Bph, Bph2F, Bph2H, Bph3CON, Bph3COO, Bph3F, Bph3H, and Bph4OMe.

[0053] 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.

[0054] <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.

[0055] 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.

[0056] 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 (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions 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.

[0057] 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.

[0058] 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.

[0059] 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").

[0060] 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.

[0061] 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").

[0062] 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.

[0063] 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."

[0064] 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.

[0065] "Peptoids" include amino acids in which a side chain is added to the amide nitrogen contained in the amino acid. The side chain is not particularly specified and may be the side chain structure of a known amino acid. In this specification, peptoids are also treated as part of unnatural amino acids.

[0066] <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 salts, and basic amino acid salts.

[0067] Examples of inorganic salts include hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates.

[0068] Examples of organic salts include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, trifluoroacetate, and p-toluenesulfonate.

[0069] 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.

[0070] Examples of organic base salts include diethylamine salt, diethanolamine salt, meglumine salt, and N,N'-dibenzylethylenediamine salt.

[0071] Examples of acidic amino acid salts are aspartate and glutamate. Examples of basic amino acid salts are arginine salt, lysine salt, and ornithine salt.

[0072] Peptides and peptide complexes containing peptides may be pharmaceutically acceptable salts or solvates. An example of a solvate is a hydrate.

[0073] In the peptide, X1 is any amino acid residue, preferably MeF, dmor, MeKCOPipzaa, MeE, KCOPipzaa, dp, or dhyp. dmor, MeKCOPipzaa, and KCOPipzaa have the structures of formulas (1), (2), and (3), respectively.

[0074] dmor(1)

[0075]

[0076] MeKCOpipzaa (2) KCOpipzaa (3)

[0077] In the peptide, X2 is an amino acid residue having an aryl group in its side chain, which may be N-alkylated or substituted, and is preferably MeF, MeF4COO, MeY, MeF4CON, MeF3T, MeF3C, F, MeF3COO, MeF3CON, Me4Py, Me3Py, MeF4F, MeF3F, MeF4C, MeF4T, MeF3Et, MeF4Et, or MeF4OMe. MeF4COO, MeF4CON, MeF3T, MeF3C, MeF3COO, MeF3CON, Me4Py, Me3Py, MeF4F, MeF3F, MeF4C, MeF4T, MeF3Et, MeF4Et, or MeF4OMe each have the structure of formulas (4) through (18).

[0078] MeF4COO(4) MeF4CON(5) MeF3T(6) MeF3C(7) MeF3COO(8)

[0079] MeF3CON(9) Me4Py(10) Me3Py(11) MeF4F(12) MeF3F(13) MeF4C (14)

[0080] MeF4T(15) MeF3Et(16) MeF4Et(17) MeF4OMe (18)

[0081] In the peptide, X3 is any amino acid residue, preferably Tic, P, T, MeT, Hyp, P4Sh, MeKAc, Pipz4Me, R, Mor, alT, dMeS, DapAc, DapMs, DapCOPipzaa, V, Gthp, N, H, A1mor, MeS, MeDapAc, MeE, MeD, MeQ, MeN, MeL, MeKCOPipzaa, MeCit, MeKMor, Aib, P4Cp, P4F2, P4RNHAAc, P4SNHAAc, P4Raao, or P4Saao. Tic, P4Sh, MeKAc, Pipz4Me, Mor, alT, dMeS, DapAc, DapMs, DapCopipzaa, Gthp, A1mor, MeDapAc, MeCit, MeKMor, Aib, P4Cp, P4F2, P4RNHAAc, P4SNHAc, P4Raao, or P4Saao each have the structure of formulas (19) to (40).

[0082] Tic (19) P4Sh (20) MeKAc (21) Pipz4Me(22) Mor (23) alT (24)

[0083] dMeS (25) DapAc (26) DapMs (27) DapCOpipzaa (28) Gthp(29) A1mor(30)

[0084] MeDapAc (31) MeCit (32) MeKMor (33) Aib (34) P4Cp(35)

[0085] P4F2(36) P4RNHAc (37) P4SNHAc (38) P4Raao (39) P4Saao (40)

[0086] In the peptide, X4 is any amino acid residue, preferably S, R, H, Q, E, Cit, 4Py, H3Py6NH2, H4Py2NH2, H3Py, or H4Py. Cit, 4Py, H3Py6NH2, H4Py2NH2, H3Py, or H4Py each have the structure of formulas (41) to (46).

[0087] Cit (41) 4Py(42) H3Py6NH2 (43) H4Py2NH2 (44) H3Py(45) H4Py(46)

[0088] In the peptide, X5 is a secondary amino acid or peptoid that may be N-alkylated, and is preferably MeG, CeG, CmG, P, P4Sh, or Mor. CeG, CmG, and P4Sh have the structures of formulas (47) to (49), respectively.

[0089] CeG (47) CmG (48) P4Sh (49)

[0090] In the peptide, X6 is a secondary amino acid or peptoid, and is preferably P, Hyp, PpG, MsapG, IeG, ImG, 3PypG, 4PypG, 2PyeG, 3PyeG, 4PyeG, 2PymG, 4PymG, P4F2, P4RNHAc, P4RO3Py, P4RO2Py, P4SPh, HypBn, P4Raao, P4RNHCONHmCOO, or P4RNCOPipzaa. Hyp, PpG, MsapG, IeG, ImG, 3PypG, 4PypG, 2PyeG, 3PyeG, 4PyeG, 2PymG, 4PymG, P4F2, P4RNHAc, P4RO3Py, P4RO2Py, P4SPh, HypBn, P4Raao, P4RNHCONHmCOO, or P4RNCOPipzaa each have the structure of formulas (50) to (67).

[0091] Hyp (50) PpG (51) MsapG (52) IeG (53) ImG (54) 3PypG (55)

[0092] 4PypG(56) 2PyeG (57) 3PyeG (58) 4PyeG (59) 2 PymG (60)

[0093] 4PymG(61) P4RO3Py (62) P4RO2Py (63) P4SPh(64) HypBn(65)

[0094] P4RNHCONHmCOO (66) P4RNCOpipzaa (67)

[0095] In the peptide, X7 is an amino acid residue having an optionally substituted aryl group in its side chain, and is preferably Y, F4aao, F4F, F3CON, Ym2Py, F34dOMe, F35dOMe, F4COO, F4OEt, F, F4C, F4OMe, F3H, F3OMe, F3C, Yph, YBn, Ym4Py, Ym3Py, F3OPh, F3OBn, Dopa, F345tOMe, F34diox, F34mdio, W, W5H, W7N, F4CON, F4T, or F3T. F4aao, F4F, F3CON, Ym2Py, F34dOMe, F35dOMe, F4COO, F4OEt, F4C, F4OMe, F3H, F3OMe, F3C, Yph, YBn, Ym4Py, Ym3Py, F3OPh, F3OBn, Dopa, F345tOMe, F34diox, F34mdio, W5H, W7N, F4CON, F4T, or F3T each have the structure of formulas (68) to (95).

[0096] F4aao(68) F4F (69) F3CON (70) Ym2Py(71) F34dOMe(72) F35dOMe(73)

[0097] F4COO (74) F4OEt(75) F4C (76) F4OMe(77) F3H (78) F3OMe(79)

[0098] F3C (80) Yph(81) YBn (82) Ym4Py(83) Ym3Py(84) F3OPh(85)

[0099] F3OBn(86) Dopa (87) F345tOMe (88) F34diox(89) F34mdio(90)

[0100] W5H (91) W7N (92) F4CON (93) F4T (94) F3T (95)

[0101] In the peptide, X8 is an amino acid residue having an optionally substituted aryl group in its side chain, and is preferably Bph, Bph4COO, Bph4NAc, Bph3COO, Bph4CON, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F44Py, F42Py, 3Py6Ph, Bph3NAc, Bph3F, pBph2F, pBph3F, pBph3C, or Nal2. Bph, Bph4COO, Bph4NAc, Bph3COO, Bph4CON, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F44Py, F42Py, 3Py6Ph, Bph3NAc, Bph3F, pBph2F, pBph3F, pBph3C, or Nal2 each have the structure of formulas (96) to (114).

[0102] Bph (96) Bph4COO(97) Bph4NAc (98) Bph3COO(99) Bph4CON(100)

[0103] Bph3CON (101) Bph4H (102) Bph3H (103) Bph4OMe (104) Bph3OMe (105)

[0104] F44Py (106) F42Py (107) 3Py6Ph (108) Bph3NAc (109) Bph3F (110)

[0105] pBph2F(111) pBph3F (112) pBph3C (113) Naal2(114)

[0106] In the peptide, X9 is glycine or any d-amino acid residue, preferably G, da, ds, or dn.

[0107] In the peptide, X10 is an amino acid residue having an aryl group in its side chain, which may be substituted, and is preferably Bph, Bph3COO, 3Py6Ph, Bph4COO, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F43Py, F42Py, Bph2F, 3Py6O4pip1aa, Bph3F, Bph2H, Bph4NAc, Bph3NAc, pBph3C, pBph3F, pBph2C, pBph2F, pBph2Me, or pBph2OMe. Bph, Bph3COO, 3Py6Ph, Bph4COO, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F43Py, F42Py, Bph2F, 3Py6O4pip1aa, Bph3F, Bph2H, Bph4NAc, Bph3NAc, pBph3C, pBph3F, pBph2C, pBph2F, pBph2Me, or pBph2OMe each have the structure of formula (115) to (XX).

[0108] F43Py (115) 3Py6O4pip1aa (116) Bph2H (117) pBph2C (118) pBph2Me(119) pBph2OMe (120)

[0109] In peptides, X11 is D, G, A, E, Hgl, R, P, or Hyp. Hgl has the structure of formula (121) below.

[0110] Hgl(121)

[0111] In peptides, X12 is N, R, Hgl, MeN, MeF, dp, dhpr, Q, Hgn, D, E, Cit, KCOPIPZAa, or dn.

[0112] dhpr and Hgn have the structures of formulas (122) and (123), respectively. dhpr(122) Hgn(123)

[0113] In peptides, X13 is D, E, or dd.

[0114] In peptides, X14 is cysteine.

[0115] Specific examples of peptides include those with peptide sequence numbers XX to XX, as listed in Table 1.

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125] The peptide in this embodiment may be a peptide containing the amino acid sequence shown in formula A2. Alternatively, the amino acid sequence shown in formula A2 may be an amino acid sequence in which at least one amino acid residue is substituted, deleted, added, or inserted among 1 to 13 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th amino acid residues. Formula A2: MeE-MeF3T-T-R-MeG-P-F35dOMe-Bph4COO-G-Bph3COO-D-N-D-C (Peptide Sequence ID No. 57)

[0126] There are no particular limitations on the three-dimensional structure of the peptide, but a cyclic peptide is preferred. Advantages of a cyclic peptide include, for example, improved metabolic stability due to enhanced protease resistance, increased rigidity due to restrictions on conformational changes, and improved membrane permeability and affinity to target proteins.

[0127] When the peptide is a cyclic peptide, it is preferable that the N-terminal amino acid residue of the peptide before cyclization is a chloroacetylated amino acid residue, that the same peptide before cyclization contains a cysteine ​​residue, and that it has a cyclic structure in which the N-terminal amino acid residue and the cysteine ​​residue are linked. The above advantages can be obtained by linking the chloroacetylated amino acid residue and the cysteine ​​residue to form a cyclic structure.

[0128] 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.

[0129] In one embodiment, the peptide has a cyclic structure in which a chloroacetylated amino acid, preferably the N-terminal amino acid (the first amino acid residue), is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which an N-terminal amino acid (the first amino acid residue) is bonded to a cysteine ​​residue (or a substituted cysteine ​​residue, or a compound containing an -SH group within its structure) contained in the peptide. In one embodiment, the peptide has a cyclic structure in which an N-terminal amino acid (the first amino acid residue) is bonded to a 14th cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which a chloroacetylated N-terminal amino acid (the first amino acid residue) is bonded to a 14th cysteine ​​residue contained in the peptide.

[0130] "Chloroacetylation" may also be "halogen acetylation" with other halogens. Furthermore, "acetylation" may also be "acylation" with acyl groups other than acetyl groups. In one embodiment, the peptide has a cyclic structure obtained by linking the first amino acid residue of the chloroacetylated amino acid sequence with a cysteine ​​residue contained in the peptide. In another embodiment, the peptide may have a structure in which the amino acid sequence described in any of SEQ ID NOs: 1-261 and cysteine ​​or a substituted version contained in the peptide are linked via an acetyl group. That is, in this specification, for example, "cyclic peptide consisting of SEQ ID NOs: XX" also includes a cyclic structure in which the amino acid sequence represented by SEQ ID NOs: XX is linked with an acetyl group attached to the first amino acid contained in the amino acid sequence and cysteine.

[0131] In this specification, some amino acids may be modified for the purpose of cyclizing peptides. Such partially modified amino acids are also included. For example, as described above, a chloroacetyl group (ClAc group) may be added to the N-terminal amino acid and then bound to a cysteine ​​residue or an amino acid residue having an -SH group in the peptide to form a cyclamen. Various (natural / unnatural) amino acids to which such chloroacetyl groups have been added are also included in the amino acids of this application.

[0132] 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.

[0133] 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 X14. One or more amino acid residues may be inserted between X1 and X14. Also, one or more of the amino acid residues X2 to X14 may be deleted.

[0134] 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 14 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 14 residues. The preferred ranges for the upper and lower limits can be combined as appropriate.

[0135] The peptide before cyclization may contain additional amino acid residues that are added to the N-terminus of X1 or the C-terminus of X14.

[0136] The number of amino acid residues added to a peptide can be appropriately selected depending on the type and purpose of the added amino acids, and is not particularly limited.

[0137] In the peptide before cyclization, it is preferable to have an additional Gly residue at the C-terminus of the amino acid residue X14. 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 residue acts as a linker or part of a linker structure when forming the peptide complex described later, allowing the peptide to be linked to another peptide.

[0138] <Method for producing peptides> A second aspect of the present invention is a method for producing peptides, comprising the steps of synthesizing the peptide according to the first aspect and cyclizing the peptide.

[0139] 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.

[0140] There are no particular limitations on the type of amino acid used as a raw material for peptides. Examples include amino acids protected with a benzyloxycarbonyl group (Cbz group) (Cbz-amino acids) and amino acids protected with a tert-butoxycarbonyl group (Boc group) (Boc-amino acids), with amino acids protected with a 9-fluorenylmethoxy group (Fmoc group) (Fmoc-amino acids) being preferred. By using Fmoc amino acids, they can be used in solid-phase synthesis.

[0141] 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.

[0142] 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 X14 of the peptide before cyclization.

[0143] 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.

[0144] <Peptide complex> The peptide complex according to the third aspect of the present invention comprises two or more peptides according to the first aspect.

[0145] The peptide complex preferably has FFFR2b agonist activity.

[0146] The peptide complex is preferably used as a substitute peptide for KGF.

[0147] The peptide complex preferably comprises two or more peptides according to the first embodiment and a linker that connects the peptides.

[0148] The peptide complex may be a homomultimer in which all the constituent peptides are the same peptide. In the 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 containing a different peptide from the peptide of the first embodiment.

[0149] When the peptide complex is a homomultimer, there are no particular limitations on the homology of amino acid sequences between the peptides contained in the peptide complex, but 90% to 100% is preferred, and 93% to 100% is more preferred.

[0150] The number of peptides contained in a peptide complex is not particularly limited.

[0151] The peptide complex may include peptides having amino acid sequences different from 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.

[0152] There are no particular limitations on the three-dimensional structure of the peptides included in the peptide complex, but it is preferable that each peptide is a cyclic peptide. Advantages of cyclic peptides include, for example, improved metabolic stability due to enhanced protease resistance, increased rigidity due to restrictions on conformational changes, and improved membrane permeability and affinity to target proteins.

[0153] Specific examples of peptide complexes include the peptide complexes (dimers) listed in Tables 5 and 6, but are not limited to these.

[0154] In the peptide complex, the peptide of the first embodiment is as described above under <Peptide>, so its explanation is omitted.

[0155] <Linker>

[0156] In this specification, "linker" refers to a structure that links two or more peptides of the first embodiment. When an additional amino acid is bound to a peptide that forms a cyclic structure, and two or more peptides are linked via the additional amino acid, the additional amino acid can also be considered a linker. Furthermore, when a linker described later is bound to the additional amino acid, the additional amino acid and the bound linker can be considered together as a linker.

[0157] Examples of linkers include amino acid linkers, chemical linkers, fatty acid linkers, nucleic acid linkers, and glycan linkers.

[0158] 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 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.

[0159] 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.

[0160] Examples of fatty acid linkers include fatty acid linkers containing a divalent chemical moiety derived from fatty acids.

[0161] 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.

[0162] 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.

[0163] Examples of linkers in this embodiment include the linkers with linker structure numbers 1 to 11 shown in Table 2. The dashed lines at the ends of the linker structures in the table indicate that they are bound to a peptide.

[0164]

[0165]

[0166] 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.

[0167] <Composition> The peptide of the first embodiment or the peptide complex of the third embodiment may have the function of binding to FFFR2b. In this case, by forming a composition in which the desired substance to be delivered to FFFR2b is bound to the peptide complex, it is possible to deliver the substance to FFFR2b.

[0168] The function of binding to FFFR2b (also called FFFR2b binding ability) refers to the activity that allows a peptide to bind to FFFR2b as described above. The method for measuring the binding ability to FFFR2b is not particularly limited, as long as the binding ability of the peptide or peptide complex to FFFR2b can be measured.

[0169] The substance is not particularly limited as long as it is a substance intended to be delivered to FGFR2b. Examples of substances include compounds, peptides different from those in the first embodiment described above, proteins, compounds containing radioisotopes (RI), and nucleic acids.

[0170] The compound may be a low-molecular-weight compound or a medium-molecular-weight compound, and examples include well-known low-molecular-weight drugs.

[0171] A peptide different from the first embodiment may be a peptide that binds to a target in the body and exerts some effect.

[0172] The 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.

[0173] Compounds containing radioisotopes are not particularly limited as long as they are 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.

[0174] There are no particular limitations on the types of nucleic acids, but examples include DNA, RNA, and DNA-RNA chimeras.

[0175] 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 also contain other compounds such as pharmaceuticals.

[0176] The substance intended to be delivered to FFFR2b may be a composite of the substances listed above.

[0177] The composition comprising the peptide complex of the third embodiment may also contain a carrier. Examples of carriers include water such as sterile water, pure water, or distilled water; physiological saline solution; glucose solution; alcohol such as ethanol; polyalcohols such as glycerol, propylene glycol, or polyethylene glycol; sterile organic solvent; or aqueous starch; and a mixture of two or more of the following.

[0178] <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.

[0179] The peptide complex of the third embodiment has FGFR2b 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 FGFR2b agonist activity. For example, mammalian cells are preferred, and human cells are more preferred.

[0180] The cell culture composition according to this embodiment may be used, for example, as a reagent or additive for a culture medium for culturing cells for the production of cultured meat.

[0181] The cell culture composition of this embodiment may be used as a culture medium reagent or additive for the purpose of differentiation, migration, and proliferation of cells expressing FGFR2b, such as epithelial cells of the epidermis, oral cavity, esophagus, stomach, large intestine, pancreas, lung, mammary gland, uterus, prostate, etc., and endoderm-derived cells such as pancreatic islet cells and hepatocytes, as well as differentiation, migration, and proliferation of these cells from pluripotent stem cells. Therefore, the peptide complex of the third embodiment having FGFR2b agonist activity can be used for the purpose of differentiation, migration, and proliferation of cells expressing FGFR2b, but is not limited to this.

[0182] The culture medium is not particularly limited as long as it is a medium for culturing cells or tissues. For example, serum medium is preferred, and serum-free medium or low-serum medium is more preferred.

[0183] Culture medium additives may be in the form of a solution or a dry solid (e.g., solid or powder). If 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 needed 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.

[0184] If the culture medium additive 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 additive may be added as needed before being used as the culture medium.

[0185] The content of peptides or peptide complexes in a culture medium for culturing cells or tissues, or in a cell culture medium 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 medium.

[0186] <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 peptide complex in the medical composition, which is the first embodiment of this aspect, is not particularly limited.

[0187] There are no particular limitations on the content of peptides or peptide complexes when actually using a medical composition; as long as it is an effective amount, there are no particular limitations. 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.

[0188] When the 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 medical composition is administered to humans, the dosage varies depending on the symptoms, the patient's age, sex, weight, sensitivity, method of administration, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but for example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered 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.

[0189] The diseases targeted by the medical compositions include any disease caused by, exacerbated by, or otherwise related to an increase or decrease in the expression or activity of FGFR2b, or any disease caused by, exacerbated by, or otherwise related to an increase or decrease in FGF / FGFR signaling or any other intracellular signaling cascade activated via FGFR2b. Examples 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.

[0190] The administration method of the medical composition is not particularly limited and may be oral or parenteral. Parenteral administration methods include, for example, injection (intramuscular injection, intravenous injection, subcutaneous injection, etc.), transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0191] Peptides in medical compositions can be modified in various ways, given their easily metabolized and excreted nature. For example, polyethylene glycol (PEG) or sugar chains can be added to polypeptides to increase their blood retention time and reduce their antigenicity. Alternatively, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., may be used as sustained-release bases, and polypeptides may be encapsulated within them. When administering transdermally, iontophoresis, which involves applying a weak electric current to the skin surface to penetrate the stratum corneum, may be used.

[0192] Medical compositions may use the active ingredient as is, or they 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, poultices, etc.

[0193] Formulation can be carried out by conventional methods, for example, by using excipients, binders, disintegrants, lubricants, solvents, solubilizers, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., as appropriate.

[0194] Examples of ingredients used in 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.

[0195] Absorption enhancers can be used to improve the absorption of poorly absorbed drugs. Absorption enhancers 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.

[0196] Pills or tablets may be coated with sugar, gastric-soluble, or enteric-soluble substances. Injectable preparations may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, humectants, emulsifiers, dispersants, stabilizers, solvents, solubilizers, preservatives, etc. may be added.

[0197] The medical composition may be administered in combination with other pharmaceuticals or therapies useful for the disease.

[0198] [Diagnostic Composition] The diagnostic composition according to the second embodiment of this aspect can also be used as a diagnostic agent for detecting FGFR2b using a peptide or peptide complex. The diagnostic agent may be a detection agent for detecting the expression level of FGFR2b, and when used as a detection agent, the peptide or peptide complex may be labeled in a detectable manner.

[0199] The content of peptides or peptide complexes in the diagnostic composition is not particularly limited, as long as the expression level of FGFR2b can be detected.

[0200] There are no particular limitations on the content of peptides or peptide complexes when actually using the diagnostic composition; as long as it is an effective amount, there are no particular limitations. The effective amount varies depending on the type of sample being examined.

[0201] [Research Composition] The research composition, which is the third embodiment of this aspect, can be used for solubility in solvents, binding ability to FFFR2b, toxicity tests on cells and tissues, and toxicity tests on experimental animals, etc.

[0202] 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.

[0203] The method for measuring the binding affinity to FGFR2b is not particularly limited, as long as the binding affinity between the peptide or peptide complex and FGFR2b can be measured. As measurement methods, known methods such as surface plasmon resonance (SPR) assay, scachard analysis, radioimmunoassay (RIA), enzyme immunoassay (EIA), and competitive binding assays can be preferably used.

[0204] 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.

[0205] The cells and tissues subject to toxicity evaluation testing can be any cells and tissues that are normally subjected to toxicity evaluation testing for pharmaceuticals; there are no particular limitations.

[0206] 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 and bioanalysis (TK / PK).

[0207] The experimental animals used in 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.).

[0208] <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 peptides with a linker.

[0209] The peptide according to the first embodiment can be obtained by the method for producing the peptide according to the second embodiment.

[0210] 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 together.

[0211] 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.

[0212] 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.

[0213] In the following examples, for convenience, a compound in which one linker molecule is bound to one peptide molecule will be referred to as a "monomer." A peptide complex in which two peptide molecules are linked via a linker may be referred to as a "dimer." A dimer contains at least one linker, and examples include a complex in which two monomer molecules are further linked by another linker molecule, and a complex in which two peptide molecules are linked by one linker molecule.

[0214] <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.

[0215] 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's Syro I, Biotage's Syro II, CEM's Liberty Blue, CEM's Liberty Blue HT12, CEM's multipep2, or CEM's Liberty Prime, following the manufacturer's manual.

[0216] 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.

[0217] The reagent cocktails used for deprotecting the side chains and cleaving them from the solid-phase resin ranged from 4 mL to 50 mL depending on the peptide, and the following solutions were used: A: TFA / H2O / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) B: TFA / H2O / TIS / DODT (90 / 2.5 / 2.5 / 5) The abbreviations and full names of the amino acids used are listed in Table 3, and the common Fmoc amino acids used in the following examples are listed in Table 4, with side-chain protecting groups indicated in parentheses. For amino acids without a CAS number, the patent application number is used.

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] 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

[0228] ​​​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 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.

[0229] <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: x1 / y1 / z1 / x2 / z2 / x.

[0230] Instrument: Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020) 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 H2O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.5 mL / min Wavelength: 225 nm PDA or gradient B (%): x1: 20-60% / 7.15 min, 60-95% / 0.3 min, 95-95% / 1.55 ​​min; y1: 40-80% / 7.15min, 80-95% / 0.3min, 95-95% / 1.55min; z1: 5-45% / 7.15min, 45-95% / 0.3min, 95-95% / 1.55min; x2: 20-60% / 54.15min, 60-95% / 0.3min, 95-95% / 1.55min; z2: 5-45% / 54.15min, 45-95% / 0.3min, 95-95% / 1.55min;

[0231] 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 H2O; Mobile phase B: 0.025% TFA in MeCN; Flow rate: 0.6 mL / min; Wavelength: 220 nm; Gradient B (%): x: 5-95% / 2.10 min, 95-95% / 0.75 min;

[0232] <Monomer Synthesis> Synthesis of peptides (peptide compounds in which linker structure number 5 is added to a cyclic peptide having the amino acid sequence described in Peptide Sequence ID No. 56, monomer linker structure number 43) Using Sieber amide resin, the target peptide was synthesized starting with the removal of the Fmoc group using the general method described above. CEM's 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, the first residue was reacted twice for 10 minutes at 90°C. The second, third, fifth, and sixth residues were reacted once for 10 minutes at 90°C. The fourth residue was reacted twice for 15 minutes at 50°C. The fourteenth residue was reacted once for 15 minutes at 50°C. The Fmoc group was removed by reacting the peptide with a 10% pyrrolidine DMF solution at room temperature for 1 minute, then removing the solution, and reacting it again with a 10% pyrrolidine DMF solution at room temperature for 1 minute. The 13th and 14th residues, and the linker structure portion (15th, 16th, and 17th residues) were reacted once at 90°C for 1 minute. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step using the method described above, and then shaking it in DMF with ClAcOH / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents) at room temperature for 30 minutes. For deprotection of the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride, and then diethyl ether, then dried under reduced pressure, and the reaction agent cocktail-A (TFA / H) was added 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 well, then shaken at room temperature for 60 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to chilled excess diisopropyl ether / hexane (1 / 1), a turbid precipitate formed, and the mixture was centrifuged and the solution decanted. The obtained solid was washed again with diethyl ether and dried. The obtained solid was used in the next cyclization reaction. For the peptide cyclization reaction, DMSO / H was used so that the final concentration of the peptide was 5 mM based on the number of moles of the solid phase resin. ２ After dissolving in O(9 / 1), triethylamine (10 equivalents) was added and the mixture was stirred for 60 minutes, then acetic acid was added. The resulting reaction solution was concentrated under reduced pressure using a Genevac EZ-2 Elite. The resulting crude product was purified under the following conditions: Column: XSelect CSH prep C18 5μm OBD 50x150mm; Mobile phase: A=0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: 40°C; Gradient (%B conc): 5%-5% over 2 minutes, 5%-26% over 1 minute, 26%-31% over 8 minutes, then 31-60% over 1 minute. Flow rate: 120 mL / min. The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 99.09%. Analytical conditions: Retention time = 3.23 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 = 923.9(M+3H)3+.

[0233] <Synthesis of Dimers> Synthesis of Peptides (Dimer structure number 46; a peptide compound in which linker structure number 8 is added to a cyclic peptide having the amino acid sequence of peptide sequence number 56) The peptide complex was synthesized by dissolving the peptide monomer (XX) (30 mg) prepared in Example 1 in DMF, then adding NHS-cPEG1c-NHS (0.45 equivalents) and DIEA (10 equivalents), stirring overnight at room temperature, and finally adding water. The resulting crude product was purified under the following conditions: Coca-Cola column: XSelect CSH prep C18 5 μm OBD 50 x 250 mm; mobile phase: A = 1% AcOH in H ２ O, B = 1% AcOH in MeCN Temperature: 50°C; Gradient (%B conc): 0%-0% over 5.1 minutes, 0%-4.2% over 1.9 minutes, 4.2%-28.6% over 3 minutes, 28.6%-33.7% over 15.5 minutes, then 33.7%-60% over 1.5 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. The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 98.87%. Analysis conditions: Retention time = 4.44 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 = 1416.9 (M + 4H) 4+.

[0234] <Monomer Synthesis> Synthesis of Peptides (cyclic peptides having the amino acid sequence of Peptide Sequence ID No. 57, with linker structure number 5, and monomer linker structure number 44) Using Sieber amide resin, the target peptide 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, the first residue was reacted twice for 10 minutes at 90°C. The fourth residue was reacted twice for 15 minutes at 50°C. The fifth and sixth residues were reacted once for 10 minutes at 90°C. The fourteenth residue was reacted once for 15 minutes at 50°C. The Fmoc group was removed by reacting the peptide with a 10% pyrrolidine DMF solution at room temperature for 1 minute, then removing the solution, and reacting it again with a 10% pyrrolidine DMF solution at room temperature for 1 minute. The 13th and 14th residues, and the linker structure portion (15th, 16th, and 17th residues) were reacted once at 90°C for 1 minute. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step using the method described above, and then shaking it in DMF with ClAcOH / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents) at room temperature for 30 minutes. For deprotection of the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride, and then diethyl ether, then dried under reduced pressure, and the reaction agent cocktail-A (TFA / H) was added 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 60 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to chilled excess diisopropyl ether / hexane (1 / 1), a turbid precipitate formed, and the mixture was centrifuged and the solution decanted. The obtained solid was washed again with diethyl ether and dried. The obtained solid was used in the next cyclization reaction. For the peptide cyclization reaction, DMSO / H was used so that the final concentration of the peptide was 5 mM based on the number of moles of the solid phase resin. ２After dissolving in O (9 / 1), triethylamine (10 equivalents) was added, the mixture was stirred for 60 minutes, and then acetic acid was added. The obtained reaction solution was concentrated under reduced pressure using a Genevac EZ-2 Elite. The obtained crude product was purified under the following conditions. Column: XBridge C18 30×150 mm; mobile phase: A = 0.1% TFA in H ２ ₂O, B = 0.1% TFA in MeCN. Temperature: 50°C; gradient (%B concentration): 5%-28% over 3 minutes, 28%-33% over 8 minutes, then 33-60% over 1 minute. Flow rate: 45 mL / min. The purity of the target product was calculated from the area ratio of an LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was found to be 96.05%. Analytical conditions: retention time = 3.58 min; column: Kinetex EVO C18 2.6 μm 2.1×150 mm, 100Å; mobile phase: A = 0.025% TFA in H ２ ₂O, B = 0.025% TFA in MeCN; temperature: 60°C; gradient (%B concentration): 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 = 962.7 (M+3H)³⁺.

[0235] <Synthesis of Dimer> Synthesis of peptide dimer (dimer structure No. 47) The synthesis of the peptide complex was carried out as follows: after dissolving the monomer (Chemical Formula XX) (30 mg) synthesized in Example 3 in DMF, NHS-cPEG1c-NHS (0.45 eq) and DIEA (10 eq) were added, the mixture was stirred at room temperature overnight, and then water was added. The obtained crude product was purified under the following conditions. Column: XSelect CSH prep C18 5 μm OBD 50×250 mm; mobile phase: A = 1% AcOH in H ２O, B = 1% AcOH in MeCN Temperature: 50°C; Gradient (%B conc): 0%-0% over 5.1 minutes, 0%-4.2% over 1.9 minutes, 4.2%-29.7% over 3 minutes, 29.7%-34.7% over 15.5 minutes, then 34.7-60% over 1.5 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. The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 99.51%. Analysis conditions: Retention time = 4.65 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 = 1180.5(M+5H)5+.

[0236] <Synthesis of Dimers> Synthesis of peptide dimers (dimer structure number 3) Using Sieber amide resin, the target peptide (a peptide compound in which linker structure number 6 is added to a cyclic peptide having the amino acid sequence described in Peptide Sequence ID No. 12) was synthesized starting with the removal of the Fmoc group using the general method described above. CEM's Liberty PRIME 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 2 minutes at 105°C in DMF. However, the first residue was reacted twice for 10 minutes at 90°C. The fourth residue was reacted twice for 15 minutes at 50°C. The fourteenth residue was reacted once for 15 minutes at 50°C. The Fmoc group was removed by reacting with a 25% pyrrolidine DMF solution at 50°C for 90 seconds. However, the reaction for the third residue was carried out twice at room temperature for 1 minute each time. The reaction for the 13th and 14th residues, and the linker structure portion consisting of the 15th, 16th, and 17th residues, was carried out once at 110°C for 1 minute each time. The introduction of the chloroacetyl group was performed by removing the Fmoc group from the α-amino group of 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). For deprotection of the side chain and cleavage from the solid resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride, and then diethyl ether, then dried under reduced pressure, and the reaction agent cocktail-A (TFA / H) was added to the reaction vessel containing the solid 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 60 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to chilled excess diisopropyl ether / hexane (1 / 1), a turbid precipitate formed, and the mixture was centrifuged and the solution decanted. The obtained solid was washed again with diethyl ether and dried. The obtained solid was used in the next cyclization reaction. For the peptide cyclization reaction, DMSO / H was used so that the final concentration of the peptide was 5 mM based on the number of moles of the solid phase resin. ２After dissolving in O(9 / 1), triethylamine (10 equivalents) was added and the mixture was stirred for 8 hours. The resulting reaction solution was concentrated under reduced pressure using Genevac HT-12. Dimerization was performed by dissolving the resulting residue in DMSO so that the final peptide concentration was 25 mM based on the number of moles of the solid phase resin, then adding NHS-cPEG1c-NHS (0.34 equivalents) and DIEA (1.6 equivalents), stirring at room temperature for 20 hours, and finally adding acetic acid.

[0237] The obtained crude product was purified using the following conditions: Column: XBridge C18 50x250 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: 50°C; Gradient (%B conc): 18.5%–15.6% over 0.1 minutes, 15.6%–15.6% over 4.9 minutes, 15.6%–18.5% over 2 minutes, 18.5%–43.9% over 3 minutes, 43.9%–48.9% over 15 minutes, then 48.9%–60% over 3 minutes. Flow rate: (118 mL / min–18 mL / min) over 0.1 minutes, 18 mL / min over 4.9 minutes, (18 mL / min–118 mL / min) over 2 minutes, then 118 mL / min. The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 90.22%. Analysis conditions: Retention time = 5.74 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 = 1215.4(M+4H)4+.

[0238] <Synthesis of Dimers> Synthesis of peptide dimers (dimer structure number 4) Using Sieber amide resin, the target peptide (a peptide compound in which linker structure number 7 is added to a cyclic peptide having the amino acid sequence described in peptide sequence number 12) was synthesized starting with the removal of the Fmoc group using the general method described above. CEM's Liberty PRIME was used as a 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 2 minutes at 105°C in DMF. However, the first residue was reacted twice for 10 minutes at 90°C. The fourth residue was reacted twice for 15 minutes at 50°C. The fourteenth residue was reacted once for 15 minutes at 50°C. The Fmoc group was removed by reacting with a 25% pyrrolidine DMF solution at 50°C for 90 seconds. However, the reaction for the third residue was carried out twice at room temperature for 1 minute each time. The reaction for the 13th and 14th residues, and the linker structure portion consisting of the 15th, 16th, and 17th residues, was carried out once at 110°C for 1 minute each time. The introduction of the chloroacetyl group was performed by removing the Fmoc group from the α-amino group of 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). For deprotection of the side chain and cleavage from the solid resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride, and then diethyl ether, then dried under reduced pressure, and the reaction agent cocktail-A (TFA / H) was added to the reaction vessel containing the solid 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 60 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to chilled excess diisopropyl ether / hexane (1 / 1), a turbid precipitate formed, and the mixture was centrifuged and the solution decanted. The obtained solid was washed again with diethyl ether and dried. The obtained solid was used in the next cyclization reaction. For the peptide cyclization reaction, DMSO / H was used so that the final concentration of the peptide was 5 mM based on the number of moles of the solid phase resin. ２After dissolving in O(9 / 1), triethylamine (10 equivalents) was added and the mixture was stirred for 8 hours. The resulting reaction solution was concentrated under reduced pressure using Genevac HT-12. Dimerization was performed by dissolving the resulting residue in DMSO so that the final peptide concentration was 25 mM based on the number of moles of the solid phase resin, then adding NHS-cPEG1c-NHS (0.31 equivalents) and DIEA (1.6 equivalents), stirring at room temperature for 20 hours, and finally adding acetic acid. The resulting crude product was purified using the following conditions: Column: XBridge C18 50x250 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN Temperature: 50°C; Gradient (%B conc): 19.5%–16.6% over 0.1 minutes, 16.6%–16.6% over 4.9 minutes, 16.6%–19.5% over 2 minutes, 19.5%–44.9% over 3 minutes, 44.9%–49.9% over 15 minutes, then 49.9%–60% over 3 minutes. Flow rate: (118 mL / min–18 mL / min) over 0.1 minutes, 18 mL / min over 4.9 minutes, (18 mL / min–118 mL / min) over 2 minutes, then 118 mL / min. The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 88.50%. Analysis conditions: Retention time = 5.81 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 = 1303.4(M+4H)4+.

[0239] <Synthesis of Dimers> Synthesis of peptide dimer (dimer structure number 53)

[0240] Using NovaPEG Link Amide resin, the synthesis began with the removal of the Fmoc group using the general method described above, followed by the introduction of Fmoc-K(Fmoc)-OH, after which the target peptide (a peptide compound in which linker structure number 10 is added to a cyclic peptide having the amino acid sequence described in Peptide Sequence ID No. 64) was synthesized. Biotage's Syro I was used as a 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 (12.6 equivalents / 11.8 equivalents / 25.2 equivalents) was used per equivalent of resin, and the reaction was carried out twice in DMF at 75°C for 30 minutes. However, for the 4th, 8th, and 14th residues, the reaction was carried out twice at 50°C for 60 minutes. For the 16th residue, the reaction was carried out twice at 25°C for 60 minutes.

[0241] 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.

[0242] 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 60 minutes with ClAcOH / HATU / DIEA (12.6 equivalents / 11.8 equivalents / 25.2 equivalents).

[0243] 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, 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 92.5:2.5:2.5:25 was added and shaken well, then shaken at room temperature for 30 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 again with a mixed solvent of diethyl ether / hexane (1 / 1) and dried. The obtained solid was used in the next cyclization reaction.

[0244] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 2.5 mM based on the number of moles of the solid phase resin, then adding triethylamine (20 equivalents) and stirring for 15 hours. The resulting reaction solution was concentrated under reduced pressure using Genevac EZ-2 Elite.

[0245] The obtained crude product was subjected to solid-phase extraction using a Gilson column in the following steps (column: Gilson ASPEC C18 50 mg 1 mL): (1) The column was used to extract solution A (0.1% TFA in 95% MeCN / H ２ Washed with 0.3 mL of 0. (2) Extract B (0.1% TFA in 5% MeCN / H ２ (3) The column was equilibrated with (0.3 mL) the above solution. (4) The column was washed with extract B (0.4 mL). (5) Extraction was performed with extract A (0.4 mL). The obtained extract was concentrated under reduced pressure using EZ-2 Elite.

[0246] The purity of one of the main peaks of the target substance was calculated to be 37% from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions: Retention time = 1.60 min; Column: Kinetex EVO C18 1.7 μm 2.1 x 50 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 = 1292.4(M+4H)4+

[0247] <Synthesis of Dimers> Synthesis of peptide dimers (dimer structure number 61)

[0248] Using Sieber amide resin, the target peptide (a peptide compound in which linker structure number 11 is added to a cyclic peptide having the amino acid sequence described in peptide sequence number 11) was synthesized starting with the removal of the Fmoc group using the general method described above. CEM's multipep2 was used as a 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 / 3.9 equivalents / 8.4 equivalents) was used per equivalent of resin, and the reaction was carried out once at 50°C for 15 minutes. However, for residues 4, 6, 7, 8, and 10, the reaction was carried out twice at 50°C for 30 minutes each. For residue 12, the reaction was carried out twice at 50°C for 15 minutes each. For residues 13-17, the reaction was carried out twice at 50°C for 60 minutes each. Furthermore, to introduce the 17th residue, which constitutes the linker structure, a mixture of Fmoc-Gpra-OH / Fmoc-KN3-OH (1.4 / 1) was used as Fmoc-AA.

[0249] The Fmoc group was removed by reacting it with a 10% pyrrolidine DMF solution at room temperature for 3 minutes.

[0250] 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 reacting it twice in DMF at room temperature for 30 minutes using ClAcOH / HATU / DIEA (4.2 equivalents / 3.9 equivalents / 8.4 equivalents).

[0251] 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, 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 90:2.5:2.5:5 was added and shaken well, then shaken at room temperature for 50 minutes. The reaction mixture was filtered and recovered through the frit. When this filtrate was added to chilled excess diethyl ether, a turbid precipitate formed, and the mixture was centrifuged and the solution decanted. The obtained solid was washed again with diethyl ether and dried. The obtained solid was used in the next cyclization reaction.

[0252] 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. ２ After dissolving in O(9 / 1), triethylamine (20 equivalents) was added and the mixture was stirred for 15 hours. The resulting reaction solution was concentrated under reduced pressure using Genevac HT-12.

[0253] The dimerization of the obtained crude product was carried out by the method described below. First, DMSO / H was used to adjust the final peptide concentration to 2.5 mM based on the number of moles of the solid phase resin. ２ After dissolving in O(3 / 1), THPTA (4 equivalents) and tetrakis(acetonitrile) copper(I) hexafluorophosphate (4 equivalents) were added and the mixture was stirred for 1 hour. Subsequently, the reaction was stopped by adding 12 equivalents of 1 M DTT aqueous solution, and the resulting solid was removed by centrifugation. The resulting reaction solution was concentrated under reduced pressure using Genevac HT-12.

[0254] The obtained crude product was subjected to solid-phase extraction using a Gilson column in the following steps (column: Gilson ASPEC C18 50 mg 1 mL): (1) The column was used to extract solution A (0.1% TFA in 95% MeCN / H ２ Washed with 0.3 mL of 0. (2) Extract B (0.1% TFA in 5% MeCN / H ２ (1) The column was equilibrated with (0.3 mL). (2) 0.02 mL of the above solution was loaded onto the column. (3) The column was washed with extractant B (0.4 mL). (4) Extraction was performed with extractant A (0.4 mL). The obtained extract was concentrated under reduced pressure.

[0255] The purity of one of the main peaks of the target substance was calculated to be 37% from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions: Retention time = 2.04 min; Column: Kinetex EVO C18 1.7 μm 2.1 x 50 mm, 100 Å; Mobile phase: A = 0.025% TFA in H ２O, 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 = 1314.3(M+4H)4+

[0256] <Monomer Synthesis> Synthesis of Peptides (Monomer Linker Structure No. 84; Peptide compounds in which linker structure No. 2 is added to a cyclic peptide having the amino acid sequence described in Peptide Sequence ID No. 84)

[0257] Using Sieber amide resin preloaded with HA tag, the target peptide was synthesized by starting with the removal of the Fmoc group using the general method described above. A Biotage 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 (4.2 equivalents / 3.9 equivalents / 8.4 equivalents) was used per equivalent of resin, and the reaction was carried out twice at 75°C for 20 minutes in DMF. However, the first residue was reacted three times at 75°C for 30 minutes. The second and fifth residues were reacted twice at 75°C for 30 minutes. The fourth residue was reacted twice at 50°C for 60 minutes. The fourteenth residue was reacted twice at 50°C for 30 minutes. The sixteenth residue, which is the linker structure, was reacted once at room temperature for 60 minutes.

[0258] The Fmoc group was removed by reacting it with a 10% pyrrolidine DMF solution at room temperature for 3 minutes.

[0259] 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 reacting it twice in DMF at room temperature for 30 minutes using ClAcOH / HATU / DIEA (4.2 equivalents / 3.9 equivalents / 8.4 equivalents).

[0260] 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, 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 well, then shaken at room temperature for 30 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 resulting solid was washed with diethyl ether / hexane (1:1) and dried. The resulting solid was used in the next cyclization reaction.

[0261] 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. ２ After dissolving in O(9 / 1), triethylamine (20 equivalents) was added and the mixture was stirred for 16 hours. The resulting reaction solution was concentrated under reduced pressure using Genevac EZ-II elite.

[0262] The obtained crude product was subjected to solid-phase extraction using a Gilson column in the following steps (column: Gilson ASPEC C18 50 mg 1 mL): (1) The column was used to extract solution A (0.1% TFA in 95% MeCN / H ２ Washed with 0.3 mL of 0. (2) Extract B (0.1% TFA in 5% MeCN / H ２ (1) The column was equilibrated with (0.3 mL). (2) 0.02 mL of the above solution was loaded onto the column. (3) The column was washed with extractant B (0.4 mL). (4) Extraction was performed with extractant A (0.4 mL). The obtained extract was concentrated under reduced pressure.

[0263] The purity of one of the main peaks of the target substance was calculated to be 53% from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions: Retention time = 1.51 min; Column: Kinetex EVO C18 1.7 μm 2.1 x 50 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 = 1210.7(M+3H)3+

[0264] <Synthesis of Various Peptide Complexes (Dimers)> In this example, the dimers shown in Tables 5 and 6 were synthesized in the same manner as described in Examples 1 to 8. The dimers listed in Table 5 were synthesized in the same manner as in Examples 1 to 6, and the dimers listed in Table 6 were synthesized in the same manner as in Example 7 or 8. In Tables 5 and 6, the peptide sequence number and linker structure number indicate the structure of the peptide and linker contained in the monomer. The monomers were analyzed under any of the analytical conditions described in Examples 1 to 8, and their structures were 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 5 and 6.

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] <Synthesis of Various Peptide Complexes (Monomers)> In this example, monomers (monomers with linker structures added) shown in Tables 7 to 9 were synthesized. Monomers shown in Table X7 were synthesized in the same manner as in Example 1. Monomers (monomers with linker structures added) shown in Table 8 were synthesized in the same manner as in Example 9. Also, monomers (monomers with linker structures added) shown in Table 9 were synthesized in the same manner as in Examples 1 and 3. In Tables 7 to 9, the peptide sequence number and linker structure number indicate the structure of the peptide and linker contained in the monomer. Furthermore, the monomers were analyzed under any of the analytical conditions described in Examples 1 to 8, and their structures were confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observations, retention time, valency, and concentration gradient (%) of mobile phase B used for analysis are shown in Tables 7 to 9.

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] Table 1 shows the amino acid sequence of the peptide contained in the synthesized monomer or dimer, and Table 2 shows the structure of the linker.

[0288] ​<Evaluation of FGFR2b agonist activity by phosphorylation of FGFR2b and ERK> To evaluate the FGFR2b agonist activity of the peptide complex (peptide dimer) of the present invention, the phosphorylation of FGFR2b and ERK1 / 2 was investigated.

[0289] Mouse proB cell line Ba / F3 cells expressing human FGFR2b (human FGFR2b-expressing Ba / F3) were generated by the following method.

[0290] After transfection of Ba / F3 cells with a human FGFR2b expression vector by electroporation, the cells were 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. 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 FGF7 protein (R&D) (hereinafter referred to as FGF7), 1 IU Heparin (Sigma), and 50 nM β-mercaptoethanol (Thermo Fisher Scientific). Subsequently, they were cultured in growth medium supplemented with 800 ng / mL Geneticin. Single clones were then obtained by limiting dilution.

[0291] <Evaluation of FGFR2b agonist activity by phosphorylation of FGFR2b> Human FGFR2b-expressing Ba / F3 cells prepared as described above were cultured in growth medium supplemented with 800 ng / mL Geneticin. After harvesting the cells, 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. The cells were incubated in a CO2 incubator at 37°C for 3 hours. Subsequently, FGF7 or the peptide dimer synthesized in Examples 1-11 was added, and the plate was incubated at room temperature for 15 minutes while permeating. The samples were then dissolved in the Lysis buffer included with the Phospho-FGF Receptor 2 (Tyr653 / 654) AlphaLISA SureFire Ultra Detection Kit (Revvity). The procedure was carried out according to the kit protocol, and SpectraMAX Paradigm (Molecular Devices) was used to detect the signal. The obtained signals were analyzed with GraphPad Prism, and % activity was calculated with the maximum signal induced by 52 nM FGF7 set to 100% and no stimulation set to 0%. The peptide dimers synthesized in Examples 1 to 11 were evaluated at concentrations of 100 nM and 1000 nM for peptide dimer structure numbers 1, 11-13, 32, 53-263, and 10 nM and 100 nM for peptide dimer structure numbers 2-10, 14-31, and 33-52. Dimers with a % activity of 80% or more at 10 nM were evaluated as A-1, those with 80% or more at 100 nM were A-2, those with 80% or more at 1000 nM were A-3, those with 50% or more but less than 80% were A-4, and those with 10% or more but less than 50% were A-5. The results are shown in Table 10. This demonstrates the phosphorylation ability of the peptide complex of the present invention to FCFR2b.

[0292] <Evaluation of FGFR2b agonist activity by ERK phosphorylation> To evaluate the ERK activating ability of the FGFR2 agonist peptide (peptide dimer) of the present invention, ERK phosphorylation in human FGFR2b-expressing Ba / F3 cells was investigated. Using the lysate prepared above, the procedure was carried out according to the AlphaLISA SureFire Ultra Human & Mouse Phosph-ERK1 / 2 (Thr202 / Tyr204) Detection Kit (Revvity) protocol, and SpectraMAX Paradigm was used for signal detection. The obtained signals were analyzed with GraphPad Prism, and % activity was calculated with the maximum value of the signal induced by FGF7 set to 100% and no stimulation set to 0%. FGF7 was evaluated at 52 nM and 10-fold dilutions at three or four points (0.052 nM to 52 nM), and Emax (100% activity) was calculated. The peptide dimers synthesized in Examples 1 to 11 were evaluated at the following concentrations: 1 nM, 10 nM, 100 nM, and 1000 nM for peptide dimer structure numbers 1 to 19; 1 nM, 10 nM, and 100 nM for peptide dimer structure numbers 20 to 31, 35, and 40 to 45; and 1 nM and 10 nM for peptide dimer structure numbers 33, 34, 36 to 39, and 46 to 52. Dimers with a % activity of 50% or more at 1 nM were evaluated as B-1, those with 50% or more at 10 nM as B-2, those with 50% or more at 100 nM as B-3, those with 50% or more at 1000 nM as B-4, and those with 10% or more but less than 50% as B-5. The results are shown in Table 10. This demonstrates the ERK phosphorylation ability of the peptide complex of the present invention.

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302] <Proliferation assay of human FFFR2b-expressing Ba / F3 cells> To evaluate the FFFR2b agonist activity of the peptide complex (peptide dimer) of the present invention, the proliferation of human FFFR2b-expressing Ba / F3 cells was investigated.

[0303] After culturing the human FGFR2b-expressing Ba / F3 cells prepared in Example 11, they were washed twice with RPMI-1640 medium (Nakarai Tesque) containing 10% FBS, 1 unit / mL Heparin, and 25 ug / mL gentamicin, suspended, and seeded into 96-well cell adhesion plates with white sides and a transparent bottom, so that there were 9000 cells per well. Immediately afterward, FGF7 or the peptide dimers synthesized in Examples 1 to 10 were added, and the cells were cultured in a CO2 incubator at 37°C for 3 days. A white seal was attached to the bottom of the plate, and cell count detection was performed according to the CellTiter-Glo (Promega) kit protocol. SpectraMAX Paradigm was used for signal detection. The obtained signals were analyzed using GraphPad Prism, and % activity was calculated with the maximum signal induced by FGF7 set to 100% and the unstimulated state to 0%. FGF7 was evaluated at five 10-fold dilutions (0.0052 nM to 52 nM) starting from 52 nM, and Emax (100% activity) was calculated. The peptide dimer was evaluated at concentrations of 1 nM and 10 nM. Those with % activity of 50% or more at 1 nM were evaluated as C-1, and those with % activity of 50% or more at 10 nM were evaluated as C-2. The results are shown in Table 11. This demonstrates the proliferative ability of the peptide complex of the present invention in human FGFR2b-expressing Ba / F3 cells.

[0304]

[0305] <Evaluation Test of Intermolecular Interactions between FFFR2b and Peptide Monomers> To evaluate the binding ability of the peptide monomers (linker-structured monomers) synthesized in Examples 1 to 11 to FFFR2b, the intermolecular interactions of the peptides with Recombinant Human FFFR2 beta (IIIb) Fc Chimera Protein, CF (R&D systems) (hereinafter referred to as βFFFR2b-Fc) were tested by surface plasmon resonance (SPR) using the method described below.

[0306] A CM3 sensor chip (Cytiva) was inserted into the BiacoreT200 (Cytiva), and several priming operations were performed with running buffer: HBS-P+, pH 7.4 (Cytiva), followed by equilibration at a flow rate of 30 μL / min. Since the FGFR2b protein used in this study is an Fc fusion protein, the Human Antibody Capture Kit, type 2 (Cytiva) was used. Immobilization of the capture molecule (0.5 mg / ml Anti-human IgG (Fc) antibody, hereafter referred to as Fc antibody, contained in the above capture kit) was performed at a flow rate of 10 μL / min. 100 μL each of 60 mM EDC solution (Cytiva) and 650 mM NHS solution (Cytiva) were mixed, and the mixture was reacted at a flow rate of 10 μL / min for 420 seconds to activate the carboxyl groups on the sensor tip. 200 μL of 0.2 μM 0.02 mg / ml Fc antibody solution was prepared by diluting with 10 mM acetic acid solution (pH 5.0), and the Fc antibody was immobilized on the CM3 sensor tip by reacting at a flow rate of 10 μL / min for 420 seconds. After immobilization, capping was performed by reacting with 1.0 M ethanolamine aqueous solution (Cytiva) at a flow rate of 10 μL / min for 420 seconds. Next, 0.1 μM βFFFR2b-Fc was reacted with the capture molecule (Fc antibody) at a flow rate of 5 μL / min for 120 seconds to capture approximately 1,200 RU. The peptide monomer (linker-structured monomer) solutions synthesized in Examples 1-11, prepared to 10 mM in DMSO solution, were diluted with running buffer to a final concentration of 10 μM peptide solution. 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 with respect to βFFFR2b-Fc were obtained by SPR measurement.

[0307] The kinetics evaluation model was Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Cytiva). The binding of the peptide to βFFFR2b-Fc was evaluated by performing curve fitting using the least squares method on the obtained sensorgrams and determining the KD value. The calculated KD values ​​are shown in Table 12. This demonstrates the binding ability of the peptide monomer of the present invention to FFFR2b.

[0308]

[0309] <Evaluation of Binding Activity to FGFR2b by ELISA Method> To evaluate the binding ability of the peptide monomers (linker-structured monomers) synthesized in Examples 1 to 11 to FGFR2b, binding activity was evaluated using the ELISA method. The specific test method is shown below.

[0310] Goat anti-Human IgG Fc Secondary Antibody, Biotin, eBiosscience® (Invitrogen) (hereinafter referred to as anti-Fc Ab), diluted 125 times with PBS-T containing 1% BSA, was added to a Nunc immunobilizer streptavidin f96 clear (Thermo; 436014) (hereinafter referred to as SA plate), and the plate was allowed to stand for 30 minutes to immobilize the anti-Fc Ab onto the SA plate. After washing three times with PBS-T, 3 pmol of βFFFR2b-Fc was added per well, and the mixture was allowed to stand for 30 minutes to bind βFFFR2b-Fc to anti-Fc Ab immobilized on the SA plate. After washing three times with PBS-T, HA-tagged fusion peptides (linker-structured monomers synthesized in Examples 1-11), diluted to 10 nM or 100 nM (amounts are shown in Table 13 or Table 14), were added and allowed to stand for 1 hour. After washing three times with PBS-T, Anti-HA-tagmAb-HRP-DirectT (MBL), diluted 5000-fold with PBS-T containing 1% BSA, was added and allowed to stand for 30 minutes. After washing three times with PBS-T, the detection reagent SureBlue® TMB 1-Component Microwell Peroxidase Substrate (SeraCare) (hereinafter referred to as TMB solution) was added and allowed to stand for 10 minutes. The reaction was then stopped by adding an equal volume of TMB Stop Solution (SeraCare) to the TMB solution. The absorbance at 450 nm was measured using Infinite M Nano (TECAN). The binding signal of each peptide was calculated by subtracting the absorbance of the well without the peptide from the absorbance of the well with the peptide added. The calculated binding signals are shown in Tables 13 and 14. This demonstrates the binding ability of the peptide monomers of the present invention to FGFR2b.

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] <Discussion> The results from Examples 12 to 15 demonstrate that the peptide complex (dimer) of the present invention has agonist activity against FFFR2b. Furthermore, the results from Examples 14 and 15 demonstrate that the peptide monomer of the present invention (including monomers to which a linker structure has been added) has binding ability to FFFR2b.

Claims

1. A peptide comprising the 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, or a pharmaceutically acceptable salt thereof. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 where, X1 is any amino acid residue, X2 is an amino acid residue having an aryl group in its side chain which may be N-alkylated or substituted, X3 is any amino acid residue, X4 is any amino acid residue, X5 is a secondary amino acid or peptoid which may be N-alkylated, X6 is a secondary amino acid or peptoid, X7 is an amino acid residue having an aryl group in its side chain which may be substituted, X8 is an amino acid residue having an aryl group in its side chain which may be substituted, X9 is glycine or any d-amino acid residue, X10 is an amino acid residue having an aliphatic hydrocarbon group in its side chain which may be substituted, X11 is any amino acid residue, X12 is any amino acid residue, X13 is an acidic amino acid residue that may be of type D, and X14 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 MeF, dmor, MeKCOPipzaa, MeE, KCOPipzaa, dp, or dhyp, which may be modified with a chloroacetyl group, and X2 is MeF, MeF4COO, MeY, MeF4CON, MeF3T, MeF3C, F, MeF3COO, MeF3CON, Me4Py, Me3Py, MeF4F, MeF3F, MeF4C, MeF4T, MeF3Et, MeF4Et, or MeF4OMe, X3 is Tic, P, T, MeT, Hyp, P4Sh, MeKAc, Pipz4Me, R, Mor, alT, dMeS, DapAc, DapMs, DapCOPipzaa, V, Gthp, N, H, A1mor, MeS, MeDapAc, MeE, MeD, MeQ, MeN, MeL, MeKCOPipzaa, MeCit, MeKMor, Aib, P4Cp, P4F2, P4RNHAAc, P4SNHAAc, P4Raao, or P4Saao, and X4 is S, R, H, Q, E, Cit, 4Py, H3Py6NH2, H4Py2NH2, H3Py, or H4Py, X5 is MeG, CeG, CmG, P, P4Sh or Mor, and X6 is P, Hyp, PpG, MsapG, IeG, ImG, 3PypG, 4PypG, 2PyeG, 3PyeG, 4PyeG, 2PymG, 4PymG, P4F2, P4RNHAc, P4RO3Py, P4RO2Py, P4SPh, HypBn, P4Raao, P4RNHCONHmCOO or P4RNCOPipzaa, X7 is Y, F4aao, F4F, F3CON, Ym2Py, F34dOMe, F35dOMe, F4COO, F4OEt, F, F4C, F4OMe, F3H, F3OMe, F3C, Yph, YBn, Ym4Py, Ym3Py, F3OPh, F3OBn, Dopa, F345tOMe, F34diox, F34mdio, W, W5H, W7N, F4CON, F4T, or F3T, X8 is Bph, Bph4COO, Bph4NAc, Bph3COO, Bph4CON, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F44Py, F42Py, 3Py6Ph, Bph3NAc, Bph3F, pBph2F, pBph3F, pBph3C, or Nal2, and X9 is G, da, ds, or dn,X10 is Bph, Bph3COO, 3Py6Ph, Bph4COO, Bph3CON, Bph4H, Bph3H, Bph4OMe, Bph3OMe, F43Py, F42Py, Bph2F, 3Py6O4pip1aa, Bph3F, Bph2H, Bph4NAc, Bph3NAc, pBph3C, pBph3F, pBph2C, pBph2F, pBph2Me, or pBph2OMe, and X11 is D, G, A, E, Hgl, R, P, or Hyp, The peptide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X12 is N, R, Hgl, MeN, MeF, dp, dhpr, Q, Hgn, D, E, Cit, KCOPIPZAa, or dn, and X13 is D, E, or dd.

3. The peptide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence represented by formula A2, or the amino acid sequence represented by formula A2, in which 1 to 13 amino acid residues arbitrarily selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th amino acid residues from the N-terminus are substituted or deleted. A2: MeE-MeF3T-T-R-MeG-P-F35dOMe-Bph4COO-G-Bph3COO-D-N-D-C (SEQ ID NO: 57) 4. A peptide comprising the amino acid sequence shown in SEQ ID NOs: 1 to 261, or a peptide according to any one of claims 1 to 3, having an amino acid sequence in which the N-terminal amino acid residue of the amino acid sequence shown in SEQ ID NOs: 1 to 261 is modified with a chloroacetyl group, or a pharmaceutically acceptable salt thereof.

5. A cyclic peptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

6. The peptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein one of the amino acid residues 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.

7. The peptide according to any one of claims 1 to 3, further having a glycine residue at its C-terminus, or a pharmaceutically acceptable salt thereof.

8. The peptide according to claim 5, or a pharmaceutically acceptable salt thereof, further comprising additional amino acid residues.

9. A peptide having the amino acid sequence of Sequence ID No. 57, wherein one amino acid residue may be substituted, and the peptide has a cyclic structure in which the first amino acid residue and the 14th cysteine ​​residue of the peptide are linked intramolecularly, according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

10. A peptide complex comprising two or more peptides described in claim 1, or pharmaceutically acceptable salts thereof.

11. The peptide complex according to claim 10, having FGFR2b agonist activity.

12. The peptide complex according to claim 10, comprising two or more peptides according to claim 1, or pharmaceutically acceptable salts thereof, and a linker connecting the peptides, wherein the amino acid sequences of the peptides may be the same or different.

13. The peptide complex according to claim 10, wherein the amino acid sequence homology between the peptides is 90% or more and 100% or less.

14. The peptide complex according to claim 10, wherein each of the two or more peptides is a cyclic peptide.

15. The peptide complex according to claim 10, 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.

16. The peptide complex according to claim 10, wherein at least one of the two or more peptides comprises an additional amino acid residue.

17. The peptide complex according to claim 12, wherein the linker is a PEG linker, or a linker consisting of PEG and amino acid residues.

18. The peptide complex according to claim 12, wherein the linker is one arbitrarily selected from linkers represented by linker structure numbers 1 to 11.

19. The peptide complex according to claim 12, wherein the C-terminuses of the peptides or the side chains of the amino acid residues are linked to each other via the linker.

20. A cell culture composition comprising the peptide complex described in claim 10, used for cell culture.

21. A composition comprising the peptide complex described in claim 10, for use in medical, diagnostic, or research purposes.

22. A method for producing a peptide complex, comprising the steps of: producing a peptide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof; and linking two or more of the peptides together with a linker.

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