Bicyclic peptide ligand specific for EphA2

Abstract

The present invention relates to polypeptides covalently linked to a non-aromatic molecular scaffold such that two or more peptide loops are interposed between the attachment points to the scaffold. In particular, the present invention describes peptides that are high-affinity binders of Eph receptor tyrosine kinase A2 (EphA2). The present invention also relates to pharmaceutical compositions comprising the peptide ligands and to the use of the peptide ligands in the prevention, suppression, or treatment of diseases or disorders characterized by overexpression of EphA2 in diseased tissues (e.g., tumors).

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to a polypeptide covalently bound to a non-aromatic molecular scaffold such that two or more peptide loops are internal to the attachment points to the scaffold. In particular, the present invention describes a peptide that is a high-affinity binder of the Eph receptor tyrosine kinase A2 (EphA2). The present invention also relates to a pharmaceutical composition comprising the peptide ligand and the use of the peptide ligand in the prevention, suppression, or treatment of a disease or disorder characterized by overexpression of EphA2 in an affected tissue (e.g., a tumor).

Background Art

[0002] (Background of the Invention) Cyclic peptides can bind to protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutic agents. Indeed, some cyclic peptides have already been successfully used in the clinic, such as the antibacterial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The excellent binding properties are due not only to the relatively large interaction surface formed between the peptide and the target, but also to the reduced conformational flexibility of the cyclic structure. Usually, macrocyclic molecules bind to surfaces of several hundred square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide having an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å 2 )(Xiong et al. (2002), Science 296(5565), 151-5), or the cyclic peptide inhibitor upain-1 (603 Å 2 ; Zhao et al. (2007), J Struct Biol 160(1), 1-10) that binds to urokinase-type plasminogen activator).

[0003] Due to this cyclic three-dimensional arrangement, the peptide macrocyclic molecule is less flexible than the linear peptide, resulting in a smaller entropy loss when binding to the target, and consequently, a higher binding affinity. The reduced flexibility also leads to the fixation of the target-specific three-dimensional structure and increases the binding specificity compared to the linear peptide. This effect is exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8) that loses its selectivity for other MMPs when its ring is opened (Cherney et al. (1998), J Med Chem 41(11), 1749-51). The favorable binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with multiple peptide rings, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously linked polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops on a synthetic scaffold for the structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for preparing candidate drug compounds, where the compound is made by linking a cysteine-containing polypeptide to a molecular scaffold such as tris(bromomethyl)benzene, are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] A phage display-based combinatorial approach has been developed for generating and screening large libraries of bicyclic peptides against a target of interest (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine residues and two random six-amino acid regions (Cys-(Xaa) 6 -Cys-(Xaa) 6 -Cys) was displayed on phage and cyclized by covalently attaching the cysteine side chains to a small molecule (tris-(bromomethyl)benzene). SUMMARY OF THE INVENTION

[0006] (Summary of the Invention) According to a first aspect of the present invention, there is provided 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-triazinane-1,3,5-triyl)triprop-2-en-1-one and which forms a covalent bond 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

Chemical formula

[0007] According to a further aspect of the present invention, a pharmaceutical composition is provided which comprises the peptide ligand defined herein in combination with one or more pharmaceutically acceptable excipients.

[0008] According to a further aspect of the present invention, there is provided a peptide ligand as defined herein for use in the prevention, suppression or treatment of cancer.

Mode for Carrying Out the Invention

[0009] (Detailed Description of the Invention) According to a first aspect of the present invention, there is provided a polypeptide comprising at least three reactive groups separated by at least two loop arrays and a molecular scaffold which is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-ene-1-one which forms a covalent bond with the reactive groups of the polypeptide, whereby at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand is

Chemical formula

Chemical formula

[0010] In one particular embodiment, the EphA2-binding bicyclic peptide ligand is selected from BCY13118, BCY12860, BCY12859, BCY13119, BCY13917, BCY13918, BCY13919, BCY13920, BCY13922, BCY13923, BCY14047, BCY14048, BCY13135, BCY12865, BCY13120, and BCY13117:.

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

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, such as in the fields of peptide chemistry, cell culture, and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for methods of molecular biology, genetics, and biochemistry (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Edition, John Wiley & Sons, which are incorporated herein by reference).

[0013] (Nomenclature) (Numbering) When referring to the positions of amino acid residues within the compounds of the present invention, cysteine residues (C i , C ii , and C iii ) are invariant and are therefore omitted from the numbering. Thus, the numbering of the amino acid residues in SEQ ID NO: 1 is referred to as follows: A-[HArg]-D-C i -[HyP] 1 -L 2 -V 3 -N 4 -P 5 -L 6 -C ii -L 7 -H 8 -P 9 -[dD] 10 -W 11 -[HArg] 12 -C iii (SEQ ID NO: 1).

[0014] For the purposes of this description, all bicyclic peptides are considered to be cyclized with 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), resulting in a trisubstituted structure. Cyclization by TATA occurs at C i 、C ii 、and C iii above.

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

[0016] (Reverse peptide sequence) In view of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, the peptide sequences disclosed herein are expected to find utility in their retro-inverso form. For example, the sequence is reversed (i.e., the N-terminal becomes the C-terminal and the C-terminal becomes the N-terminal), and its stereochemistry is similarly reversed (i.e., D-amino acids become L-amino acids and L-amino acids become D-amino acids). To avoid misunderstanding, any reference to an amino acid, either by its formal name or by its one-letter or three-letter notation of the amino acid, is intended to be represented as an L-amino acid in this specification, unless otherwise specified. When such an amino acid is intended to be represented as a D-amino acid, a small letter d is prefixed to the amino acid within square brackets, e.g., [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0017] (Advantages of peptide ligands) Certain bicyclic peptides of the invention have several advantageous properties that can be considered suitable drug-like molecules for injection, inhalation, nasal, ocular, oral, or topical administration. Such advantageous properties include the following: - Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations; - Protease stability. Bicyclic peptide ligands should exhibit stability against plasma proteases, epithelial ("membrane-bound") proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. in most situations. Protease stability should be maintained across different species so that bicyclic peptide lead candidates can be developed in animal models and administered to humans with confidence; - Desirable solubility profile. This is a function of the ratio of charged and hydrophilic residues to hydrophobic residues and intramolecular / intermolecular H-bonds, which is important for formulation and absorption purposes; - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides with short-term or long-term in vivo exposure times for the management of either chronic or acute disease states. The optimal exposure time is determined by the requirement for sustained exposure (for maximum therapeutic efficiency) compared to the requirement for short exposure times to minimize toxicological effects due to sustained exposure to the drug; - Selectivity. Certain peptide ligands of the invention exhibit superior selectivity over other Eph receptor tyrosine kinases, such as EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, and EphB1, as well as factor XIIA, carbonic anhydrase 9, and CD38. It should also be noted that the selected peptide ligands of the invention exhibit cross-reactivity with other species (e.g., mice and rats) that permit testing in animal models; and - Safety. Bleeding events have been reported in preclinical in vivo models and clinical trials using EphA2 antibody-drug conjugates. For example, a Phase 1 open-label trial using MEDI-547 was terminated due to bleeding and coagulation events that occurred in 5 out of 6 patients (Annunziata et al., Invest New Drugs (2013) 31:77-84). The bleeding events observed in the patients were consistent with the effects on the coagulation system observed in preclinical trials in rats and monkeys: an increase in activated partial thromboplastin time and an increase in fibrinogen / fibrin degradation products (Annunziata et al., ibid.). Apparent bleeding events have been reported to be seen in toxicology trials in monkeys (Annunziata et al., ibid.). Collectively, these results imply that MEDI-547 causes disseminated intravascular coagulation (DIC) in both preclinical species and patients.

[0018] (Peptide ligand) As used herein, a peptide ligand refers to a peptide covalently attached to a molecular scaffold. Typically, such a peptide includes two or more reactive groups (i.e., cysteine residues) capable of forming a covalent bond with the scaffold, and an internal sequence between the reactive groups, which is called a loop sequence because it forms a loop when the peptide binds to the scaffold. In this case, the peptide contains at least three reactive groups selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine, and forms at least two loops on the scaffold.

[0019] (Pharmaceutically acceptable salts) Salt forms are within the scope of the present invention, and it will be understood that references to peptide ligands include salt forms of the ligand.

[0020] The salts of the present invention can be synthesized from the parent compounds containing basic or acidic moieties by the methods described in conventional chemical methods, for example, Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Usually, such salts can be prepared by reacting the free acid or base forms of these compounds with appropriate bases or acids in water, in an organic solvent, or in a mixture of the two.

[0021] Acid addition salts (mono- or di-salts) can be formed with a wide variety of both inorganic and organic acids. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid, etc.), glutamic acid (e.g., L-glutamic acid, etc.), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (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, malonic acid, (±)-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, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, and mono- or di-salts formed with an acid selected from the group consisting of acylated amino acids and cation exchange resins.

[0022] One particular group of salts consists of salts formed with 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, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0023] If the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO - ), salts can be formed with organic or inorganic bases to generate suitable cations. Examples of suitable inorganic cations include alkali metal ions such as Li + , Na + , and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + , but are not limited thereto. Examples of suitable organic cations include ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ), but are not limited thereto. Examples of some suitable substituted ammonium ions include those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .

[0024] If the compounds of the present invention contain an amine function, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0025] (Reactive group) The molecular scaffold of the present invention may be attached to a polypeptide via a functional group or a reactive group on the polypeptide. These are typically formed from the side chains of specific amino acids found in polypeptide polymers. Such reactive groups may be cysteine side chains, lysine side chains, or N-terminal amino groups, or any other suitable reactive group, for example, penicillamine. Details of suitable reactive groups can be found in WO 2009 / 098450.

[0026] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartic acid or glutamic acid, the guanidium group of arginine, the phenol group of tyrosine, or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including azide, keto-carbonyl, alkyne, vinyl, or aryl halide groups. The terminal amino and carboxyl groups of the polypeptide can also serve as reactive groups for forming covalent bonds with the molecular scaffold / molecular core.

[0027] The polypeptide of the present invention contains at least three reactive groups. The polypeptide can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.

[0028] In a preferred embodiment, a polypeptide having three reactive groups is produced. By reacting the polypeptide with a molecular scaffold / molecular core having three-fold rotational symmetry, a single product isomer is produced. 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 and do not encode the molecular species of the heterogeneous states formed upon reaction of the polypeptide with the molecular core. When only one product isomer can be formed, the attribution of the nucleic acid to the product isomer is clearly defined. When multiple product isomers are formed, the nucleic acid cannot provide information regarding the nature of the product isomers isolated in a screening or selection process. Information on a single product isomer is also advantageous when a particular member of the library of the present invention is synthesized. In this case, a chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.

[0029] In another embodiment, a polypeptide having four reactive groups is produced. By reacting the polypeptide with a molecular scaffold / molecular core having tetrahedral symmetry, two product isomers are produced. Even if two different product isomers are encoded by one and the same nucleic acid, the nature of the isolated isomers can be determined by chemically synthesizing both isomers, separating the two isomers, and testing both isomers for binding to a target ligand.

[0030] In one embodiment of the present invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the orthogonal reactive groups to be directed to specific sites on the molecular core. By using a ligation strategy involving orthogonal reactive groups, the number of product isomers formed can be limited. In other words, by selecting a reactive group selected relative to the remaining ones of at least three bonds and a distinct or different reactive group for one or more of at least three bonds, a specific order of binding or orientation of a specific reactive group of the polypeptide to a specific position on the molecular scaffold can be effectively achieved.

[0031] In another embodiment, the reactive group of the polypeptide of the present invention reacts with a molecular linker, in which case the linker can react with the molecular scaffold such that the linker enters between the molecular scaffold in the final bound state and the polypeptide.

[0032] In some embodiments, the amino acids of the members of a library or set of polypeptides can be exchanged for any natural or non-natural amino acid. Those having a functional group for cross-linking the polypeptide to the molecular core are excluded from these exchangeable amino acids so that only the loop sequences are exchangeable. The exchangeable polypeptide sequences can have either a random sequence, a defined sequence, or a sequence having both random and defined amino acids. Since the positions of these amino acids determine the loop size, any amino acid having a reactive group is in a defined position within the polypeptide.

[0033] In one embodiment, a 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, X represents a random amino acid, m and n represent numbers from 3 to 6 that define the length of intervening polypeptide segments (which may be the same or different), and l and o represent numbers from 0 to 20 that define the length of adjacent polypeptide segments.

[0034] Using an alternative to thiol-mediated conjugation, a molecular scaffold can be attached to a peptide via a covalent interaction. Alternatively, these techniques can be used in the modification or attachment of such additional moieties (e.g., small molecules other than the molecular scaffold) to polypeptides after they have been selected or isolated according to the present invention - in this embodiment, clearly, the attachment need not be covalent and can include non-covalent linkages. These methods can be by producing phage presenting proteins and peptides having unnatural amino acids with the necessary chemical reactive groups in combination with small molecules having complementary reactive groups, or by incorporating unnatural amino acids chemically or recombinantly synthesized polypeptides when the molecule is made after the selection / isolation step, can be used instead of (or in combination with) the thiol-mediated method. Further details can be found in WO 2009 / 098450 or in the literature of Heinis et al., Nat Chem Biol 2009, 5(7), 502-7.

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

[0036] (Modified derivative) Modified derivatives of the peptide ligands defined herein will be understood to be within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); 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 amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within a bicyclic peptide ligand; substitution of one or more peptide bonds with surrogate bonds; modification of the peptide backbone length; substitution of hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid, and tyrosine with suitable amine, thiol, carboxylic acid, and phenol-reactive reagents to functionalize the amino acid, and introduction or substitution of an amino acid having an azide or alkyne group that enables functionalization with a moiety having an alkyne or azide, respectively, to introduce orthogonal reactivity suitable for functionalization: one or more modifications selected from the foregoing.

[0037] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amine-reactive chemistry and / or a C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group including, but not limited to, a cytotoxic agent, a radiochelator, or a chromophore.

[0038] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (designated herein as C iThe group (referred to as such) is capped with acetic anhydride or other suitable reagents during peptide synthesis, resulting in a molecule with an acetylated N-terminus. This embodiment provides the advantage of removing potential recognition points for aminopeptidases and avoiding the possibility of degradation of the bicyclic peptide.

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

[0040] In a further embodiment, the modified derivative includes a C-terminal modification. In a further embodiment, the C-terminal modification includes an amide group. In this embodiment, the C-terminal cysteine group (herein, C iii The group (referred to as such) is synthesized as an amide during peptide synthesis, resulting in a molecule with an amidated C-terminus. This embodiment provides the advantage of removing potential recognition points for carboxypeptidases and reducing the possibility of proteolysis of the bicyclic peptide.

[0041] In one embodiment, the modified derivative includes substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids with isosteric / isoelectronic side chains that are not recognized by degradative proteases and do not have any detrimental effects on target potency may be selected.

[0042] Alternatively, non-natural amino acids with constrained amino acid side chains may be used such that proteolytic hydrolysis of nearby peptide bonds is sterically and conformationally hindered. In particular, these relate to proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., amino isobutyric acid, Aib), and cyclic amino acids that are simple derivatives of aminocyclopropylcarboxylic acid.

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

[0044] In one embodiment, the modified derivative comprises substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises substitution of tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0045] In one embodiment, the modified derivative comprises substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged and hydrophobic amino acid residues is an important feature of the bicyclic peptide ligand. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. These two combinations can affect the half-life, volume of distribution, and exposure of the peptide drug and can be adjusted according to clinical endpoints. Furthermore, the correct combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0046] In one embodiment, the modified derivative comprises substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is thought to enhance proteolytic stability due to steric hindrance and the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0047] In one embodiment, the modified derivative includes removal of any amino acid residue and substitution with alanine. This embodiment has the advantage of removing potential proteolytic attack sites.

[0048] It should be noted that each of the above modifications serves to intentionally improve the potency or stability of the peptide. Further potency improvements based on modifications can be achieved by the following mechanisms: - Incorporating hydrophobic moieties that utilize the hydrophobic effect to achieve higher affinity and result in a lower dissociation rate; - Incorporating charged groups that utilize long-range ionic interactions to result in a faster association rate and higher affinity (see, for example, Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - Incorporating additional constraints into the peptide, for example, by correctly constraining the side chains of amino acids, constraining the backbone torsion angles, and introducing additional cyclization within the molecule so that the loss of entropy is minimized upon target binding (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0049] (Isotope Variation) The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present invention in which one or more atoms are replaced by atoms having the same atomic number but a different atomic mass or mass number from the atomic mass or mass number normally found in nature, and peptide ligands of the present invention (referred to as "effectors") to which a metal chelate group capable of retaining the relevant (radio)isotope is attached, and peptide ligands of the present invention in which specific functional groups are covalently replaced by relevant (radio)isotopes or isotope-labeled functional groups.

[0050] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention are isotopes of hydrogen, such as 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C, 13 C and 14 C, isotopes of chlorine, such as 36 Cl, isotopes of fluorine, such as 18 F, isotopes of iodine, such as 123 I, 125 I, and 131 I, isotopes of nitrogen, such as 13 N and 15 N, isotopes of oxygen, such as 15 O, 17 O, and 18 O, isotopes of phosphorus, such as 32 P, isotopes of sulfur, such as 35 S, isotopes of copper, such as 64 Cu, isotopes of gallium, such as 67 Ga or 68 Ga, isotopes of yttrium, such as 90 Y, and isotopes of lutetium, such as 177 Lu, and isotopes of bismuth, such as 213 Bi.

[0051] Certain isotope-labeled peptide ligands of the present invention, for example, those incorporating a radioisotope, are useful in the study of the tissue distribution of drugs and / or substrates and in clinically evaluating the presence and / or absence of nectin-4 targets on diseased tissue. The peptide ligands of the present invention can further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods can use compounds labeled with labeling agents such as, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). Tritium, a radioisotope, i.e., 3 H(T) and carbon-14, i.e., 14 C are particularly useful for this purpose in view of the ease of their incorporation and the availability of means for detection.

[0052] Substitution with heavier isotopes such as deuterium, i.e., 2 H(D) can result in certain therapeutic advantages obtained as a result of greater metabolic stability, e.g., increased in vivo half-life or reduced required dosage, and may therefore be preferred in some situations.

[0053] 11 C, 18 F, 15 O, and 13 N, etc., substitution with positron-emitting isotopes can be useful in Positron Emission Topography (PET) tests for examining target occupancy.

[0054] Isotope-labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by a process similar to that described in the appended examples using appropriate isotope-labeled reagents in place of the unlabeled reagents previously utilized.

[0055] (Synthesis) The peptides of the present invention can be synthetically produced by standard techniques and then reacted with a molecular scaffold in vitro. When doing this, standard chemistry can be used. This enables the rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistry such as that disclosed in the literature of Timmerman et al. (supra).

[0056] Accordingly, the present invention also relates to the manufacture of a polypeptide or conjugate selected as described herein, where the manufacture includes any further steps as described below. In one embodiment, these steps are performed on the polypeptide conjugate of the final product made by chemical synthesis.

[0057] Optionally, the amino acid residues in the polypeptide of interest may be substituted when producing the conjugate or complex.

[0058] The peptide can also be extended to incorporate, for example, another loop and thus introduce multiple specificities.

[0059] To extend the peptide, it may simply be chemically extended at its N- or C-terminus or within a loop using orthogonally protected lysine (and analogs) using standard solid or liquid phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively, the addition may be effected enzymatically, for example, by fragment condensation or native chemical ligation as described in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or using sortase as described, for example, in (Chang et al., Proc Natl Acad Sci U S A. 1994 Dec 20; 91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, November 15, 2008, pp. 6000-6003).

[0060] Alternatively, the peptide may be extended or modified by further conjugation via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other within the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with three cysteine groups; subsequently, additional cysteine or thiol can be added to the N or C-terminus of the first peptide such that this cysteine or thiol reacts only with the free cysteine or thiol of the second peptide to form a disulfide-bonded bicyclic peptide-peptide conjugate.

[0061] Similar techniques are equally applicable to the synthesis / coupling of two bicyclic bispecific macrocyclic molecules that potentially give rise to a quadruple specific molecule.

[0062] Furthermore, the addition of other functional or effector groups may be achieved in the same manner by coupling at the N- or C-terminus, or via the side chain, using appropriate chemistry. In one embodiment, the coupling is carried out in such a way as not to block the activity of either entity.

[0063] (Pharmaceutical composition) According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0064] Generally, the peptide ligand is utilized in a purified form together with a pharmaceutically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, and lactated Ringer's. Suitable physiologically acceptable adjuvants may be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginate, if necessary to keep the polypeptide complex in suspension.

[0065] Parenteral vehicles include fluids and nutrient replenishers and electrolyte replenishers, for example, those based on Ringer's dextrose. Also, preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may be present (Mack (1982), Remington's Pharmaceutical Sciences, 16th edition).

[0066] The peptide ligands of the invention may be used as separately administered compositions or in combination with other agents. These include antibodies, antibody fragments, and various immunotherapeutic agents, such as Cyclosporine, such as methotrexate, adriamycin, or cisplatin, and immunotoxins. The pharmaceutical composition can be a "cocktail" of various cytotoxic agents or other drugs in combination with the protein ligand of the present invention, or a combination of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether pooled or not prior to administration.

[0067] The route of administration of the pharmaceutical composition according to the present invention can be any generally known to those skilled in the art. For therapy, the peptide ligand of the present invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, via the pulmonary route, or, equally appropriately, by direct injection using a catheter. Preferably, the pharmaceutical composition according to the present invention is administered by inhalation. The dosage and frequency of administration are determined by the patient's age, sex, and condition, the simultaneous administration of other drugs, contraindications, and other parameters considered by the clinician.

[0068] The peptide ligand of the present invention can be lyophilized before storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective and can utilize lyophilization and reconstitution techniques known in the art. It will be understood by those skilled in the art that lyophilization and reconstitution can result in varying degrees of loss of activity and that it may be necessary to adjust the levels upward to compensate.

[0069] The composition containing the peptide ligand of the present invention or a cocktail thereof can be administered for prophylactic and / or therapeutic treatment. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, regulation, killing, or some other measurable parameter of a selected population of cells is defined as a "therapeutically effective dose". The amount required to achieve this dosage depends on the severity of the disease and the general state of the patient's own immune system, but generally ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, and a dosage of 0.05 to 2.0 mg / kg is more commonly used. For prophylactic use, the composition containing the present peptide ligand or a cocktail thereof may also be administered at a similar or slightly lower dosage.

[0070] The composition containing the peptide ligand according to the present invention can be utilized in prophylactic and therapeutic settings to assist in the alteration, inactivation, killing, or removal of a selected target cell population in a mammal. Furthermore, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove a target cell population from a heterogeneous cell aggregate. Blood derived from a mammal can be combined ex vivo with a selected peptide ligand, thereby killing unwanted cells or removing them from the blood in another form for return to the mammal according to standard techniques.

[0071] (Therapeutic use) According to a further aspect of the present invention, there is provided a heterotandem bicyclic peptide complex as defined herein for use in the prevention, suppression, or treatment of cancer.

[0072] Examples of cancers (and their benign counterparts) that can be treated (or suppressed) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other carcinomas), such as those of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary tract, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma), head and neck (e.g., cancers of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and appendage cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematological malignancies (i.e., leukemias, lymphomas) and borderline malignancies including pre-malignant blood disorders and hematological malignancies and related diseases of the lymphoid lineage (e.g., acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphoma, e.g., diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and related diseases of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilic syndrome, myeloproliferative disorders, e.g., polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, Ewing sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumor, benign and malignant histiocytoma, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma);Endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid); tumors of the eye and its appendages (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, undifferentiated embryonal cell tumor, cystic teratoma, and choriocarcinoma); and pediatric and fetal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and undifferentiated neuroectodermal tumor); or congenital or other syndromes (e.g., xeroderma pigmentosum) that predispose a patient to malignancy, but are not limited thereto.;

[0073] In a further embodiment, the cancer is selected from hematopoietic malignancies selected from, for example, non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).;

[0074] The mention in this specification of the term "prevention" includes the administration of a prophylactic composition before the induction of the disease. "Suppression" refers to the administration of a composition after an inductive event but before the clinical appearance of the disease. "Treatment" includes the administration of a prophylactic composition after the manifestation of the disease symptoms.;

[0075] Animal model systems are available that can be used to screen for the effectiveness of peptide ligands in the prevention of disease or the treatment of disease. The use of animal model systems is facilitated by the present invention, which enables the development of polypeptide ligands that can cross-react with human and animal targets.;

[0076] The present invention will be further described below with reference to the following examples.;

Examples

[0077] (Example) (Materials and Methods) (Peptide Synthesis) The peptide was synthesized by solid-phase synthesis. Rink amide MBHA resin was used. DMF was added to a mixture containing Rink amide MBHA (0.4 - 0.45 mmol / g) and Fmoc-Cys(Trt)-OH (3.0 equivalents), and then DIC (3 equivalents) and HOAt (3 equivalents) were added and mixed for 1 hour. 20% piperidine in DMF was used for deblocking. Each subsequent amino acid was coupled in 3 equivalents using the activator reagents DIC (3.0 equivalents) and HOAT (3.0 equivalents) in DMF. The reaction was monitored by ninhydrin color reaction or tetrachloride color reaction. After completion of the synthesis, the peptide resin was washed with DMF×3 and MeOH×3, and then 2 dried overnight under bubbling. Then, the peptide resin was treated with 92.5% TFA / 2.5% TIS / 2.5% EDT / 2.5% H 2 O for 3 hours. The peptide was precipitated with cold isopropyl ether and centrifuged (3000 rpm for 3 minutes). The pellet was washed twice with isopropyl ether, and the crude peptide was dried under vacuum for 2 hours and then lyophilized. The lyophilized powder was dissolved in ACN / H 2 O (50:50), an ACN solution of 100 mM TATA was added, and then ammonium bicarbonate (1 M) in H 2 O was added and the solution was mixed for 1 hour. When cyclization was completed, the reaction solution was quenched with 1 M aqueous cysteine hydrochloride (10 equivalents relative to TATA), then mixed and allowed to stand for 1 hour. Freeze-drying the solution gave a crude product. The crude peptide was purified by preparative HPLC and lyophilized to give the product.

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

[0079] (Biological data) Peptides without a fluorescent tag were tested by competing with a peptide having a fluorescent tag and a known Kd. The fluorescent tracer used had the sequence [Chemical formula] (Here, Fl is 5 / 6-carboxyfluorescein and Sar is sarcosine) of BCY90 (Kd = 2 nM).

[0080] The peptide was diluted to an appropriate concentration in assay buffer as described in a direct binding assay using up to 5% DMSO and then serially diluted 1:2. 5 μL of the diluted peptide was added to the plate, followed by 10 μL of human EphA2 at a concentration of 25 nM and then 10 μL of the fluorescent peptide (final concentration 0.8 nM). Measurements were taken, and the gain was determined prior to the first measurement. Data analysis was performed with Systat Sigmaplot version 12.0, in which case the mP values were fitted to a user-defined cubic equation to give the Ki value: f = ymax+(ymin - ymax) / Lig*((Lig*((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*Kcomp)-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 - 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*Kcomp)-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 - Prot*c))))。 「Lig」, 「KLig」, and 「Prot」 were all defined values related to the fluorescent peptide concentration, Kd of the fluorescent peptide, and EphA2 concentration, respectively.

[0081] The specific bicyclic peptides of the present invention were tested in the above-described competitive binding assay. The results are shown in Table 1: Table 1: Competitive Binding Assay of Selected Bicyclic Peptides of the Present Invention [Table 1] TIFF0007682852000013.tif255170 This application provides an invention in the following aspects. (Aspect 1) A polypeptide ligand specific for EphA2, comprising at least three reactive groups separated by at least two loop arrays and a molecular scaffold that is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one that forms a covalent bond with the reactive groups of the polypeptide, resulting in the formation of at least two polypeptide loops on the molecular scaffold, wherein the peptide ligand is (Chemical Formula 1) TIFF0007682852000014.tif190170TIFF0007682852000015.tif246170TIFF0007682852000016.tif245170TIFF0007682852000017.tif124170 (where Ac represents acetyl, HyP represents hydroxyproline, HArg represents homoarginine, PYA represents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, 1Nal represents 1-naphthylalanine, NMeAla represents N-methyl-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 4ThiAz represents β-(4-thiazolyl)-alanine, Thi represents 2-thienyl-alanine, 3Thi represents 3-thienylalanine, palmitoyl-Glu-LysN 3 represents N2-((S)-4-carboxy-4-palmitamidobutanoyl)-N6-diazo-L-lysine: (Chemical Formula 2) TIFF0007682852000018.tif106170 represents p-carboxy-phenylalanine, hGlu represents homoglutamic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, Nle represents norleucine, and [MerPro] i 、C i 、C ii 、C iii , and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, 3-mercaptopropionic acid (MerPro), and cysteamine (Cysam)) : an amino acid sequence selected from, or a pharmaceutically acceptable salt thereof, the peptide ligand specific for EphA2 as described above. (Aspect 2) (Chemical Formula 3) TIFF0007682852000019.tif14170 The peptide ligand according to Aspect 1, or a pharmaceutically acceptable salt thereof. (Aspect 3) (Chemical Formula 4) TIFF0007682852000020.tif15170 The peptide ligand according to Aspect 1, or a pharmaceutically acceptable salt thereof. (Aspect 4) The peptideligand according to any one of aspects 1 to 3, wherein the pharmaceutically acceptable salt is selected from the free acid or sodium, potassium, calcium, ammonium salts. (Aspect 5) The peptideligand according to any one of aspects 1 to 4, wherein the EphA2 is human EphA2. (Aspect 6) A pharmaceutical composition comprising the peptideligand according to any one of aspects 1 to 5 in combination with one or more pharmaceutically acceptable excipients. (Aspect 7) The peptideligand according to any one of aspects 1 to 6 for use in the prevention, suppression, or treatment of a disease or disorder characterized by overexpression of EphA2 in diseased tissue. (Aspect 8) The peptideligand according to any one of aspects 1 to 7 for use in the prevention, suppression, or treatment of cancer. (Aspect 9) The peptideligand for use according to aspect 8, wherein the cancer is selected from prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), breast cancer (e.g., triple-negative breast cancer), gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma, and fibrosarcoma.

Claims

1. A peptide ligand specific for EphA2, comprising a polypeptide containing at least three reactive groups separated by at least two loop arrays, and a molecular scaffold that is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one and forms a covalent bond with the reactive groups of the polypeptide, whereby at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide is 【Chemical 1】 【Chem.】 [Chemical] 【Chem.】 (Here, Ac represents acetyl, HyP represents hydroxyproline, HArg represents homoarginine, PYA represents 4-pentenoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, 1Nal represents 1-naphthylalanine, NMeAla represents N-methyl-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 4ThiAz represents β-(4-thiazolyl)-alanine, Thi represents 2-thienyl-alanine, 3Thi represents 3-thienylalanine, Nle represents norleucine, and [MerPro] i , C i , C ii , C iii , and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, 3-mercaptopropionic acid (MerPro), and cysteamine (Cysam)) an amino acid sequence selected from: or a pharmaceutically acceptable salt thereof, the peptide ligand specific for EphA2.

2. The polypeptide is [Chemical Formula 3] or a pharmaceutically acceptable salt thereof, the peptide ligand according to Claim 1.

3. The pharmaceutically acceptable salt is selected from sodium, potassium, calcium, and ammonium salts, the peptide ligand according to any one of Claims 1 to 2.

4. The EphA2 is human EphA2, the peptide ligand according to any one of Claims 1 to 3.

5. The peptide ligand according to any one of Claims 1 to 4, further comprising a cytotoxic agent, a radioactive chelating agent, or a chromophore.

6. A pharmaceutical composition comprising the peptide ligand according to any one of Claims 1 to 5.

7. A medicament for preventing, suppressing, or treating a disease or disorder characterized by overexpression of EphA2 in diseased tissue, comprising, as an active ingredient, the peptide ligand according to any one of Claims 1 to 5.

8. A medicament for preventing, suppressing, or treating cancer, comprising, as an active ingredient, the peptide ligand according to any one of Claims 1 to 5.

9. The cancer is selected from prostate cancer, lung cancer, breast cancer, gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma, and fibrosarcoma, the medicament according to Claim 8.

10. The lung cancer is non-small cell lung cancer (NSCLC), and / or the breast cancer is triple-negative breast cancer, the medicament according to Claim 9.

11. Use of the peptide ligand according to any one of Claims 1 to 5 in the manufacture of a medicament for preventing, suppressing, or treating a disease or disorder characterized by overexpression of EphA2 in diseased tissue. Use of the peptide ligand according to any one of claims 1 to 5 in the manufacture of a medicament for preventing, suppressing or treating cancer. Use according to claim 12, wherein the cancer is selected from prostate cancer, lung cancer, breast cancer, gastric cancer, ovarian cancer, esophageal cancer, multiple myeloma, and fibrosarcoma. Use according to claim 13, 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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