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

Abstract

This invention relates to peptide complexes comprising a first peptide and a second peptide. The first peptide is a peptide comprising the amino acid sequence shown in Formula A1, or an amino acid sequence formed by substitution, deletion, addition, or insertion of one or more amino acid residues in the amino acid sequence shown in Formula A1. The second peptide is a peptide comprising the amino acid sequence shown in Formula B1, or an amino acid sequence formed by substitution, deletion, addition, or insertion of one or more amino acid residues in the amino acid sequence shown in Formula B1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14; B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480083790.6 (22) Application Date 2024.11.08 (30) Priority Data 2023-192587 2023.11.10 JP (85) PCT International Application Entering National Phase Date 2026.07.03 (86) PCT International Application Application Data PCT / JP2024 / 039749 2024.11.08 (87) PCT International Application Publication Data WO2025 / 100520 JA 2025.05.15 (71) Applicant: Pepperdim Co., Ltd. Address: Kanagawa Prefecture, Japan (72) Inventors: Yoshinori Suzuki, Norihiro Shibata, Kenta Kamiyaku (74) Patent Agency: Beijing Gecheng Intellectual Property Agency Co., Ltd. 11314 Patent Attorney Cheng Wei Han Wenhua (51) Int.Cl. C07K 7 / 08 (2006.01) A61K 38 / 10 (2006.01) A61K 38 / 12 (2006.01) A61K 47 / 65 (2006.01) A61P 35 / 00 (2006.01) A61P 43 / 00 (2006.01) C07K 1 / 06 (2006.01) C12N 5 / 00 (2006.01) (54) Invention Title Peptide, Peptide Complex, Pharmaceutical Composition, Composition for Cell Culture and Composition for Medical, Diagnostic or Research Use (57) Abstract This invention relates to peptide complexes comprising a first peptide and a second peptide. Wherein, the first peptide is a peptide comprising the amino acid sequence shown in Formula A1 or a peptide comprising an amino acid sequence formed by substitution, deletion, addition, or insertion of one or more amino acid residues in the amino acid sequence shown in Formula A1; the second peptide is a peptide comprising the amino acid sequence shown in Formula B1 or a peptide comprising an amino acid sequence formed by substitution, deletion, addition, or insertion of one or more amino acid residues in the amino acid sequence shown in Formula B1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14; B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14. Claims (19 pages), Description (110 pages), Sequence Listing (electronic publication) CN 122555715 A 2026.08.11 CN 1 22 55 57 15 A 1. A peptide comprising the amino acid sequence shown in 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 shown in Formula A1.A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 Where X1 is any D-amino acid residue, X2 and X3 are each independently any amino acid residue, X4 and X5 are each independently an amino acid residue with a substituted aliphatic hydrocarbon group on its side chain, X6 is an amino acid residue with a substituted aryl group on its side chain, X7 is an N-alkyl amino acid residue with a substituted aryl or substituted cycloalkyl group on its side chain, X8 is any amino acid residue, X9 is an L-threonine (T) residue, X10 is any N-alkylated amino acid residue, X11 is any amino acid residue, X12 is an amino acid residue with an aliphatic hydrocarbon group on its side chain, X13 is an amino acid residue with a substituted aliphatic hydrocarbon group on its side chain, and X14 is an L-cysteine ​​(C) residue. In Formula A1, the amino acid sequence is described from the N-terminus to the C-terminus. 2. The peptide according to claim 1, wherein, in Formula A1, X1 is a D-phenylalanine (df) residue, (R)-2-amino-3-(pyridin-4-yl)propionic acid (d4py) residue, (R)-2-amino-3-(pyridin-3-yl)propionic acid (d3py) residue, (R)-2-amino-3-(3-methoxyphenyl)propionic acid (df3OMe) residue, (R)-2-amino-3-(4-methoxyphenyl)propionic acid (df4OMe) residue, (R)-2-amino-5-ureidovalerate (dcit) residue, D-tyrosine (dy) residue, or (R)-2-amino-3-(4-fluorophenyl)propionic acid (df4F) residue. X2 is an L-serine (S) residue, L-asparagine (N) residue, L-aspartic acid (D) residue, L-2-aminoadipic acid (Hgl) residue, L-citrulline (Cit) residue, or (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue; X3 is an L-aspartic acid (D) residue, L-arginine (R) residue, L-citrulline (Cit) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue, L-asparagine (N) residue, or L-serine (S) residue; X4 is an L-isoleucine (I) residue or O-methyl-L-threonine (TMe) residue. X5 is an L-arginine (R) residue, an L-glutamic acid (E) residue, a (S)-2-aminoheptanoic acid (Ahp) residue, an L-serine (S) residue, an L-homoserine (Hse) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, a (S)-2-amino-4-ureidobutyric acid (Ncit) residue, or a (S)-5-acetamido-2-aminovaleric acid (OrnAc) residue.X6 is an L-tyrosine (Y) residue, a methyl-L-tyrosine (MeY) residue, a (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, a (S)-2-amino-3-(4-chlorophenyl)propionic acid (F4C) residue, a (S)-2-amino-3-(pyridin-4-yl)propionic acid (4Py) residue, or a (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue. X7 is a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue; X8 is an L-glutamine (Q) residue, L-valine (V) residue, L-2-aminoadipic acid (Hgl) residue, 1-(carboxymethyl)- (Claims 1 / 19 page 2 CN 122555715 A) L-tryptophan (W1aa) residue, (S)-2-aminoheptanoic acid (Ahp) residue, L-high tyrosine (Hty) residue, L-threonine (T) residue, allothreonine (alT) residue, L-citrulline (Cit) residue, N6-(4-(carboxymethyl)piperazin-1-carbonyl)-L-lysine (KCOpipzaa) residue, (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (Gthp) residue, (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, (S)-2-amino-2-cyclohexylacetic acid (Chg) residue, or O-methyl-threonine (TMe) residue. X10 is a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)hexanoic acid (MeNle) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue; X11 is an L-isoleucine (I) residue, L-tyrosine (Y) residue, (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (F4aao) residue, or O-methyl-L-threonine (TMe) residue.X12 is an L-valine (V) residue, a methyl-L-valine (MeV) residue, or an N-methyl-L-cyclohexylglycine (MeChg) residue, and X13 is a glycine (G) residue, a D-alanine (da) residue, or a D-serine (ds) residue. 3. A peptide comprising an amino acid sequence of formula A2 or an amino acid sequence in which at least one amino acid residue selected from the group consisting of amino acid residues at positions 1 to 12 of the amino acid sequence of formula A2 has been substituted, deleted, added or inserted, wherein A2: df-SDIRY-MeF-QT-MeF-IVGC (serum number 164) wherein df is a D-phenylalanine residue, S is an L-serine residue, D is an L-aspartic acid residue, I is an L-isoleucine residue, R is an L-arginine residue, Y is an L-tyrosine residue, MeF is a methyl-L-phenylalanine residue, Q is an L-glutamine residue, T is an L-threonine residue, V is an L-valine residue, G is a glycine residue, and C is an L-cysteine ​​residue. 4. The peptide according to any one of claims 1 to 3, wherein the C-terminus further comprises a glycine residue. 5. The peptide according to any one of claims 1 to 3, wherein it is a cyclic peptide. 6. The peptide according to claim 4, wherein it is a cyclic peptide. 7. The peptide according to any one of claims 1 to 3, wherein it has a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide. 8. The peptide according to any one of claims 1 to 3, further comprising an added amino acid residue. 9. The peptide according to any one of claims 1 to 3, wherein it has binding affinity to an Fzd receptor. Claims 2 / 19 Page 3 CN 122555715 A 10. The peptide according to claim 9, wherein it has binding affinity to at least one selected from the group consisting of Fzd receptor 2, Fzd receptor 7, and Fzd receptor 8. 11. The peptide according to any one of claims 1 to 3, comprising the amino acid sequence shown in formula A3, A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein ClAc is a chloroacetyl group, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are the amino acid residues shown in Table 1, wherein in Table 1, df is a D-phenylalanine residue, d4py is a (R)-2-amino-3-(pyridin-4-yl)propionic acid residue, and d3py is a (R)-2-amino-3-(pyridin-3-yl)propionic acid residue.df3OMe represents (R)-2-amino-3-(3-methoxyphenyl)propionic acid residue, df4OMe represents (R)-2-amino-3-(4-methoxyphenyl)propionic acid residue, dcit represents (R)-2-amino-5-ureidovalerate residue, dy represents D-tyrosine residue, df4F represents (R)-2-amino-3-(4-fluorophenyl)propionic acid residue, S represents L-serine residue, N represents L-asparagine residue, D represents L-aspartic acid residue, Hgl represents L-2-aminoadipic acid residue, Cit represents L-citrulline residue, Kmor represents (S)-2-amino-6-morpholinohexanoic acid residue, R represents L-arginine residue, and KCOpipzaa represents N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue. Atp is a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid residue, I is an L-isoleucine residue, TMe is an O-methyl-L-threonine residue, E is an L-glutamic acid residue, Ahp is a (S)-2-aminoheptanoic acid residue, Hse is an L-homoserine residue, Hcit is an N6-carbamoyl-L-lysine residue, Ncit is a (S)-2-amino-4-ureidobutyric acid residue, OrnAc is a (S)-5-acetamido-2-aminovaleric acid residue, Y is an L-tyrosine residue, MeY is a methyl-L-tyrosine residue, F4F is a (S)-2-amino-3-(4-fluorophenyl)propionic acid residue, F4C is a (S)-2-amino-3-(4-chlorophenyl)propionic acid residue, and 4Py is a (S)-2-amino-3-(pyridin-4-yl)propionic acid residue. F3COO is a (S)-3-(2-amino-2-carboxyethyl)benzoic acid residue, MeF is a methyl-L-phenylalanine residue, Me3Py is a (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid residue, claims 3 / 19, page 4, CN 122555715 A, Me4Py is a (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid residue, MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeAtp is a (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid residue, Q is an L-glutamine residue, and V is an L-valine residue. W1aa is a 1-(carboxymethyl)-L-tryptophan residue, Hty is an L-homotyrosine residue, T is an L-threonine residue, alT is an allothreonine residue, Gthp is a (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid residue, and G4pipaa is a (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid residue.Cbg is a (S)-2-amino-2-cyclobutylacetic acid residue, Chg is a (S)-2-amino-2-cyclohexylacetic acid residue, MeNle is a (S)-2-(methylamino)hexanoic acid residue, F4aao is a (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid residue, MeV is a methyl-L-valine residue, MeChg is an N-methyl-L-cyclohexylglycine residue, G is a glycine residue, C is an L-cysteine ​​residue. [Table 1A] Claims 4 / 19, page 5, CN 122555715 A [Table 1B] Claims 5 / 19, page 6, CN 122555715 A [Table 1C] Claims 6 / 19, page 7, CN 122555715 A [Table 1D] Claims 7 / 19, page 8, CN 122555715 A 12. A peptide comprising the amino acid sequence shown in Formula B1 or an amino acid sequence in which one or more amino acid residues are substituted, deleted, added, or inserted. Claims 8 / 19, page 9, CN 122555715 A B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 wherein, Y1 is a substituted L-phenylalanine (F) residue, Y2 is a substituted L-arginine (R) residue, Y3 is a substituted L-tryptophan (W) residue or a substituted (S)-2-amino-3-(naphthyl-1-yl)propionic acid (Nal1) residue, Y4 is an acidic amino acid residue or a neutral amino acid residue, Y5 is a substituted aromatic amino acid residue, an aliphatic amino acid residue, or an L-proline (P) residue, and Y6 is a substituted aliphatic amino acid residue. Y7 is an N-alkylated aromatic amino acid residue, Y8 is an amino acid residue derived from an amino acid having a hydroxyl, carboxyl, or amide group on its side chain, Y9 is any amino acid residue, Y10 is a substituted L-tryptophan (W) residue, Y11 is a substituted L-asparagine (N) residue or glutamine (Q) residue, Y12 is a substituted (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, Y13 is a substituted neutral or acidic amino acid residue, and Y14 is an L-cysteine ​​(C) residue. In Formula B1, the amino acid sequence is described from the N-terminus to the C-terminus. 13. The peptide according to claim 12, wherein, in Formula B1, Y1 is an L-phenylalanine (F) residue, (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, (S)-2-amino-3-Y2 is an L-arginine (R) residue or an N6-carbamoyl-L-lysine (Hcit) residue, and Y3 is an L-tryptophan (W) residue, a methyl-L-tryptophan (MeW) residue, or a (S)-2-amino-3-(1H-pyrrolo[2,3-b] residue. The residues are: (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyrrolo-3-yl)propionic acid (W7N), (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyrrolo-3-yl)propionic acid (W1Me7N), (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyrrolo-3-yl)propionic acid (W7N5Me), (S)-2-amino-3-(naphthyl-1-yl)propionic acid (Nal1) or (S)-2-amino-3-(quinolin-4-yl)propionic acid (Nal14N). Y4 is an L-aspartic acid (D) residue, L-glutamic acid (E) residue, L-asparagine (N) residue, or L-glutamine (Q) residue; Y5 is an L-valine (V) residue, methyl-L-valine (MeV) residue, 1-(carboxymethyl)-L-tryptophan (W1aa) residue, (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, (S)-2-amino-3-(naphth-1-yl)propionic acid (Nal1) residue, (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid (3Py5Ph) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, or L-proline (P) residue; Y6 is an L-arginine (R) residue, methyl-L-arginine (MeR) residue, or (S)-2-amino-6-morpholinohexanoic acid (Kmor). The residues are: L-citrulline (Cit) residue, N6-carbamoyl-L-lysine (Hcit) residue, (S)-2-amino-4-ureidobutyric acid (Ncit) residue, or (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue; Y7 is methyl-L-phenylalanine (MeF) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, methyl-L-tyrosine (MeY) residue, (S)-2-(methylamino)-3-(p-tolyl)propionic acid (MeF4Me) residue, (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid (MeF4C) residue, (S)-3-(2-carboxy ... (S)-2-(methylamino)-3-(m-tolyl)propionic acid (MeF3COO) residues, (S)-2-(methylamino)-3-(m-tolyl)propionic acid (MeF3Me) residuesOr (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid (MeF3C) residue, Y8 is L-serine (S) residue, 1-(carboxymethyl)-L-tryptophan (W1aa) residue, L-homotyrosine (Hty) residue, allothreonine (alT) residue, L-threonine (T) residue, L-asparagine (N) residue, L-glutamine (Q) residue, L-aspartic acid (D) residue or L-glutamic acid (E) residue, Y9 is L-asparagine (N) residue, methyl-L-asparagine (MeN) residue, L-2-aminoadipic acid (Hgl) residue, (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, L-homotyrosine (Hty) residue, (S)-2-amino-4-(1H-pyrrolo[2] [3-b]pyridin-3-yl)butyric acid (Hw7N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, L-glutamine (Q) residue, L-aspartic acid (D) residue, or L-glutamic acid (E) residue. Y10 is an L-tryptophan (W) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (W6N) residue, (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W1Me7N) residue, or (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N5Me) residue; Y11 is an L-asparagine (N) residue, L-glutamine (Q) residue, or (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue; Y12 is (S)-1 Y13 is a residue of 2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C), (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) or (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C), and Y13 is a residue of L-asparagine (N), L-aspartic acid (D), L-glutamine (Q), (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) or N4,N4-dimethyl-L-asparagine (Ndm). 14. A peptide comprising the amino acid sequence shown in Formula B2, or selected from the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and 13th positions of the amino acid sequence shown in Formula B2.The amino acid sequence comprising at least one amino acid residue of the group consisting of 1 to 13 amino acid residues has undergone substitution, addition, deletion, or insertion, B2: FRWDVR-MeF-SNWN-Tic-NC (Sequence No. 165), wherein F is an L-phenylalanine residue, R is an L-arginine residue, W is an L-tryptophan residue, D is an L-aspartic acid residue, V is an L-valine residue, MeF is a methyl-L-phenylalanine residue, S is an L-serine residue, Tic is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, N is an L-asparagine residue, and C is an L-cysteine ​​residue. 15. The peptide according to any one of claims 12 to 14, wherein the C-terminus further comprises a glycine residue. 16. The peptide according to any one of claims 12 to 14, wherein it is a cyclic peptide. 17. The peptide according to claim 15, wherein it is a cyclic peptide. Claims 10 / 19, page 11, CN 122555715 A 18. The peptide according to any one of claims 12-14, having a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide. 19. The peptide according to any one of claims 12-14, further comprising added amino acid residues. 20. The peptide according to any one of claims 12-14, having binding affinity for LRP. 21. The peptide according to claim 20, having binding affinity for at least one selected from the group consisting of LRP5 and LRP6. 22. The peptide according to any one of claims 12-14, having DKK1-like activity. 23. The peptide according to any one of claims 12 to 14, comprising the amino acid sequence shown in formula B3, B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14, wherein ClAc is a chloroacetyl group, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, and Y14 are the amino acid residues shown in Table 2, wherein in Table 2, F is an L-phenylalanine residue, F4F is a (S)-2-amino-3-(4-fluorophenyl)propionic acid residue, F4Me is a (S)-2-amino-3-(p-tolyl)propionic acid residue, F3Me is a (S)-2-amino-3-(m-tolyl)propionic acid residue, and R is an L-arginine residue. Hcit represents N6-carbamoyl-L-lysine residue, W represents L-tryptophan residue, MeW represents methyl-L-tryptophan residue, and W7N represents (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue.W5N represents (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propionic acid residue; W1Me7N represents (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue; W7N5Me represents (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue; Nal1 represents (S)-2-amino-3-(naphthyl-1-yl)propionic acid residue; Nal14N represents (S)-2-amino-3-(quinoline-4-yl)propionic acid residue; D represents L-aspartic acid residue; E represents L-glutamic acid residue; N represents L-asparagine residue; Q represents L-glutamine residue; V represents L-valine residue; and MeV represents methyl-L-valine residue. W1aa is a 1-(carboxymethyl)-L-tryptophan residue, Cha is a (S)-2-amino-3-cyclohexylpropionic acid residue, 3Py5Ph is a (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid residue, Cbg is a (S)-2-amino-2-cyclobutylacetic acid residue, P is an L-proline residue, MeR is a methyl-L-arginine residue, Kmor is a (S)-2-amino-6-morpholinohexanoic acid residue, Cit is an L-citrulline residue, claims 11 / 19, page 12, CN 122555715 A, Ncit is a (S)-2-amino-4-ureidobutyric acid residue, OrnAc is a (S)-5-acetamido-2-aminovaleric acid residue, MeF is a methyl-L-phenylalanine residue, MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue. MeY represents a methyl-L-tyrosine residue, MeF4Me represents a (S)-2-(methylamino)-3-(p-tolyl)propionic acid residue, MeF4C represents a (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid residue, MeF3COO represents a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF3Me represents a (S)-2-(methylamino)-3-(m-tolyl)propionic acid residue, MeF3C represents a (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid residue, S represents an L-serine residue, Hty represents an L-homotyrosine residue, alT represents an allothreonine residue, T represents an L-threonine residue, MeN represents a methyl-L-asparagine residue, and Hgl represents an L-2-aminoadipic acid residue. Hw7N is a (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid residue, KCOpipzaa is an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue, and W6N is a (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid residue.Nmm is a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid residue, Tic is a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic6C is a (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7H is a (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7C is a (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Ndm is an N4,N4-dimethyl-L-asparagine residue, C is an L-cysteine ​​residue, G is a glycine residue. [Table 2A] Claims 12 / 19, page 13, CN 122555715 A [Table 2B] Claims 13 / 19, page 14, CN 122555715 A [Table 2C] Claims 14 / 19, page 15, CN 122555715 A [Table 2D] Claims 15 / 19, page 16, CN 122555715 A [Table 2E] Claims 16 / 19, page 17, CN 122555715 A. 24. A peptide complex comprising a first peptide and a second peptide, wherein the first peptide is the peptide of claim 1, and the second peptide is the peptide of claim 12. Claims 17 / 19, page 18, CN 122555715 A. 25. The peptide complex of claim 24, wherein the peptide complex comprises the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide. 26. The peptide complex of claim 25, wherein the C-terminus of the first peptide and the C-terminus of the second peptide are connected by the linker. 27. The peptide complex according to claim 25, wherein the linker is selected from the linkers listed in Table 3, wherein -click- in Table 3 represents a bond formed by click chemistry, [Table 3]. 28. The peptide complex according to claim 25, wherein the first peptide is a peptide composed of the amino acid sequence shown in Formula A1, and the second peptide is a peptide composed of the amino acid sequence shown in Formula B1, wherein: A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 wherein, in Formula A1, X1 is a D-phenylalanine (df) residue, X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue, and X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue, or a (2S)-2-amino-3-(oxacyclohexane-4-yl) residue.Propionic acid (Atp) residue, X4 is L-isoleucine (I) residue, claims 18 / 19, page 19, CN 122555715 A, X5 is L-arginine (R) residue or L-citrulline (Cit) residue, X6 is L-tyrosine (Y) residue, X7 is methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, X8 is L-glutamine (Q) residue, L-valine (V) residue, L-high tyrosine (Hty) residue, L-threonine (T) residue, L-citrulline (Cit) residue or O-methyl-L-threonine (TMe) residue, X10 is methyl-L-phenylalanine (MeF) residue or (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, X11 is an L-isoleucine (I) residue or an L-tyrosine (Y) residue, X12 is an L-valine (V) residue or a methyl-L-valine (MeV) residue, X13 is a glycine (G) residue or a D-serine (ds) residue, and in Formula B1, Y1 is an L-phenylalanine (F) residue, Y2 is an L-arginine (R) residue, Y3 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, Y4 is an L-aspartic acid (D) residue, Y5 is an L-valine (V) residue, and Y6 is an L-arginine (R) residue or (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue. Y7 is a methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue; Y8 is an L-serine (S) residue or L-glutamic acid (E) residue; Y9 is an L-asparagine (N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue or L-glutamic acid (E) residue; Y10 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue; Y11 is an L-asparagine (N) residue; Y12 is a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue; and Y13 is an L-asparagine (N) residue or L-aspartic acid (D) residue. 29. The peptide complex of claim 25, which activates Wnt signaling. 30. A pharmaceutical composition comprising the peptide of any one of claims 1-3 and 12-14. 31. A pharmaceutical composition comprising the peptide complex of claim 25. 32. A cell culture composition for use in cell culture, comprising the peptide complex of claim 25.33. A composition for medical, diagnostic, or research use, comprising the peptide complex of claim 25. 34. A composition for medical, diagnostic, or research use, comprising any one of peptides 1-3 and 12-14. Claims 19 / 19 pages 20 CN 122555715 A Peptide, Peptide Complex, Pharmaceutical Composition, Cell Culture Composition and Composition for Medical, Diagnostic, or Research Use Technical Field

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

[0002] This application claims priority based on Japanese Patent Application No. 2023-192587 filed on November 10, 2023, the contents of which are incorporated herein by reference. Background Art

[0003] Wnt is a secretory glycoprotein with a molecular weight of about 40,000, which controls early development and morphogenesis, and also controls proliferation, differentiation, cell movement, polarity, etc. after birth. Nineteen types of Wnt have been identified in humans and mice. Wnt3a, a member of the Wnt family, activates the Wnt signaling pathway by binding to the 7-transmembrane Frizzled receptor and the co-receptor 1-transmembrane LRP5 or LRP6. When this pathway is chronically activated, cell proliferation and survival become uncontrolled, resulting in proliferative diseases such as cancer. Alternatively, significant inhibition of this pathway can lead to osteoporosis and other bone diseases. Therefore, various drugs and antibodies targeting the Wnt signaling pathway have been reported for the treatment of these cancers and bone diseases (Patent Literature 1-9, Non-Patent Literature 1-2).

[0004] On the other hand, activation of the Wnt signaling pathway plays an important role in early development, the maintenance, proliferation, and differentiation of normal tissue stem cells. In particular, Wnt3a has been used to prepare various organoids such as the small intestine, large intestine, stomach, pancreas, and lung.

[0005] Prior art documents: Patent document 1: Japanese Patent Application Publication No. 2022-544308; Patent document 2: Japanese Patent Application Publication No. 2022-544307; Patent document 3: Japanese Patent Application Publication No. 2022-551380; Patent document 4: Japanese Patent Application Publication No. 2022-137092; Patent document 5: Japanese Patent Application Publication No. 2020-178701; Patent document 6: Japanese Patent Application Publication No. 2020-0632. Patent Document No. 62; Patent Document 7: Japanese Patent Publication No. 2013-530929; Patent Document 8: Japanese Patent No. 6093692; Patent Document 9: Japanese Patent Application Publication No. 2009-142274; Non-Patent Document 1: Shinji Matsumoto et al., “Development of a New Anticancer Agent Based on Research on Wnt Sci-Fi”, Regional Integration Review, December 27, 2018, Volume 7, e009Non-Patent Literature 2: Neiheisel, A. et al., “Wnt pathway modulators in cancer therapeutics: An update on completed and ongoing clinical trials”, International Journal of Cancer, March 2022, Vol. 150, No. 5, pp. 727-740. Specification 1 / 110 pages 21 CN 122555715 A Summary of the Invention

[0006]

Problem to be Solved by the Invention

[0007] The problem of the present invention is to provide a peptide complex that can activate the Wnt signaling pathway.

[0008]

Means for Solving the Problem

[0009] That is, the present invention includes the following scheme.

[0010] [1] A peptide comprising an amino acid sequence of formula A1 or an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted.

[0011] A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 Wherein, X1 is any D-amino acid residue, X2 and X3 are each independently any amino acid residue, X4 and X5 are each independently an amino acid residue with a substituted aliphatic hydrocarbon group on its side chain, X6 is an amino acid residue with a substituted aryl group on its side chain, X7 is an N-alkyl amino acid residue with a substituted aryl or substituted cycloalkyl group on its side chain, X8 is any amino acid residue, X9 is an L-threonine (T) residue, X10 is any N-alkylated amino acid residue, X11 is any amino acid residue, X12 is an amino acid residue with an aliphatic hydrocarbon group on its side chain, X13 is an amino acid residue with a substituted aliphatic hydrocarbon group on its side chain, and X14 is an L-cysteine ​​(C) residue. In Formula A1, the amino acid sequence is recorded from the N-terminus to the C-terminus.

[0012] [2] According to the peptide described in [1], wherein, in Formula A1, X1 is a D-phenylalanine (df) residue, (R)-2-amino-3-(pyridin-4-yl)propionic acid (d4py) residue, (R)-2-amino-3-(pyridin-3-yl)propionic acid (d3py) residue, (R)-2-amino-3-(3-methoxyphenyl)propionic acid (df3OMe) residue,(R)-2-amino-3-(4-methoxyphenyl)propionic acid (df4OMe) residue, (R)-2-amino-5-ureidovalerate (dcit) residue, D-tyrosine (dy) residue, or (R)-2-amino-3-(4-fluorophenyl)propionic acid (df4F) residue, X2 is L-serine (S) residue, L-asparagine (N) residue, L-aspartic acid (D) residue, L-2-aminoadipic acid (Hgl) residue, L-citrulline (Cit) residue, or (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue. X3 is an L-aspartic acid (D) residue, L-arginine (R) residue, L-citrulline (Cit) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue, L-asparagine (N) residue, or L-serine (S) residue; X4 is an L-isoleucine (I) residue or O-methyl-L-threonine (TMe) residue; X5 is an L-arginine (R) residue, L-glutamic acid (E) residue, (S)-2-aminoheptanoic acid (Ahp) residue, L-serine (S) residue, L-homoserine (Hse) residue, L-citrulline (Cit) residue, or N6-carbamoyl-L-lysine (Hcit) residue. (Instructions for use 2 / 110, page 22, CN 122555715 A) The residues are: (S)-2-amino-4-ureidobutyric acid (Ncit) residue or (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue; X6 is an L-tyrosine (Y) residue, methyl-L-tyrosine (MeY) residue, (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, (S)-2-amino-3-(4-chlorophenyl)propionic acid (F4C) residue, (S)-2-amino-3-(pyridin-4-yl)propionic acid (4Py) residue or (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue. X7 is a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue; X8 is an L-glutamine (Q) residue, L-valine (V) residue, L-2-aminoadipic acid (Hgl) residue, 1-(carboxymethyl)propionic acid (Q) residue, or L-phenylalanine (Q) residue.(S)-L-tryptophan (W1aa) residue, (S)-2-aminoheptanoic acid (Ahp) residue, L-high tyrosine (Hty) residue, L-threonine (T) residue, allothreonine (alT) residue, L-citrulline (Cit) residue, N6-(4-(carboxymethyl)piperazin-1-carbonyl)-L-lysine (KCOpipzaa) residue, (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (Gthp) residue, (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, (S)-2-amino-2-cyclohexylacetic acid (Chg) residue, or O-methyl-threonine (TMe) residue. X10 is a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)hexanoic acid (MeNle) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue; X11 is an L-isoleucine (I) residue, L-tyrosine (Y) residue, (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (F4aao) residue, or O-methyl-L-threonine (TMe) residue. X12 is an L-valine (V) residue, a methyl-L-valine (MeV) residue, or an N-methyl-L-cyclohexylglycine (MeChg) residue, and X13 is a glycine (G) residue, a D-alanine (da) residue, or a D-serine (ds) residue.

[0013] [3] A peptide comprising an amino acid sequence of formula A2 or an amino acid sequence of formula A2 in which at least one of 1 to 12 amino acid residues selected from the group consisting of amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, and 13 has been substituted, deleted, added, or inserted.

[0014] A2: df-SDIRY-MeF-QT-MeF-IVGC (Serial No. 164) where df is a D-phenylalanine residue, S is an L-serine residue, D is an L-aspartic acid residue, I is an L-isoleucine residue, R is an L-arginine residue, Y is an L-tyrosine residue, MeF is a methyl-L-phenylalanine residue, Q is an L-glutamine residue, T is an L-threonine residue, V is an L-valine residue, (Specification 3 / 110 pages 23 CN)122555715 AG is a glycine residue, and C is an L-cysteine ​​residue.

[0015] [4] The peptide according to any one of [1] to [3], wherein the C-terminus further has a glycine residue.

[0016] [5] The peptide according to any one of [1] to [3] is a cyclic peptide.

[0017] [6] The peptide according to [4] is a cyclic peptide.

[0018] [7] The peptide according to any one of [1] to [6] has a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the above peptide.

[0019] [8] The peptide according to any one of [1] to [7] further comprises an added amino acid residue.

[0020] [9] The peptide according to any one of [1] to [8] has binding affinity to the Fzd receptor.

[0021]

[10] The peptide according to [9] has the ability to bind to at least one of the group consisting of Fzd receptor 2, Fzd receptor 7 and Fzd receptor 8.

[0022]

[11] The peptide according to any one of [1] to [3] is composed of the amino acid sequence shown in Formula A3.

[0023] A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 Wherein, ClAc is chloroacetyl, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are the amino acid residues shown in Table 1. In Table 1, df is a D-phenylalanine residue, d4py is a (R)-2-amino-3-(pyridin-4-yl)propionic acid residue, d3py is a (R)-2-amino-3-(pyridin-3-yl)propionic acid residue, df3OMe is a (R)-2-amino-3-(3-methoxyphenyl)propionic acid residue, and df4OMe is a (R)-2-amino-3-(4-methoxyphenyl)propionic acid residue. dcit represents (R)-2-amino-5-ureidovalerate, dy represents D-tyrosine, df4F represents (R)-2-amino-3-(4-fluorophenyl)propionic acid, S represents L-serine, N represents L-asparagine, D represents L-aspartic acid, Hgl represents L-2-aminoadipic acid, Cit represents L-citrulline, Kmor represents (S)-2-amino-6-morpholinohexanoic acid, R represents L-arginine, KCOpipzaa represents N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine, Atp represents (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid, I represents L-isoleucine, TMe represents O-methyl-L-threonine, E represents L-glutamic acid, and Ahp represents (S)-2-aminoheptanoic acid. Hse is an L-homoserine residue.Hcit is N6-carbamoyl-L-lysine residue, Ncit is (S)-2-amino-4-ureidobutyric acid residue, Specification 4 / 110 page 24 CN 122555715 A OrnAc is (S)-5-acetamido-2-aminovaleric acid residue, Y is L-tyrosine residue, MeY is methyl-L-tyrosine residue, F4F is (S)-2-amino-3-(4-fluorophenyl)propionic acid residue, F4C is (S)-2-amino-3-(4-chlorophenyl)propionic acid residue, 4Py is (S)-2-amino-3-(pyridin-4-yl)propionic acid residue, F3COO is (S)-3-(2-amino-2-carboxyethyl)benzoic acid residue, MeF is methyl-L-phenylalanine residue, Me3Py is (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid residue. Me4Py is a (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid residue, MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeAtp is a (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid residue, Q is an L-glutamine residue, V is an L-valine residue, W1aa is a 1-(carboxymethyl)-L-tryptophan residue, Hty is an L-homotyrosine residue, T is an L-threonine residue, alT is an allothreonine residue, and Gthp is a (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid residue. G4pipaa is a (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid residue, Cbg is a (S)-2-amino-2-cyclobutylacetic acid residue, Chg is a (S)-2-amino-2-cyclohexylacetic acid residue, MeNle is a (S)-2-(methylamino)hexanoic acid residue, F4aao is a (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid residue, MeV is a methyl-L-valine residue, MeChg is an N-methyl-L-cyclohexylglycine residue, G is a glycine residue, and C is an L-cysteine ​​residue.

[0024]

[12] A peptide comprising the amino acid sequence of Formula B1 or an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted in the amino acid sequence of Formula B1.

[0025] B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 Wherein, Y1 is a substituted L-phenylalanine (F) residue, Y2 is a substituted L-arginine (R) residue, and Y3 is a substituted L-tryptophan (W) residue or a substituted (S)-2-amino-3-(naphth-1-yl)propionic acid.(Nal1) residue, Y4 is an acidic amino acid residue or a neutral amino acid residue, Y5 is a substituted aromatic amino acid residue, aliphatic amino acid residue or L-proline (P) residue, Specification 5 / 110 page 25 CN 122555715 A Y6 is a substituted aliphatic amino acid residue, Y7 is an N-alkylated aromatic amino acid residue, Y8 is an amino acid residue derived from an amino acid with a hydroxyl, carboxyl or amide group on the side chain, Y9 is any amino acid residue, Y10 is a substituted L-tryptophan (W) residue, Y11 is a substituted L-asparagine (N) residue or L-glutamine (Q) residue, Y12 is a substituted (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, Y13 is a substituted neutral amino acid residue or an acidic amino acid residue, Y14 is an L-cysteine ​​(C) residue, In Formula B1, the amino acid sequence is recorded from the N-terminus to the C-terminus.

[0026]

[13] According to the peptide of

[12] , wherein, in formula B1, Y1 is an L-phenylalanine (F) residue, (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, (S)-2-amino-3-(p-tolyl)propionic acid (F4Me) residue or (S)-2-amino-3-(m-tolyl)propionic acid (F3Me) residue, Y2 is an L-arginine (R) residue or N6-carbamoyl-L-lysine (Hcit) residue, Y3 is an L-tryptophan (W) residue, methyl-L-tryptophan (MeW) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propionic acid (W5N) residue. The residues are: (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W1Me7N) residue, (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N5Me) residue, (S)-2-amino-3-(naphthyl-1-yl)propionic acid (Nal1) residue, or (S)-2-amino-3-(quinolin-4-yl)propionic acid (Nal14N) residue; Y4 is an L-aspartic acid (D) residue, L-glutamic acid (E) residue, L-asparagine (N) residue, or L-glutamine (Q) residue; Y5 is an L-valine (V) residue, methyl-L-valine (MeV) residue, or 1-(carboxymethyl)-L-tryptophan (W1aa). Residues, (S)-2-amino-3-cyclohexylpropionic acid (Cha) residues, (S)-2-amino-3-(naphth-1-yl)propionic acid (Nal1) residues,(S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid (3Py5Ph) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, or L-proline (P) residue, Y6 being an L-arginine (R) residue, methyl-L-arginine (MeR) residue, (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, L-citrulline (Cit) residue, N6-carbamoyl-L-lysine (Hcit) residue, (S)-2-amino-4-ureobutyric acid (Ncit) residue, or (S)-5-acetamido-2-aminovaleric acid (OrnAc) residue. Y7 is a methyl-L-phenylalanine (MeF) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, methyl-L-tyrosine (MeY) residue, (S)-2-(methylamino)-3-(p-tolyl)propionic acid (MeF4Me) residue, (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid (MeF4C) residue, (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, (S)-2-(methylamino)-3-(m-tolyl)propionic acid (MeF3Me) residue, or (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid (MeF3C) residue. Y8 is an L-serine (S) residue, 1-(carboxymethyl)-L-tryptophan (W1aa) residue, L-high tyrosine (Hty) residue, allothreonine (alT) residue, L-threonine (T) residue, L-asparagine (N) residue, L-glutamine (Q) residue, L-aspartic acid (D) residue, or L-glutamic acid (E) residue; Y9 is an L-asparagine (N) residue, methyl-L-asparagine (MeN) residue, L-2-aminoadipic acid (Hgl) residue, (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, L-high tyrosine (Hty) residue, (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid (Hw7N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine. (Instructions for use, page 6 / 110, 26 CN) 122555715 A (KCOpipzaa) residue, L-glutamine (Q) residue, L-aspartic acid (D) residue or L-glutamic acid (E) residue, Y10 is L-tryptophan (W) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (W6N) residue, (S)-2-amino-3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W1Me7N) residue or (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N5Me) residue, Y11 is L-asparagine (N) residue, L-glutamine (Q) residue or (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue, Y12 is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C) residue, (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) residue or (S)-7- Chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C) residue, Y13 is an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-glutamine (Q) residue, a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue or an N4,N4-dimethyl-L-asparagine (Ndm) residue.

[0027]

[14] A peptide comprising an amino acid sequence of formula B2 or an amino acid sequence of formula B2 in which at least one amino acid residue selected from the group consisting of amino acid residues at positions 1 to 13 has been substituted, added, deleted or inserted.

[0028] B2: FRWDVR-MeF-SNWN-Tic-NC (Serial No. 165) wherein F is an L-phenylalanine residue, R is an L-arginine residue, W is an L-tryptophan residue, D is an L-aspartic acid residue, V is an L-valine residue, MeF is a methyl-L-phenylalanine residue, S is an L-serine residue, Tic is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, N is an L-asparagine residue, and C is an L-cysteine ​​residue.

[0029]

[15] The peptide according to any one of

[12] to

[14] , wherein the C-terminus further comprises a glycine residue.

[0030]

[16] The peptide according to any one of

[12] to

[14] is a cyclic peptide.

[0031]

[17] The peptide according to

[15] is a cyclic peptide.

[0032]

[18] The peptide according to any one of

[12] to

[17] has a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide.

[0033]

[19] The peptide according to any one of

[12] to

[18] further comprises added amino acid residues.

[0034]

[20] The peptide according to any one of

[12] to

[19] has binding affinity for LRP.

[0035]

[21] The peptide according to

[20] has binding affinity for at least one of the group consisting of LRP5 and LRP6.

[0036]

[22] The peptide according to any one of

[12] to

[19] has DKK1-like activity.

[0037]

[23] The peptide according to any one of

[12] to

[14] is composed of the amino acid sequence shown in Formula B3. Instructions 7 / 110, page 27, CN 122555715 A

[0038] B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 Wherein, ClAc is chloroacetyl, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13 and Y14 are the amino acid residues shown in Table 2. In Table 2, F is L-phenylalanine residue, F4F is (S)-2-amino-3-(4-fluorophenyl)propionic acid residue, F4Me is (S)-2-amino-3-(p-tolyl)propionic acid residue, F3Me is (S)-2-amino-3-(m-tolyl)propionic acid residue, and R is L-arginine residue. Hcit represents N6-carbamoyl-L-lysine residue, W represents L-tryptophan residue, MeW represents methyl-L-tryptophan residue, W7N represents (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue, W5N represents (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propionic acid residue, W1Me7N represents (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue, W7N5Me represents (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue, and Nal1 represents (S)-2-amino-3-(naphthyl-1-yl)propionic acid residue. Nal14N represents (S)-2-amino-3-(quinoline-4-yl)propionic acid residue, D represents L-aspartic acid residue, E represents L-glutamic acid residue, N represents L-asparagine residue, Q represents L-glutamine residue, V represents L-valine residue, MeV represents methyl-L-valine residue, W1aa represents 1-(carboxymethyl)-L-tryptophan residue, Cha represents (S)-2-amino-3-cyclohexylpropionic acid residue, 3Py5Ph represents (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid residue, Cbg represents (S)-2-amino-2-cyclobutylacetic acid residue, P represents L-proline residue, MeR represents methyl-L-arginine residue, Kmor represents (S)-2-amino-6-morpholinohexanoic acid residue, and Cit represents L-citrulline residue.Ncit is a (S)-2-amino-4-ureidobutyric acid residue, OrnAc is a (S)-5-acetamido-2-aminovaleric acid residue, MeF is a methyl-L-phenylalanine residue, MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeY is a methyl-L-tyrosine residue, MeF4Me is a (S)-2-(methylamino)-3-(p-tolyl)propionic acid residue, MeF4C is a (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid residue, MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, and MeF3Me is a (S)-2-(methylamino)-3-(m-tolyl)propionic acid residue. (Specification 8 / 110, page 28, CN 122555715 A) MeF3C represents (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid residue, S represents L-serine residue, Hty represents L-homotyrosine residue, alT represents allothreonine residue, T represents L-threonine residue, MeN represents methyl-L-asparagine residue, Hgl represents L-2-aminoadipic acid residue, Hw7N represents (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid residue, KCOpipzaa represents N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue, W6N represents (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid residue, and Nmm represents (2S)-2-amino-4-(methylamino)-4-oxobutyric acid residue. Tic is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic6C is (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7H is (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7C is (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Ndm is N4,N4-dimethyl-L-asparagine residue, C is L-cysteine ​​residue, and G is glycine residue.

[0039]

[24] A peptide complex comprising a first peptide and a second peptide, wherein the first peptide is any one of [1] to

[11] , and the second peptide is any one of

[12] to

[23] .

[0040]

[25] According to

[24] , the peptide complex comprises the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

[0041]

[26] According to

[25] , the C-terminus of the first peptide and the C-terminus of the second peptide are connected by the linker.

[0042]

[27] According to

[25] , the linker is selected from the linkers listed in Table 3.

[0043] Wherein, -click- in Table 3 represents a bond formed by click chemistry.

[0044]

[28] The peptide complex according to any one of

[24] to

[27] , wherein the first peptide is a peptide composed of the amino acid sequence shown in Formula A1, and the second peptide is a peptide composed of the amino acid sequence shown in Formula B1.

[0045] A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 Wherein, in formula A1, X1 is a D-phenylalanine (df) residue, X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue, X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue or a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue, X4 is an L-isoleucine (I) residue. (See specification 9 / 110 page 29 CN 122555715 A) X5 is an L-arginine (R) residue or an L-citrulline (Cit) residue; X6 is an L-tyrosine (Y) residue; X7 is a methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue; X8 is an L-glutamine (Q) residue, L-valine (V) residue, L-homotyrosine (Hty) residue, L-threonine (T) residue, L-citrulline (Cit) residue, or O-methyl-L-threonine (TMe) residue; X10 is a methyl-L-phenylalanine (MeF) residue or (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue; X11 is an L-isoleucine (I) residue or an L-tyrosine (Y) residue; X12 is an L-valine (V) residue or a methyl-L-valine (MeV) residue. X13 is a glycine (G) residue or a D-serine (ds) residue. In formula B1, Y1 is an L-phenylalanine (F) residue, Y2 is an L-arginine (R) residue, Y3 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, Y4 is an L-aspartic acid (D) residue, Y5 is an L-valine (V) residue, Y6 is an L-arginine (R) residue or (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, Y7 is a methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, and Y8 is an L-serine (S) residue or an L-glutamic acid (E) residue.Y9 is an L-asparagine (N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue or L-glutamic acid (E) residue, Y10 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, Y11 is an L-asparagine (N) residue, Y12 is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, and Y13 is an L-asparagine (N) residue or L-aspartic acid (D) residue.

[0046]

[29] The peptide complex according to any one of

[24] to

[28] is capable of activating the Wnt signal.

[0047]

[30] A pharmaceutical composition comprising the peptide according to any one of [1] to

[23] .

[0048]

[31] A pharmaceutical composition comprising the peptide complex described in any one of

[24] to

[29] .

[0049]

[32] A cell culture composition for use in cell culture comprising the peptide complex described in any one of

[24] to

[29] .

[0050]

[33] A composition for medical, diagnostic or research use comprising the peptide complex described in any one of

[24] to

[29] .

[0051]

[34] A composition for medical, diagnostic or research use comprising the peptide described in any one of [1] to

[11] and

[12] to

[23] . Specification 10 / 110 pages 30 CN 122555715 A

[0052]

[35] The peptide according to any one of [1] to

[23] is used for the treatment or prevention of cancers such as breast cancer, colorectal cancer, lung cancer or gastric cancer or inflammatory bowel disease.

[0053]

[36] The peptide complex according to any one of

[24] to

[29] is used for the treatment or prevention of bone metabolic diseases such as osteoporosis.

[0054]

[37] Use of the peptide according to any one of [1] to

[23] in the manufacture of a pharmaceutical composition for the treatment or prevention of cancers such as breast cancer, colorectal cancer, lung cancer or gastric cancer or inflammatory bowel disease.

[0055]

[38] Use of the peptide complex according to any one of

[24] to

[29] in the manufacture of a pharmaceutical composition for the treatment or prevention of bone metabolic diseases such as osteoporosis.

[0056]

[39] A method for the treatment or prevention of cancers such as breast cancer, colorectal cancer, lung cancer or gastric cancer or inflammatory bowel disease, comprising the step of administering an effective amount of the peptide according to any one of [1] to

[23] to a subject in need of the treatment or prevention.

[0057]

[40] A method for the treatment or prevention of bone metabolic diseases such as osteoporosis, comprising the step of administering an effective amount of the peptide complex according to any one of

[24] to

[29] to a subject in need of the treatment or prevention.

[0058] Use of the peptide complex described in any one of

[41]

[24] ~

[29] in the preparation of organoids such as small intestine, large intestine, stomach, pancreas or lung.

[0059]

Effects of the Invention

[0060] The embodiments of the peptides, peptide complexes, pharmaceutical compositions, cell culture compositions and compositions for medical, diagnostic or research purposes of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described below, and various modifications may be made without departing from the spirit of the invention.

[0061] [Abbreviation (Conventional)] Å: angstrom (unit, 10-10m) AcOH: Acetic acid BSA: Bovine serum albumin Boc: tert-Butyloxycarbonyl ClAc: Chloroacetyl ClAc OSu: (2,5-dioxopyrrolidone-1-yl)2-chloroacetate (CAS No.: 27243-15-8) [Cu(CH3CN)4]PF6: Copper hexafluorophosphate tetraacetonitrile (I) (CAS No.: 64443-05-6) DCM: Dichloromethane or dichloroalkanes; DIPCI or DIC: N,N'-diisopropylcarbodiimide; DIPEA or DIEA: N,N-diisopropylethylamine; DMA: N,N-dimethylacetamido; Dmb: dimethoxybenzyl; DMEM: Dulbecco modified Eagle medium; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; DODT: 3,6-dioxa-1,8-octanedithiol; EC50: half-maximal effective concentration. (Instructions for use, page 11 / 110, 31, CN 122555715 A) FBS: Fetal bovine serum; Fmoc: 9-fluorenylmethoxycarbonyl; Fmoc-Lys(Fmoc)-OH: N2,N6-bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine; g: grams (units). HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate HPLC: High-performance liquid chromatography LC-MS or LC / MS: Liquid chromatography-mass spectrometry M: Mole (unit) MeCN: Acetonitrile mg: Milligram (unit) min: Minute (unit) mL: Milliliter (unit) mM: Millimole (unit) mm: Millimeter (unit) Mpe group: 3-Methyl-pentan-3-yl NHS: N-hydroxysuccinimide nm: Nanometer (unit) μL: Microliter (unit) OSu: Oxysuccinimide Pbf: 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl PEG: Polyethylene glycol rpm: Revolutions per minute (unit) Sub: Dibenzocycloheptayl TBTA: Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (CAS No.: 510758-28-8) tBu: tert-butyl TEAA: Triethylamine acetateTFA: Trifluoroacetic acid; TIS: Triisopropylsilane; TRT or Tr: Triphenylmethyl [abbreviation (non-natural amino acid)]; 3Py5Ph: (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid (CAS No.: 2973752-28-0); 3Py6NH2: (S)-2-amino-3-(6-aminopyridin-3-yl)propionic acid (CAS No.: 1269968-61-7); 4Py: (S)-2-amino-3-(pyridin-4-yl)propionic acid (CAS No.: 37535-49-2); Ahp: (S)-2-aminoheptanoic acid (CAS No.: 44902-02-5); alT: Allothreonine (CAS No.: 28954-12-3) Atb: (S)-2-amino-4,4-dimethylvaleric acid (CAS No.: 57224-50-7) Atp: (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (CAS No.: 1344910-91-3) AZ(L08)-P12-PS: 2,5-dioxopyrrolidone-1-yl 40-(33-azido-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatritriatridecanoamide)-40-(2,5,8,11,14,17,20,23,26,29,32,35, ... Specification 12 / 110 pages 32 CN 122555715 A 38-Tridecano(3,4,3)-O-2,5,8,11,14,17,20,23,26,29,32,35,38,42-Tetradecano(4,4,3,4,4)-Zaza-7-heptadecane-4,7-ester (Nippon Oil Co., Ltd.) Cbg: (S)-2-amino-2-cyclobutyric acid (CAS No.: 49607-08-1) Cha: (S)-2-amino-3-cyclohexylpropionic acid (CAS No.: 27527-05-5) Chg: (S)-2-amino-2-cyclohexylacetic acid (CAS No.: 14328-51-9) Cit: L-citrulline (CAS No.: 372-75-8) cPEG9c: 4,7,10,13,16,19,22,25,28-Nonoxatrahexadecanoic acid (CAS No.: 1268488-70-5) cPEG13c: 4,7,10,13,16,19,22,25,28,31,34,37,40-Tetraoxatrahexadecanoic acid (CAS No.: 2225903-66-0) cPEG17c: 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-Heptaoxapentadecanoic acid (CAS No.: 2226897-74-9) d3py: (R)-2-Amino-3-(pyridin-3-yl)propionic acid (CAS No.: 70702-47-5)d4py: (R)-2-amino-3-(pyridin-4-yl)propionic acid (CAS No.: 37535-50-5) da: D-alanine (CAS No.: 338-69-2) dcit: (R)-2-amino-5-ureidovalerate (CAS No.: 13594-51-9) df: D-phenylalanine (CAS No.: 673-06-3) df3OMe: (R)-2-amino-3-(3-methoxyphenyl)propionic acid (CAS No.: 145306-65-6) df4F: (R)-2-amino-3-(4-fluorophenyl)propionic acid (CAS No.: 18125-46-7) df4OMe: (R)-2-amino-3-(4-methoxyphenyl)propionic acid (CAS No.: 39878-65-4) dkCOpipzaa: N6-(4-(carboxymethyl)piperazine-1-carbonyl)-D-lysine (CAS No.: 2973757-42-3) ds: D-serine (CAS No.: 312-84-5) dy: D-tyrosine (CAS No.: 556-02-5) F3COO: (S)-3-(2-amino-2-carboxyethyl)benzoic acid (CAS No.: 13861-02-4) F3Me: (S)-2-amino-3-(m-tolyl)propionic acid (CAS No.: 114926-37-3) F4aao: (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (CAS No.: 24558-63-2) F4C: (S)-2-amino-3-(4-chlorophenyl)propionic acid (CAS No.: 14173-39-8) F4F: (S)-2-amino-3-(4-fluorophenyl)propionic acid (CAS No.: 1132-68-9) F4Me: (S)-2-amino-3-(p-tolyl)propionic acid (CAS No.: 1991-87-3) G4pipaa: (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (CAS No.: 2973756-12-4) Gpra: (S)-2-aminopentan-4-ynyl acid (CAS No.: 23235-01-0) Gthp: (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (CAS No.: 811842-25-8) Hcit: N6-Carbamoyl-L-Lysine (CAS No.: 1190-49-4) Hgl: L-2-Aminoadipic acid (CAS No.: 1118-90-7) Hse: L-Homoserine (CAS No.: 672-15-1) Hty: L-Homotyrosine (CAS No.: 221243-01-2) Hw7N: (S)-2-Amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid (CAS No.: 2349860-05-3)KCOpipzaa: N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (CAS No.: 2913141-21-4) Kmor: (S)-2-amino-6-morpholinohexanoic acid (CAS No.: 960135-14-2) Specification 13 / 110 Page 33 CN 122555715 A KN3: N6-diaza-L-lysine (CAS No.: 159610-92-1) Me3Py: (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (CAS No.: 2651172-69-7) Me4Py: (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (CAS No.: 2913141-25-8) MeAtp: (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (CAS No.: 2973752-56-4) MeChg: (S)-2-cyclohexyl-2-(methylamino)acetic acid (CAS No.: 145963-18-4) MeF: Methyl-L-phenylalanine (CAS No.: 2566-30-5) MeF3C: (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid (CAS No.: 2255324-91-3) MeF3COO: (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (CAS No.: 2913141-26-9) MeF3Me: (S)-2-(methylamino)-3-(m-tolyl)propionic acid; MeF4C: (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid (CAS No.: 347851-70-1); MeF4COO: (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (CAS No.: 2973752-03-1); MeF4Me: (S)-2-(methylamino)-3-(p-tolyl)propionic acid (CAS No.: 2307782-25-6); MeN: Methyl-L-asparagine (CAS No.: 19026―58-5); MeNle: (S)-2-(methylamino)hexanoic acid (CAS No.: 17343-27-0); MeR: Methyl-L-arginine (CAS No.: 2480-28-6). MeV: Methyl-L-valine (CAS No.: 2480-23-1) MeW: Methyl-L-tryptophan (CAS No.: 526-31-8) MeY: Methyl-L-tyrosine (CAS No.: 537-49-5) Nal1: (S)-2-amino-3-(naphth-1-yl)propionic acid (CAS No.: 55516-54-6) Nal14N: (S)-2-amino-3-(quinoline-4-yl)propionic acid (CAS No.: 137855-78-8) Ncit: (S)-2-amino-4-ureobutyric acid (CAS No.: 1190-47-2)Nmm: (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (CAS No.: 7175-34-0) Ndm: (2S)-2-amino-4-(dimethylamino)-4-oxobutyric acid (CAS No.: 62937-43-3) OrnAc: (S)-5-acetamido-2-aminopentanoic acid (CAS No.: 2185-16-2) PEG4c: 1-amino-3,6,9,12-tetraoxapentadecano-15-acid (CAS No.: 663921-15-1) PEG8c: 1-amino-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-acid (CAS No.: 756526-04-2) PEG12c: 1-Amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecoxane-39-acid (CAS No.: 1415408-69-3) SMe: O-methyl-L-serine (CAS No.: 32620-11-4) Tbg: (S)-2-Amino-3,3-dimethylbutyric acid (CAS No.: 20859-02-3) Tic: (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS No.: 74163-81-8) Tic6C: (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS No.: 1344407-69-7) Tic7C: (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS No.: 1344492-44-9) Tic7H: (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS No.: 128502-56-7) TMe: O-methyl-L-threonine (CAS No.: 4144-02-9) W1aa: 1-(carboxymethyl)-L-tryptophan (CAS No.: 773823-50-0) W1Me7N: (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (CAS No.: 1632971-18-6) Specification 14 / 110 Page 34 CN 122555715 A W5N: (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propionic acid (CAS No.: 149704-62-1) W6N: (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (CAS No.: 149704-63-2) W7N: (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (CAS No.: 49758-35-2) W7N5Me: (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (CAS No.:1270158-20-7) [Wnt] Wnt is a secretory glycoprotein with a molecular weight of approximately 40,000, which controls early development and morphogenesis. Additionally, it controls postnatal proliferation, differentiation, cell movement, and polarity. Nineteen types of Wnt (Wnt1-19) have been identified in humans and mice.

[0062] Wnt activates the Wnt signaling pathway by binding to members of the Frizzled (Fzd) receptor family and co-receptors LRP5, LRP6, ROR, or RYK.

[0063] The peptide complex of the present invention activates the Wnt signaling pathway by binding to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), and therefore can be used as an alternative to naturally occurring Wnt families. Examples of Wnt families that can be replaced by the peptide complex of the present invention include Wnt3 and Wnt1, with Wnt3 being preferred.

[0064] [Wnt signaling activation capacity] Wnt signaling activation capacity refers to the ability to produce effects similar to those produced by naturally occurring Wnt. It also refers to the ability to specifically activate the Wnt signaling pathway.

[0065] Wnt signaling activation capacity can be evaluated, for example, by activation of the Wnt-β-catenin pathway. For example, non-limitingly, it can be evaluated by nuclear migration of β-catenin in cells, or by the TCF / LEF reporter assay. Non-limitingly, examples include evaluations using the PathHunter eXpress β-Catenin Nuclear Translocation Assay (DiscoverX), the TCF / LEF Reporter-HEK 293 cell line (BPS Bioscience), etc. In any evaluation system, when Wnt signaling activation capacity can be detected using the optimal concentration of peptide or peptide complex under optimal conditions according to standard procedures, it is said to possess Wnt signaling activation capacity.

[0066] [Wnt inhibitory activity] Wnt inhibitory activity refers to the activity that inhibits the Wnt signaling pathway. For example, the peptide of the present invention binds to LRP5 and / or LRP6, which are co-receptors of Wnt, inhibiting the binding of Wnt to the co-receptors, thereby inhibiting the Wnt signaling pathway.

[0067] Wnt inhibitory activity can be evaluated, for example, by the inhibition of activation of the Wnt-β-catenin pathway. For example, non-limitingly, it can be evaluated by nuclear migration of β-catenin in cells, or by the TCF / LEF reporter assay. Non-limitingly, examples include the PathHunter eXpress β-Catenin Nuclear Translocation Assay (DiscoverX), the TCF / LEF Reporter-HEK 293 cell line (BPS).Evaluations by companies such as Bioscience. In any evaluation system, Wnt inhibitory activity is defined as the ability to detect Wnt inhibitory activity when the peptide or peptide complex is used at the optimal concentration under optimal conditions according to standard procedures.

[0068] [Classical and Non-classical Wnt Signaling Pathways] See Neiheisel, A., and four others, “Wnt pathway modulators in cancer therapeutics: An update on completed and ongoing clinical trials”, International Journal of Cancer, March 2022, Vol. 150, No. 5, pp. 727-740.

[0069] Signaling pathways generally refer to biochemical causal relationships initiated by protein-protein interactions, such as the binding of growth factors to receptors, which lead to the transmission of signals from one part of the cell to other parts of the cell. The Wnt signaling pathway refers to the biochemical causal relationship that occurs when the aforementioned growth factor is Wnt.

[0070] Wnt signaling pathways can be divided into classical and non-classical Wnt signaling pathways. Instructions 15 / 110, page 35, CN 122555715 A

[0071] <Classical Wnt Signaling Pathway> In the absence of Wnt ligands, the degradation complex composed of APC, Axin, CK1, and GSK3β undergoes phosphooxidation, ubiquitination, and degradation in the proteasome. As a result, the level of β-annexin in the cytoplasm remains low, and the transcription of genes targeted by Wnt is inhibited. On the other hand, when Wnt ligands bind to Frizzled receptors and co-receptors LRP5 or LRP6, Dvl protein is attracted to the cell membrane, and the β-annexin degradation complex becomes unstable. The binding of Wnt ligands to the above-mentioned receptors leads to the accumulation of unphosphorylated β-annexin in the cytoplasm, and β-annexin migrates into the nucleus, forming a complex with TCF / LEF, causing transcription of genes targeted by Wnt. It should be noted that it has been reported that the binding of R-spondin to LGR5 can also activate the above-mentioned classical Wnt signaling pathway.

[0072] The Wnt signaling pathway refers to the binding of Wnt family members, which are secretory protein ligands, to the receptor complexes of LRP and Frizzled (Fzd), thereby enabling β-linkin to move into the nucleus and interact with TCF / LEF transcription factors to activate the expression of target genes, i.e., the classical Wnt signaling pathway.

[0073] <Non-classical Wnt signaling pathway>There are at least two non-classical Wnt signaling pathways: the PCP pathway, which controls planar cell polarity (PCP); and the Ca2+ pathway, which promotes intracellular calcium ion mobilization.

[0074] (PCP pathway) When Wnt ligands bind to Frizzled receptors and their co-receptors ROR or RYK, they attract Dvl proteins, activating ROCK, which controls the cytoskeleton, or JNK, which activates the transcription factor Jun, leading to cellular responses.

[0075] (Ca2+ pathway) When Wnt ligands interact with Frizzled receptors and their co-receptors ROR, they activate PLC, a membrane-bound enzyme, converting PIP2, a membrane-bound phospholipid, into IP3 and DAG. IP3 causes the release of calcium ions from the endoplasmic reticulum into the cytoplasm. Subsequently, calcium ions activate calmodulin and PKC. Activation of PKC leads to cellular responses.

[0076] [Fzd Receptor] The Fzd receptor is a 7-transmembrane domain protein of the G protein-coupled receptor (GPCR) superfamily, containing a large extracellular N-terminal ligand-binding domain with 10 conserved cysteine ​​residues, known as a cysteine-rich domain (CRD) or Fri domain. Ten Fzd receptors (Fzd1-10) have been identified in humans, participating in various important biological processes such as development, cell proliferation, survival, migration, and stem cell maintenance.

[0077] In adult animals, the Wnt signaling pathway is activated through the interaction of Wnt ligands with members of the Fzd receptor family, controlling embryonic development, regeneration of stem cells and progenitor cells in tissue homeostasis, and cell differentiation.

[0078] [Fzd Binding Activity] Fzd (Frizzled) binding activity refers to the activity of specifically binding to Fzd (Frizzled) receptors, preferably the activity of binding to Fzd2, Fzd7, and / or Fzd8 receptors.

[0079] [LRP5 / 6] LRP is short for low-density lipoprotein receptor-associated protein, including LRP1~6, LRP1B, LRP8, and LRP10~12. LRP5 and LRP6, as members of the LRP family, are single-transmembrane receptors present on the cell surface, forming part of the LRP5 / LRP6 / Fzd co-receptor group involved in the classical Wnt signaling pathway.

[0080] LRP5 and LRP6 have high homology, with their extracellular and intracellular domains sharing 73% and 64% similarity, respectively. They are widely co-expressed during embryonic development and in mature tissues, and their functions overlap.

[0081] [LRP binding activity] LRP binding activity refers to the activity of specifically binding to LRP, preferably the activity of binding to LRP5 and / or LRP6.

[0082] [Dkk]Dkk (Dikkopff) refers to nucleic acids and proteins belonging to the Dkk family, including Dkk1-4, Soggy, and related Dkk proteins. In this application, Dkk1 is a preferred example, but it is not limited thereto.

[0083] Dkk1 binds to the co-receptors LRP5 and LRP6 of Wnt and inhibits the binding of Wnt to these co-receptors, thereby inhibiting the Wnt signaling pathway. It has been established that Dkk1 inhibits both Wnt1-mediated signaling and Wnt3a-mediated signaling. In this specification, "DKK1-like activity" refers to the inhibition of the activity of the Wnt signaling pathway through the above mechanism.

[0084] [Conservative amino acid substitution] In the peptides and peptide complexes of the present invention, when one, two, or three amino acid residues are substituted, deleted, added, or inserted from a specific amino acid sequence, a conservative amino acid substitution is preferably performed.

[0085] "Conservative amino acid substitution" refers to the substitution with a functionally equivalent or similar amino acid.

[0086] Conserved amino acid substitutions in a peptide result in a static change in the peptide's amino acid sequence. For example, one or more amino acids of the same polarity function equivalently, resulting in a static change in the peptide's amino acid sequence. Generally, substitutions within a certain group are considered conserved in terms of structure and function.

[0087] However, it will be apparent to those skilled in the art that the role of a particular amino acid residue may depend on the three-dimensional structure of the molecule containing that amino acid.

[0088] Cysteine ​​residues can form an oxidized (disulfide bond) form with lower polarity than the reduced (thiol) form.

[0089] Long aliphatic portions of arginine side chains may constitute structurally and functionally important features.

[0090] Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) may contribute to ion-aromatic interactions or cation-pi interactions. In such cases, even if these amino acids with side chains are substituted with amino acids belonging to the acidic or nonpolar groups, the structure and function may still be conserved.

[0091] Residues such as proline, glycine, and cysteine ​​(in disulfide bond form) can directly affect the stereostructure of the main chain and are generally not replaceable without causing structural distortion.

[0092] Conserved amino acid substitutions are shown below, including specific substitutions based on side chain similarity (Lehninger, Biochemistry, 2nd edition, revised, 1975, pp. 73-75; Worth Publisher, New York (1975)) and typical substitutions.

[0093] In addition, the preferred method of conservative amino acid substitution is, for example, replacing a certain amino acid with an amino acid from the same group as the group to which it belongs, in a group where natural amino acids are grouped based on their common side chain properties, as described below.

[0094] Hydrophobic (also known as nonpolar) amino acids: Amino acids that exhibit hydrophobicity (nonpolarity), including, for example, L-alanine (A), glycine (G), L-valine (V), L-leucine (L), L-isoleucine (I), L-proline (P), L-phenylalanine (F), L-tryptophan (W), L-tyrosine (Y), and L-methionine (M).

[0095] It should be noted that hydrophobic amino acids can also be further divided into the following groups.

[0096] Aliphatic amino acids: Amino acids whose side chains have an aliphatic hydrocarbon group or hydrogen atom, including, for example, L-alanine (A or Ala), glycine (G or Gly), L-valine (V or Val), L-isoleucine (I or Ile), and L-leucine (L or Leu).

[0097] Aliphatic branched-chain amino acids: Amino acids with branched aliphatic hydrocarbon groups in their side chains, including, for example, L-valine (V), L-isoleucine (I), and L-leucine (L).

[0098] Aromatic amino acids: Amino acids with aromatic rings in their side chains, including, for example, L-tryptophan (W), L-tyrosine (Y), and L-phenylalanine (F).

[0099] Hydrophilic (also known as polar) amino acids: Amino acids exhibiting hydrophilic (polar) properties, including, for example, L-serine (S), L-threonine (T), L-cysteine ​​(C), L-asparagine (N), L-glutamine (Q), L-aspartic acid (D), L-glutamic acid (E), L-lysine (K), L-arginine (R), and L-histidine (H).

[0100] It should be noted that hydrophilic amino acids can also be further divided into the following groups.

[0101] Acidic amino acids: Amino acids whose side chains exhibit acidity, including, for example, L-aspartic acid (D) and L-glutamic acid (E).

[0102] Basic amino acids: Amino acids whose side chains exhibit basicity, including, for example, L-lysine (K), arginine (R), and L-histidine (H).

[0103] Neutral amino acids: Amino acids whose side chains exhibit neutrality, including, for example, L-serine (S), L-threonine (T), L-asparagine (N), L-glutamine (Q), and L-cysteine ​​(C).

[0104] Glycine (G) and L-proline (P) can also be classified as "amino acids that affect the orientation of the main chain".

[0105] L-cysteine ​​(C) and L-methionine (M), whose side chains contain sulfur molecules, can also be classified as "sulfur-containing amino acids".

[0106] In this specification, "amino acid" includes not only natural amino acids but also non-natural amino acids. Non-natural amino acidsThis includes, for example, N-alkyl amino acids that have undergone N-alkylation of natural amino acids as described above, and amino acids whose nitrogen atoms forming peptide bonds are branched or straight-chain modified by lower-order (e.g., C1-C5, preferably C1-C3, more preferably C1) alkyl groups. N-alkyl amino acids are preferably N-ethyl amino acids, N-butyl amino acids, or N-methyl amino acids, and more preferably N-methyl amino acids.

[0107] In addition, non-natural amino acids include chemically modified amino acids such as D-type amino acids (also referred to as D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, and amino acid derivatives, as well as amino acids such as ortholeucine and ornithine that cannot serve as protein building blocks in organisms.

[0108] This also includes amino acids in which functional groups have been further added to or replaced with other functional groups on the side chains of natural amino acids (e.g., amino acids with substituted or added arylene, alkylene, etc. portions of the side chain; amino acids with increased C numbers in the arylene, alkylene, or alkyl groups of the side chain; amino acids with substituted aromatic rings in the side chain; and heterocyclic or fused-ring amino acids, etc.).

[0109] It should be noted that by adding or substituting functional groups or other structures into the side chains of natural amino acids, properties different from those of natural amino acids can be imparted. For example, (S)-2-amino-3-(pyridin-4-yl)propionic acid (4Py) is alanine with a pyridinium group (pyridinium ring) added to its side chain. Unlike alanine, which belongs to the nonpolar amino acid group, it exhibits basic polarity due to the addition of the pyridinium group.

[0110] That is, in the above-mentioned group in which natural amino acids are divided based on their common side chain properties, non-natural amino acids with the same side chain properties may be included. For example, methyl-L-arginine (MeR), an N-methylated amino acid of arginine, which belongs to the basic amino acid group, is a non-natural amino acid and can be classified as a basic amino acid because it exhibits basicity. Therefore, non-natural amino acids that exhibit the same side chain properties as a certain amino acid can also be included as objects of conservative amino acid substitution.

[0111] Non-limitingly, non-natural amino acids include N-methyl amino acids, (S)-2-amino-3-(pyridin-4-yl)propionic acid (4Py), allothreonine (alT), L-citrulline (Cit), O-methyl-L-serine (SMe), (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp), L-2-aminoadipic acid (Hgl), (S)-2-amino-3-(naphthyl-1-yl)propionic acid (Nal1), (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (W6N) and (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N), etc. It should be noted that N-methyl amino acids can be classified as N-alkyl amino acids, or they can be classified according to the side chain properties of the original amino acid without N-methylation.

[0112] [Peptides and their pharmaceutically acceptable salts, esters or solvates] Specification 18 / 110 pages 38 CN 122555715 A Examples of pharmaceutically acceptable salts are inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, and acidic or basic amino acid salts. However, they are not limited to these.

[0113] Examples of inorganic acid salts are hydrochlorides, hydrobromic acid salts, sulfates, nitrates, and phosphates. However, they are not limited to these.

[0114] Examples of organic acid salts are acetates, succinates, fumarates, maleates, tartrates, citrates, lactates, stearates, benzoates, methanesulfonates, and p-toluenesulfonates. However, they are not limited to these.

[0115] Examples of the above-mentioned inorganic base salts are alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, aluminum salts, and ammonium salts. However, they are not limited to these.

[0116] Examples of the above-mentioned organic base salts are diethylamine salts, diethanolamine salts, meglumine salts, and N,N'-dibenzylethylenediamine salts. However, they are not limited to these.

[0117] Examples of the above-mentioned acidic amino acid salts are aspartic acid salts and glutamate salts. Examples of basic amino acid salts are arginine salts, lysine salts, and ornithine salts. However, they are not limited to these.

[0118] Examples of pharmaceutically acceptable esters are acetate esters and sulfate esters. However, they are not limited to these.

[0119] Examples of pharmaceutically acceptable solvates are hydrates. However, they are not limited to these.

[0120] [Peptide] The peptides of the present invention can be pharmaceutically acceptable salts, esters, or solvates. Examples of solvates are hydrates.

[0121] <First Embodiment> The first embodiment of the peptide of the present invention is a peptide comprising the amino acid sequence represented by Formula A1 or an amino acid sequence formed by substitution, deletion, addition, or insertion of one or more amino acid residues in the amino acid sequence represented by Formula A1. It should be noted that in Formula A1, the amino acid sequence is recorded from the N-terminus to the C-terminus.

[0122] A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 Wherein, X1 is any D-amino acid residue, X2 and X3 are each independently any amino acid residue, X4 and X5 are each independently an amino acid residue with a substituted aliphatic hydrocarbon group on its side chain, X6 is an amino acid residue with a substituted aryl group on its side chain, X7 is an N-alkyl amino acid residue with a substituted aryl group or a substituted cycloalkyl group on its side chain, X8 is any amino acid residue, X9 is an L-threonine (T) residue, X10 is any N-alkylated amino acid residue, X11 is any amino acid residue, X12 is an amino acid residue with an aliphatic hydrocarbon group on its side chain, X13 is an amino acid residue with a substituted aliphatic hydrocarbon group on its side chain, and X14 is an L-cysteine ​​(C) residue.

[0123] In the peptide of the first embodiment described above, X1 is preferably a D-phenylalanine (df) residue, (R)-2-amino-3-(pyridin-4-yl)propionic acid (d4py) residue, (R)-2-amino-3-(pyridin-3-yl)propionic acid (d3py) residue, (R)-2-amino-3-(3-methoxyphenyl)propionic acid (df3OMe) residue, (R)-2-amino-3-(4-methoxyphenyl)propionic acid (df4OMe) residue, (R)-2-amino-5-ureidovalerate (dcit) residue, D-tyrosine (dy) residue, or (R)-2-amino-3-(4-fluorophenyl)propionic acid (df4F) residue.

[0124] The structures of d4py, d3py, df4F, df3OMe, df4OMe, dcit, df, and dy are shown below. Specification 19 / 110 Page 39 CN 122555715 A

[0125]

Chemical Formula 1

[0126] The structures of Kmor, Hgl, and Cit are shown below.

[0127] [Chemical Formula 2] Specification 20 / 110 pages 40 CN 122555715 A In the peptide of the first embodiment described above, X3 is preferably an L-aspartic acid (D) residue, an L-arginine (R) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue, an L-asparagine (N) residue, or an L-serine (S) residue.

[0128] The structures of Cit, Atp, and KCOpipzaa are shown below.

[0129] [Chemical Formula 3] In the peptide of the first embodiment described above, X4 is preferably an L-isoleucine (I) residue or an O-methyl-L-threonine (TMe) residue.

[0130] The structure of TMe is shown below.

[0131] [Chemical Formula 4] Specification 21 / 110 Page 41 CN 122555715 A In the peptide of the first embodiment described above, X5 is preferably an L-arginine (R) residue, an L-glutamic acid (E) residue, a (S)-2-aminoheptanoic acid (Ahp) residue, an L-serine (S) residue, an L-homoserine (Hse) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, a (S)-2-amino-4-ureidobutyric acid (Ncit) residue, or a (S)-5-acetylamine.2-aminopentanoic acid (OrnAc) residue.

[0132] The structures of Ahp, Hse, Ncit, Hcit, Cit and OrnAc are shown below.

[0133] [Chemical Formula 5] Specification 22 / 110 Page 42 CN 122555715 A In the peptide of the first embodiment above, X6 is preferably an L-tyrosine (Y) residue, a methyl-L-tyrosine (MeY) residue, (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, (S)-2-amino-3-(4-chlorophenyl)propionic acid (F4C) residue, (S)-2-amino-3-(pyridin-4-yl)propionic acid (4Py) residue or (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue.

[0134] The structures of F4F, F4C, F3COO and 4Py are shown below.

[0135] [Chemical Formula 6] In the peptide of the first embodiment described above, X7 is preferably a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue.

[0136] The structures of Me3Py, Me4Py, MeF3COO, MeF4COO, and MeAtp are shown below.

[0137] [Chemical Formula 7] In the peptide of the first embodiment described above, X8 is preferably an L-glutamine (Q) residue, an L-valine (V) residue, an L-2-aminoadipic acid (Hgl) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, a (S)-2-aminoheptanoic acid (Ahp) residue, an L-high tyrosine (Hty) residue, an L-threonine (T) residue, an allothreonine (alT) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, or a (S)-2-amino-2-(tetrahydro-2H-pyran-4- (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, (S)-2-amino-2-cyclohexylacetic acid (Chg) residue, or O-methyl-threonine (TMe) residue.

[0138] The structures of Hgl, Ahp, Hty, Cit, W1aa, KCOpipzaa, TMe, alT, Cbg, Gthp, G4pipaa, and Chg are shown below.

[0139] [Chemical Formula 8A] Specification 24 / 110 Page 44 CN 122555715 A In the peptide of the first embodiment described above, X10 is preferably a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)hexanoic acid (MeNle) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue.

[0140] The structures of Me3Py, Me4Py, MeF4COO, MeAtp and MeNle are shown below.

[0141] [Chemical Formula 9] In the peptide of the first embodiment described above, X11 is preferably an L-isoleucine (I) residue, an L-tyrosine (Y) residue, (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (F4aao) residue, or an O-methyl-L-threonine (TMe) residue.

[0142] The structures of F4aao and TMe are shown below.

[0143] [Chemical Formula 10] Specification 25 / 110 pages 45 CN 122555715 A In the peptide of the first embodiment described above, X12 is preferably an L-valine (V) residue, a methyl-L-valine (MeV) residue, or an N-methyl-L-cyclohexylglycine (MeChg) residue.

[0144] The structure of MeChg is shown below.

[0145] [Chemical Formula 11] In the peptide of the first embodiment described above, X13 is preferably a glycine (G) residue, a D-alanine (da) residue, or a D-serine (ds) residue.

[0146] In the peptide of the first embodiment described above, it is preferable to be a peptide comprising the amino acid sequence shown in Formula A2 or an amino acid sequence in which at least one amino acid residue selected from the group consisting of amino acid residues at positions 1 to 12 of the amino acid sequence shown in Formula A2 has been substituted, deleted, added, or inserted. It should be noted that in Formula A2, the amino acid sequence is described from the N-terminus to the C-terminus.

[0147] A2: df-SDIRY-MeF-QT-MeF-IVGC (Sequence No. 164) where df is a D-phenylalanine residue, and S is an L-serine residue.D is an L-aspartic acid residue, I is an L-isoleucine residue, R is an L-arginine residue, Y is an L-tyrosine residue, MeF is a methyl-L-phenylalanine residue, Q is an L-glutamine residue, T is an L-threonine residue, V is an L-valine residue, G is a glycine residue, and C is an L-cysteine ​​residue.

[0148] In the peptide of the first embodiment described above, it is preferable that the C-terminus also has a glycine residue.

[0149] In the peptide of the first embodiment described above, a cyclic peptide is preferred. Specification 26 / 110 pages 46 CN 122555715 A

[0150] In the peptide of the first embodiment described above, when it is a cyclic peptide, it is preferable that it has a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and the L-cysteine ​​residue contained in the peptide.

[0151] A cyclic peptide refers to a peptide in which two amino acids are combined, and all or part of them form a ring. It should be noted that this specification includes peptides in which amino acids form a bridge structure, peptides in which a cyclic structure is formed through the formation of a lactam ring or a macrocyclization reaction, and peptides in which a lasso peptide structure is formed. That is, in this specification, a cyclic peptide is a peptide in which a cyclic structure is formed as a part, and it may have a linear chain portion.

[0152] In order to cyclize the peptide of this embodiment, sometimes a part of the amino acid is modified. The peptide of this embodiment also includes peptides containing such modified amino acids. As an example of modification for cyclization, it includes the addition of a chloroacetyl group to the amino acid located at the N-terminus, which binds to the cysteine ​​residue in the peptide and cyclizes it. Peptides containing various (natural / non-natural) amino acids in which a chloroacetyl group is added are also included in the peptide of this specification.

[0153] Peptides generally have poor metabolic stability in organisms and are large in size, so they are not easy to penetrate cell membranes. For such problems, the method of cyclizing peptides has been adopted. When peptides are cyclized, protease resistance is increased, metabolic stability is increased, and conformational changes are restricted and rigidity is increased, thereby showing improved membrane permeability and affinity for target proteins.

[0154] Regarding peptide cyclization, it can be performed according to known methods. It is not limited to any particular method; for example, the peptide can be designed to contain two or more cysteine ​​residues, thereby forming a cyclic structure via disulfide bonds post-translation. Alternatively, peptides with a chloroacetyl group at the N-terminus can be synthesized using genetic code reprogramming techniques, as described by Goto et al. (Goto, Y. et al., ACS Chem. Biol., 2008, Vol. 3, pp. 120-129), and cysteine ​​residues can be pre-configured in the peptide for cyclization. Thus, post-translation, the thiol group actively nucleophilically attacks the chloroacetyl group, and the peptide cyclizes via a thioether bond. Alternatively, combinations of other amino acids capable of bonding and forming a cyclic structure can be incorporated into the peptide using genetic code reprogramming techniques for cyclization.Alternatively, a peptide with a cyclic amide at the N-terminus can be synthesized, and L-2-aminoadipic acid residues can be incorporated into the peptide and linked together to form a cyclization. Therefore, any known cyclization method can be used without particular limitation.

[0155] The peptide of the first embodiment described above preferably also contains added amino acid residues. In this case, the added amino acid residues are not particularly limited.

[0156] In the peptide of the first embodiment described above, the added amino acid residues may be included in the peptide forming a cyclic structure, or amino acid residues may be added further from the cyclic peptide in a connector form. The number of peptides and the number of amide bonds (number of amino acids, length) at the peptide site are not particularly limited, and the total number of amino acid residues (referring to the number of amino acid residues contained in the peptide forming a cyclic structure, excluding these amino acids when amino acid residues are added further from the cyclic peptide in a connector form) is preferably 20 residues or less. Regarding the preferred peptide length, the number of amino acid residues is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, and preferably 19 or less or 18 or less. Regarding a further preferred peptide length, the number of amino acid residues is 13 or more and 16 or less, with 14 or 15 amino acids being most preferred.

[0157] The peptide of the first embodiment described above preferably has binding affinity to the Fzd receptor.

[0158] The Fzd receptor described above is preferably at least one selected from the group consisting of Fzd receptor 2, Fzd receptor 7, and Fzd receptor 8.

[0159] The peptide of the first embodiment described above is preferably a peptide composed of the amino acid sequence shown in Formula A3.

[0160] A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 Wherein, ClAc is chloroacetyl, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are the amino acid residues shown in Table 1. The abbreviations of the amino acid residues in Table 1 are as described above.

[0161]

Table 1A

Table 1B

Table 1C

Table 1D

[0162] B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 Wherein, Y1 is a substituted L-phenylalanine (F) residue, Y2 is a substituted L-arginine (R) residue, Y3 is a substituted L-tryptophan (W) residue or a substituted (S)-2-amino-3-(naphthyl-1-yl)propionic acid (Nal1) residue, Y4 is an acidic amino acid residue or a neutral amino acid residue, Y5 is a substituted aromatic amino acid residue, aliphatic amino acid residue or L-proline (P) residue, Y6 is a substituted aliphatic amino acid residue, Y7 is an N-alkylated aromatic amino acid residue, Y8 is an amino acid residue derived from an amino acid with a hydroxyl, carboxyl or amide group on its side chain, Y9 is any amino acid residue, and Y10 is a substituted L-tryptophan (W) residue. Y11 is a substituted L-asparagine (N) residue or glutamine (Q) residue, Y12 is a substituted (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, Y13 is a substituted neutral amino acid residue or acidic amino acid residue, and Y14 is an L-cysteine ​​(C) residue.

[0163] In the peptide of the second embodiment described above, Y1 is preferably an L-phenylalanine (F) residue, (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, (S)-2-amino-3-(p-tolyl)propionic acid (F4Me) residue, or (S)-2-amino-3-(m-tolyl)propionic acid (F3Me) residue.

[0164] The structures of F3Me, F4F, and F4Me are shown below.

[0165] [Chemical Formula 12] In the peptide of the second embodiment described above, Y2 is preferably an L-arginine (R) residue or an N6-carbamoyl-L-lysine (Hcit) residue.

[0166] The structure of Hcit is shown below.

[0167]

Chemical Formula 13

[0168] The structures of W7N, W5N, W1Me7N, W7N5Me, Nal1, and Nal14N are shown below.

[0169] [Chemical Formula 14A] [Chemical Formula 14B] In the peptide of the second embodiment described above, Y4 is preferably an L-aspartic acid (D) residue, an L-glutamic acid (E) residue, an L-asparagine (N) residue, or an L-glutamine (Q) residue. Specification 33 / 110 pages 53 CN 122555715 A

[0170] In the peptide of the second embodiment described above, Y5 is preferably an L-valine (V) residue, a methyl-L-valine (MeV) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, a (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, a (S)-2-amino-3-(naphth-1-yl)propionic acid (Nal1) residue, a (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid (3Py5Ph) residue, a (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, or an L-proline (P) residue.

[0171] The structures of Na1, 3Py5Ph, W1aa, Cha, and Cbg are shown below.

[0172] [Chemical Formula 15] In the peptide of the second embodiment described above, Y6 is preferably an L-arginine (R) residue, a methyl-L-arginine (MeR) residue, a (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, a (S)-2-amino-4-ureidobutyric acid (Ncit) residue, or a (S)-5-acetamido-2-aminovaleric acid (OrnAc) residue.

[0173] The structures of Kmor, Cit, Hcit, Ncit, and OrnAc are shown below.

[0174]

Chemical Formula 16

[0175] The structures of MeF4COO, MeF4C, MeF4Me, MeF3Me, MeF3COO and MeF3C are shown below.

[0176] [Chemical Formula 17] Specification 35 / 110 pages 55 CN 122555715 A In the peptide of the second embodiment above, Y8 is preferably an L-serine (S) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an L-high tyrosine (Hty) residue, an allothreonine (alT) residue, an L-threonine (T) residue, an L-asparagine (N) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue or an L-glutamic acid (E) residue.

[0177] The structures of alT, Hty and W1aa are shown below.

[0178] [Chemical Formula 18] Specification 36 / 110 pages 56 CN 122555715 A In the peptide of the second embodiment described above, Y9 is preferably an L-asparagine (N) residue, a methyl-L-asparagine (MeN) residue, an L-2-aminoadipic acid (Hgl) residue, a (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, an L-high tyrosine (Hty) residue, a (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid (Hw7N) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue, or an L-glutamic acid (E) residue.

[0179] The structures of Cha, Hty, Hgl, Hw7N and KCOpipzaa are shown below.

[0180] [Chemical Formula 19] Specification 37 / 110 pages 57 CN 122555715 A In the peptide of the second embodiment described above, Y10 is preferably an L-tryptophan (W) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (W6N) residue, (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W1Me7N) residue or (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N5Me) residue.

[0181] The structures of W1Me7N, W7N, W7N5Me and W6N are shown below.

[0182] [Chemical Formula 20] Specification 38 / 110 pages 58 CN 122555715 AIn the peptide of the second embodiment described above, Y11 is preferably an L-asparagine (N) residue, an L-glutamine (Q) residue, or a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue.

[0183] The structure of Nmm is shown below.

[0184] [Chemical Formula 21] In the peptide of the second embodiment described above, Y12 is preferably a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, a (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C) residue, a (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) residue, or a (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C) residue.

[0185] The structures of Tic, Tic6C, Tic7C and Tic7H are shown below.

[0186] [Chemical Formula 22] Specification 39 / 110 pages 59 CN 122555715 A In the peptide of the second embodiment described above, Y13 is preferably an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-glutamine (Q) residue, a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue or an N4,N4-dimethyl-L-asparagine (Ndm) residue.

[0187] The structures of Nmm and Ndm are shown below.

[0188]

Chemical Formula 23

[0189] B2: FRWDVR-MeF-SNWN-Tic-NC (Serial No. 165) Wherein, F is an L-phenylalanine residue, R is an L-arginine residue, W is an L-tryptophan residue, D is an L-aspartic acid residue, V is an L-valine residue, MeF is a methyl-L-phenylalanine residue, S is an L-serine residue, Tic is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, N is an L-asparagine residue, and C is an L-cysteine ​​residue. Specification 40 / 110 pages 60 CN 122555715 A

[0190] In the peptide of the second embodiment described above, it is preferable that the C-terminus also has a glycine residue. The number of glycine residues is only one or more, and there is no particular limitation.

[0191] In the peptide of the second embodiment described above, a cyclic peptide is preferred.

[0192] In the peptide of the second embodiment described above, when it is a cyclic peptide, it is preferable to have a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide.

[0193] In the peptide of the second embodiment described above, it is preferable to further include added amino acid residues.

[0194] The added amino acid residues are not particularly limited.

[0195] In the peptide of the second embodiment described above, it is preferable to have binding to LRP.

[0196] The LRP is preferably at least one selected from the group consisting of LRP5 and LRP6.

[0197] In the peptide of the second embodiment described above, it is preferable to have DKK1-like activity.

[0198] The peptide of the second embodiment described above is preferably composed of the amino acid sequence shown in Formula B3.

[0199] B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 Wherein, ClAc is chloroacetyl, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, and Y14 are the amino acid residues shown in Table 2. The abbreviations of the amino acid residues in Table 2 are as described above.

[0200]

Table 2A

Table 2B

Table 2C

Table 2D

Table 2E

[0201] The above-mentioned peptide complex is a peptide containing a first peptide and other peptides, a peptide containing a compound, a peptide compound, or a pharmaceutically acceptable salt thereof. Specification 46 / 110 pages 66 CN 122555715 A The aforementioned peptide complex may contain one or more (three or more or four or more) first peptides.

[0202] The aforementioned peptide complex may also contain one or more partial peptides different from the first peptide. In the aforementioned peptide complex, the first peptide and partial peptides are preferably linked by a linker. The aforementioned peptide complex may be a homopolymer containing only peptides having the same amino acid sequence. The aforementioned peptide complex may be a heteropolymer containing peptides with different amino acid sequences. The aforementioned peptide complex preferably contains a first peptide and a second peptide with different amino acid sequences, the first peptide and the second peptide being linked by a linker.A heteropolymer connected by a linker. As shown in the examples, the first peptide and the second peptide exhibit Wnt signal activation ability by forming a peptide complex structure through the linker.

[0203] The peptide complex of the present invention may be composed of a first peptide, a second peptide, and a linker connecting the first peptide and the second peptide. In this case, the second peptide and the first peptide may be the same or different.

[0204] One embodiment of the peptide complex of the present invention is a peptide complex comprising a first peptide and a second peptide, wherein the first peptide is the peptide of the first embodiment described above, and the second peptide is the peptide of the second embodiment described above.

[0205] The peptide complex is preferably composed of the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

[0206] The linker may be a structure that connects multiple peptides in the peptide complex to each other. Examples of the linker include, for example, amino acid linkers (peptide linkers), chemical linkers, fatty acid linkers, nucleic acid linkers, and glycolinkers. In addition, it may be a complex of, for example, a chemical linker and a peptide linker.

[0207] An example of a chemical linker is a PEG (Polyethylene glycol) linker.

[0208] The PEG connector can be a connector composed of 1 to 36 ethylene glycol units.

[0209] In addition, the above-mentioned connector can be a fatty acid connector containing a divalent chemical moiety derived from a fatty acid. The amino acid (peptide) connector is a connector containing at least one amino acid, and can be, for example, a peptide rich in glycine such as the peptide having the sequence [GGGGS]n (where n is 1, 2, 3, 4, 5, or 6) as described in U.S. Patent No. 7,271,149, or a peptide connector rich in serine as described in U.S. Patent No. 5,525,491.

[0210] Non-limitingly, sometimes the physical properties of the peptide (e.g., solubility) may change due to the addition of a connector. Furthermore, the above-mentioned connector can be a combination of the above-mentioned connectors. For example, as an amino acid connector, it can be in the form of glycine (G)-L-lysine (K) bound together, and then a PEG connector bound to the side chain end of the Lys. In addition, the connector can have a structure in which amino acids and PEG are alternately bound, such as PEG-amino acid-PEG. Here, PEG refers to the PEG connector.

[0211] Another example of a linker is a linker in which 1 to 6 amino acids are added to the PEG linker portion. This linker may have a structure in which an amino acid is added to one end of the PEG linker portion, or it may have a structure in which amino acids are added to both ends of the PEG linker portion. A preferred example of the amino acid added to the PEG portion is L-lysine (K), but it is not limited to L-lysine (K) and may be other amino acids.

[0212] Regarding the location of the linker addition, it can be added at any position on the peptide. For example, it can be added at the C-terminus of the peptide, or at the...The linker can be formed by binding the L-cysteine ​​(C) at the C-terminus of the amino acid at position 1 to the amino acid at position 14 (i.e., L-cysteine ​​(C)) or the amino acid contained in the cyclic peptide. This is not a limitation, but the linker is preferably bound to the L-cysteine ​​(C) at the C-terminus or to the side chain of the amino acid contained in the cyclic peptide.

[0213] It should be noted that, for example, in the amino acid sequences shown in sequences 1-163, the glycine (G) at position 15 can be considered to be included in the linker. Preferably, the glycine (G) at position 15 is included in the linker. When the cyclic peptide formed by the combination of the amino acid at position 1 and the amino acid at position 14 (i.e., L-cysteine ​​(C)) in the amino acid sequences shown in sequences 1-163 forms a dimer structure through the structure shown in the following formula (in which the glycine at position 15 of the amino acid at position 14 (i.e., L-cysteine ​​(C)) is added), the linker structure is the structure shown in the following formula. The dimer is preferably a dimer formed by linking the C-terminus of the first peptide with the C-terminus of the second peptide via a linker.

[0214] [Chemical Formula 25A] [Chemical Formula 25B] [Chemical Formula 25C] [Chemical Formula 25A] The linkers described above are also preferably selected from those listed in Table 3. In Table 3, -click- indicates a bond formed by click chemistry.

[0215] [Table 3] As an example of click chemistry, the Wheatstone reaction (the following formula) in which alkynes undergo addition cyclization with azide compounds to form a 1,2,3-triazole ring can be cited.

[0216] [Chemical Formula 26] In the peptide complex of this embodiment, it is preferable that the first peptide is a peptide composed of the amino acid sequence shown in Formula A1 and the second peptide is a peptide composed of the amino acid sequence shown in Formula B1.

[0217] A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 Wherein, in formula A1, X1 is a D-phenylalanine (df) residue, X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue, X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue or a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue, X4 is an L-isoleucine (I) residue, X5 is an L-arginine (R) residue or an L-citrulline (Cit) residue, and X6 is an L-tyrosine (Y) residue. X7 is a methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue.X8 is an L-glutamine (Q) residue, L-valine (V) residue, L-high tyrosine (Hty) residue, L-threonine (T) residue, L-citrulline (Cit) residue, or O-methyl-L-threonine (TMe) residue; X9 is an L-threonine (T) residue; X10 is a methyl-L-phenylalanine (MeF) residue or (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue; X11 is an L-isoleucine (I) residue or L-tyrosine (Y) residue; X12 is an L-valine (V) residue or methyl-L-valine (MeV) residue; X13 is a glycine (G) residue or D-serine (ds) residue; and X14 is an L-cysteine ​​(C) residue.

[0218] B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 Wherein, in formula B1, Y1 is an L-phenylalanine (F) residue, Y2 is an L-arginine (R) residue, Y3 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, Y4 is an L-aspartic acid (D) residue, Y5 is an L-valine (V) residue, Y6 is an L-arginine (R) residue or (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, Y7 is a methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, Y8 is an L-serine (S) residue or an L-glutamic acid (E) residue; Y9 is an L-asparagine (N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue or an L-glutamic acid (E) residue; Y10 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue; Y11 is an L-asparagine (N) residue; Y12 is (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue; Y13 is an L-asparagine (N) residue or an L-aspartic acid (D) residue; and Y14 is an L-cysteine ​​(C) residue.

[0219] The peptide complex of this embodiment is preferably capable of activating the Wnt signal.

[0220] [Pharmaceutical Composition] Another embodiment of the present invention is a pharmaceutical composition.

[0221] One embodiment of the pharmaceutical composition is a drug comprising at least one (hereinafter also referred to as "the peptide of the present invention") selected from the group consisting of the above-described peptides and their pharmaceutically acceptable salts, esters or solvates.Another embodiment of the composition is a pharmaceutical composition comprising at least one selected from the group consisting of the above-described peptide complexes and their pharmaceutically acceptable salts, esters or solvates (hereinafter also referred to as "the peptide complex of the present invention" for convenience). Yet another embodiment is a pharmaceutical composition comprising at least one selected from the group consisting of the above-described peptides and peptide complexes and their pharmaceutically acceptable salts, esters or solvates.

[0222] Preferably, the above-described pharmaceutical compositions comprise an effective amount of the peptides or peptide complexes of the present invention as the active ingredient. Instructions for Use, Page 52 / 110, 72 CN 122555715 A

[0223] The disease that is the object of the above-described pharmaceutical composition refers to all diseases that are aggravated or otherwise related to the increase or decrease in the expression or activity of Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6); or, all diseases that are aggravated or otherwise related to the increase or decrease in Wnt signaling or any other intracellular signaling cascades activated by the above-described Frizzled receptors and / or the above-described LRPs. For example, it refers to bone metabolic diseases such as osteoporosis (see Manolagas, SC, "Wnt signaling and osteoporosis", Maturitas, 2014, Vol. 78, No. 3, pp. 233-237), inflammatory bowel disease (see Moparthi, L., and one other, "Wnt signaling in intestinal inflammation", Differentiation, 2019, Vol. 108, pp. 24-32), and cancers such as breast cancer, colorectal cancer, lung cancer, and gastric cancer (see Park, W.-J. and one other, "A New Wave of Targeting 'Undruggable' Wnt Signaling for Cancer Therapy: Challenges and Opportunities", Cells, 2023, Vol. 12, pp. 1110), but is not limited thereto.

[0224] The administration method of the above-mentioned drug composition is not particularly limited, and it can be administered orally or non-orally. Examples of non-oral administration methods include intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, and transmucosal administration (via nose, mouth, eye, lung, vagina, or rectum).

[0225] Considering that the peptides in the above-mentioned pharmaceutical compositions are easily metabolized and excreted, they can be modified in various ways. For example, they can be modified...Polyethylene glycol (PEG) and sugar chains are added to peptides to prolong their residence time in the blood and reduce their antigenicity. Alternatively, biodegradable polymers such as polylactic-co-glycolic acid (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., can be used as sustained-release bases to encapsulate peptides. When administering the drug transdermally, applying a weak current to the skin surface allows it to penetrate the stratum corneum (iontophoresis).

[0226] The above-mentioned drug composition can use its active ingredient directly, or it can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquid preparations (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, capsules, syrups, tablets, inhalers, ointments, eye drops, nasal drops, ear drops, and creams.

[0227] The above-mentioned formulation can be carried out by using excipients, binders, disintegrants, lubricants, solvents, dissolving aids, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc., through conventional methods.

[0228] Examples of the ingredients used in the above formulation include, but are not limited to, pharmaceutically acceptable organic solvents such as purified water, saline, phosphate buffer, dextran, glycerol, and ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicate, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tamarind gum, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, petrolatum, paraffin, octyl dodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, trehalose, and polysorbate.

[0229] The above-mentioned absorption enhancers can be used to improve the absorption of poorly absorbed drugs. As absorption enhancers, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins can be used; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as hexanoic acid, decanoic 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.

[0230] The above-mentioned pills or tablets can also be coated with sugar coatings, gastric-soluble substances, or enteric-soluble substances. The above-mentioned injections can be described in the instructions on page 53 / 110, 73 CN 122555715 A.It contains distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, dissolving aids, preservatives, etc., can be added.

[0231] The pharmaceutical composition of the present invention can also be administered in combination with other drugs and treatment methods that are effective against the above-mentioned diseases.

[0232] When administering the pharmaceutical composition of the present invention to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), especially humans, the dosage varies depending on the symptoms, the patient's age, sex, weight, sensitivity differences, method of administration, dosing interval, type of active ingredient, and type of preparation, and is not particularly limited. For example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or divided into several doses. In the case of injection administration, the dosage can be 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg, depending on the patient's weight, administered once or divided into several doses.

[0233] [Composition for Cell Culture] Another embodiment of the present invention is a composition for cell culture comprising the above-described peptide complex.

[0234] The above-described peptide complex has Wnt signal activation ability, and therefore can be used as a reagent or additive for culture media for cell culture, preferably for culturing mammalian cells, and more preferably as a reagent or additive for culture media for human cell culture. The above-described reagent or additive for culture media can be a reagent or additive for culture media for culturing cells in order to produce cultured meat.

[0235] In addition, the above-described peptide complex can also be used as a reagent or additive for preparing digestive tract organoids, lung organoids, etc. Non-limitingly, the above-described organoids are prepared from pluripotent stem cells through differentiation induction into cells of the endoderm and then into various tissues. Wnt signaling plays an important role in this differentiation induction. Therefore, the above-mentioned peptide complex can be used for the differentiation induction from pluripotent stem cells to the endoderm and to various cells, but is not limited thereto (see Yiangou, L., and three others, "Human Pluripotent Stem Cell-Derived Endoderm for Modeling Development and Clinical Applications", Cell Stem Cell, 2018, Vol. 22, No. 4, pp. 485-499).

[0236] The above-mentioned culture medium is not particularly limited as long as it is a culture medium used for culturing cells or tissues. The above-mentioned culture medium can be a serum culture medium, preferably a serum-free culture medium or a low-serum culture medium.

[0237] The above-mentioned culture medium additives can be in the form of a solution or a dry solid (e.g., solid form,In the form of powder, etc. When it is in solution form, it can be used directly as a culture medium for culture, or the solution diluted with a solvent and the above-mentioned additives added as needed can be used as a culture medium for culture. Examples of solvents used for dilution include water, buffer, physiological saline, and various culture media used in cell and tissue culture. These can be used alone or in combination of two or more.

[0238] When the above-mentioned culture medium additives are in the form of dry solids, for example, they can be dissolved in solvents such as water, buffer, physiological saline, and various culture media used in cell or tissue culture, and the above-mentioned additives can be added as needed, and used as a culture medium for culture.

[0239] The content of the peptide complex of the present invention in the culture medium for culturing cells or tissues or in the culture medium for cells obtained therefrom can be, for example, about 0.01 to about 10000 nmol / L, preferably about 0.1 to about 1000 nmol / L, more preferably about 0.5 to about 1000 nmol / L, and even more preferably about 1 to about 100 nmol / L, based on the final concentration relative to the total amount of the composition or the total amount of the culture medium.

[0240] [Composition for Medical Use, Diagnostic Use, or Research Use] Another embodiment of the present invention is a composition for medical use, diagnostic use, or research use. The composition for medical use, diagnostic use, or research use of this embodiment is a pharmaceutical composition comprising at least one selected from the group consisting of the above-described peptides and their pharmaceutically acceptable salts, esters, or solvates (hereinafter, for convenience, it is also simply referred to as "the peptide of the present invention"). Another embodiment is a composition comprising at least one selected from the group consisting of the above-described peptide complexes and their pharmaceutically acceptable salts, esters, or solvates (hereinafter, for convenience, it is also simply referred to as "the peptide complex of the present invention").

[0241] <Composition for Medical Use> The composition for medical use of this embodiment (hereinafter also simply referred to as "medical composition") comprises at least one selected from the group consisting of the peptides and peptide complexes of the present invention.

[0242] The above-described medical composition preferably comprises an effective amount of the peptide of the present invention or the peptide complex of the present invention as an active ingredient.

[0243] The disease to which the above-described medical composition is a disease caused by or aggravated by an increase or decrease in the expression or activity of Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), or any other disease related thereto; or a disease caused by or aggravated by an increase or decrease in Wnt signaling or any other intracellular signaling cascade activated by the above-described Frizzled receptors and / or LRPs, or any other disease related thereto.All diseases. For example, osteoporosis and other bone metabolic diseases (see Manolagas, SC, "Wnt signaling and osteoporosis", Maturitas, 2014, Vol. 78, No. 3, pp. 233-237), inflammatory bowel disease (see Moparthi, L., and one other, "Wnt signaling in intestinal inflammation", Differentiation, 2019, Vol. 108, pp. 24-32), breast cancer, colorectal cancer, lung cancer, stomach cancer and other cancers (see Park, W.-J. and one other, "A New Wave of Targeting 'Undruggable' Wnt Signaling for Cancer Therapy: Challenges and Opportunities", Cells, 2023, Vol. 12, pp. 1110), etc., but not limited thereto.

[0244] The administration method of the above medical composition is not particularly limited, and it can be administered orally or non-orally. Examples of non-oral administration methods include intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0245] Considering that the peptides in the above-mentioned medical composition are easily metabolized and excreted, they can be modified in various ways. For example, polyethylene glycol (PEG) and sugar chains can be added to the peptides to prolong their residence time in the blood and reduce their antigenicity. In addition, biodegradable polymers such as polylactic-co-glycolic acid (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, and nanospheres can be used as sustained-release bases to encapsulate peptides. When performing transdermal administration, applying a weak current to the skin surface can allow it to penetrate the stratum corneum (iontophoresis).

[0246] The above-mentioned medical composition can be used directly for its active ingredients, or it can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include, for example, liquid preparations (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, tablets, inhalers, ointments, eye drops, nasal drops, ear drops, and ointments.

[0247] The above-mentioned formulations can be carried out using excipients, binders, disintegrants, lubricants, solubilizers, dissolving aids, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc., through conventional methods.

[0248] Examples of ingredients used in the above formulations include, but are not limited to, pharmaceutically acceptable organic solvents such as purified water, saline, phosphate buffer, dextran, glycerol, and ethanol; animal and vegetable oils; lactose; mannitol; glucose; sorbitol; crystalline cellulose; hydroxypropyl cellulose; starch; corn starch; anhydrous silicate; magnesium aluminum silicate; collagen; polyvinyl alcohol; polyvinylpyrrolidone; carboxyvinyl polymers; sodium carboxymethyl cellulose; sodium polyacrylate; sodium alginate; water-soluble dextran; sodium carboxymethyl starch; pectin; methyl cellulose; ethyl cellulose; xanthan gum; gum arabic; tamarind gum; casein; agar; polyethylene glycol; diglyceride; glycerol; propylene glycol; petrolatum; paraffin; octyl dodecyl myristate; isopropyl myristate; higher alcohols; stearyl alcohol; stearic acid; human serum albumin; trehalose; and polysorbate.

[0249] The above-mentioned absorption enhancers can be used to improve the absorption of poorly absorbed drugs. As the above-mentioned absorption enhancers, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins can be used; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acid; fatty acids such as hexanoic acid, decanoic 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.

[0250] The above-mentioned pills or tablets can also be coated with sugar coating, gastric-soluble substances, or enteric-soluble substances. The above-mentioned injections can contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, dissolving aids, preservatives, etc., can be added.

[0251] The medical composition of the present invention can also be administered in combination with other drugs or treatments effective against the above-mentioned diseases.

[0252] When administering the medical composition of the present invention to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), especially humans, the dosage varies depending on the symptoms, the patient's age, sex, weight, sensitivity differences, method of administration, dosing interval, type of active ingredient, and type of preparation, and is not particularly limited. For example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or divided into several doses. In the case of injection administration, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg can be administered once or divided into several doses according to the patient's weight.

[0253] <Composition for Diagnostic Use> The composition for diagnostic use in this embodiment (hereinafter also simply referred to as "diagnostic composition") comprises at least one peptide selected from the group consisting of peptides and peptide complexes of the present invention.

[0254] The aforementioned peptides and peptide complexes can bind to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6). Therefore, they can also be used as diagnostic agents for detecting the aforementioned Frizzled receptors and / or the aforementioned LRPs. As such diagnostic agents, they can be used as detection agents for detecting the expression levels of the aforementioned Frizzled receptors and / or the aforementioned LRPs. When used as detection agents, the peptides or peptide complexes of the present invention can be labeled to enable detection. Thus, the aforementioned peptides or peptide complexes, or compositions comprising them, can be used as diagnostic agents for detecting the aforementioned Frizzled receptors and / or the aforementioned LRPs.

[0255] <Compositions for Research Use> The compositions for research use in this embodiment (hereinafter also simply referred to as "research compositions") comprise at least one selected from the group consisting of peptides and peptide complexes of the present invention.

[0256] The above-described peptides and peptide complexes are capable of binding to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6). Therefore, they are preferably used in studies related to the above-described Frizzled receptors and / or LRPs.

[0257] The research composition of this embodiment, for the above-described peptides or peptide complexes, and / or complexes containing substances further bound to the above-described peptides or peptide complexes via linkers, is preferably used in experimental methods for detecting at least one of the following:

[0258] a) Solubility in solvents.

[0259] b) Binding ability to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6).

[0260] c) Toxicity to cells and / or tissues.

[0261] d) Toxicity to experimental animals. Instructions for Use, Page 56 / 110, CN 122555715 A

[0262] Regarding the above test methods, the test for the solubility of the above peptide or the above peptide complex, and / or a complex containing a substance further bound to the above peptide or the above peptide complex via a linker, in a solvent can be a solubility determination. When determining solubility, the solvent is not limited and can be freely selected according to the purpose. Furthermore, regarding the solubility determination method, a known method can be appropriately selected according to the type of solvent.

[0263] The test for the binding ability with the above Frizzled receptor and / or the above LRP can be a determination of the binding ability with the above Frizzled receptor and / or the above LRP, without limitation, and known methods can preferably be used, such as surface plasmon resonance (SPR) assay, scanning analysis and / or radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EIA), and sandwich competitive binding assays, etc.

[0264] The test for toxicity to cells and / or tissues can be a test using a known toxicity evaluation assay for cells and / or tissues.The test, for example, can be a method performed in vitro. Cells and tissues can be cells and / or tissues commonly used for toxicity evaluation tests of pharmaceuticals, without limitation.

[0265] The toxicity test method for laboratory animals can be a well-known toxicity evaluation test using laboratory animals. Regarding laboratory animals, there are no particular limitations as long as they are commonly used laboratory animals; examples include mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cattle, poultry (e.g., chickens, quails, etc.), monkeys, and primates other than humans (e.g., cynomolgus monkeys, marmosets, orangutans, etc.). The above-mentioned toxicity evaluation tests are not limited and can be safety-related tests commonly conducted in non-clinical trials of pharmaceuticals. Examples include general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames test / chromosomal abnormality test / in vitro micronucleus test), carcinogenicity tests, reproductive 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), chemical analysis, and biological analysis (TK / PK), etc.

[0266] [Nucleic Acids] Another embodiment of the present invention is the above-mentioned peptide or nucleic acid encoding the peptide.

[0267] The nucleic acid of this embodiment can be natural or non-natural. Furthermore, nucleic acids include DNA, RNA, and chimeras thereof, but are not limited to these. The nucleic acid of this embodiment can be designed and manufactured based on the amino acid sequence of the above-mentioned peptide or peptide complex using known methods.

[0268] [Peptide-Drug Conjugate (PDC)] Another embodiment of the present invention comprises the above-described peptide or peptide complex, a desired substance to be delivered to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), and a complex (peptide-drug conjugate, PDC) for binding the above-described peptide or peptide complex to the above-described substance.

[0269] The above-described peptide is capable of binding to the above-described Frizzled receptor or the above-described LRP. The above-described peptide complex is capable of binding to the above-described Frizzled receptor and the above-described LRP. Therefore, the above-described peptide or peptide complex is capable of delivering a substance to the above-described Frizzled receptor and / or the above-described LRP.

[0270] The above-described substance is any substance that is desired to be delivered to the above-described Frizzled receptor and / or the above-described LRP, and can be any substance desired by those skilled in the art.

[0271] The above-described substance is not particularly limited, and examples include compounds, peptides, RI (radioisotopes), proteins, nucleic acids, and molecules used in DDS (drug delivery systems).

[0272] The above-mentioned compounds are not particularly limited, but low-molecular-weight compounds or medium-molecular-weight compounds are preferred, for example, well-known low-molecular-weight pharmaceutical agents.

[0273] The peptides mentioned above are not particularly limited, but are preferably peptides that bind to targets in vivo and exhibit certain effects, such as cyclic peptides.

[0274] The RI mentioned above is not particularly limited, and can be a low-molecular-weight compound, a medium-molecular-weight compound, or an antibody labeled with a radioactive isotope, such as compounds used in PET (positron emission tomography) examinations.

[0275] The proteins mentioned above are not particularly limited, and can be any protein that exhibits useful functions in vivo, such as antibodies or enzymes. Examples include enzymes used in enzyme replacement therapy.

[0276] The nucleic acids mentioned above can be DNA, RNA, or chimeras thereof, and are not particularly limited. Examples include nucleic acid drugs.

[0277] The molecules used in the DDS mentioned above are not particularly limited, but are preferably known molecules that can be used in DDS, such as liposomes or micelles. The molecules used in the DDS mentioned above may further contain compounds such as drugs.

[0278] Additionally, the substance desired to be delivered to the above-mentioned Frizzled receptor and / or the above-mentioned LRP may be a complex of the substances listed above.

[0279] Examples The present invention will be described in more detail below based on examples, but the present invention is not limited to the examples described below. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and these modifications are also included within the technical scope of the present invention.

[0280] [Chemical Synthesis] All raw materials, building blocks, reagents, acids, bases, solid resins and solvents used in the chemical synthesis in the following examples may be commercially available or may be synthesized by those skilled in the art using organic chemical methods. It should be noted that amino acids containing protecting groups are commercially available unless otherwise stated.

[0281] The elongation of peptide chains in the solid resin is carried out by using the resins described in each example as starting materials and conventional peptide coupling reaction conditions and Fmoc removal reaction conditions. The reaction was performed using Biotage's Syro I, Biotage's Syro II, CEM's Liberty Blue, CEM's Liberty Blue HT12, or CEM's Liberty Prime peptide synthesizers according to the manufacturer's manual.

[0282] Regarding the resin used, NovaPEG Rink amide resin, HMPB-MBHA resin, or Seiber amide resin were used, in amounts ranging from 5 mg to 2 g depending on the peptide.

[0283] The reactant mixture used for deprotection of the side chains and cleavage from the solid-phase resin was 4 mL to 50 mL depending on the peptide, using a solution with the following composition.

[0284] 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 commonly used Fmoc amino acids are listed in Table 4, and the side chain protecting groups are shown in parentheses.

[0285]

Table 4A

Table 4B

Table 4C

Table 4D

[0286] A) Shimadzu prep-HPLC system (LC-20AP, SPD-M20A, CTO-20AC, and CBM-20A).

[0287] B) Waters AutoPurification System.

[0288] C) Waters AutoPurification System with SQD.

[0289] D) Waters Preparative HPLC System. Specification 62 / 110 pages 82 CN 122555715 A

[0290] E) YMC Contichrom CUBE.

[0291] Regarding the column used, unless otherwise specified, any of the following a) to k) shall be used.

[0292] a) Jeanious One-Column 20mm I.D. ×150mmL.

[0293] b) Kinetex EVO C18 5μm 21.2×150mm. c) Waters XBridge C18 5μm 19×150mm. d) Waters XBridge C18 5μm 30×150mm. e) Waters XBridge C18 5μm 50×150mm. f) Waters XBridge C18 5μm 50×250mm. g) Waters XSelect C18 30×150mm. h) Waters XSelect C18 5μm 19×150mm. i) Waters XSelect C18 5μm30×150mm.

[0301] j) Waters XSelect CSH Prep C18 5μm OBD 50×250mm.

[0302] k) YMC-Triart Prep C18-S 10μm 10×250mm.

[0303] In the structural identification of chemically synthesized peptides, the molecular weight was confirmed by ESI-MS(+) mass spectrometry, taking into account the amino acids used according to the target sequence and the building blocks used as required. It should be noted that "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. The detected mass is reported in "m / z" unit symbol. It should be noted that compounds with a molecular weight greater than about 1000 are detected as multivalent ions at a higher frequency.

[0304] Basic analytical apparatus and basic conditions In the mass spectrometry analysis of the peptides synthesized in the following examples, the following basic analytical apparatus and basic conditions were used unless otherwise specified. The gradient B(%) is analyzed using any of the conditions in w / x / y / z.

[0305] (Basic Analytical Apparatus) Waters AutoPurification System-SQD2 single quadruple mass spectrometer (Basic Conditions) Column: Kinetex EVO C18 (1.7 μm, 2.1 × 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 (%): w) 5~95% / 2.10 min, 95~95% / 0.75 min (Basic Analytical Apparatus) Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020) (Basic Conditions) Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Column temperature: 60℃ Mobile phase A: 0.025% TFA, H2O (Instructions for Use, page 63 / 110, CN 122555715 A) Mobile phase B: 0.025% TFA, MeCN Flow rate: 0.5 mL / min Wavelength: 225 nm PDA gradient B (%): x) 5~45% / 7.15 min, 45~95% / 0.3 min, 95~95% / 1.55 ​​min y) 20~60% / 7.15 min, 60~95% / 0.3 min, 95~95% / 1.55 ​​minz) 20~60% / 54.15 min, 60~95% / 0.3 min, 95~95% / 1.55 ​​min [Example 1] Synthesis of a peptide complex (a peptide complex with linker structure number 9 added to peptide sequence number 66) [Chemical Formula 27] Specification 64 / 110 pages 84 CN 122555715 A The target peptide was synthesized using Sieber amide resin, starting with the removal of the Fmoc group by the conventional method described above. At this time, the Biotage Syro II was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. When each residue of Specification 65 / 110 pages 85 CN 122555715 A was introduced, Fmoc-AA / HATU / DIEA (8.4 equivalents / 7.84 equivalents / 16.8 equivalents) was used relative to 1 equivalent of resin, and the reaction was carried out twice in DMF at 75°C for 20 minutes. Specifically, residues 5 and 14 were reacted twice at 50°C for 30 minutes. Residues 6, 7, 8, 9, 10, 11, and 12 were reacted three times at 75°C for 20 minutes. The introduction of Fmoc-PEG10c at residue 16 was carried out once at 25°C for 60 minutes.

[0306] The removal of the Fmoc group was carried out by reacting with a 10% pyrrolidine DMF solution at 25°C for 3 minutes.

[0307] The introduction of the chloroacetyl group was carried out as follows: For the solid resin holding the Fmoc protected peptide obtained by the previous process, after removing the α-amino Fmoc group using the above method, a DMF solution of ClAcOSu (5 equivalents) was added to the solid resin and shaken at room temperature for 60 minutes.

[0308] Regarding the deprotection of the side chains and the cleavage from the solid resin, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to a reaction vessel containing the solid resin, and the mixture was shaken thoroughly for 60 minutes at room temperature. The reaction solution was then filtered and recovered using a filter screen. When the filtrate was added to a cold excess of diisopropyl ether, a white turbid precipitate was formed. The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with a cold diisopropyl ether / hexane (1 / 1) mixed solvent and dried for 60 minutes. The resulting solid was used in the subsequent cyclization reaction.

[0309] Regarding the cyclization reaction of the peptide, the peptide was dissolved in DMSO / H2O (7 / 3) to achieve a final concentration of 1.25 mM based on the molar amount of solid resin, and then triethylamine (10 equivalents) was added and left to stand overnight at room temperature. The resulting reaction solution was concentrated under reduced pressure using Genevac EZ-2 Elite, and DMSO was added to achieve a peptide concentration of 12.5 mM.

[0310] The crude product was subjected to solid-phase extraction using a Gilson column (column: Gilson ASPEC C18, 50 mg, 1 mL). (1) The column was washed with extraction buffer A (0.1% TFA, in 95% MeCN / H2O, 0.3 mL).

[0311] (2) The column was equilibrated with extraction buffer B (0.1% TFA, in 5% MeCN / H2O, 0.3 mL).

[0312] (3) 0.02 mL of the above solution was loaded onto the column.

[0313] (4) The column was washed with extraction buffer B (0.4 mL).

[0314] (5) Extraction was performed with extraction buffer A (0.4 mL). The resulting extract was concentrated under reduced pressure using EZ-2 Elite.

[0315] The purity of a major peak of the target analyte was calculated based on the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions, and the result was 55%.

[0316] (Analytical conditions) Retention time: 1.52 min Column: Kinetex EVO C18 (1.7 μm 2.1 × 50 mm, 100 Å) Mobile phase: A) 0.025% TFA in H2O, B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 5-95% for 2.10 min, then 95-95% for 0.75 min Flow rate: 0.6 mL / min ESI-MS(+) Observation m / z = 1144(M+3H)3+ [Example 2] Synthesis of peptide complex (peptide complex with adapter structure number 9 added to peptide sequence number 99) [Chemical Formula 28] Specification 66 / 110 pages 86 CN 122555715 A The target peptide was synthesized using Sieber amide resin, starting from the removal of the Fmoc group by the conventional method described above. At this time, a solid-phase synthesizer, Biotage's Syro II, was used, and the synthesis was performed according to the manufacturer's manual. When introducing residues from each of the manuals, pages 67 / 110, 87 CN 122555715 A, Fmoc-AA / HATU / DIEA (4.2 equivalents / 3.92 equivalents / 8.4 equivalents) were used relative to 1 equivalent of resin, and the reaction was carried out twice in DMF at 75°C for 20 minutes. Specifically, for residues 2 and 14, the reaction was carried out twice at 50°C for 30 minutes. For residue 6, the reaction was carried out twice at 50°C for 60 minutes. For residue 7, the reaction was carried out twice at 75°C for 30 minutes. For the introduction of Fmoc-PEG10c for residue 16, the reaction was carried out once at room temperature for 60 minutes.

[0317] Regarding the removal of the Fmoc group, after reacting with a 20% piperidine DMF solution at room temperature for 5 minutes, the solution was removed, and then...The reaction was repeated at room temperature with a 20% piperidine DMF solution for 15 minutes.

[0318] The introduction of chloroacetyl groups was carried out as follows: For the solid resin containing the Fmoc protected peptide obtained in the previous process, after removing the α-amino Fmoc group using the above method, a DMF solution of ClAcOSu (5 equivalents) was added to the solid resin, and the mixture was shaken at room temperature for 60 minutes.

[0319] Regarding the deprotection of the side chain and the cleavage from the solid resin, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid resin, and the mixture was shaken thoroughly and then shaken at room temperature for 90 minutes. The reaction solution was filtered and recovered using a filter screen. When the filtrate was added to a cold excess of diisopropyl ether, a white turbid precipitate was formed. The mixture was centrifuged and the solution was decanted. The obtained solid was washed again with a cold diisopropyl ether / hexane (1 / 1) mixed solvent and dried for 60 minutes. The obtained solid was used in the subsequent cyclization reaction.

[0320] Regarding the cyclization reaction of the peptide, the peptide was dissolved in DMSO / H2O (7 / 3) to achieve a final concentration of 2.5 mM based on the number of moles of solid-phase resin. Triethylamine (20 equivalents) was added and stirred, and the mixture was left to stand overnight at room temperature. The resulting reaction solution was concentrated under reduced pressure using Genevac EZ-2 Elite to achieve 12.5 mM.

[0321] The crude product was subjected to solid-phase extraction using a Gilson column (column: Gilson ASPEC C18, 50 mg, 1 mL). (1) The column was washed with extraction solution A (0.1% TFA, in 95% MeCN / H2O, 0.3 mL).

[0322] (2) The column was equilibrated with extraction solution B (0.1% TFA, in 5% MeCN / H2O, 0.3 mL).

[0323] (3) 0.02 mL of the above solution was loaded onto the column.

[0324] (4) Wash the column with extraction buffer B (0.4 mL).

[0325] (5) Extract with extraction buffer A (0.4 mL). The resulting extract was concentrated under reduced pressure using EZ-2 Elite.

[0326] The purity of a major peak of the target analyte was calculated based on the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions, and the result was 50%.

[0327] (Analytical conditions) Retention time: 1.24 min Column: Kinetex EVO C18 (1.7 μm, 2.1 × 50 mm, 100 Å) Mobile phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 5-95% for the first 2.10 min, then 95-95% for the next 0.75 min Flow rate: 0.6 mL / min ESI-MS(+)Observed value m / z = 1204(M+3H)3+ [Example 3] Synthesis of peptide complex (dimer of structure number 62) I. Peptide complex (peptide complex with a linker structure number 2 added to peptide of peptide sequence number 74) Instructions 68 / 110 pages 88 CN 122555715 A Synthesis [Chemical Formula 29] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.6 mmol / g) starting from the removal of the Fmoc group by the conventional method described above. At this time, the Liberty Blue HT of CEM Corporation was used as the solid phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. When introducing each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used relative to 1 equivalent of resin, and the reaction was carried out once in DMF at 90°C for 3 minutes. Specifically, for residue 5, the reaction was carried out twice at 50°C for 15 minutes. For residues 6 and 9, the reaction was carried out twice at 90°C for 10 minutes. For residue 11, the reaction was carried out twice at 75°C for 30 minutes. For residues 12, 13, 15, and 16 (Fmoc-PEG4c), and residue 17 (Fmoc-Gpra-OH), the reaction was carried out once at 75°C for 10 minutes. For residue 14, the reaction was carried out once at 50°C for 15 minutes. For residue 10, the reaction was carried out once at 40°C for 30 minutes, using the condensation conditions of Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents).

[0328] Regarding the removal of Fmoc, the following conditions were used: reaction with a 10% pyrrolidine DMF solution at 90°C for 1 minute, reaction at 50°C for 90 seconds, reaction at 75°C for 3 minutes, or reaction at room temperature for 1 minute.

[0329] The introduction of chloroacetyl groups was carried out as follows: a DMF solution of ClAcOSu (5 equivalents) was added to the solid resin and shaken at room temperature for 60 minutes.

[0330] Regarding the deprotection of the side chains and the cleavage from the solid resin, firstly, the resin obtained after the chloroacetyl group introduction process was washed in DMF, then washed with dichloromethane, then washed with diethyl ether, and then dried under reduced pressure. A reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid resin and shaken at room temperature for 60 minutes. The reaction solution was then recovered by filtration using a filter screen. When the filtrate is added to a cold, excess mixture of diisopropyl ether / hexane (1 / 1), a white turbid precipitate forms. The mixture is then centrifuged, and the precipitate is decanted. (Instructions for use, pages 69 / 110, 89)CN 122555715 A The obtained solid was washed again with cold diethyl ether and dried under reduced pressure. The obtained solid was used in the subsequent cyclization reaction.

[0331] Regarding the cyclization reaction of the peptide, the peptide was dissolved in MeCN / H2O (1 / 1) to achieve a final concentration of 3.1 mM based on the molar amount of solid resin, and then triethylamine (10 equivalents) was added. After shaking at room temperature for 1 hour, acetic acid was added. The resulting reaction solution was concentrated using Genevac HT-12.

[0332] The obtained crude product was purified under the following conditions.

[0333] (Purification conditions) Column: Waters XBridge C18 (5μm, 50×150mm) Mobile phase: A) 0.1% TFA in H2O B) 0.1% TFA in MeCN Temperature: 40℃ Gradient (%B concentration): 5% for 2 minutes, 5~28% for 1 minute, 28~33% for the next 8 minutes, and 33~60% for the next 1 minute Flow rate: 20mL / min for 1 minute, 20mL / min~120mL / min for the next 1 minute, and 120mL / min after freeze-drying to obtain a peptide complex (190mg) with a portion of adapter structure number 2 added to peptide sequence number 74.

[0334] II. Synthesis of the peptide complex (a peptide complex with a linker structure number 2 added to the peptide of peptide sequence number 162) [Chemical Formula 30] Specification 70 / 110 pages 90 CN 122555715 A The target peptide was synthesized by removing the Fmoc group using the conventional method described above, starting with Sieber amide resin (Watanabe Chemical, 0.54 mmol / g). At this time, Liberty Blue from CEM Corporation was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. When introducing each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used relative to 1 equivalent of resin, and the reaction was carried out once in DMF at 90°C for 3 minutes. Among them, with respect to the second residue, the reaction was carried out twice at 50°C for 15 minutes. Specification 71 / 110 pages 91 CN 122555715 A For residues 11 and 12, the reaction was carried out twice at 90°C for 10 minutes. For residue 14, the reaction was carried out twice at 50°C for 15 minutes. For residue 6, the reaction was carried out twice at room temperature for 60 minutes, using the condensation conditions of Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents). Additionally, when introducing Fmoc-W7N-OH, Fmoc-Kmor-OH, and Fmoc-KN3-OH, these amino acids were dissolved...The liquid is formed in NMP. The removal of Fmoc is carried out under the following conditions: a 10% pyrrolidine DMF solution is used, reacting at 90°C for 1 minute; a 90-second reaction is carried out at 50°C; or a 1-minute reaction is performed twice consecutively at room temperature.

[0335] The introduction of chloroacetyl groups is performed as follows: a DMF / DCM (1 / 1) solution of ClAcOSu (5 equivalents) is added to the solid resin, and the mixture is shaken at room temperature for 30 minutes.

[0336] Regarding the deprotection of the side chains and the cleavage from the solid resin, firstly, the resin obtained after the chloroacetyl group introduction process is washed in DMF, then washed with dichloromethane, then washed with ether, and then dried under reduced pressure. The reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) is added to the reaction vessel containing the solid resin, and the mixture is shaken at room temperature for 60 minutes. The reaction solution is then filtered and recovered using a filter screen. When the filtrate was added to a cold, excess mixture of diisopropyl ether / hexane (1 / 1), a turbid precipitate was formed. The mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cold diethyl ether and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.

[0337] For the peptide cyclization reaction, the peptide was dissolved in MeCN / H2O (1 / 1) to achieve a final concentration of 5 mM based on the molar amount of solid resin. Triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 1 hour. Acetic acid was then added. The resulting reaction solution was concentrated using Genevac HT-12.

[0338] The crude product was purified under the following conditions.

[0339] (Purification conditions) Column: Waters XBridge C18 (5μm, 50×150mm) Mobile phase: A) 0.1% TFA in H2O B) 0.1% TFA in MeCN Temperature: 40℃ Gradient (%B concentration): 5% for 2 minutes, 5-21% for 1 minute, 21-26% for the next 8 minutes, and 26-60% for the next 1 minute Flow rate: 20mL / min for 1 minute, 20mL / min-120mL / min for the next 1 minute, and then 120mL / min After freeze-drying, a peptide complex (161mg) with a portion of adapter structure number 2 added to peptide sequence number 162 was obtained.

[0340] III. Synthesis of the peptide complex (dimer of structure number 62) [Chemical Formula 31] Specification 72 / 110 pages 92 CN 122555715 A Regarding the synthesis of the peptide complex, the peptide complex (a peptide complex with a portion of linker structure number 2 added to the peptide of peptide sequence number 162) (30 mg) was dissolved in DMF / H2O (3 / 1), and then the peptide complex (a peptide complex with a portion of linker structure number 2 added to the peptide of peptide sequence number 74) was added. Specification 73 / 110 pages 93 CN122555715 A peptide complex containing part of adapter structure number 2 (27 mg, 1.1 equivalents) and [Cu(CH3CN)4]PF6 (4 equivalents) were added and stirred at room temperature for 30 minutes.

[0341] The crude product obtained was purified under the following conditions.

[0342] (Purification Conditions) Column: Waters XSelect CSH Prep C18 (5μm, OBD, 50×250mm) Mobile Phase: A) 1% AcOH in H2O B) 1% AcOH in MeCN C) 0.2M TEAA in H2O D) MeCN Temperature: 50℃; Main Pump Gradient (%A Concentration): 0.1% for the first 5.0 minutes, 0.1~100% for the next 0.1 minutes, and 100% after 5.1 minutes; %B (%B Concentration): 0% for the first 5.1 minutes, 0~4.2% for the next 1.9 minutes, 4.2~50% for the next 20 minutes, and 50~90% for the next 4 minutes; %C Concentration: 99.9% for the first 5.0 minutes, 99.9~0% for the next 0.1 minutes, and 0% after 5.1 minutes. (%D concentration): 0%. Flow rate: 18 mL / min for 5.1 minutes, then 18 mL / min to 118 mL / min for 1.9 minutes, then 118 mL / min. At-column dilution pump: 1% AcOH in H2O / MeCN (1 / 1). Flow rate: 2 mL / min. The purity of the target analyte was calculated based on the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 95.06%.

[0343] (Analytical conditions) Retention time: 5.80 min Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 5-45% for 7.15 min, 45-95% for 0.30 min, and 95-95% for 1.55 min Flow rate: 0.5 mL / min ESI-MS(+) observation m / z = 1186(M+4H)4+ [Example 4] Synthesis of peptide complex (dimer of structure number 46) I. Synthesis of peptide complex (peptide complex with a linker structure number 8 added to peptide of peptide sequence number 35) [Chemical Formula 32] Specification 74 / 110 pages 94 CN 122555715 A The Fmoc group was removed using Sieber amide resin (Watanabe Chemicals, 0.54 mmol / g) via the conventional method described above.The target peptide was synthesized. At this time, a Liberty PRIME from CEM Corporation was used as a solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. When introducing each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used relative to 1 equivalent of resin, and the reaction was carried out once in DMF at 105°C for 2 minutes. Among them, for residue 5, the reaction was carried out twice at 50°C for 15 minutes. For residues 6, 9, and 11, the reaction was carried out twice at 75°C for 30 minutes. For residue 14, the reaction was carried out once at 50°C for 15 minutes.

[0344] The removal of Fmoc was carried out as follows: with the basic conditions of 83 mM Oxyma pure in 4% pyrrolidine DMF solution and reaction at 110°C for 90 seconds, a 10% pyrrolidine DMF solution was also used and the reaction was carried out twice consecutively at room temperature for 1 minute.

[0345] The introduction of chloroacetyl groups was carried out as follows: a DMF solution of ClAcOSu (5 equivalents) was added to the solid resin and shaken for 60 minutes at room temperature.

[0346] Regarding the deprotection of the side chains and the cleavage from the solid resin, the resin obtained after the chloroacetyl group introduction process was first washed in DMF, then washed with dichloromethane, then washed with diethyl ether, and then dried under reduced pressure. A reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid resin and shaken for 60 minutes at room temperature. The reaction solution was filtered and recovered using a filter screen. When the filtrate was added to a cold excess of a diisopropyl ether / hexane (1 / 1) mixed solvent, a white turbid precipitate was produced. The mixture was centrifuged, and the solution was decanted. The obtained solid was washed again with cold diethyl ether and dried under reduced pressure. The obtained solid was used in the subsequent cyclization reaction.

[0347] Regarding the cyclization reaction of the peptide, the peptide was dissolved in MeCN / H2O (1 / 1) to achieve a final concentration of 2.5 mM based on the molar amount of solid resin. Triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 4 hours. Acetic acid was then added. The resulting reaction solution was concentrated using Genevac EZ-2 Elite. The crude product was purified under the following conditions to obtain a peptide complex (a peptide complex with a portion of linker structure number 8 added to peptide sequence number 35).

[0348] (Purification conditions) Specification 75 / 110 pages 95 CN 122555715 A Column: Waters XBridge C18 (5 μm, 50 × 150 mm) Mobile phase: A) 0.1% TFA in H2O B) 0.1% TFA in MeCN Temperature: 40 °CGradient (%B concentration): 9% within 2 minutes, 9-34% within 1 minute, 34-39% within 8 minutes, and 39-60% within 1 minute. Flow rate: 20 mL / min within 1 minute, 20 mL / min to 120 mL / min within 1 minute, and then 120 mL / min. The purity of the target analyte was calculated based on the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 97.80%.

[0349] (Analytical conditions) Retention time: 4.25 min Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 20-60% for 7.15 min, 60-95% for 0.30 min, and 95-95% for 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observation m / z = 988 (M+2H)2+ ​​II. Synthesis of peptide complex (a peptide complex with a linker structure number 8 added to peptide sequence number 79) [Chemical Formula 33] Specification 76 / 110 pages 96 CN 122555715 A Using Sieber The target peptide was synthesized using an amide resin (Watanabe Chemical, 0.54 mmol / g) by removing the Fmoc group using the conventional method described above. A Liberty PRIME from CEM Corporation was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalences / 8 equivalences / 4 equivalences) was used relative to 1 equivalence of resin, and the reaction was carried out once in DMF at 105°C for 2 minutes. Specifically, residues 2 and 6 were reacted twice at 50°C for 15 minutes. Residues 11 and 12 were reacted twice at 105°C for 3 minutes. Residue 14 was reacted once at 50°C for 15 minutes.

[0350] The removal of Fmoc was carried out as follows: Based on the conditions of reacting with a 4% pyrrolidine DMF solution of 83 mM Oxyma pure at 110°C for 90 seconds, a 10% pyrrolidine DMF solution was also used, with the reaction being carried out twice consecutively at room temperature for 1 minute, or a 10% pyrrolidine DMF solution was used, with the reaction being carried out at 50°C for 90 seconds.

[0351] The introduction of chloroacetyl groups was carried out as follows: A DMF solution of ClAcOSu (5 equivalents) was added to the solid resin, and the mixture was shaken at room temperature for 60 minutes.

[0352] Regarding the deprotection of the side chains and the cleavage from the solid resin, firstly, the chloroacetyl groups were introduced into the resin obtained after the process...The resin was washed in DMF, then with dichloromethane, followed by washing with diethyl ether, and then dried under reduced pressure. A reaction mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to a reaction vessel containing the solid resin, and the mixture was shaken for 60 minutes at room temperature. The reaction solution was recovered by filtration. Adding the filtrate to a cold, excess diisopropyl ether / hexane (1 / 1) mixture produced a white turbid precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cold diethyl ether and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. Instructions 77 / 110, page 97, CN 122555715 A

[0353] Regarding the cyclization reaction of the peptide, the peptide was dissolved in MeCN / H2O (1 / 1) to achieve a final concentration of 2.5 mM based on the molar amount of solid resin, followed by the addition of triethylamine (10 equivalents), and shaking at room temperature for 4 hours, followed by the addition of acetic acid. The resulting reaction solution was concentrated using Genevac EZ-2 Elite. The crude product was purified under the following conditions to obtain a peptide complex (a peptide complex with a linker structure number 8 added to the peptide of peptide sequence number 79).

[0354] (Purification conditions) Column: Waters XBridge C18 (5μm, 50×150mm) Mobile phase: A) 0.1% TFA in H2O B) 0.1% TFA in MeCN Temperature: 40℃ Gradient (%B concentration): 5% for 2 minutes, 5~27% for 1 minute, 27~32% for the next 8 minutes, and 32~60% for the next 1 minute Flow rate: 20mL / min for 1 minute, 20mL / min~120mL / min for the next 1 minute, and then 120mL / min The purity of the target analyte was calculated based on the area ratio of the LC / MS (UV wavelength 225nm) chromatogram under the following analytical conditions, and was 95.95%.

[0355] (Analytical conditions) Retention time: 5.85 min Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 5-45% for 7.15 min, 45-95% for 0.30 min, and 95-95% for 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observation m / z = 1055 (M+2H)2+ ​​III. Synthesis of peptide complex (dimer structure number 46) [Chemical Formula 34] Specification 78 / 110 pages 98 CN 122555715 ARegarding the synthesis of the peptide complex, 0.0175M DMA solution of AZ(L08)-P12-PS (0.36mL) and DIPEA (0.012mL) were added to a 20mg peptide complex (with a portion of linker structure number 8 added to peptide sequence number 35) at 40°C. After stirring at the same temperature for 2 hours, another 0.0175M DMA solution of AZ(L08)-P12-PS (0.13mL) was added, and the mixture was stirred at the same temperature for 1 hour. Water (0.36mL), a DMA solution of 20.05mg peptide complex (with a portion of linker structure number 8 added to peptide sequence number 79), TBTA (7.2mg), and [Cu(CH3CN)4]PF6 (3.4mg) were added to the reaction mixture. After stirring at room temperature for 1 hour, acetic acid was added. The crude product was purified under the following conditions to obtain the target analyte (17 mg).

[0356] (Purification conditions) Column: Waters XBridge C18 (5 μm, 19 × 150 mm) Mobile phase: A) 0.1% TFA in H2O B) 0.1% TFA in MeCN Temperature: 40 °C Gradient (%B concentration): 14-39% for 3 minutes, 39-44% for 8 minutes, and 44-60% for 1 minute Flow rate: 17 mL / min The purity of the target analyte was calculated to be 94.64% based on the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions.

[0357] (Analytical conditions) Retention time = 5.40 Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 20-60% for 7.15 minutes, 60-95% for 0.30 minutes, and 95-95% for 1.55 minutes Flow rate: 0.5 mL / min ESI-MS (+) observation m / z = 1484(M+4H)4+ [Example 5] Synthesis of peptide complex (dimer structure number 63) [Compound 35] Specification 80 / 110 pages 100 CN 122555715 A The target compound was obtained by synthesis using the same method as in Example 3.

[0358] The purity of the target analyte was calculated based on the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 97.49% as specified in document 81 / 110, page 101, CN 122555715 A.

[0359] (Analytical Conditions) Retention Time: 4.10 min Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile Phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 20–60% for 7.15 min, 60–95% for the next 0.30 min, and 95–95% for the next 1.55 min Flow Rate: 0.5 mL / min ESI-MS (+) Observation m / z = 1242(M+5H)5+ [Example 6] Synthesis of a peptide complex (dimer structure number 66) [Chemical Formula 36] Specification 82 / 110 pages 102 CN 122555715 A HMPB-MBHA resin (Novabiochem, 0.69 mmol / g) and Fmoc-Gpra-OH / DIC / DMAP (4 equivalents / 4 equivalents / 0.5 equivalents) relative to 1 equivalent of the resin were reacted once in DMF / DCM (1 / 2) at room temperature for 1 hour. The peptide complex synthesized in the same manner as in Example 3 (a peptide complex with a linker structure number 3 added to the peptide of peptide sequence number 163) and HMPB-MBHA resin (Novabiochem, 0.69 mmol / g) and Fmoc-KN3-OH / DIC / DMAP (4 equivalents / 4 equivalents / 0.5 equivalents) relative to 1 equivalent of the resin were reacted once in DMF / DCM (1 / 2) at room temperature for 1 hour. (See page 83 / 110 of the specification, CN 122555715 A). The reaction was carried out once at room temperature for 1 hour in DCM (1 / 2), using the synthesized Fmoc-KN3-HMPB-MBHA resin and the peptide complex synthesized in the same way as in Example 3 (a peptide complex with a linker structure number 3 added to the peptide of peptide sequence number 76), and the target compound was obtained by the same method as in Example 3.

[0360] The purity of the target compound was calculated based on the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 95.04%.

[0361] (Analytical Conditions) Retention Time: 2.80 min Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile Phase: A) 0.025% TFA in H2O B) 0.025% TFA in MeCN Temperature: 60 °C Gradient (%B concentration): 20–60% for 7.15 min, 60–95% for the next 0.30 min, and 95–95% for the next 1.55 min Flow Rate: 0.5 mL / min ESI-MS (+) Observation m / z = 1176(M+4H)4+[Example 7] Various peptide synthesis In this example, various peptide complexes were chemically synthesized in the same manner as in Examples 1 to 6. The amino acid sequences of the synthesized cyclic peptides are shown in Tables 5 and 6, the structures of the linkers are shown in the following formula, and the peptide complexes dimerized by the linkers are shown in Table 7.

[0362] The synthesized peptide complexes were analyzed under any of the analytical conditions described in Examples 1 to 6, and the structures were confirmed by ESI-MS(+) mass spectrometry. The obtained ESI-MS(+) observations, retention times, valences, and concentration gradients (%) of mobile phase B used in the analysis are shown in Tables 5, 6, and 7.

[0363] It should be noted that the analytical results recorded in Tables 5 and 6 are the analytical results of the peptide complexes with the linker structure number 9 added to the peptides shown by the peptide sequence numbers.

[0364]

Table 5A

Table 5B

Table 5C

Table 5D

Table 6A

Table 6B

Table 6C

Table 6D

Table 6E

[0365] 200 μg of avidin-labeled polyclonal anti-human IgG (eBioscience; 13-4998) was added to each well of a streptavidin-coated 96-well plate (Thermo; 436014) and allowed to stand for 30 minutes.

[0366] After washing three times with PBS-T, 3 pmol of Fc-tagged fusion Frizzled-2 (R&D systems; 1307-FZ), Frizzled-7 (R&D systems; 6178-FZ), or Frizzled-8 (R&D systems; 6129-FZ) was added to each well and allowed to stand for 30 minutes, thereby immobilizing each Frizzled on the plate.

[0367] After washing three times with PBS-T, peptide complexes diluted to 10 nM were added to Frizzled-2 and Frizzled-7, and peptide complexes diluted to 500 nM were added to Frizzled-8. The mixture was allowed to stand for 1 hour.

[0368] After washing three times with PBS-T, Anti-HA-tag mAb-HRP-DirecT (MBL; M180-7) diluted 5000 times with PBS-T was added, and the mixture was allowed to stand for 30 minutes.

[0369] After washing three times with PBS-T, SureBlue TMB single-component microplate peroxidase substrate (SeraCare; 5120-0077) was added, and the mixture was allowed to stand for 10 minutes. Then, an equal volume of TMB stop solution (SeraCare; 5150-0021) was added to stop the reaction.

[0370] The absorbance at 450 nm was measured using an Infinite M Nano* (TECAN). The binding signal of each peptide complex was calculated by subtracting the absorbance of the pore without peptide complex from the absorbance of the pore with added peptide.

[0371] The results are shown in Table 8. This shows that the synthesized peptide complexes have binding activity for Frizzled-2 (FZD2), Frizzled-7 (FZD7), and Frizzled-8 (FZD8).

[0372]

Table 8A

Table 8B

Table 8C

[0373] I. Evaluation of the binding activity of the peptide complex to LRP5 3 pmol of avidin-labeled LRP5 (ACROBiosystems; LR5-H82E6) was added to each well of a 96-well plate (Thermo; 436014) coated with streptavidin, and allowed to stand for 30 minutes to immobilize LRP5 on the plate.

[0374] After washing three times with PBS-T, the peptide complex diluted to 10 nM was added, and allowed to stand for 1 hour.

[0375] After washing three times with PBS-T, Anti-HA-tag mAb-HRP-DirecT (MBL; M180-7) diluted 5000 times with PBS-T was added, and allowed to stand for 30 minutes.

[0376] After washing three times with PBS-T, SureBlue TMB single-component microporous peroxidase substrate (SeraCare; 5120-0077) was added for detection. After standing for 10 minutes, an equal volume of TMB stop solution (SeraCare; 5150-0021) was added to stop the reaction.

[0377] The absorbance at 450 nm was measured using an Infinite M Nano* (TECAN). The binding signal of each peptide complex was calculated by subtracting the absorbance of the pores without peptide complex from the absorbance of the pores with each peptide complex.

[0378] II. Evaluation of the binding activity of the peptide complex to LRP6 (Instructions for use, pages 102 / 110, 122 CN 122555715 A) 200 μg of avidin-labeled polyclonal anti-human IgG (eBioscience; 13-4998) was added to each well of a 96-well plate (Thermo; 436014) coated with streptavidin, and allowed to stand for 30 minutes.

[0379] After washing three times with PBS-T, 3 pmol of Fc-tagged fused LRP6 (R&D systems; 1505-LR) was added to each well, thereby immobilizing LRP6 on the plate.

[0380] After washing three times with PBS-T, the peptide complex diluted to 100 nM was added, and allowed to stand for 1 hour. Antibody reaction and absorbance detection were then performed using the same method as in "I. Evaluation of the binding activity of the peptide complex to LRP5".

[0381] The results are shown in Table 9. This indicates that the synthesized peptide complex has binding activity for LRP5 and LRP6.

[0382] [Table 9A] [Table 9B] Specification 103 / 110 pages 123 CN 122555715 A [Table 9C] Specification 104 / 110 pages 124 CN 122555715 A [Example 10] Evaluation of Wnt3a inhibitory activity In order to evaluate the Wnt3a inhibitory activity of the synthesized peptides (peptide sequence numbers 79, 81, 83, 85~96, 98~100), the transcriptional activity inhibition was evaluated by TCF / LEF reporter assay.

[0383] TCF / LEF Reporter-HEK 293 cells (BPS Bioscience) were cultured in EMEM (Fuji FILM) containing 10% FBS (Thermo Fisher Scientific), 50 μg / mL gentamicin (Nacalai tesque), and 400 μg / mL genimycin (Thermo Fisher Scientific).

[0384] After detaching the cells using a TrypLE (Thermo Fisher Scientific), they were resuspended in EMEM containing 10% FBS, 50 μg / mL gentamicin, and 10 mM LiCl (Sigma).

[0385] 35,000 cells were seeded per well in white 96-well plates (Thermo Fisher Scientific) for cell adhesion detection and cultured overnight. Instructions for Use, pages 105 / 110, CN 122555715 A

[0386] After adding the peptide, add 1 nM recombinant human R-spondin 1 (R&D systems) and 10 nM Wnt3a. Stimulate in a CO2 incubator at 37°C for 5 hours and 30 minutes.

[0387] Add ONE-Glo luciferase assay system (Peomega), shake for 4 minutes, and detect the luminescence signal using a SpectraMax Paradigm multimode microplate reader (Molecular Devices). Analyze the obtained signals using GraphPad Prism, setting the signal values ​​induced by Wnt3a and R-spondin to 0% inhibition, and the signal values ​​without added Wnt3a and R-spondin to 100% inhibition, and calculate the inhibition percentage of each peptide.

[0388] Experiments were conducted at peptide concentrations of 1 nM, 10 nM, and 100 nM.

[0389] Regarding the inhibition percentage, those with more than 50% inhibitory activity when 1 nM peptide was added were designated as 1-a, those with more than 50% inhibitory activity when 10 nM peptide was added were designated as 1-b, those with more than 50% inhibitory activity when 100 nM peptide was added were designated as 1-c, and those with more than 1% but less than 50% inhibitory activity when 100 nM peptide was added were designated as 1-d.

[0390] The results are shown in Table 10. This indicates that the synthesized peptides have Wnt3a inhibitory activity.

[0391] [Table 10] [Example 11] Evaluation of Wnt signal activation ability In order to evaluate the Wnt signal activation ability of the synthesized peptide complexes (dimer structures numbered 1~67), the transcriptional activity was evaluated using the TCF / LEF reporter assay.

[0392] TCF / LEF Reporter-HEK 293 cells (BPS) were used.The cells were cultured in EMEM (Fuji FILM) containing 10% FBS (Thermo Fisher Scientific, 106 / 110 pages, CN 122555715 A), 50 μg / mL gentamicin (Nacalai tesque), and 400 μg / mL genimycin (Thermo Fisher Scientific).

[0393] After peeling the cells using a TrypLE (Thermo Fisher Scientific), they were resuspended in EMEM containing 10% FBS, 50 μg / mL gentamicin, and 10 mM LiCl (Sigma).

[0394] The cells were seeded at a rate of 35,000 cells per well in white 96-well plates for cell adhesion detection (Thermo Fisher Scientific) and cultured overnight.

[0395] Subsequently, in the presence of 1 nM recombinant human R-spondin 1 (R&D systems), recombinant human Wnt-3a protein (R&D systems) or peptide complex was added, and the mixture was stimulated in a CO2 incubator at 37°C for 5 hours and 30 minutes.

[0396] An ONE-Glo luciferase assay system (Promega) was added, and the mixture was shaken for 4 minutes. The luminescence signal was detected using a SpectraMax Paradigm multimode microplate reader (Molecular Devices).

[0397] The obtained signals were analyzed using a GraphPad Prism. The maximum value of the signal induced by Wnt-3a was set to 100%, and no stimulation was set to 0%. The activity was calculated. Evaluation was performed at Wnt concentrations of 0.03–30 nM, and the 100% value was determined.

[0398] Regarding the numbering of each dimer structure, experiments were conducted at the peptide complex addition concentrations shown below.

[0399] a) For dimer structure number 1, the values ​​are 1 nM, 10 nM, 100 nM, and 1000 nM. b) For dimer structure numbers 2-4, the values ​​are 1 nM, 10 nM, and 100 nM. c) For dimer structure numbers 5-14, the values ​​are 0.1 nM, 1 nM, 10 nM, and 100 nM. d) For dimer structure numbers 15-43, the values ​​are 0.1 nM, 1 nM, and 10 nM. e) For dimer structure numbers 44-56, a common dilution of 1 / 4 starting from 30 nM is performed, resulting in 6 dilutions (0.03 nM, 0.12 nM, 0.47 nM, 1.9 nM, 7.5 nM, and 30 nM). f) For peptide sequence numbers 57-67, a common dilution of 1 / 5 starting from 30 nM is performed, resulting in 6 dilutions (0.0096, ... 0.048, 0.24, 1.2, 6.0, 30nM)Regarding the percentage of activity, those with more than 50% activity when 0.1 nM of peptide complex is added are designated as 2-A, those with more than 50% activity when 1 nM of peptide complex is added are designated as 2-B, those with more than 50% activity when 10 nM of peptide complex is added are designated as 2-C, those with more than 50% activity when 100 nM of peptide complex is added are designated as 2-D, and those with more than 1% but less than 50% activity when 100 nM of peptide complex is added are designated as 2-E.

[0400] Those exhibiting more than 50% activity when the peptide complex is added at 0.03 nM are designated as 3-A, those exhibiting more than 50% activity when the peptide complex is added at 0.12 nM are designated as 3-B, those exhibiting more than 50% activity when the peptide complex is added at 0.47 nM are designated as 3-C, those exhibiting more than 50% activity when the peptide complex is added at 1.9 nM are designated as 3-D, those exhibiting more than 50% activity when the peptide complex is added at 7.5 nM are designated as 3-E, those exhibiting more than 50% activity when the peptide complex is added at 30 nM are designated as 3-F, and those exhibiting more than 1% but less than 50% activity when the peptide complex is added at 30 nM are designated as 3-G.

[0401] Peptide complexes exhibiting ≥50% activity at 0.0096 nM were designated as 4-A; peptide complexes exhibiting ≥50% activity at 0.048 nM were designated as 4-B; peptide complexes exhibiting ≥50% activity at 0.24 nM were designated as 4-C; peptide complexes exhibiting ≥50% activity at 1.2 nM were designated as 4-D; peptide complexes exhibiting ≥50% activity at 6 nM were designated as 4-E; peptide complexes exhibiting ≥50% activity at 30 nM were designated as 4-F; and peptide complexes exhibiting ≥1% but <50% activity at 30 nM were designated as 4-G.

[0402] The results are shown in Table 11. This indicates that the synthesized peptide complexes, like the recombinant protein Wnt3a, possess Wnt signal activation capabilities.

[0403]

Table 11A

Table 11B

Table 11C

Claims

1. A peptide comprising the amino acid sequence of Formula A1 or an amino acid sequence comprising substitutions, deletions, additions, or insertions of one or more amino acid residues in the amino acid sequence of Formula A1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 in, X1 is any D-amino acid residue. X2 and X3 can each be any amino acid residue independently. X4 and X5 are each an amino acid residue with a substitutable aliphatic hydrocarbon group on its side chain. X6 is an amino acid residue with a substituted aryl group on its side chain. X7 is an N-alkyl amino acid residue with a substituted aryl or substituted cycloalkyl group on its side chain. X8 can be any amino acid residue. X9 is an L-threonine (T) residue. X10 is any N-alkylated amino acid residue. X11 can be any amino acid residue. X12 is an amino acid residue with an aliphatic hydrocarbon group on its side chain. X13 is an amino acid residue with a substitutable aliphatic hydrocarbon group on its side chain. X14 is an L-cysteine ​​(C) residue. In Formula A1, the amino acid sequence is recorded from the N-terminus to the C-terminus.

2. The peptide of claim 1, wherein, In equation A1, X1 is a D-phenylalanine (df) residue, (R)-2-amino-3-(pyridin-4-yl)propionic acid (d4py) residue, (R)-2-amino-3-(pyridin-3-yl)propionic acid (d3py) residue, (R)-2-amino-3-(3-methoxyphenyl)propionic acid (df3OMe) residue, (R)-2-amino-3-(4-methoxyphenyl)propionic acid (df4OMe) residue, (R)-2-amino-5-ureidovalerate (dcit) residue, D-tyrosine (dy) residue, or (R)-2-amino-3-(4-fluorophenyl)propionic acid (df4F) residue. X2 is an L-serine (S) residue, an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-2-aminoadipic acid (Hgl) residue, an L-citrulline (Cit) residue, or a (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue. X3 is an L-aspartic acid (D) residue, an L-arginine (R) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue, an L-asparagine (N) residue, or an L-serine (S) residue. X4 is an L-isoleucine (I) residue or an O-methyl-L-threonine (TMe) residue. X5 is an L-arginine (R) residue, an L-glutamic acid (E) residue, a (S)-2-aminoheptanoic acid (Ahp) residue, an L-serine (S) residue, an L-homoserine (Hse) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, a (S)-2-amino-4-ureidobutyric acid (Ncit) residue, or a (S)-5-acetamido-2-aminovaleric acid (OrnAc) residue. X6 is an L-tyrosine (Y) residue, a methyl-L-tyrosine (MeY) residue, a (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, a (S)-2-amino-3-(4-chlorophenyl)propionic acid (F4C) residue, a (S)-2-amino-3-(pyridin-4-yl)propionic acid (4Py) residue, or a (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue. X7 is a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue. X8 represents L-glutamine (Q) residue, L-valine (V) residue, L-2-aminoadipic acid (Hgl) residue, 1-(carboxymethyl)-L-tryptophan (W1aa) residue, (S)-2-aminoheptanoic acid (Ahp) residue, L-high tyrosine (Hty) residue, L-threonine (T) residue, allothreonine (alT) residue, L-citrulline (Cit) residue, and N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine. The residues include: (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (Gthp), (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa), (S)-2-amino-2-cyclobutylacetic acid (Cbg), (S)-2-amino-2-cyclohexylacetic acid (Chg), or O-methyl-threonine (TMe). X10 is a methyl-L-phenylalanine (MeF) residue, (S)-2-(methylamino)hexanoic acid (MeNle) residue, (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid (Me3Py) residue, (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid (MeAtp) residue. X11 is an L-isoleucine (I) residue, an L-tyrosine (Y) residue, (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (F4aao) residue, or an O-methyl-L-threonine (TMe) residue. X12 is an L-valine (V) residue, a methyl-L-valine (MeV) residue, or an N-methyl-L-cyclohexylglycine (MeChg) residue. X13 is a glycine (G) residue, a D-alanine (da) residue, or a D-serine (ds) residue.

3. A peptide comprising an amino acid sequence of Formula A2, or an amino acid sequence comprising at least one amino acid residue selected from the group consisting of amino acid residues at positions 1 to 12 of the amino acid sequence of Formula A2, wherein at least one amino acid residue has been substituted, deleted, added, or inserted. A2: df-SDIRY-MeF-QT-MeF-IVGC (Serial Number 164) in, df represents a D-phenylalanine residue. S is an L-serine residue. D stands for L-aspartic acid residue. I represents an L-isoleucine residue. R is an L-arginine residue. Y represents an L-tyrosine residue. MeF is a methyl-L-phenylalanine residue. Q is an L-glutamine residue. T stands for an L-threonine residue. V represents an L-valine residue. G stands for a glycine residue. C stands for L-cysteine ​​residue.

4. The peptide according to any one of claims 1 to 3, wherein, The C-terminus also contains a glycine residue.

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

6. The peptide according to claim 4, wherein it is a cyclic peptide.

7. The peptide according to any one of claims 1 to 3, having a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide.

8. The peptide according to any one of claims 1 to 3, further comprising added amino acid residues.

9. The peptide according to any one of claims 1 to 3, wherein it has binding affinity to the Fzd receptor.

10. The peptide according to claim 9, wherein it has binding capacity to at least one selected from the group consisting of Fzd receptor 2, Fzd receptor 7 and Fzd receptor 8.

11. The peptide according to any one of claims 1 to 3, wherein it comprises the amino acid sequence shown in formula A3. A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 wherein, ClAc represents chloroacetyl, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are the amino acid residues shown in Table 1. In Table 1, df represents a D-phenylalanine residue. d4py is an (R)-2-amino-3-(pyridin-4-yl)propionic acid residue. d3py is an (R)-2-amino-3-(pyridin-3-yl)propionic acid residue. df3OMe represents (R)-2-amino-3-(3-methoxyphenyl)propionic acid residues. df4OMe represents (R)-2-amino-3-(4-methoxyphenyl)propionic acid residues. dcit is a residue of (R)-2-amino-5-ureidovalerate. dy represents a D-tyrosine residue. df4F is a (R)-2-amino-3-(4-fluorophenyl)propionic acid residue. S is an L-serine residue. N represents an L-asparagine residue. D stands for L-aspartic acid residue. Hgl is an L-2-aminohexanoic acid residue. Cit is an L-citrulline residue. Kmor is a (S)-2-amino-6-morpholinohexanoic acid residue. R is an L-arginine residue. KCOpipzaa is an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue. Atp is a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid residue. I represents an L-isoleucine residue. TMe is an O-methyl-L-threonine residue. E represents an L-glutamic acid residue. Ahp is a (S)-2-aminoheptanoic acid residue. Hse is an L-homoserine residue. Hcit is an N6-carbamoyl-L-lysine residue. Ncit is a (S)-2-amino-4-ureobutyric acid residue. OrnAc is a (S)-5-acetamido-2-aminovaleric acid residue. Y represents an L-tyrosine residue. MeY is a methyl-L-tyrosine residue. F4F is a (S)-2-amino-3-(4-fluorophenyl)propionic acid residue. F4C is a (S)-2-amino-3-(4-chlorophenyl)propionic acid residue. 4Py is a (S)-2-amino-3-(pyridin-4-yl)propionic acid residue. F3COO is a (S)-3-(2-amino-2-carboxyethyl)benzoic acid residue. MeF is a methyl-L-phenylalanine residue. Me3Py is a (S)-2-(methylamino)-3-(pyridin-3-yl)propionic acid residue. Me4Py is a (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid residue. MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue. MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue. MeAtp is a (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propionic acid residue. Q is an L-glutamine residue. V represents an L-valine residue. W1aa is a 1-(carboxymethyl)-L-tryptophan residue. Hty is an L-high tyrosine residue. T stands for an L-threonine residue. alT is an allothreonine residue. Gthp is a (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid residue. G4pipaa is a (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid residue. Cbg is a (S)-2-amino-2-cyclobutylacetic acid residue. Chg represents (S)-2-amino-2-cyclohexylacetic acid residues. MeNle is a (S)-2-(methylamino)hexanoic acid residue. F4aao is a (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid residue. MeV is a methyl-L-valine residue. MeChg is an N-methyl-L-cyclohexylglycine residue. G stands for a glycine residue. C represents an L-cysteine ​​residue. Table 1A Table 1B 【Table 1C】 【Table 1D】 。 12. A peptide comprising the amino acid sequence of Formula B1 or an amino acid sequence in which one or more amino acid residues are substituted, deleted, added, or inserted in the amino acid sequence of Formula B1. B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 in, Y1 is a substituted L-phenylalanine (F) residue. Y2 is a substituted L-arginine (R) residue. Y3 is a substituted L-tryptophan (W) residue or a substituted (S)-2-amino-3-(naphth-1-yl)propionic acid (Nal1) residue. Y4 is an acidic amino acid residue or a neutral amino acid residue. Y5 is a substituted aromatic amino acid residue, aliphatic amino acid residue, or L-proline (P) residue. Y6 is a replaceable aliphatic amino acid residue. Y7 is an N-alkylated aromatic amino acid residue. Y8 is an amino acid residue derived from an amino acid with a hydroxyl, carboxyl, or amide group on its side chain. Y9 can be any amino acid residue. Y10 is a substituted L-tryptophan (W) residue. Y11 is a substituted L-asparagine (N) residue or glutamine (Q) residue. Y12 is a substituted (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue. Y13 is a substituted neutral or acidic amino acid residue. Y14 is an L-cysteine ​​(C) residue. In Formula B1, the amino acid sequence is recorded from the N-terminus to the C-terminus.

13. The peptide of claim 12, wherein, In equation B1, Y1 is an L-phenylalanine (F) residue, (S)-2-amino-3-(4-fluorophenyl)propionic acid (F4F) residue, (S)-2-amino-3-(p-tolyl)propionic acid (F4Me) residue, or (S)-2-amino-3-(m-tolyl)propionic acid (F3Me) residue. Y2 is an L-arginine (R) residue or an N6-carbamoyl-L-lysine (Hcit) residue. Y3 represents an L-tryptophan (W) residue, a methyl-L-tryptophan (MeW) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propionic acid (W5N) residue, or (S)-2-amino-3-(1-methyl-1H-pyrrolo)propionic acid (W7N) residue. [2,3-b]pyridin-3-yl)propionic acid (W1Me7N) residue, (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N5Me) residue, (S)-2-amino-3-(naphth-1-yl)propionic acid (Nal1) residue, or (S)-2-amino-3-(quinolin-4-yl)propionic acid (Nal14N) residue, Y4 is an L-aspartic acid (D) residue, an L-glutamic acid (E) residue, an L-asparagine (N) residue, or an L-glutamine (Q) residue. Y5 can be an L-valine (V) residue, a methyl-L-valine (MeV) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, a (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, a (S)-2-amino-3-(naphth-1-yl)propionic acid (Nal1) residue, a (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid (3Py5Ph) residue, a (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, or an L-proline (P) residue. Y6 is an L-arginine (R) residue, a methyl-L-arginine (MeR) residue, a (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, a (S)-2-amino-4-ureidobutyric acid (Ncit) residue, or a (S)-5-acetamido-2-aminovaleric acid (OrnAc) residue. Y7 is a methyl-L-phenylalanine (MeF) residue, (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, methyl-L-tyrosine (MeY) residue, (S)-2-(methylamino)-3-(p-tolyl)propionic acid (MeF4Me) residue, (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid (MeF4C) residue, (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, (S)-2-(methylamino)-3-(m-tolyl)propionic acid (MeF3Me) residue, or (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid (MeF3C) residue. Y8 is an L-serine (S) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an L-homotyrosine (Hty) residue, an allothreonine (alT) residue, an L-threonine (T) residue, an L-asparagine (N) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue, or an L-glutamate (E) residue. Y9 is an L-asparagine (N) residue, methyl-L-asparagine (MeN) residue, L-2-aminoadipic acid (Hgl) residue, (S)-2-amino-3-cyclohexylpropionic acid (Cha) residue, L-high tyrosine (Hty) residue, (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid (Hw7N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, L-glutamine (Q) residue, L-aspartic acid (D) residue, or L-glutamic acid (E) residue. Y10 is an L-tryptophan (W) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue, (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (W6N) residue, (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W1Me7N) residue, or (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N5Me) residue. Y11 is an L-asparagine (N) residue, an L-glutamine (Q) residue, or a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue. Y12 is a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C) residue, (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) residue, or (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C) residue. Y13 is an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-glutamine (Q) residue, a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid (Nmm) residue, or an N4,N4-dimethyl-L-asparagine (Ndm) residue.

14. A peptide comprising the amino acid sequence of Formula B2, or an amino acid sequence comprising at least one amino acid residue selected from the group consisting of amino acid residues at positions 1 to 13 of the amino acid sequence of Formula B2, wherein at least one amino acid residue has been substituted, added, deleted, or inserted. B2: FRWDVR-MeF-SNWN-Tic-NC (Serial Number 165) in, F represents an L-phenylalanine residue. R is an L-arginine residue. W represents an L-tryptophan residue. D stands for L-aspartic acid residue. V represents an L-valine residue. MeF is a methyl-L-phenylalanine residue. S is an L-serine residue. Tic is a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue. N represents an L-asparagine residue. C stands for L-cysteine ​​residue.

15. The peptide according to any one of claims 12 to 14, wherein, The C-terminus also contains a glycine residue.

16. The peptide according to any one of claims 12 to 14, wherein it is a cyclic peptide.

17. The peptide according to claim 15, wherein it is a cyclic peptide.

18. The peptide according to any one of claims 12 to 14, having a cyclic structure formed by the combination of an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide.

19. The peptide according to any one of claims 12 to 14, further comprising added amino acid residues.

20. The peptide according to any one of claims 12 to 14, wherein it has binding affinity to LRP.

21. The peptide of claim 20, wherein it has binding capacity to at least one selected from the group consisting of LRP5 and LRP6.

22. The peptide according to any one of claims 12 to 14, having DKK1-like activity.

23. The peptide according to any one of claims 12-14, comprising the amino acid sequence shown in formula B3. B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 in, ClAc represents chloroacetyl, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, and Y14 are the amino acid residues shown in Table 2. In Table 2, F represents an L-phenylalanine residue. F4F is a (S)-2-amino-3-(4-fluorophenyl)propionic acid residue. F4Me is a (S)-2-amino-3-(p-tolyl)propionic acid residue. F3Me is a (S)-2-amino-3-(m-tolyl)propionic acid residue. R is an L-arginine residue. Hcit is an N6-carbamoyl-L-lysine residue. W represents an L-tryptophan residue. MeW stands for methyl-L-tryptophan residue. W7N is a (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue. W5N is a (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propionic acid residue. W1Me7N is a (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue. W7N5Me is a (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid residue. Nal1 is a (S)-2-amino-3-(naphth-1-yl)propionic acid residue. Nal14N is a (S)-2-amino-3-(quinolin-4-yl)propionic acid residue. D stands for L-aspartic acid residue. E represents an L-glutamic acid residue. N represents an L-asparagine residue. Q is an L-glutamine residue. V represents an L-valine residue. MeV is a methyl-L-valine residue. W1aa is a 1-(carboxymethyl)-L-tryptophan residue. Cha is a (S)-2-amino-3-cyclohexylpropionic acid residue. 3Py5Ph represents (S)-2-amino-3-(5-phenylpyridin-3-yl)propionic acid residues. Cbg is a (S)-2-amino-2-cyclobutylacetic acid residue. P is an L-proline residue. MeR is a methyl-L-arginine residue. Kmor is a (S)-2-amino-6-morpholinohexanoic acid residue. Cit is an L-citrulline residue. Ncit is a (S)-2-amino-4-ureobutyric acid residue. OrnAc is a (S)-5-acetamido-2-aminovaleric acid residue. MeF is a methyl-L-phenylalanine residue. MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue. MeY is a methyl-L-tyrosine residue. MeF4Me is a (S)-2-(methylamino)-3-(p-tolyl)propionic acid residue. MeF4C is a (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid residue. MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue. MeF3Me is a (S)-2-(methylamino)-3-(m-tolyl)propionic acid residue. MeF3C is a (S)-3-(3-chlorophenyl)-2-(methylamino)propionic acid residue. S is an L-serine residue. Hty is an L-high tyrosine residue. alT is an allothreonine residue. T stands for an L-threonine residue. MeN is a methyl-L-asparagine residue. Hgl is an L-2-aminohexanoic acid residue. Hw7N is a (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butyric acid residue. KCOpipzaa is an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue. W6N is a (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid residue. Nmm represents a (2S)-2-amino-4-(methylamino)-4-oxobutyric acid residue. Tic is a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue. Tic6C is a (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue. Tic7H is a (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue. Tic7C is a (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue. Ndm represents N4,N4-dimethyl-L-asparagine residues. C is an L-cysteine ​​residue. G stands for a glycine residue. Table 2A Table 2B Table 2C 【Table 2D】 Table 2E 。 24. A peptide complex comprising a first peptide and a second peptide, wherein... The first peptide is the peptide described in claim 1. The second peptide is the peptide described in claim 12.

25. The peptide complex according to claim 24, wherein, The peptide complex consists of the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

26. The peptide complex according to claim 25, wherein, The C-terminus of the first peptide is connected to the C-terminus of the second peptide via the connector.

27. The peptide complex according to claim 25, wherein, The connector is selected from the connectors listed in Table 3. In Table 3, -click- indicates a bond formed through click chemistry. Table 3 。 28. The peptide complex according to claim 25, wherein, The first peptide is a peptide composed of the amino acid sequence shown in Formula A1. The second peptide is a peptide composed of the amino acid sequence shown in Formula B1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 in, In formula A1, X1 is a D-phenylalanine (df) residue. X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue. X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue, or a (2S)-2-amino-3-(oxacyclohexane-4-yl)propionic acid (Atp) residue. X4 is an L-isoleucine (I) residue. X5 is an L-arginine (R) residue or an L-citrulline (Cit) residue. X6 is an L-tyrosine (Y) residue. X7 is a methyl-L-phenylalanine (MeF) residue or (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue. X8 is an L-glutamine (Q) residue, an L-valine (V) residue, an L-homotyrosine (Hty) residue, an L-threonine (T) residue, an L-citrulline (Cit) residue, or an O-methyl-L-threonine (TMe) residue. X10 is a methyl-L-phenylalanine (MeF) residue or a (S)-2-(methylamino)-3-(pyridin-4-yl)propionic acid (Me4Py) residue. X11 is an L-isoleucine (I) residue or an L-tyrosine (Y) residue. X12 is an L-valine (V) residue or a methyl-L-valine (MeV) residue. X13 is a glycine (G) residue or a D-serine (ds) residue. In equation B1, Y1 is an L-phenylalanine (F) residue. Y2 is an L-arginine (R) residue. Y3 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue. Y4 is an L-aspartic acid (D) residue. Y5 is an L-valine (V) residue. Y6 is an L-arginine (R) residue or a (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue. Y7 is a methyl-L-phenylalanine (MeF) residue or a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue. Y8 is an L-serine (S) residue or an L-glutamic acid (E) residue. Y9 is an L-asparagine (N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, or L-glutamic acid (E) residue. Y10 is an L-tryptophan (W) residue or (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (W7N) residue. Y11 is an L-asparagine (N) residue. Y12 is a (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue. Y13 is an L-asparagine (N) residue or an L-aspartic acid (D) residue.

29. The peptide complex according to claim 25, which can activate the Wnt signal.

30. A pharmaceutical composition comprising the peptide according to any one of claims 1-3 and 12-14.

31. A pharmaceutical composition comprising the peptide complex of claim 25.

32. A cell culture composition for use in cell culture, comprising the peptide complex of claim 25.

33. A composition for medical, diagnostic or research use, comprising the peptide complex of claim 25.

34. A composition for medical, diagnostic or research use, comprising any one of peptides 1 to 3 and 12 to 14.