Bicyclic peptide ligands specific for psma

Abstract

To provide peptides which are high affinity binders of prostate-specific membrane antigen (PSMA).SOLUTION: The present invention relates to polypeptides which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of prostrate specific membrane antigen (PSMA). The invention also includes drug conjugates comprising the peptides, conjugated to one or more effector and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and the use of the peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder mediated by PSMA.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides a method for preparing a peptide having two or more peptide loops interspersed between attachment points to a scaffold. In particular, the present invention relates to a polypeptide covalently attached to a molecular scaffold. Peptides that are high affinity binders of prostate specific membrane antigen (PSMA) are described. a drug comprising said peptide conjugated to one or more effectors and / or functional groups; and a pharmaceutical composition comprising said peptide ligand and drug conjugate. and the use of said peptide ligases in the prevention, inhibition, or treatment of a disease or disorder mediated by PSMA. This also includes the use of bonds and drug conjugates. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are capable of binding to protein targets with high affinity and target specificity; It is therefore an attractive class of molecules for therapeutic development. Indeed, several cyclic peptides The tides include, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer 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 of the peptide and the target The conformational flexibility of the ring structure as well as the relatively large interaction surface formed between the ring and the target Generally, the macrocycle is, for example, the cyclic peptide CXCR4 antagonist CVX 15(400Å 2 (Wu et al., 2007), Science 330, 1066-71), A, which binds to integrin αVb3 Cyclic peptide with rg-Gly-Asp motif (355 Å 2 )(Xiong et al. (2002), Science 296( 5565), 151-5), or a cyclic peptide that binds to urokinase-type plasminogen activator Inhibitor upain-1 (603Å 2 (Zhao et al., 2007), J Struct Biol 160(1), 1-10) , binding to surfaces of several hundred square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides. This results in a smaller entropy loss upon binding to the target, resulting in higher binding The reduced flexibility also leads to the fixation of the target-specific conformation, and the linear peptide This effect is due to the fact that when the ring is opened, it binds to other MM peptides. A potent and selective matrix metalloproteinase 8 (MMP-8) loses its selectivity for P This has been 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-containing drugs. In polycyclic peptides with multiple peptide rings, such as actinomycin and 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 have reported that tris(bromomethyl)benzene and related molecules Synthesis 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 is a cysteine-containing polypeptide, e.g., tris(bromomethyl (b) by linking to a molecular scaffold such as benzene The method is disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] Creating 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 chains were then covalently attached to a small molecule scaffold to effect cyclization. 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 cysteine ​​residues and a polypeptide having the cysteine ​​residues The present invention relates to a method for producing a polypeptide comprising the steps of: A peptide ligand specific for PSMA is provided, on which a droop is formed on the molecular scaffold. Provided.

[0007] According to a further aspect of the invention, the compound is conjugated to one or more effectors and / or functional groups. and a drug conjugate comprising the peptide ligand as defined herein. .

[0008] According to a further aspect of the invention, a peptide ligand or drug conjugate as defined herein is Pharmaceutical compositions are provided that include the adjugate in combination with one or more pharma- ceutical acceptable excipients. can be.

[0009] According to a further aspect of the present invention, a method for preventing, inhibiting, or preventing a disease or disorder mediated by PSMA is provided. or a peptide ligand or drug conjugate as defined herein for use in therapy. A gate is provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention In one embodiment, the loop sequence comprises 2 or 7 amino acids.

[0011] In a further embodiment, the loop sequence is one of two amino acids, The other has three cysteines separated by two loop sequences of seven amino acids. Contains amine residues.

[0012] In one embodiment, the peptide ligand is [ka] (wherein X represents any amino acid residue, C i , C ii , and C iii are the first and second and a second and third cysteine ​​residue): Including acceptable salts.

[0013] In one embodiment, the peptide ligand is [ka] (wherein X represents any amino acid residue, Nle represents norleucine, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively): or a pharma- ceutically acceptable salt thereof.

[0014] In a further embodiment, [ka] The peptide ligands are represented by SEQ ID NOs: 1 to 14: [ka] (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. and Nle represents norleucine) or a pharmaceutical composition thereof. This includes pharma- ceutically acceptable salts.

[0015] In a further embodiment, [ka] The peptide ligand is A-(SEQ ID NO:1)-A (referred to herein as 21-31-N002); Ac-(SEQ ID NO:1) (referred to herein as 21-31-N003); (SEQ ID NO:1)-AGASPAAPSAPP (referred to herein as 21-31-N004; SEQ ID NO:17); (SEQ ID NO:2)-A (referred to herein as 21-31-01-N001); (SEQ ID NO:3)-A (referred to herein as 21-31-02-N001); (SEQ ID NO:4)-A (referred to herein as 21-31-03-N001); EV-(SEQ ID NO:5)-A (referred to herein as 21-31-04-T01-N001); (D-Gln)V-(SEQ ID NO:5)-A-Sar6-K (referred to herein as 21-31-04-T01-N002); β-Ala-Sar5-EV-(SEQ ID NO:5) (referred to herein as 21-31-04-T01-N003); Ac-(D-Gln)-V-(SEQ ID NO:5)-A-Sar6-K-Ac (referred to herein as 21-31-04-T01-N004) )); Ac-(D-Gln)-V-(SEQ ID NO:6)-A-Sar6-K (referred to herein as 21-31-04-T01-N005) ); Ac-(D-Gln)-V-(SEQ ID NO:5)-A-Sar6-(D-Lys) (referred to herein as 21-31-04-T01-N007) will be)); Ac-(D-Gln)-V-(SEQ ID NO: 7)-A-Sar6-(D-Lys) (referred to herein as 21-31-04-T01-N008) will be)); Ac-(D-Gln)-V-(SEQ ID NO:8)-A-Sar6-(D-Lys) (referred to herein as 21-31-04-T01-N009) will be)); Ac-(D-Gln)-V-(SEQ ID NO:9)-A-Sar6-(D-Lys) (referred to herein as 21-31-04-T01-N010) will be); SV-(SEQ ID NO:10)-A (referred to herein as 21-31-07-T01-N001); F-(SEQ ID NO:11)-A (referred to herein as 21-31-11-T01-N001); L-(SEQ ID NO:12)-A (referred to herein as 21-31-12-T01-N001); (D-Val)-(SEQ ID NO:13)-A-Sar6-K (referred to herein as 21-31-14-T01-N002); β-Ala-Sar5-V-(SEQ ID NO: 13)-A-Sar6-K (referred to herein as 21-31-14-T01-N003) ; Ac-(SEQ ID NO:1)-A-Sar6-K (referred to herein as 21-31-N005); β-Ala-Sar5-A-(SEQ ID NO:1) (referred to herein as 21-31-N006); Ac-(SEQ ID NO:14)-A-Sar6-K (referred to herein as 21-31-N007); and β-Ala-Sar5-A-(SEQ ID NO:14) (referred to herein as 21-31-N008) The amino acid sequence is selected from:

[0016] In a further embodiment, [ka] The peptide ligand is EV-(SEQ ID NO:5)-A (referred to herein as 21-31-04-T01-N001); Ac-(D-Gln)-V-(SEQ ID NO:8)-A-Sar6-(D-Lys) (referred to herein as 21-31-04-T01-N009) will be)); F-(SEQ ID NO:11)-A (referred to herein as 21-31-11-T01-N001); and L-(SEQ ID NO:12)-A (referred to herein as 21-31-12-T01-N001) The peptide ligand of this embodiment comprises an amino acid sequence selected from the group consisting of: , which showed excellent PSMA binding as shown in Table 1.

[0017] 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 commonly understood by those skilled in the art. are used in methods of molecular biology, genetics, and biochemistry (incorporated herein by reference). 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).

[0018] (Nomenclature) (Numbering) When referring to amino acid residue positions within the peptides of the invention, a cysteine ​​residue (C i , C ii , and C iii ) are invariant, they are omitted from the numbering and therefore are not included in the peptides of the present invention. The numbering of amino acid residues in is referred to as follows: -C i -M1-V2-C ii -H3-M4-M5-E6-D7-G8-T9-C iii -(SEQ ID NO:1) .

[0019] For the purposes of this description, all bicyclic peptides are designated TBMB (1,3,5-tris(bromomethyl)benzene). It is believed that TBMB is cyclized with 1,3,5-trimethylbenzene to give a trisubstituted 1,3,5-trimethylbenzene structure. Cyclization by C i , C ii , and C iii This occurs above.

[0020] (Molecular Format) N- or C-terminal extensions to the bicyclic core sequence may be placed to the left or right of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO:X) This is expressed as:

[0021] (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, it is envisaged that the sequence may be reversed (i.e., the N-terminus becomes the C-terminus, and so on). the terminus becomes N-terminus), and the stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids). acids, and L-amino acids become D-amino acids).

[0022] (Peptide Ligand) The peptide ligands referred to herein are covalently attached to a molecular scaffold. Typically, such peptides are covalently bonded to a scaffold. The peptide has two or more reactive groups (i.e., cysteine ​​residues) that can form a sucrose residue. The sequence between the reactive groups is called a loop sequence because it forms a loop when it binds to the nucleic acid structure. In this case, the peptide contains at least three cysteine ​​residues (as in the present invention). In the fine print, C i , C ii , and C iii (called the "scaffold") and Both form two loops.

[0023] (Advantages of peptide ligands) Certain bicyclic peptides of the present invention can be administered by injection, inhalation, nasal, ocular, oral, or topical administration. These compounds 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. The bicyclic peptide ligands are ideally suited to inhibit plasma proteases, epidermal ("membrane-anchored") proteases, gastrointestinal proteases, pulmonary surface proteases, intracellular proteases Protease stability should be demonstrated for bicyclic lead candidates. Not only can we develop them in animal models, but we can also confidently administer them to humans. should be maintained among different species; - Desirable solubility profile. This is important for formulation and absorption purposes, as it does not contain charged residues and and the ratio of hydrophilic to hydrophobic residues and intra- / inter-molecular H-bonds; - Optimal plasma half-life in the circulation. Depending on clinical indications and treatment regimens, acute disease management settings Develop bicyclic peptides for short-term exposure at constant doses or with enhanced retention in the circulation, thus It may be necessary to develop bicyclic peptides that are more optimal for managing chronic disease states. Other factors driving a desirable plasma half-life are sustained exposure for maximum therapeutic efficacy and drug durability. The requirement for concomitant toxicity due to continued exposure; and -Selectivity: Certain peptide ligands of the invention exhibit better selectivity than other membrane antigens.

[0024] (Pharmaceutical acceptable salts) Salt forms are within the scope of the present invention and a reference to a peptide ligand includes the salt form of that ligand. It will be understood that

[0025] The salts of the present invention can be prepared in a manner similar to that described in 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 that contains a basic or acidic moiety by the following methods: Typically, such salts are prepared by reacting the free acid or base form of these compounds with an appropriate base or acid and water. They can be prepared by reacting them in aqueous or organic solvents, or in a mixture of the two. This can be done.

[0026] 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, ascorbyl acetate, and the like. Ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid 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 Hydrochloric 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, oro Tonic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyrogenic acid 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, undecylenic acid and valeric acid, and acylated amino acids and cation exchange resins. Examples of the salt include mono-salts and di-salts formed with acids.

[0027] 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 a salt formed from biotic acid. One particular salt is the hydrochloride salt. Another particular salt is , acetate.

[0028] The compound is anionic or has a functional group which may be anionic (e.g., -CO OH is -COO - In the case where the cation is a cation of the formula (I), the salt is formed with an organic or inorganic base to provide a suitable cation. 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 + And more 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 are methylamine, ethylamine, diethylamine, 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: N(C H3)4 + It is.

[0029] When the peptides of the invention contain amine functions, these can be, for example, substituted or unsubstituted amines, according to methods well known to those skilled in the art. By reaction with alkylating agents according to the method, quaternary ammonium salts can be formed. Such quaternary ammonium compounds are within the scope of the peptides of the present invention.

[0030] (Modified derivative) Modified derivatives of the peptide ligands defined herein are considered to be within the scope of the present invention. It will be appreciated that suitable examples of such modified derivatives include N-terminal and / or C-terminal End modifications; substitution of one or more amino acid residues with one or more non-naturally occurring amino acid residues (e.g., one or more Substitution of one or more polar amino acid residues with isosteric or isoelectronic amino acids; replacement of the amino acid residues with other non-natural isosteric or isoelectronic amino acids; addition of spacer groups; 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 at one or more amide bonds in the bicyclic peptide ligand; N-alkylation of one or more peptide Replacement of doped bonds with surrogate bonds; modification of peptide backbone length; modification of the α-carbon of one or more amino acid residues replacement of hydrogen with another chemical group, cysteine, lysine, glutamic acid / aspartic acid, and the appropriate amine, thiol, etc. of the amino acid, such as tyrosine, to functionalize the amino acid. Direct modification and functionalization with phenol-, carboxylic acid-, and phenol-reactive reagents The amino acid that introduces the cross-linking activity, e.g., an alkyne or azide-containing moiety, respectively, may be 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 modifications:

[0031] 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.

[0032] 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 cyclic group (referred to herein as C i The group called the aryl group is reacted with acetic anhydride or other suitable This embodiment results in a molecule that is capped with a reagent and acetylated at the N-terminus. This offers the advantage of eliminating potential recognition points for aminopeptidases, and is a promising method for the synthesis of bicyclic peptides. Avoid possible decomposition.

[0033] In an alternative embodiment, the N-terminal modification comprises conjugation of an effector group and Includes the addition of a molecular spacer group that facilitates retention of potency of the bicyclic peptide against its target. .

[0034] 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 C-terminated group, called 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 decrease.

[0035] 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 substitution includes a group that is recognized by a degradative protease. have isosteric / isoelectronic side chains that are not toxic or have any adverse effect on targeting efficacy Unnatural amino acids may also be selected.

[0036] Alternatively, proteolytic hydrolysis of nearby peptide bonds may result in conformational and steric To be disruptive, unnatural amino acids with constrained amino acid side chains may be used. In particular, these include proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisopropyl ethers, butyric acid, Aib), and cycloamino, a simple derivative of amino-cyclopropyl carboxylic acid. Concerning acids.

[0037] In one embodiment, the modified derivative comprises the addition of a spacer group. In the present invention, the modified derivatives are i ) and / or a C-terminal cysteine ​​(C iii ) to This includes the addition of a spacer group.

[0038] In one embodiment, the modified derivatives include one or more oxidation-resistant modifications of one or more oxidation-sensitive amino acid residues. In a further embodiment, the modified derivative comprises a substitution with a tripeptide-binding amino acid residue. This embodiment includes the replacement of the tophan residue with a naphthylalanine or alanine residue. The resulting bicyclic peptide ligands provide the advantage of improved pharmaceutical stability profiles. do.

[0039] In one embodiment, the modified derivatives include one or more hydrophobic amino acids of one or more charged amino acid residues. In an alternative embodiment, the modified derivatives include one or more hydrophobic acid residues. The amino acid residues are replaced by one or more charged amino acid residues. A proper 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. This affects the concentration of the isolated fraction, whereas charged amino acid residues (especially arginine) affect the concentration of the peptide. The combination of these two may affect the interaction of the phospholipid membranes on the cell surface. This may affect the half-life, volume of distribution, and exposure of peptide drugs, and may have clinical implications. In addition, the amount of charged and hydrophobic amino acids can be adjusted according to the endpoint. The appropriate combination and number of residues reduces irritation at the injection site (if the peptide drug is administered subcutaneously). It can be reduced.

[0040] In one embodiment, the modified derivative comprises one or more D-amino acid residues of one or more L-amino acid residues. This embodiment includes 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).

[0041] 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.

[0042] Each of the above modifications serves to purposefully improve the potency or stability of the peptide. It should be noted that further improvement in potency based on the modification is achieved by the following mechanism: Can: -Utilizes the hydrophobic effect, resulting in a lower dissociation rate so that higher affinity is achieved Incorporating a hydrophobic moiety; -Long-range ionic interactions are utilized, resulting in faster association rates and higher affinity Incorporating charged groups into proteins (e.g., Schreiber et al., Rapid Electrostatic Assisted Synthesis of Proteins) Rapid, electrostatically assisted association of proteins (1996), Nature St. ruct. Biol. 3, 427-31); and - For example, the side chains of amino acids are appropriately oriented so that entropy loss is minimized upon target binding. and constraining the torsion angles of the backbone 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, 31 85-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0043] (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 amounts commonly found in nature. is replaced by an atom having a different atomic mass or mass number from that of the present invention. All known pharma- ceutically acceptable (radio)isotope-labeled peptide ligands, as well as related The peptide of the present invention is provided with a metal chelating group capable of carrying a (radioactive) isotope. A peptide ligand (called an "effector"), as well as a specific functional group associated with it (radioactive Peptides of the invention covalently substituted with isotopes or isotopically labeled functional groups Contains a ligand.

[0044] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include 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, Galli Isotopes of uranium, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y and Ru Isotopes of tethium, e.g. 177 Lu, as well as isotopes of bismuth, e.g. 213 Contains Bi.

[0045] Certain isotopically labeled peptide ligands of the invention, e.g., incorporate a radioisotope. These include in drug and / or substrate tissue distribution studies and in the presence of PSMA targets on diseased tissues. The peptide ligands of the present invention are useful for clinically assessing the presence and / or absence of Formation of a complex between a labeled compound and another molecule, peptide, protein, enzyme, or receptor It also has valuable diagnostic properties in that it can be used to detect or identify Detection or identification methods may include, for example, radioisotopes, enzymes, fluorescent substances, luminescent Luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferin) Compounds that are labeled with a labeling agent such as a radioisotope may be used. Tritium in the body, i.e. 3 H(T) and carbon-14, i.e. 14 C is easy to implement It is particularly useful for this purpose given the means for detection and detection available.

[0046] 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.

[0047] 11 C. 18 F, 15 O, and 13 Substitution with positron emitting isotopes such as N is a promising method to investigate target occupancy. It may be useful in positron emission tomography (PET) studies for

[0048] Isotopically labeled compounds of the peptide ligands of the present invention are typically prepared using conventional techniques known to those of skill in the art. or by using appropriate isotopically labeled reagents instead of the previously utilized non-labeled reagents. These compounds can be prepared by processes similar to those described in the accompanying examples.

[0049] (Molecular scaffolds) Molecular scaffolds can be prepared, for example, as described in WO 2009 / 098450 and the references cited therein. It is described in the literature, in particular in WO 2004 / 077062 and WO 2006 / 078161.

[0050] As described in the aforementioned documents, molecular scaffolds are small molecules, such as small organic molecules. It may be a child.

[0051] In one embodiment, the molecular scaffold may be a polymer. In the present invention, the molecular scaffold is composed of amino acids, nucleotides, or carbohydrates. It is a polymer.

[0052] In one embodiment, the molecular scaffold reacts with a functional group of the polypeptide to form a covalent bond. It contains a reactive group capable of forming a bond.

[0053] The molecular scaffold contains chemical groups that form bonds with peptides, e.g., amines, thiol groups, etc. alcohols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, Alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and halides. It may contain acyl halogenides.

[0054] In one embodiment, the molecular scaffold is tris(bromomethyl)benzene, particularly , 1,3,5-tris(bromomethyl)benzene ("TBMB"), or a derivative thereof; Or it may consist of these.

[0055] In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesityle. The molecule is similar to 1,3,5-tris(bromomethyl)benzene, but It contains three additional methyl groups attached to the cyclic ring. This is because the additional methyl groups The advantage of this is that it can form additional contacts with the nuclei and therefore provide additional structural constraints. Yes.

[0056] The molecular scaffolds of the invention are capable of binding to the polypeptides of the encoded libraries of the invention. The functional group of the amide comprises a chemical group that enables the functional group to form a covalent bond with the molecular scaffold. The chemical groups include amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylates, and carboxylates. carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides The functional groups may be selected from a wide range of groups including alkyl, alkyl halides, and acyl halides.

[0057] Can be used on molecular scaffolds to react with cysteine ​​thiol groups The scaffold reactive groups that can be used are alkyl halides (or halogenoalkanes or (also called haloalkanes).

[0058] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups that can be used to couple are maleimide, αβ-substituted saturated carbonyl-containing compounds, and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimides) and tris-(2-maleimides). Imidoethyl)amine, Tris-(2-maleimidoethyl)benzene, Tris-(maleimido)benzene Examples of αβ-unsaturated carbonyl-containing compounds include 1,1',1''-(1,3,5-triphenylphosphine) (1,3,5-diamino-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, In International Edition(2014), 53(6), 1602-1606). α-Halomethylcarbonyl-containing compounds An example is N,N',N''-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Lenocysteine ​​is a natural amino acid with similar reactivity to cysteine ​​and can be involved in the same reactions. Therefore, whenever cysteine ​​is mentioned, the context Unless otherwise indicated, selenocysteine ​​is generally permitted to be substituted. will be done.

[0059] (Effectors and Functional Groups) According to a further aspect of the invention, the compound is conjugated to one or more effectors and / or functional groups. and a drug conjugate comprising the peptide ligand as defined herein. .

[0060] The effector and / or functional group can be, for example, a polypeptide at the N- and / or C-terminus of the polypeptide. It can be attached to an amino acid within a polypeptide or to a molecular scaffold.

[0061] Suitable effector groups include antibodies and portions or fragments thereof. For example, the effector group In addition to one or more constant region domains, the antibody light chain constant region (CL), antibody CH1 heavy chain domain, The antibody may comprise an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof. The effector group is attached to the hinge region of the antibody (between the CH1 and CH2 domains of the IgG molecule). Such regions may include those commonly found in

[0062] In a further embodiment of this aspect of the invention the effector group according to the invention is Advantageously, the peptide ligand-effector group according to the invention is tβ half-life of ≥ 2 days, ≥ 3 days, ≥ 4 days, ≥ 5 days, ≥ 6 days, or ≥ 7 days Most preferably, the peptide ligand Fc fusion comprises or consists of a peptide ligand Fc fusion having the The peptide ligand of the present invention is a peptide ligand Fc fusion protein having a tβ half-life of one day or more. It comprises or consists of a compound.

[0063] Functional groups generally include linking groups, reactive groups for the attachment of drugs or other entities, macrocyclic phenyl groups, and the like. and functional groups that aid in the uptake of the peptide into cells.

[0064] The ability of a peptide to penetrate into cells determines the effectiveness of the peptide against its intracellular target. This allows the target to be accessed by peptides that have the ability to penetrate into cells. In particular, transcription factors, tyrosine kinases, and other intracellular signaling molecules, as well as the apoptotic pathway, are involved in the pathogenesis of apoptosis. Functional groups that allow cell penetration include peptides or These include chemical groups attached to either the peptide or the molecular scaffold. See, for example, Chen and Harrison, Biochemical Society Transactions (2007), Vol. 35, no. 4, p. 821; Gupta et al., Advanced Drug Discovery Reviews (2004), Vol. 57, p. 9637 For example, VP22, HIV-Tat, Drosophila homeobox protein ( Peptides such as those derived from the ubiquitin complex (Antennapedia) that are efficient at moving through cell membranes Examples of short peptides that have been shown to The 16 amino acid penetratin peptide derived from protein (Derossi et al., 1994, J. Biol. Chem. 2003, 144:1311-1323) m. vol. 269, p. 10444), and the 18 amino acid "model amphipathic peptide" (Oehlke et al., 1998 ) Biochim Biophys Acts, Vol. 1414, p. 127) The arginine-rich region of the HIV TAT protein Non-peptide approaches include the use of peptides that can be easily attached to biomolecules. Examples include the use of small molecule mimetics, or SMOCs, that can s, vol. 4, p. 153). Another chemical strategy, adding guanidinium groups to molecules, also enhances cell penetration. (Elson-Scwab et al., 2007, J. Biol. Chem., vol. 282, p. 13585). Steroids, etc. It is possible to add low molecular weight molecules to the molecular scaffold to enhance their uptake into cells. can.

[0065] One class of functional groups that can be attached to peptide ligands is antibodies and Binding fragments thereof include, for example, Fab, Fv, or single domain fragments. In particular, peptides Using antibodies that bind to proteins that can increase the half-life of ligands in vivo It is possible.

[0066] In one embodiment, a peptide ligand effector group according to the invention is administered for: 12 hours or more; 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more Above, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, or 20 days or more Advantageously, the peptide ligand-enantiomer according to the invention has a tβ half-life selected from the group consisting of: The effector group or composition has a tβ half-life ranging from 12 hours to 60 hours. In an embodiment, it has a tβ half-life of 1 day or more. It ranges from 12 to 26 hours.

[0067] In one particular embodiment of the invention, the functional group complexes a medicamentously relevant metal radioisotope. The metal chelating agent is selected from those suitable for chelating the metal.

[0068] Possible effector groups include, for example, peptide ligands that act as substitutes for antibodies in ADEPT. Alternative Enzyme / Prodrug Therapy for Use with Carboxypeptidase G2 and Other Enzymes Any enzyme can be mentioned.

[0069] In one particular embodiment of the invention, the functional group is a cytotoxic agent, such as a cytotoxic agent for cancer therapy. Suitable examples include: cisplatin and carboplatin, and oxaliplatin. Saliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide alkylating agents such as the purine analogs azathioprine and mercaptopurine or pyrimidinyl Antimetabolites, including vinoxacin analogs; vincristine, vinblastine, vinorelbine, and vinoxacin analogs. Plant alkaloids and terpenoids, including vinca alkaloids such as desine; podophyllo Toxin and its derivatives etoposide and teniposide; originally known as taxol , taxanes, including paclitaxel; topoisomerase inhibitors, including camptothecin; Notecan and Topototecan, as well as Amsacrine, Etoposide, Etoposide Phosphate, and Further drugs include the immunosuppressant dactyly. cerebrospinal fluid (used in kidney transplants), doxorubicin, epirubicin, bleomycin, Examples of such antitumor antibiotics include cyclosporine, calicheamicin, and the like.

[0070] In one further specific embodiment of the invention, the cytotoxic agent is a maytansinoid (e.g. For example, DM1) or monomethyl auristatin (for example, MMAE).

[0071] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure: do: [ka] .

[0072] Monomethylauristatin E (MMAE) is a synthetic anti-neoplastic agent having the following structure: [ka] .

[0073] In one embodiment, the cytotoxic agent is a cytotoxic agent that is a bond such as a disulfide bond or a protease sensitive bond. In a further embodiment, The groups adjacent to the disulfide bond are responsible for disruption of the disulfide bond and, therefore, cytotoxicity. The active agent may be modified to control the rate of cleavage and concomitant release of the active agent.

[0074] Published studies have demonstrated that by introducing steric hindrance on either side of the disulfide bond, has established the possibility of modifying the susceptibility of disulfide bonds to reduction (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance may result in increased cell It slows down the rate of reduction by endogenous glutathione and also by extracellular (systemic) reducing agents, resulting in This reduces the ease with which toxins are released both inside and outside the cell. , efficient release in the circulation versus the intracellular environment, which maximizes therapeutic efficacy. Optimal conditions for disulfide stability, which minimizes unwanted side effects of the toxin The selection of is determined by careful selection of the degree of disorder on either side of the disulfide bond. can be achieved.

[0075] Disorders on either side of the disulfide bond allow the targeting entity (here, bicyclic peptide by introducing one or more methyl groups into either the cytosine thiol (peptide) or the toxin side of the molecular construct. The voltage is adjusted accordingly.

[0076] In one embodiment, the cytotoxic agent and the linker are described in WO 2016 / 067035. (The cytotoxic agents and their linkers are incorporated herein by reference in their entirety.) (Incorporated herein).

[0077] (synthesis) The peptides of the invention can be synthetically produced by standard techniques and then subjected to in vitro molecular sequencing. This can be accomplished using standard chemistries. This allows for rapid release of soluble material for further downstream experimentation or validation. Such a method is disclosed in Timmerman et al. (supra). This can be accomplished using conventional chemistries such as those described in

[0078] Thus, the present invention also relates to 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 by chemical synthesis. The procedure is carried out on the final product polypeptide / conjugate produced by the method of the present invention.

[0079] Optionally, the amino acid residues in the polypeptide of interest are capable of forming a conjugate or complex. They may be replaced when manufacturing.

[0080] The peptide can be extended, for example, to incorporate additional loops and thus introduce multiple specificities. You can also enter.

[0081] To extend the peptide, it is simply performed using standard solid or solution phase chemistry: Orthogonally protected lysines (and analogues) 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, for example, by introducing a modified or activatable N- or C-terminus. awson et al., 1994, Synthesis of Proteins by Native Chemical Ligation (Synthesis of Proteins by Native Chemical Ligation). Hesis of Proteins by Native Chemical Ligation. Science 266:776-779) by fragment condensation or native chemical ligation, as described, for example, in Chang et al. Proc Natl Acad Sci US 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 carried out enzymatically using subtiligase, as described in US Pat. No. 6,319,992 (pp. 6000-6003).

[0082] Alternatively, the peptides can be conjugated via further disulfide bonds. This is because the first and second peptides may be extended or modified by one another in the reducing environment of the cell. In this case, the molecular scaffold has the additional advantage of allowing the scaffold to dissociate into A peptide (e.g., TBMB) is added to the first peptide chemical synthesis 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 of a second It reacts only with free cysteines or thiols of the peptide to form disulfide-bonded bicyclic rings. A peptide-peptide conjugate of the formula was formed.

[0083] 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.

[0084] Furthermore, the addition of other functional or effector groups can be accomplished by N- or C-addition using appropriate chemistry. Coupling at the terminus or via a side chain may be accomplished 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.

[0085] Pharmaceutical Composition According to a further aspect of the invention, a peptide ligand or drug conjugate as defined herein is Pharmaceutical compositions are provided that include the adjugate in combination with one or more pharma- ceutical acceptable excipients. can be.

[0086] 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 The medium may include an aqueous or alcoholic / aqueous solution, an emulsion, or a suspension. Oral vehicles include sodium chloride solution, Ringer's dextrose, dextrose , and sodium chloride, and lactated Ringer's. Adjuvants, such as carboxymethylcellulose, may be used as needed to keep the polypeptide complex in suspension. From thickeners such as ethyl cellulose, polyvinylpyrrolidone, gelatin, and alginates may be selected.

[0087] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as Ringer's fluid replenishers, Also included are those based on tallow. They contain 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.

[0088] The peptide ligands of the invention may be administered either as a separate composition or in combination with other agents. These may be used include antibodies, antibody fragments, as well as various immunotherapeutic agents, e.g. , cyclosporine, methotrexate, adriamycin, or cisplatin, and immunotherapy Pharmaceutical compositions include a variety of toxins in combination with the protein ligands of the present invention. "Cocktails" of cytotoxic or other drugs, or drugs that are pooled prior to administration or pooled polypeptides selected using different targeting ligands, whether or not they are It may even include a combination of selected polypeptides according to the invention having different specificities. Cut.

[0089] The route of administration of the pharmaceutical composition according to the present invention may be any of those commonly 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. 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 on other medications. The clinical outcome is determined by the concurrent administration of drugs, contraindications, and other parameters considered by the clinician. circle.

[0090] The peptide ligands of the present invention may be lyophilized for 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. Freeze-drying and reconstitution techniques can be used. Freeze-drying and reconstitution can result in varying degrees of activity. This may result in losses and the levels may need to be adjusted upwards to compensate. It will be understood by those skilled in the art.

[0091] 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, regulation, 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 depends on the individual, but generally, 0.005-5.0 mg of the selected peptide ligase per kilogram of body weight is used. The range is from 0.05 to 2.0 mg / kg / dose, with doses of 0.05 to 2.0 mg / kg / dose being more commonly used. Therefore, compositions containing the peptide ligands or cocktails thereof may also be used to treat similar or similar A much smaller dosage may be administered.

[0092] Compositions containing peptide ligands according to the invention can be used in prophylactic and therapeutic settings. Assisting in the alteration, inactivation, killing, or elimination of selected target cell populations in mammals In addition, the peptide ligands described herein can be used in vitro or in vivo. selectively killing, depleting, or isolating a target cell population from a heterogeneous collection of cells The peptides can be effectively removed in other ways. The antibody can be combined in vitro with the antibody, which can then be administered to a mammal according to standard techniques. To return, the unwanted cells are killed or otherwise removed from the blood.

[0093] (therapeutic use) The bicyclic peptides of the invention have particular utility as PSMA binding agents.

[0094] Prostate-specific membrane antigen (PSMA) (glutamic acid carboxypeptidase II (GCPII), N-acetylglutamate N-L-aspartyl-L-glutamic acid peptidase I (NAALADase I), and NAAG peptidase In humans, folate hydrolase 1 (FOLH1) is encoded by the gene Human GCPII contains 750 amino acids and weighs approximately 84 kDa.

[0095] Human PSMA is highly expressed in the prostate, approximately 100-fold more than in most other tissues. In some prostate cancers, PSMA was the second most frequently upregulated gene product, and This is 8-12 times higher than the levels in cancerous prostate cells. It is being developed as a potential biomarker for treatment and imaging of several cancers. In human prostate cancer, tumors with higher expression are associated with faster progression times and larger It is associated with a high percentage of patients suffering from recurrence.

[0096] Polypeptide ligands selected by the methods of the invention can be used in in vivo therapeutic and prophylactic applications. It can be used for in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, etc. Ligands with a selected level of specificity can be used to target non-human immunocytes for which cross-reactivity is desired. For applications involving testing in human animals or where cross-reactivity with homologs or paralogs is of concern, It is useful in diagnostic applications where the concentration of the ion channel is carefully controlled. In this application, the ability to induce an immune response to a range of antigens is exploited to develop a vaccine. The treatment can be tailored to specific diseases and pathogens.

[0097] Substantially pure peptide ligands having at least 90-95% homogeneity are administered to mammals. A homogeneity of 98-99% or more is preferred for pharmaceutical use, particularly for administration to mammals. Once refined, either partially or until homogeneous, as desired, it is most preferred. Once produced, the selected polypeptides may be used diagnostically or therapeutically (including in vitro), or It can be used in the development and implementation of assay procedures, immunofluorescence staining, etc. (Lefkovi te and Pernis (1979 and 1981), Immunological Methods, Vols. I and II, Academic Press. ic Press, NY).

[0098] According to a further aspect of the invention, a method for preventing, inhibiting or treating a disease or disorder mediated by PSMA is provided. The present invention relates to a peptide ligand or drug conjugate as defined herein for use in therapy. A gate is provided.

[0099] According to a further aspect of the invention, a method for preventing, inhibiting or treating a disease or disorder mediated by PSMA is provided. The present invention relates to a method of treatment comprising administering to a patient in need thereof a peptide linker as defined herein. and administering to the subject a drug conjugate and an effector group thereof. do.

[0100] In one embodiment, the PSMA is mammalian PSMA. The animal PSMA is human PSMA.

[0101] In one embodiment, the disease or disorder mediated by PSMA is cancer, neuropathic and inflammatory. Inflammatory pain, head injury, stroke, schizophrenia, diabetic neuropathy, drug addiction, amyotrophic lateral sclerosis and neurodegenerative diseases such as Parkinson's disease and Huntington's disease.

[0102] In further embodiments, the PSMA-mediated disease or disorder is selected from cancer. do.

[0103] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include tumors of epithelial origin (adenocarcinoma Various types of adenomas and carcinomas, including squamous cell carcinoma, transitional cell carcinoma, and other carcinomas, such as 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 (e.g., thyroid follicles) cancer), adrenal gland, prostate, skin, and adnexal cancer (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, Myeloid leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large 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, multiple myeloma, and post-transplant lymphoproliferative disorders), and hematological malignancies and related disorders of the myeloid lineage (e.g. For example, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), Hypereosinophilic syndromes, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia); mesenchymal Tumor of origin, e.g., soft tissue, bone, or cartilage sarcoma, e.g., osteosarcoma, fibrosarcoma, Chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma , synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and protruding Dermatofibrosarcoma; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma) endocrine tumors (e.g., pituitary tumors, meningiomas, ependymomas, pineal tumors, and schwannomas); 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:

[0104] In further embodiments, the cancer is selected from prostate, breast, kidney, and colon cancer. .

[0105] In still further embodiments, the cancer is selected from prostate and breast cancer.

[0106] In yet a further embodiment, the cancer is selected from cancer of the prostate.

[0107] References herein to the term "prophylaxis" refer 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 clinical manifestation of the disease. "Treatment" includes administration of a protective composition after disease symptoms are manifest.

[0108] Screening for the efficacy of peptide ligands in protecting against or treating disease There are animal model systems available that can be used to study the effects of Allows for the development of polypeptide ligands that can cross-react with human and animal targets This is facilitated by the present invention.

[0109] The invention will now be further described with reference to the following examples. EXAMPLES

[0110] (Example) Materials and Methods (Peptide synthesis) Peptide synthesis was performed using a Symphony peptide synthesizer manufactured by Peptide Instruments. The synthesis was based on Fmoc chemistry using a MultiSynTech Syro II synthesizer. Acids (Sigma, Merck) with appropriate side-chain protecting groups were used: standard coupling Ring-protection conditions were used in each case, followed by deprotection using standard methodology. The peptide was purified and isolated using HPLC, and then dissolved in 1,3,5-tris(bromomethyl)benzene (T For this, the linear peptide was diluted to approximately 35 mL with H2O and added acetonitrile (BMB, Sigma). Add approximately 500 μL of 100 mM TBMB in nitrile and start the reaction with 5 mL of 1 M NH4HCO3 in HO. The reaction was allowed to proceed at RT for approximately 30-60 min and was then allowed to proceed until the reaction was complete (as determined by MALDI). After lyophilization, the modified peptide was purified as described above, while The Luna C8 was replaced with a Gemini C18 column (Phenomenex) and the acid was changed to 0.1% trifluoroacetic acid. Pure fractions containing the appropriate TBMB modified material were pooled, lyophilized, and stored for storage. It was kept at -20°C.

[0111] Unless otherwise noted, all amino acids were used in the L-configuration.

[0112] (Biological Data) (PSMA binding and enzyme inhibition assays) PSMA binding was analyzed using the fluorescence polarization assay described in Shen et al. (2013) PLOS ONE 8(7), e68339. The serum was measured using the Say method, and PSMA enzyme inhibition was measured using the method described in WO 2009 / 070302. Measured.

[0113] The peptide ligands of the invention were tested in the PSMA binding and enzyme inhibition assays described above. , as shown in Table 1: Table 1: Biological assay data for peptide ligands of the invention [Table 1] TIFF2025011099000010.tif241170TIFF2025011099000011.tif46170

Claims

1. 1. A peptide ligand specific for PSMA, or a pharma- tically acceptable salt thereof, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, the molecular scaffold is 1,3,5-tris(bromomethyl)benzene; and The polypeptide, 【Chemistry 1】 (wherein X represents any amino acid residue, Nle represents norleucine, and C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. comprising an amino acid sequence selected from However, the peptide ligand is not a peptide ligand consisting of EVCWDCFMMEDGTCA as a polypeptide and 1,3,5-tris(bromomethyl)benzene as a molecular scaffold, and is a PSMA-specific peptide ligand or a pharma- ceutical acceptable salt thereof.

2. The polypeptide is selected from SEQ ID NOs: 1-14: 【Chemistry 2】 (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively, and Nle represents norleucine.

2. The peptide ligand according to claim 1, or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from any one of the following:

3. The polypeptide, A-(SEQ ID NO:1)-A; Ac- (SEQ ID NO: 1); A-(SEQ ID NO:1)-AGASPAAPSAPP; (SEQ ID NO:2)-A; (SEQ ID NO:3)-A; (SEQ ID NO:4)-A; EV-(SEQ ID NO:5)-A; (DE)V-(SEQ ID NO:5)-A-Sar 6 -K; β-Ala-Sar 5 -EV- (SEQ ID NO: 5); Ac-(DE)-V-(SEQ ID NO:5)-A-Sar 6 -K-Ac; Ac-(DE)-V-(SEQ ID NO:6)-A-Sar 6 -K; Ac-(DE)-V-(SEQ ID NO:5)-A-Sar 6 -(DK); Ac-(DE)-V-(SEQ ID NO: 7)-A-Sar 6 -(DK); Ac-(DE)-V-(SEQ ID NO:8)-A-Sar 6 -(DK); Ac-(DE)-V-(SEQ ID NO:9)-A-Sar 6 -(DK); SV-(SEQ ID NO:10)-A; F-(SEQ ID NO:11)-A; L-(SEQ ID NO:12)-A; (DV)-(SEQ ID NO:13)-A-Sar 6 -K; β-Ala-Sar 5 -V- (SEQ ID NO: 13); Ac-(SEQ ID NO:1)-A-Sar 6 -K; β-Ala-Sar 5 -A- (SEQ ID NO: 1); Ac-(SEQ ID NO:14)-A-Sar 6 -K; and β-Ala-Sar 5 -A- (SEQ ID NO: 14) 3. The peptide ligand or a pharma- ceutical acceptable salt thereof according to claim 1 or 2, comprising an amino acid sequence selected from the group consisting of:

4. The polypeptide, EV-(SEQ ID NO:5)-A; Ac-(DE)-V-(SEQ ID NO:8)-A-Sar 6 -(DK); F-(SEQ ID NO:11)-A; and L-(SEQ ID NO:12)-A The peptide ligand or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3, comprising an amino acid sequence selected from the group consisting of:

5. The peptide ligand or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4, wherein the pharma- ceutical acceptable salt is selected from a sodium salt, a potassium salt, a calcium salt, or an ammonium salt.

6. The peptide ligand or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 5, wherein the PSMA is human PSMA.

7. 1. A drug conjugate comprising a peptide ligand specific for PSMA, or a pharma- ceutically acceptable salt thereof, conjugated to one or more effector groups and / or functional groups, the peptide ligand comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold; the molecular scaffold is 1,3,5-tris(bromomethyl)benzene; and The polypeptide, 【Chemistry 3】 (wherein X represents any amino acid residue, Nle represents norleucine, and C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. The drug conjugate comprising an amino acid sequence selected from:

8. (a) the polypeptide is according to any one of claims 2 to 4; and / or (b) the pharma- ceutically acceptable salt is as defined in claim 5; and / or (c) the PSMA is as defined in claim 6; The drug conjugate of claim 7.

9. The drug conjugate of claim 7 or 8, wherein the one or more effector groups and / or functional groups are metal chelators.

10. 10. The drug conjugate of claim 9, wherein the metal chelator is complexed to a metal radioisotope.

11. The metal radioisotope is 64 Cu, 67 Ga, 68 Ga, 177 Lu, 90 Y, and 213 The drug conjugate of claim 10, wherein said compound is selected from Bi.

12. (i) a peptide ligand specific for PSMA, or a pharma- ceutically acceptable salt thereof, conjugated to one or more effector groups and / or functional groups; the peptide ligand comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold; the molecular scaffold is 1,3,5-tris(bromomethyl)benzene; and The polypeptide, 【Chemistry 4】 (wherein X represents any amino acid residue, Nle represents norleucine, and C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof; or (ii) A drug conjugate according to any one of claims 7 to 11.

13. A pharmaceutical composition comprising:

13. 1. A pharmaceutical composition for use in therapy comprising: (i) a peptide ligand specific for PSMA, or a pharma- ceutically acceptable salt thereof, conjugated to one or more effector groups and / or functional groups; the peptide ligand comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold; the molecular scaffold is 1,3,5-tris(bromomethyl)benzene; and The polypeptide, 【Chemistry 5】 (wherein X represents any amino acid residue, Nle represents norleucine, and C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- tically acceptable salt thereof, conjugated to one or more effector groups and / or functional groups, comprising an amino acid sequence selected from (ii) A drug conjugate according to any one of claims 7 to 11. The pharmaceutical composition comprising:

14. 1. A pharmaceutical composition for use in the prevention, inhibition, or treatment of a disease or disorder mediated by PSMA, comprising: (i) a peptide ligand specific for PSMA, or a pharma- ceutically acceptable salt thereof, conjugated to one or more effector groups and / or functional groups; the peptide ligand comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold; the molecular scaffold is 1,3,5-tris(bromomethyl)benzene; and The polypeptide, 【Chemistry 6】 (wherein X represents any amino acid residue, Nle represents norleucine, and C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- tically acceptable salt thereof, conjugated to one or more effector groups and / or functional groups, comprising an amino acid sequence selected from (ii) A drug conjugate according to any one of claims 7 to 11. The pharmaceutical composition comprising:

15. (a) the polypeptide is according to any one of claims 2 to 4; and / or (b) the pharma- ceutically acceptable salt is as defined in claim 5; and / or (c) the PSMA is as defined in claim 6; The pharmaceutical composition according to any one of claims 12 to 14.

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