BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR EphA2

Abstract

To provide a polypeptide covalently bound to a non-aromatic molecular scaffold such that two or more peptide loops are embedded between attachment points to the scaffold.SOLUTION: The present invention provides a peptide that is a high-affinity binder of Eph receptor tyrosine kinase A2 (EphA2). The present invention also provides a pharmaceutical composition comprising the peptide ligand and the use of the peptide ligand in preventing, suppressing, or treating a disease or disorder characterized by overexpression of EphA2 in an affected tissue (for example, a tumor).SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides a method for preparing a peptide having two or more peptide loops interposed between attachment points to a scaffold. The present invention relates to polypeptides covalently attached to non-aromatic molecular scaffolds, In particular, the present invention provides a peptide that is a high affinity binder of Eph receptor tyrosine kinase A2 (EphA2). The present invention also describes pharmaceutical compositions and diseased tissues containing the peptide ligands. and the prevention, suppression, or administration of EphA2-specific antibodies to a disease or disorder characterized by overexpression of EphA2 in tissues (e.g., tumors). relates to the use of said peptide ligands in therapy. [Background technology]

[0002] BACKGROUND OF THE INVENTION Cyclic peptides can bind to protein targets with high affinity and target specificity, Therefore, they are an attractive class of molecules for therapeutic development. The antimicrobial peptides include, for example, vancomycin, the immunosuppressant cyclosporine, or the anti-inflammatory drug Like the cancer drug octreotide, it is already being used successfully in the clinic (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24. The excellent binding properties are due to the The conformational flexibility of the ring structure as well as the relatively large interaction surface formed between the target and the ring Generally, macrocycles are used in combination with the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 (Wu et al. (2007), Science 330, 1066-71), Arg-Gly binding to integrin αVb3 -Asp motif cyclic peptide (355 Å 2)(Xiong et al. (2002), Science 296(5565), 151-5), or cyclic peptide inhibitors that bind to urokinase-type plasminogen activator Upain-1 (603Å 2 (2007), J Struct Biol 160(1), 1-10), hundreds of It bonds to a surface of square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides. This results in a lower entropy loss when binding to the target, resulting in higher binding The reduced flexibility also leads to the fixation of target-specific conformations, and the linear peptide This effect is due to the fact that when the ring is opened, it binds to other MM peptides. Potent and selective matrix metalloproteinase 8 (MMP-8) loses its selectivity for P This is exemplified by specific inhibitors (Cherney et al. (1998), J Med Chem 41(11), 1749- 51) The advantageous binding properties achieved by macrocyclization have been demonstrated in, for example, vancomycin, nicotinamide, and nicotinamide. In polycyclic peptides with multiple peptide rings, such as actinomycin and actinomycin, Even more pronounced.

[0004] Various research teams have previously synthesized polypeptides containing cysteine residues into synthetic molecular structures. (Kemp and McNamara, 1985, J. Org. Chem; Timmerman et al., 2005, Meloen and coworkers reported that tris(bromomethyl)benzene and related molecules of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces It was used for rapid and quantitative cyclization (Timmerman et al., 2005, ChemBioChem). a compound (wherein the compound reacts with a cysteine-containing polypeptide by, for example, tris(bromomethyl) (i) by linking to a molecular scaffold such as benzene) The method is disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] Generating and screening large libraries of bicyclic peptides against targets of interest A phage display-based combinatorial approach has been developed to (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). This results in a linear peptide containing three cysteine residues and two random six-amino acid regions. A combinatorial library of peptides (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage. The cysteine side chain was covalently attached to a small molecule (tris-(bromomethyl)benzene) It was further cyclized. Summary of the Invention

[0006] (Summary of the Invention) According to a first aspect of the present invention, at least two loop sequences separated by at least two loop sequences are provided. A polypeptide containing three reactive groups and a covalent bond formed with the reactive groups of the polypeptide. , a molecule that is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one scaffold, such that at least two polypeptide loops are attached to the molecular scaffold. The peptide ligand is formed on the [ka] TIFF2025122030000002.tif245170TIFF2025122030000003.tif247170TIFF2025122030000004.tif205170 (where Ac stands for acetyl, HyP stands for hydroxyproline, and HArg stands for homoarginine) PYA stands for 4-pentynoic acid, 3,3-DPA stands for 3,3-diphenylalanine, and Cba stands for β- 1Nal stands for 1-naphthylalanine, and NMeAla stands for N-methyl-alanine. His1Me stands for N1-methyl-L-histidine, His3Me stands for N3-methyl-L-histidine, 4ThiAz represents β-(4-thiazolyl)-alanine, and Thi represents 2-thienyl-alanine. 3Thi represents 3-thienylalanine, and palmitoyl-Glu-LysN3 represents N2-((S)-4-carboxy -4-Palmitamidobutanoyl)-N6-diazo-L-lysine: [ka] pCoPhe represents para-carboxy-phenylalanine, and hGlu represents homoglutamic acid. B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units, and Nle represents norleu Represents Shin and [MerPro] i , C i , C ii , C iii , and [Cysam] iii is cysteine, 3-mercapto the first (i) and second (ii) selected from propanoic acid (MerPro) and cysteamine (Cysam); and a third (iii) reactive group. or a pharmaceutically acceptable salt thereof. A peptide ligand is provided.

[0007] According to a further aspect of the present invention, the peptide ligand as defined herein is administered to one or more pharmaceutical agents. Pharmaceutical compositions containing the compounds in combination with pharmaceutically acceptable excipients are provided.

[0008] According to a further aspect of the present invention there is provided a method for preventing, suppressing or treating cancer. Peptide ligands as defined herein are provided. DETAILED DESCRIPTION OF THE INVENTION

[0009] (Detailed Description of the Invention) According to a first aspect of the present invention, at least two loop sequences separated by at least two loop sequences are provided. A polypeptide containing three reactive groups and a covalent bond formed with the reactive groups of the polypeptide. , a molecule that is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one scaffold, such that at least two polypeptide loops are attached to the molecular scaffold. The peptide ligand is formed on the [ka] TIFF2025122030000007.tif245170TIFF2025122030000008.tif248170TIFF2025122030000009.tif56170 (where Ac stands for acetyl, HyP stands for hydroxyproline, and HArg stands for homoarginine) PYA stands for 4-pentynoic acid, 3,3-DPA stands for 3,3-diphenylalanine, and Cba stands for β- 1Nal stands for 1-naphthylalanine, and NMeAla stands for N-methyl-alanine. His1Me stands for N1-methyl-L-histidine, His3Me stands for N3-methyl-L-histidine, 4ThiAz represents β-(4-thiazolyl)-alanine, and Thi represents 2-thienyl-alanine. 3Thi represents 3-thienylalanine, and palmitoyl-Glu-LysN3 represents N2-((S)-4-carboxy -4-Palmitamidobutanoyl)-N6-diazo-L-lysine: [ka] pCoPhe represents para-carboxy-phenylalanine, and hGlu represents homoglutamic acid. B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units, and Nle represents norleu Represents Shin and [MerPro] i , C i , C ii , C iii , and [Cysam] iii is cysteine, 3-mercapto the first (i) and second (ii) selected from propanoic acid (MerPro) and cysteamine (Cysam); and a third (iii) reactive group. or a pharmaceutically acceptable salt thereof. A peptide ligand is provided.

[0010] In one particular embodiment, the EphA2-binding bicyclic peptide ligand is BCY13118, BCY1 2860, BCY12859, BCY13119, BCY13917, BCY13918, BCY13919, BCY13920, BCY13922, BCY1 3923, BCY14047, BCY14048, BCY13135, BCY12865, BCY13120, and BCY13117. can be.

[0011] In one particular embodiment, the EphA2-binding bicyclic peptide ligand is BCY13118 or and pharmaceutically acceptable salts thereof.

[0012] Unless otherwise defined, all technical and scientific terms used herein are understood to be within the skill of the art. , e.g., peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. have the same meaning as generally understood by practitioners in the field of science and technology. are used in methods of molecular biology, genetics, and biochemistry (incorporated herein by reference). Included in the article by Sambrook et al., Molecular Cloning: A Laboratory Manual A Laboratory Manual), 3rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Sp Ring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology in Molecular Biology (1999) 4th ed., John Wiley & Sons).

[0013] (Nomenclature) (Numbering) When referring to amino acid residue positions within the compounds of the invention, a cysteine residue (C i , C ii , and C iii ) are invariant, they are omitted from the numbering and therefore the amino acids in SEQ ID NO: 1 Residue numbering is referred to as follows: A-[HArg]-DC i -[HyP]1-L2-V3-N4-P5-L6-C ii -L7-H8-P9-[dD] 10 -W 11 -[HArg]12 -C iii (array Number: 1).

[0014] For purposes of this description, all bicyclic peptides are designated as 1,1',1''-(1,3,5-triazinane-1,3,5- It is thought that the cyclization with TATA (triyl) triprop-2-en-1-one gives rise to a trisubstituted structure. TATA-mediated cyclization is i , C ii , and C iii occurs above.

[0015] (Molecular format) N- or C-terminal extensions to the bicyclic core sequence may be added to the left or right side of the sequence, separated by a hyphen. It is added to the right side. For example, an N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO: X) It is expressed as:

[0016] (reverse peptide sequence) In view of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, The peptide sequences disclosed in also have utility in their retro-inverso forms. For example, if the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and The stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids). (These amino acids become L-amino acids, and L-amino acids become D-amino acids.) or a reference to an amino acid either as the one-letter or three-letter code for that amino acid Unless otherwise specified, amino acids are intended to be represented herein as L-amino acids. When such an amino acid is intended to be represented as a D-amino acid, the amino acid , for example, [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc., are prefixed with a lowercase d in square brackets. will be done.

[0017] (Advantages of peptide ligands) Certain bicyclic peptides of the present invention can be administered by injection, inhalation, nasal, ocular, oral, or topical administration. They have several advantageous properties that make them suitable drug-like molecules for administration. Such advantageous properties include: -Species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation ; -Protease stability. Bicyclic peptide ligands are stable against plasma proteases under most circumstances. proteases, epithelial ("membrane-anchored") proteases, gastrointestinal proteases, lung surface proteases, intracellular Stability against proteases etc. should be demonstrated. Protease stability is Not only can we develop our lead candidates in animal models, but we can also confidently administer them to humans. should be maintained among different species so that they can; - Desirable solubility profile. This is important for formulation and absorption purposes, as it contains charged residues and and the ratio of hydrophilic to hydrophobic residues as well as intra- / inter-molecular H-bonds; - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be used in chronic disease states or Bicyclic steroids with short or long in vivo exposure times for the management of either acute disease states It may be necessary to develop a peptide with a specific effect. The optimal exposure time results in sustained exposure of the drug. Compared to the requirement for short exposure times to minimize toxicological effects (for maximum therapeutic efficacy) Determined by the requirement for sustained exposure; -Selectivity. Certain peptide ligands of the present invention may bind to other Eph receptor tyrosine kinases, e.g. For example, EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, and EphB1, as well as factor XIIA, calcitonin, and cytochrome P450. The selected compounds of the present invention exhibit superior selectivity to those of nicotinic anhydrase 9 and CD38. Peptide ligands may be used in other species (e.g., mice and rats) that allow for testing in animal models. It should also be noted that cross-reactivity with -Safety. Bleeding events were assessed in a preclinical in vivo model using EphA2 antibody-drug conjugates. For example, a Phase 1 open-label study using MEDI-547 included six patients. The study was discontinued due to bleeding and clotting events in five of the patients (Annunziata et al., In Vest New Drugs (2013) 31:77-84). The bleeding events observed in patients were similar to those observed in rats and monkeys. Effects on the coagulation system observed in preclinical studies: Increased activated partial thromboplastin time and fibrinogen / fibrin degradation products (Annunziata et al., Ibid.) Overt bleeding events were reported in toxicology studies in monkeys (An (Nunziata et al., supra). Collectively, these results demonstrate that MEDI-547 is effective in treating rheumatoid arthritis in preclinical and patient populations. Both are associated with disseminated intravascular coagulation (DIC).

[0018] (peptide ligand) The peptide ligands referred to herein are those that are covalently attached to a molecular scaffold. Typically, such peptides are covalently bonded to a scaffold. and two or more reactive groups (i.e., cysteine residues) that can form a symmetric peptide. The sequence between the reactive groups is called a loop sequence because it forms a loop when it binds to the backbone. In this case, the peptide comprises a sequence inherent in and / or cysteamine, and a scaffold comprising at least three reactive groups selected from the group consisting of a hydroxypropyl methylcellulose, ... Form at least two loops on the fold.

[0019] (Pharmaceutically acceptable salts) Salt forms are within the scope of the present invention and reference to a peptide ligand includes the salt form of that ligand. It will be understood that

[0020] The salts of the present invention can be prepared by conventional chemical methods, e.g., Pharmaceutical Salts: Properties, Selection, and Uses ical Salts: Properties, Selection, and Use), P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002 They can be synthesized from a parent compound containing a basic or acidic moiety by the following methods: Typically, such salts are prepared by converting the free acid or base forms of these compounds into salts by the addition of a suitable base or acid to a water solution. They can be prepared by reacting them in aqueous or organic solvents, or in a mixture of the two. This can be done.

[0021] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, and the like. ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoin Acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphorsulfonic acid, (+) -(1S)-Camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, Enoic acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptan D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid, α-oxoglutaric acid, glycolic acid, hippuric acid, halogenated water hydrobromic acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L- Lactic acid, (±)-DL-lactic acid, lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malo (±)-DL-Mandelic acid, Methanesulfonic acid, Naphthalene-2-sulfonic acid, Naphthalene- 1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, Acetic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglycerin glutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, Sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenate and valeric acid, and acylated amino acids and cation exchange resins. Examples of the salt include mono- and di-salts formed with the acid.

[0022] One particular group of salts is acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, Lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid , toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphtha Sulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactate It comprises salts formed from biotic acid. One particular salt is the hydrochloride salt. Another particular salt is , acetate.

[0023] The compound is anionic or has a functional group that can be anionic (e.g., —CO OH is -COO - In the case where the salt is formed with an organic or inorganic base, a suitable cation is formed. Examples of suitable inorganic cations include Li + , Na + , and K + Alkali such as Metal ions, Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Others Examples of suitable organic cations include, but are not limited to, the cations: Ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R 2 + , NHR3 + , NR4 + Some suitable substituted ammonium salts include, but are not limited to: Examples of nium ions include methylamine, ethylamine, diethylamine, and propylamine. amine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, Ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzene amine, choline, meglumine, and tromethamine, as well as lysine and arginine Examples of common quaternary ammonium ions include those derived from amino acids. H3)4 + is.

[0024] When the compounds of the invention contain amine functions, they can be prepared, for example, by methods well known to those skilled in the art. By reaction with an alkylating agent according to the formula: Quaternary ammonium compounds are within the scope of the present invention.

[0025] (reactive group) The molecular scaffolds of the present invention can be attached to polypeptides via functional or reactive groups on the polypeptide. These may be attached to peptides, which are typically found in polypeptide polymers. Such reactive groups are formed from the side chains of certain amino acids. side chain, or N-terminal amino group, or any other suitable reactive group, e.g., penicillamine Details of suitable reactive groups can be found in WO 2009 / 098450.

[0026] Examples of reactive groups in natural amino acids are the thiol group of cysteine, the amino group of lysine, and the amino group of asparagine. Carboxyl group of guanine or glutamic acid, guanidinium group of arginine, tyrosine The phenolic group of α-amino acids is the phenolic group of α-amino acids, and the hydroxyl group of serine. -Provides a wide range of reactive groups including carbonyl, alkyne, vinyl, or aryl halide groups The amino and carboxyl groups at the ends of the polypeptide can also be used as molecular scaffolds. It can serve as a reactive group to form a covalent bond with the bond / molecular core.

[0027] The polypeptides of the present invention contain at least three reactive groups. The more reactive groups used, the more Loops can be formed in the molecular scaffold.

[0028] In a preferred embodiment, a polypeptide is produced that has three reactive groups. Reaction of peptides with molecular scaffolds / cores with three-fold symmetry results in the formation of single The production of a single product isomer is preferred for several reasons. The nucleic acids of the compound library encode only the primary sequence of the polypeptide, but It does not code for the isomeric state of the molecule formed upon reaction of the dode with the molecular core. Where isomers can be formed, the assignment of nucleic acids to product isomers is unambiguously defined. If multiple product isomers are formed, the nucleic acid may be used in a screening or selection process. No information can be given about the properties of the isolated product isomers. Single product isomers This information is also advantageous when specific members of the libraries of the invention are synthesized. In the case of a mixture of isomers, the chemical reaction between the polypeptide and the molecular scaffold results in Instead, a single product isomer is produced.

[0029] In another embodiment, a polypeptide having four reactive groups is produced. The reaction of tides with molecular scaffolds / molecular cores with tetrahedral symmetry gives two products Isomers are produced when two different product isomers are encoded by the same nucleic acid. However, it is possible to chemically synthesize both isomers, separate the two isomers, and then target both isomers with the target ligand. The properties of the isolated isomers can be determined by testing for binding to the do.

[0030] In one embodiment of the invention, at least one of the reactive groups of the polypeptide is The use of orthogonal reactive groups allows the orthogonal reactive groups to be attached to specific portions of the molecular core. Using a linking strategy involving orthogonal reactive groups, the resulting product can be directed to the desired position. In other words, the number of product isomers can be limited by at least three bonds. Attach at least three reactive groups that are separate or different from the reactive groups selected for the remaining ones. By selecting for one or more of the following, specific positions on the molecular scaffold can be obtained. This effectively achieves a specific order of attachment or orientation of specific reactive groups on a polypeptide to the desired position. This can be done.

[0031] In another embodiment, the reactive group of the polypeptide of the invention reacts with a molecular linker, thereby In this case, the linker is a molecular scaffold and a polypeptide in the final conjugated state. The peptides can react with the molecular scaffold to intercalate.

[0032] In some embodiments, the identity of the members of a library or set of polypeptides is The amino acids can be replaced with any natural or unnatural amino acid. Only the loop sequence is replaced. functional groups for cross-linking the polypeptide to the molecular core so that it is exchangeable; These replaceable amino acids are excluded. The replaceable polypeptide sequences are random. Either a regular sequence, a regular sequence, or a sequence with random and regular amino acids The position of these amino acids determines the loop size, so the amino acids with reactive groups Each of the amino acids is at a defined position within the polypeptide.

[0033] In one embodiment, the polypeptide having three reactive groups has the sequence (X) l Y(X) m Y(X) n Y(X) o where Y represents an amino acid having a reactive group and X represents a random amino acid. , m and n are the intervening polypeptide segments (which may be the same or different) represents a number of 3 to 6 that defines the length of the adjacent polypeptide segments; Represents a number between 0 and 20 that specifies the length.

[0034] An alternative to thiol-mediated conjugation is via covalent interactions. Alternatively, these techniques can be used to attach molecular scaffolds to peptides. The present invention provides a method for preparing a small molecule of interest that is different from the molecular scaffold and further comprises: Thus, after selection or isolation, modification or attachment of said further moiety to the polypeptide may be performed. can be used - in this embodiment, obviously, the bond is covalent These methods involve the use of complementary reactive groups. Proteins with unnatural amino acids having the required chemically reactive groups in combination with small molecules and by producing peptide-displaying phage or by selecting molecules after a selection / isolation step. When produced, unnatural amino acids are incorporated into chemically or recombinantly synthesized polynucleotides. Incorporation into peptides can be used instead of (or in combination with) thiol-mediated methods. Further details can be found in WO 2009 / 098450 or Heinis et al., Nat Ch em Biol 2009, 5(7), 502-7.

[0035] In one embodiment, the reactive group is cysteine, 3-mercaptopropionic acid, and / or cysteamine residues.

[0036] (Modified derivative) Modified derivatives of the peptide ligands defined herein are considered to be within the scope of the present invention. It will be understood that examples of such suitable modified derivatives include N-terminal and / or C-terminal End modification; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., one or more Substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of the amino acid residue with another non-natural isosteric or isoelectronic amino acid; addition of a spacer group; Substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more L-amino acid residues by one or more D-amino acid residues; substitution; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; Replacement of a bond with a surrogate bond; modification of peptide backbone length; on the α-carbon of one or more amino acid residues substitution of hydrogen with another chemical group, cysteine, lysine, glutamic acid / aspartic acid, and suitable amines of the amino acids, such as tyrosine, to functionalize the amino acids, thiol, etc. Directly suitable for modification and functionalization with phenol-, carboxylic acid-, and phenol-reactive reagents. Amino acids that introduce cross-linking activity, e.g., alkyne or azide-bearing moieties, respectively, are used. Introduction or substitution of amino acids with azide or alkyne groups that allow functionalization of The modifications include one or more of the following:

[0037] In one embodiment, the modified derivatives include N-terminal and / or C-terminal modifications. In embodiments, the modified derivatives include N-terminal modifications using suitable amino reaction chemistries, and and / or C-terminal modification using suitable carboxy reaction chemistry. The N- or C-terminal modifications may include, but are not limited to, cytotoxic agents, radioactive chelators, or It involves the addition of effector groups, including chromophores.

[0038] In a further embodiment, the modified derivative comprises an N-terminal modification. In this embodiment, the N-terminal modification comprises an N-terminal acetyl group. A phenyl group (referred to herein as C i The group called acetic anhydride or other suitable hydroxyl group is reacted with acetic anhydride or other suitable hydroxyl group during peptide synthesis. The resulting molecule is capped with a reagent and acetylated at the N-terminus. This offers the advantage of eliminating potential recognition points for aminopeptidases, and allows for the synthesis of bicyclic peptides. Avoid possible decomposition.

[0039] In an alternative embodiment, the N-terminal modification is for conjugation of an effector group and Includes the addition of a molecular spacer group that promotes retention of the potency of the bicyclic peptide against its target. .

[0040] In a further embodiment, the modified derivative comprises a C-terminal modification. In this embodiment, the C-terminal modification comprises an amide group. In the specification, C iii The group called C- This embodiment results in a terminally amidated molecule. This offers the advantage of eliminating potential recognition points and reducing the proteolytic potential of bicyclic peptides. To lower.

[0041] In one embodiment, the modified derivatives include one or more unnatural amino acid residues of one or more amino acid residues. In this embodiment, the cleavage site is substituted with a group that is recognized by a degradative protease. have isosteric / isoelectronic side chains that are not detrimental to or have any adverse effect on targeting efficacy Unnatural amino acids may also be selected.

[0042] Alternatively, proteolytic hydrolysis of nearby peptide bonds may result in conformational and steric Non-natural amino acids with constrained amino acid side chains may also be used to hinder. In particular, these include proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisopropyl methyl esters, butyric acid, Aib), and cycloamino, a simple derivative of amino-cyclopropylcarboxylic acid. Regarding acids.

[0043] In one embodiment, the modified derivative comprises the addition of a spacer group. In the modified derivative, the N-terminal cysteine (C i ) and / or a C-terminal cysteine (C iii ) to This includes the addition of a spacer group.

[0044] In one embodiment, the modified derivatives comprise one or more oxidation-resistant amino acid residues of one or more oxidation-sensitive amino acid residues. In a further embodiment, the modified derivative comprises a substitution with a tripeptide amino acid residue. This embodiment includes the replacement of the naphthylalanine residue with a naphthylalanine or alanine residue. Offers the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligands do.

[0045] In one embodiment, the modified derivatives comprise one or more hydrophobic amino acids of one or more charged amino acid residues. In an alternative embodiment, the modified derivatives include substitution with one or more hydrophobic acid residues. The substitution of one or more amino acid residues with one or more charged amino acid residues. The correct balance of amino acid residues is an important feature of bicyclic peptide ligands. For example, Hydrophobic amino acid residues affect the degree of plasma protein binding and therefore the available free radicals in plasma. The concentration of the isolated fraction is affected by the charge of the amino acid residues, while the charged amino acid residues (especially arginine) affect the concentration of the isolated fraction. This combination may affect the interaction of the phospholipid membranes on the cell surface with the This may affect the half-life, volume of distribution, and exposure of peptide drugs, potentially affecting clinical outcomes. Furthermore, the amount of charged and hydrophobic amino acids can be adjusted depending on the endpoint. The correct combination and number of residues reduces irritation at the injection site (if the peptide drug is administered subcutaneously). It can be reduced.

[0046] In one embodiment, the modified derivatives are one or more D-amino acid residues of one or more L-amino acid residues. This embodiment involves substitution by steric hindrance and stabilization of the β-turn conformation. The tendency of D-amino acids to form complexes is thought to enhance proteolytic stability (Tugyi et al. Reference (2005) PNAS, 102(2), 413-418).

[0047] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine. This embodiment has the advantage of eliminating potential proteolytic attack sites. do.

[0048] Each of the above modifications may serve to purposefully improve the potency or stability of the peptide. It should be noted that further improvement in potency based on modifications is achieved by the following mechanism: Can: -Utilizes the hydrophobic effect, resulting in a lower dissociation rate, so that higher affinity is achieved Incorporating hydrophobic moieties; - Utilizes long-range ionic interactions, resulting in faster association rates and higher affinity Incorporating charged groups (e.g., Schreiber et al., Rapid Electrostatic Assisted Protein Synthesis) Rapid, electrostatically assisted association of proteins (1996), Nature St. ruct. Biol. 3, 427-31); and For example, amino acid side chains should be correctly oriented so that entropy loss is minimized upon target binding. The torsion angles of the backbone are tightly constrained so that entropy loss is minimized upon target binding. By restricting the degree of cyclization and for the same reason introducing further cyclization into the molecule, Incorporating additional constraints into peptides (For a review, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 318 5-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0049] (Isotopic Variation) The present invention relates to a compound in which one or more atoms have the same atomic number but different atomic masses or nuclei that are commonly found in nature. is replaced by an atom having an atomic mass or mass number different from the mass number of the present invention. All known pharmaceutically acceptable (radio)isotope-labeled peptide ligands, as well as related The present invention relates to a compound having a metal chelating group attached thereto that can carry a (radioactive) isotope. Peptide ligands (called "effectors"), as well as specific functional groups associated with them (radioactive Peptides of the invention covalently substituted with isotopes or isotopically labeled functional groups Contains a ligand.

[0050] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention are isotopes of hydrogen, e.g. , 2 H(D) and 3 H(T), an isotope of carbon, e.g. 11 C. 13 C and 14 C, an isotope of chlorine, e.g. 3 6 Cl, isotopes of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I, and 131 I, Isotopes of nitrogen, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O, and 18 O, Lin Isotopes of, e.g., 32 P, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, Gari Isotopes of uranium, e.g., 67 Ga or 68 Ga, isotopes of yttrium, e.g. 90 Y and Ru Tethium isotopes, e.g. 177 Lu, as well as isotopes of bismuth, e.g., 213 Contains Bi.

[0051] Certain isotope-labeled peptide ligands of the invention, e.g., incorporate a radioisotope. The present invention relates to the use of Nectin-4 in drug and / or substrate tissue distribution studies and in the detection of Nectin-4 targets on diseased tissues. The peptide ligands of the present invention are useful for clinically evaluating the presence and / or absence of a target. The marker is a complex between a labeled compound and another molecule, peptide, protein, enzyme, or receptor. These compounds have valuable diagnostic properties in that they can be used to detect or identify the formation of Further detection or identification methods may include, for example, radioisotopes, enzymes, fluorescent substances, etc. substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and Compounds labeled with labeling agents such as fluorophores (e.g., fluorophores and luciferase) can be used. The isotope tritium, i.e. 3 H(T) and carbon-14, i.e., 14 C uses its built-in It is particularly useful for this purpose given the ease and means of detection available. do.

[0052] Deuterium, i.e., 2 Substitution with heavier isotopes such as H(D) results in greater metabolic stability , for example, as a result of increased in vivo half-life or reduced dosage requirements. may provide additional therapeutic benefits and may therefore be preferred in some circumstances. do.

[0053] 11 C. 18 F, 15 O, and 13 Substitution with positron-emitting isotopes such as N is useful for investigating target occupancy. It is useful in Positron Emission Topography (PET) studies for It is possible.

[0054] Isotopically labeled compounds of the peptide ligands of the present invention are typically prepared using conventional techniques known to those skilled in the art. or by using appropriate isotopically labeled reagents in place of previously utilized unlabeled reagents. These compounds can be prepared by processes similar to those described in the accompanying examples.

[0055] (synthesis) The peptides of the present invention may be synthetically produced by standard techniques and then subjected to in vitro molecular spectroscopy. This can be done using standard chemistry. This allows for rapid production of soluble materials for further downstream experimentation or validation. Such a method is disclosed in Timmerman et al. (supra). This can be achieved using conventional chemistries such as those used in

[0056] Thus, the present invention also provides a polypeptide selected as described herein. or the preparation of a conjugate, wherein the preparation is carried out as described below. In one embodiment, these steps include any further steps such as: The method is performed on the final polypeptide conjugate produced by the method described above.

[0057] Optionally, the amino acid residues in the polypeptide of interest are selected to form a conjugate or complex. may be replaced when manufacturing.

[0058] The peptide can be extended, for example, to incorporate additional loops and thus introduce multiple specificities. You can also enter.

[0059] To extend the peptide, it is simply performed using standard solid or solution phase chemistry: Orthogonally protected lysines (and analogs) are used at their N- or C-termini or within loops. Standard (bio)conjugation techniques can be used to chemically extend the active Alternatively, additions may be made by introducing a modified or activatable N- or C-terminus, e.g., (D awson et al., 1994, Protein Synthesis by Native Chemical Ligation (Synthesis) (Thesis of Proteins by Native Chemical Ligation). Science 266:776-779) by fragment condensation or native chemical ligation, as described in, for example, Chang et al. Proc Natl Acad Sci U S A. 1994 Dec 20; 91(26):12544-8 or Hikari et al. Literature, Bioorganic & Medicinal Chemistry Letters, Volume 18, Issue 22, November 15, 2008, This may also be done enzymatically using subtiligase as described in the US Pat. No. 6,600,000 (pp. 6000-6003).

[0060] Alternatively, the peptides may be further conjugated via disulfide bonds. This allows the first and second peptides to react with each other in the reducing environment of the cell. In this case, the molecular scaffold The nucleotide sequence (e.g., TATA) is selected during the chemical synthesis of the first peptide to react with the three cysteine groups. then, additional cysteines or thiols can be added to the first peptide. can be added to the N- or C-terminus of the cysteine or thiol, so that the cysteine or thiol is reacts only with free cysteines or thiols of the peptide to form disulfide-bonded bicyclic A peptide-peptide conjugate of the formula:

[0061] A similar technique can be used to synthesize two bicyclic bispecific macrocycles, potentially giving rise to tetraspecific molecules. Applies equally to the synthesis / coupling of molecules.

[0062] Furthermore, the addition of other functional groups or effector groups can be accomplished using appropriate chemistry via N- or C- Coupling at the terminal end or through a side chain may be achieved in the same manner. In this manner, the coupling is performed in such a way as not to block the activity of either entity. do.

[0063] (Pharmaceutical composition) According to a further aspect of the present invention, the peptide ligand as defined herein is administered to one or more pharmaceutical agents. Pharmaceutical compositions containing the compounds in combination with pharmaceutically acceptable excipients are provided.

[0064] Typically, the peptide ligand is administered in purified form together with a pharmacologically appropriate excipient or carrier. Typically, these excipients or carriers are saline and / or buffered solutions. The medium includes an aqueous or alcoholic / aqueous solution, an emulsion, or a suspension. Oral vehicles include sodium chloride solution, Ringer's dextrose, and dextrose. , and sodium chloride, and lactated Ringer's. Adjuvants, such as carboxymethylcellulose, may be used to keep the polypeptide complex in suspension. from thickeners such as cellulose acetate, polyvinylpyrrolidone, gelatin, and alginate may be selected.

[0065] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as Ringer's Also included are those based on dextrose. Preservatives and other additives, such as antimicrobials, Biological agents, antioxidants, chelating agents, and inert gases may also be present (Mack, 1982; Remington's Pharmaceutical Sciences, 16th ed.

[0066] The peptide ligands of the present invention may be administered as separate compositions or in combination with other agents. These may include antibodies, antibody fragments, and various immunotherapeutic agents, e.g. , cyclosporine, methotrexate, adriamycin, or cisplatin Pharmaceutical compositions include the protein ligands of the present invention. "Cocktails" of various cytotoxic or other drugs in combination with other drugs, or drugs pooled together before administration Polypeptides selected with different target ligands, whether pooled or not, Combinations of selected polypeptides according to the invention with different specificities, such as peptides. It can also include.

[0067] The route of administration of the pharmaceutical composition according to the present invention may be any of those generally known to those skilled in the art. For therapy, the peptide ligands of the invention may be administered to any patient according to standard techniques. Administration can be parenteral, intravenous, intramuscular, intraperitoneal, transdermal, or pulmonary route. any method, including via a catheter or, equally appropriately, by direct injection using a catheter. Preferably, the pharmaceutical composition according to the present invention is The dosage and frequency of administration depend on the age, sex, and condition of the patient, as well as other medications. The clinical significance of the drug is determined by the concurrent administration of drugs, contraindications, and other parameters considered by the clinician. circle.

[0068] The peptide ligands of the present invention are lyophilized prior to storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and is compatible with freezing methods known in the art. Lyophilization and reconstitution techniques are available. Lyophilization and reconstitution can result in varying degrees of activity. This may result in losses and the level may need to be adjusted upward to compensate. It will be understood by those skilled in the art.

[0069] Compositions containing the peptide ligands of the present invention or a cocktail thereof are useful for prophylactic and / or therapeutic purposes. In certain therapeutic applications, the concentration of selected cells can be increased. At least partial inhibition, suppression, modulation, killing, or some other measurable parameter of the group. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose." The amount required to achieve this will depend on the severity of the disease and the general state of the patient's own immune system. The dose will depend on the individual, but generally, 0.005-5.0 mg of the selected peptide ligase per kilogram of body weight is recommended. The range is 0.05-2.0 mg / kg / day, with doses of 0.05-2.0 mg / kg / day being more commonly used. In addition, compositions containing the present peptide ligands or cocktails thereof also exhibit similar or slightly Small dosages may be administered.

[0070] Compositions containing peptide ligands according to the invention can be used in prophylactic and therapeutic settings. , to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in mammals Furthermore, the peptide ligands described herein can be selected ex vivo or in vitro. selectively used to selectively kill or deplete a target cell population from a heterogeneous collection of cells. The blood from the mammal can be filtered or otherwise effectively removed. The peptide ligand can be combined in vitro and then administered to mammals according to standard techniques. Killing or otherwise removing unwanted cells from the blood for return to the animal .

[0071] (therapeutic use) According to a further aspect of the present invention there is provided a method for preventing, suppressing or treating cancer. There is provided a heterotandem bicyclic peptide conjugate as defined herein.

[0072] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include tumors of epithelial origin (adenocarcinomas, various types of adenomas and carcinomas, including squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), e.g. Examples include bladder and urinary tract, breast, gastrointestinal tract (esophagus, stomach (gastric), small intestine, colon, rectum). , and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidneys, lungs (e.g. , adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g. Cancer of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovaries, fallopian tubes, Peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid gland (e.g., thyroid follicles) cancer), adrenal gland, prostate, skin, and adnexal cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratinocyte carcinoma) acanthoma, dysplastic nevi); hematologic malignancies (i.e., leukemia, lymphoma) and premalignant hematologic disorders borderline malignancies, including hematologic malignancies of the lymphoid lineage and related diseases (e.g., acute lymphoblastic leukemia, lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large cell lymphoma Follicular B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma , hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multifocal myeloma, and post-transplant lymphoproliferative disorders), and hematologic malignancies and related diseases of the myeloid lineage (e.g., For example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), Hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia); mesenchymal Tumors of origin, e.g., sarcomas of the soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, Chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma , synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumor, benign and malignant histiocytoma, and protruding Dermatofibrosarcoma; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma) cysts, meningiomas, ependymomas, pineal tumors, and schwannomas); endocrine tumors (e.g., pituitary tumors) tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid gland ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, spermatozoa) epithelioma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and pediatric and embryonal tumors (e.g., , medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or Congenital or other syndromes that predispose to tumors (e.g., xeroderma pigmentosum) These include, but are not limited to:

[0073] In further embodiments, the cancer is, for example, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BLL), or lymphoma (BL), multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia Leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hematopoietic malignancies selected from: Johns Hopkins lymphoma (HL) and chronic myeloid leukemia (CML) be selected.

[0074] Reference herein to the term "prevention" refers to the administration of a protective composition prior to the induction of disease. "Suppression" includes administration of a composition after an inductive event but before the clinical appearance of the disease. "Treatment" includes administration of a protective composition after disease symptoms have become manifest.

[0075] Screening for the efficacy of peptide ligands in protecting against or treating disease Animal model systems are available that can be used to study the effects of steroids on the immune system. Allows for the development of polypeptide ligands that can cross-react with human and animal targets This is facilitated by the present invention.

[0076] The invention will now be further described with reference to the following examples. [Example]

[0077] (Example) Materials and Methods (Peptide synthesis) The peptide was synthesized by solid phase synthesis using Rink amide MBHA resin. DMF was added to a mixture containing Fmoc-Cys(Trt)-OH (3.0 equivalents) and 0.4-0.45 mmol / g of methyl ... Then DIC (3 eq.) and HOAt (3 eq.) were added and mixed for 1 hour. 20% piperidine in DMF was used for deblocking. Each subsequent amino acid was deblocked with the activator reagent DIC in DMF. (3.0 equiv.) and HOAT (3.0 equiv.). After synthesis, the peptide was monitored by the tetrachloroethylene color reaction. The resin was washed with DMF x 3 and MeOH x 3, then dried under N bubbling overnight. The peptide resin was treated with 92.5% TFA / 2.5% TIS / 2.5% EDT / 2.5% H2O for 3 hours. The mixture was precipitated with isopropyl ether and centrifuged (3000 rpm for 3 min). The crude peptide was washed twice with diethyl ether, dried under vacuum for 2 hours, and then lyophilized. The lyophilized powder was dissolved in ACN / HO (50:50), followed by a 100 mM solution of TATA in ACN, followed by a heavy dilution in HO. Ammonium carbonate (1M) was added and the solution was mixed for 1 hour. Once cyclization was complete, the reaction was diluted to 1M Quench with aqueous cysteine hydrochloride (10 equivalents relative to TATA), then mix and let stand for 1 hour. The solution was lyophilized to give the crude product. The crude peptide was purified by preparative HPLC. and freeze-dried to give the product.

[0078] Unless otherwise stated, all amino acids were used in the L-configuration.

[0079] (biological data) A peptide without a fluorescent tag was competed with a peptide with a fluorescent tag and a known Kd. The fluorescent tracer used was the sequence [ka] where Fl is 5 / 6-carboxyfluorescein and Sar is sarcosine 90 (Kd=2nM).

[0080] Peptides were assayed as described in the direct binding assay with up to 5% DMSO. Dilute to the appropriate concentration in PBS buffer, then serially dilute 1:2. The plate was then added with 10 μL of human EphA2 at a concentration of 25 nM, followed by 10 μL of fluorescent The peptide was added (final concentration 0.8 nM). Measurements were performed, but the gain was determined before the first measurement. Data analysis was performed with Systat Sigmaplot version 12.0, where mP values were calculated by the user. The Ki values were obtained by fitting the cubic equation defined by the user: f=ymax+(ymin-ymax) / Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-P rot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-P rot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+K lig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*((((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kc omp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+C omp-Prot*c))) / ((3*Klig)+((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c )+Klig*(Comp-Prot*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c )^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*K comp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*((((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*( Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp-P rot*c)))). "Lig", "KLig", and "Prot" are all fluorescent peptide concentrations, fluorescent peptides, respectively. These were the defined values for Kd and EphA2 concentration.

[0081] Certain bicyclic peptides of the present invention were tested in the competitive binding assay described above. The results are shown in Table 1. Seen on: Table 1: Competitive binding assays of selected bicyclic peptides of the invention [Table 1] TIFF2025122030000013.tif255170The present application provides the following aspects of the invention. (Aspect 1) A polypeptide containing at least three reactive groups separated by at least two loop sequences. 1,1',1''-(1,3,5-triazinane) which forms a covalent bond with the reactive group of the polypeptide and The resulting product comprises a molecular scaffold that is (1,3,5-triyl)triprop-2-en-1-one. As a result, at least two polypeptide loops are formed on the molecular scaffold. A peptide ligand specific for A2, the peptide ligand comprising: (chemical 1) TIFF2025122030000014.tif190170TIFF2025122030000015.tif246170TIFF2025122030000016.tif245170TIFF2025122030000017.tif124170 (where Ac stands for acetyl, HyP stands for hydroxyproline, and HArg stands for homoarginine) PYA stands for 4-pentynoic acid, 3,3-DPA stands for 3,3-diphenylalanine, and Cba stands for β- 1Nal stands for 1-naphthylalanine, and NMeAla stands for N-methyl-alanine. His1Me stands for N1-methyl-L-histidine, His3Me stands for N3-methyl-L-histidine, 4ThiAz represents β-(4-thiazolyl)-alanine, and Thi represents 2-thienyl-alanine. 3Thi represents 3-thienylalanine, and palmitoyl-Glu-LysN3 represents N2-((S)-4-carboxy -4-Palmitamidobutanoyl)-N6-diazo-L-lysine: (chemical 2) TIFF2025122030000018.tif106170, pCoPhe represents para-carboxy-phenylalanine, and hGlu represents homoglutamic acid. B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units, and Nle represents norleu Represents Shin and [MerPro] i , C i , C ii , C iii , and [Cysam] iii is cysteine, 3-mercapto the first (i) and second (ii) selected from propanoic acid (MerPro) and cysteamine (Cysam); and a third (iii) reactive group. or a pharmaceutically acceptable salt thereof. Unusual peptide ligands. (Aspect 2) (C3) 2. The peptide ligand of embodiment 1, which is TIFF2025122030000019.tif14170 or a pharmaceutically acceptable salt thereof. (Aspect 3) (C4) 2. The peptide ligand of embodiment 1, which is TIFF2025122030000020.tif15170 or a pharmaceutically acceptable salt thereof. (Aspect 4) The pharmaceutically acceptable salts may be the free acid or salts of sodium, potassium, calcium, ammonium, 4. The peptide ligand according to any one of embodiments 1 to 3, wherein the peptide ligand is selected from the group consisting of ammonium salts. (Aspect 5) The peptide ligand according to any one of embodiments 1 to 4, wherein the EphA2 is human EphA2. (Aspect 6) The peptide ligand according to any one of aspects 1 to 5, A pharmaceutical composition comprising the compound in combination with an agent. (Aspect 7) Prevention, suppression, or treatment of diseases or disorders characterized by overexpression of EphA2 in affected tissues 7. The peptide ligand according to any one of embodiments 1 to 6, for use in (Aspect 8) A composition according to any one of aspects 1 to 7 for use in the prevention, suppression or treatment of cancer. Peptide ligand. (Aspect 9) The cancer is prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), breast cancer (e.g., triple myeloma), or selected from: gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma, and fibrosarcoma; 9. A peptide ligand for use according to embodiment 8.

Claims

1. A peptide ligand specific for EphA2, comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences, and a molecular scaffold which is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)triplop-2-en-1-one that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand is 【Chemistry 1】 (wherein HyP represents hydroxyproline, HArg represents homoarginine, 1Nal represents 1-naphthylalanine, NMeAla represents N-methyl-alanine, and C i , C ii , and C iii represent first (i), second (ii), and third (iii) reactive groups selected from cysteine, 3-mercaptopropionic acid (MerPro), and cysteamine (Cysam). or a pharmaceutically acceptable salt thereof. 【Request Item 2】 【Chemistry 2】 or a pharmaceutically acceptable salt thereof.

3. 3. The peptide ligand of claim 1 or 2, wherein the polypeptide comprises a C-terminal amide group.

4. 4. The peptide ligand of any one of claims 1 to 3, wherein the peptide ligand is in the form of a free acid or a pharmaceutically acceptable salt selected from sodium, potassium, calcium, or ammonium salts.

5. The peptide ligand of any one of claims 1 to 4, wherein the EphA2 is human EphA2.

6. The peptide ligand of any one of claims 1 to 5, further comprising a cytotoxic agent, a radiochelator, or a chromophore.

7. A pharmaceutical composition comprising the peptide ligand of any one of claims 1 to 6.

8. A pharmaceutical composition comprising the peptide ligand of any one of claims 1 to 6 for use in preventing, suppressing, or treating a disease or disorder characterized by overexpression of EphA2 in affected tissue.

9. A pharmaceutical composition comprising the peptide ligand of any one of claims 1 to 6 for use in preventing, suppressing or treating cancer.

10. 10. The pharmaceutical composition of claim 9, wherein the cancer is selected from prostate cancer, lung cancer, breast cancer, gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma, and fibrosarcoma.

11. 11. The pharmaceutical composition according to claim 10, wherein the lung cancer is non-small cell lung cancer (NSCLC) and / or the breast cancer is triple-negative breast cancer.

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