Bicyclic peptide ligands specific for PD-L1

Abstract

The present invention relates to polypeptides covalently attached to an 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 PD-L1. The present invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and uses of the peptide ligands and drug conjugates in the prevention, inhibition, or treatment of diseases or disorders mediated by PD-L1.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to polypeptides covalently attached to an 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 PD-L1. The present invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and the use of the peptide ligands and drug conjugates in the prevention, inhibition, or treatment of diseases or disorders mediated by PD-L1. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are able to bind protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutic drugs. Indeed, several cyclic peptides have already been successfully used in the clinic, such as, for example, the antimicrobial 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. Typically, macrocycles are used with a high molecular weight, e.g., the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 (Wu et al., 2007), Science 330, 1066-71), a cyclic peptide (355 Å) with an Arg-Gly-Asp motif that binds to integrin αVb3 2 ) (Xiong et al., 2002, Science 296(5565), 151-5), or upain-1 (603 Å), a cyclic peptide inhibitor that binds to urokinase-type plasminogen activator. 2; Zhao et al. (2007), J Struct Biol 160(1), 1-10), which bind to surfaces measuring several hundred square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides and experience less entropy loss when binding to targets, resulting in higher binding affinity. The reduced flexibility also results in a target-specific conformational fixation, increasing binding specificity compared to linear peptides. This effect is exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity against other MMPs when its ring is opened (Cherney et al., 1998, J Med Chem 41(11), 1749-51). The advantageous 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 tethered polypeptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara, 1985, J. Org. Chem; Timmerman et al., 2005, ChemBioChem). Meloen and coworkers used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclization of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al., 2005, ChemBioChem). Methods for the generation of candidate drug compounds, where the compounds are generated by linking cysteine-containing polypeptides to molecular scaffolds 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 to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a combinatorial library of linear peptides (Cys-(Xaa)6-Cys-(Xaa)6-Cys) containing three cysteine ​​residues and two random six amino acid tracts was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the Invention

[0006] (Summary of the invention) According to a first aspect of the invention there is provided a peptide ligand specific for PD-L1 comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and an aromatic molecular scaffold which forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

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

[0008] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0009] According to a further aspect of the invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by PD-L1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention In one embodiment, the loop sequence comprises 2, 3, or 6 amino acids.

[0011] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of two amino acids.

[0012] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of three amino acids.

[0013] In one embodiment, the peptide ligand is [ka] (wherein X1 represents any amino acid residue, 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 one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of three amino acids, and the peptide ligand comprises [ka] (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof.

[0015] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of three amino acids, and the peptide ligand comprises [ka] (wherein X1 represents any amino acid residue, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof.

[0016] In one embodiment, X 1 is selected from S, G, and N.

[0017] In a further embodiment, [ka] The peptide ligands are represented by SEQ ID NOs: 1 to 3: [ka] (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof.

[0018] In a further embodiment, the peptide ligand [ka] teeth, A-(SEQ ID NO:1)-A (referred to herein as BCY507); A-(SEQ ID NO:1)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY543); A-(SEQ ID NO:2)-A (referred to herein as BCY508); [B-Ala][Sar]5A-(SEQ ID NO:2)-A (the fluoresced derivative thereof is hereafter referred to as BCY544); A-(SEQ ID NO:3)-A (referred to herein as BCY509); [B-Ala][Sar]5A-(SEQ ID NO:3)-A (the fluoresced derivative thereof is hereafter referred to as BCY545); and A-(SEQ ID NO:3)-A[Sar]6 (the fluorescent derivative of which is hereafter referred to as BCY546) The amino acid sequence is selected from:

[0019] In a further embodiment, [ka] The peptide ligands are represented by SEQ ID NOs: 4 to 6: [ka] (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutical acceptable salt thereof.

[0020] In a further embodiment, [ka] The peptide ligand of A-(SEQ ID NO:4)-A (referred to herein as BCY514); A-(SEQ ID NO:4)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY550); G[Sar]5A-(SEQ ID NO:4)-A (the fluoresced derivative of which is hereafter referred to as BCY583); A-(SEQ ID NO:5)-A (referred to herein as BCY515); G[Sar]5A-(SEQ ID NO:5)-A (the fluoresced derivative of which is hereafter referred to as BCY584); A-(SEQ ID NO:6)-A (referred to herein as BCY516); and G[Sar]5A-(SEQ ID NO:6)-A (fluoresced derivative of which is hereafter referred to as BCY585) The amino acid sequence is selected from:

[0021] In a further embodiment, the peptide ligand [ka] teeth, A-(SEQ ID NO:9)-A[Sar]6 (fluorescein derivative thereof hereafter referred to as BCY551) The amino acid sequence is selected from:

[0022] In one embodiment, the peptide ligand is a peptide listed in any of Tables 1-3.

[0023] In one embodiment, the molecular scaffold is selected from 1,3,5-tris(bromomethyl)benzene (TBMB).

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, for example, peptide chemistry, cell culture, and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetics, and biochemistry methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc., incorporated herein by reference).

[0025] (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 the numbering of amino acid residues within the peptides of the invention is referred to as follows: -C i -P1-F2-P3-V4-E5-W6-C ii -S7-R8C iii - (SEQ ID NO: 1) .

[0026] For the purposes of this description, all bicyclic peptides are considered to be cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) to give trisubstituted structures. Cyclization with TBMB results in the C i , C ii , and C iii This occurs above.

[0027] (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, an N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO:X) This is expressed as:

[0028] (Reverse peptide sequence) In view of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, it is envisioned that the peptide sequences disclosed herein will also find utility in their retro-inverso forms, for example where the sequence is reversed (i.e., N-terminus becomes C-terminus and C-terminus becomes N-terminus) and the stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids and L-amino acids become D-amino acids).

[0029] (Peptide Ligand) A peptide ligand as referred to herein refers to a peptide covalently attached to a molecular scaffold. Typically, such a peptide contains two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond with the scaffold, and an internal sequence between the reactive groups, called a loop sequence, because the peptide forms a loop when it is attached to the scaffold. In this case, the peptide contains at least three cysteine ​​residues (herein referred to as C i , C ii , and C iii ), and forms at least two loops on the scaffold.

[0030] (Advantages of peptide ligands) Certain bicyclic peptides of the present invention have several advantageous properties that make them suitable drug-like molecules for injection, inhalation, nasal, ocular, oral, or topical administration. Such advantageous properties include: -Species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability. Bicyclic peptide ligands should ideally demonstrate stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastrointestinal proteases, pulmonary surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic lead candidates can not only be developed in animal models but also be administered with confidence to humans; - a desirable solubility profile, which is a function of charged residues and the ratio of hydrophilic to hydrophobic residues and intra / inter-molecular H-bonds, which is important for formulation and absorption purposes; - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides for short-term exposure in acute disease management settings or to develop bicyclic peptides with enhanced retention in the circulation and therefore optimal for management of more chronic disease states. Other factors driving a desirable plasma half-life are the requirement for sustained exposure for maximum therapeutic efficacy and the associated toxicity of sustained exposure of the drug; and -Selectivity: Certain peptide ligands of the invention exhibit better selectivity than other transmembrane proteins.

[0031] (Pharmaceutical acceptable salts) It will be understood that the salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of that ligand.

[0032] The salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0033] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (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), glutamic acid (e.g., L-glutamic acid), 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, Examples of suitable salts include mono- or di-salts formed with acids selected from the group consisting of acetic 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, as well as acylated amino acids and cation exchange resins.

[0034] One particular group of salts consists of salts formed from 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.

[0035] The compound is anionic or has a functional group which may be anionic (e.g., -COOH can be -COO - In the case of cations such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29 + , Na + , and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4+ ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are included, but are not limited to. Some suitable examples of substituted ammonium ions include those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0036] When the peptides of the invention contain amine functions, they may form quaternary ammonium salts, for example, by reaction with alkylating agents by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

[0037] (Modified derivative) It will be understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with surrogate bonds; modification of the peptide backbone length; Modifications include one or more selected from: replacement of the 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 acids; and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, e.g., amino acids with azide or alkyne groups that allow functionalization with alkyne- or azide-bearing moieties, respectively.

[0038] In one embodiment, the modified derivative comprises an N-terminal and / or a C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using a suitable amino reaction chemistry and / or a C-terminal modification using a suitable carboxy reaction 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 radioactive chelator, or a chromophore.

[0039] 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, an N-terminal cysteine ​​group (referred to herein as C iThe N-terminal acetyl group (referred to as "acetyl group") is capped with acetic anhydride or other suitable reagent during peptide synthesis to yield an N-terminally acetylated molecule. This embodiment offers the advantage of eliminating a potential recognition point for aminopeptidases, avoiding possible degradation of the bicyclic peptide.

[0040] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates the conjugation of an effector group and retention of potency of the bicyclic peptide against its target.

[0041] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine ​​group (herein referred to as C iii The C-terminally amidated group, termed the C-terminally amidated group, is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment offers the advantage of eliminating a potential recognition point for carboxypeptidases, reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0042] In one embodiment, the modified derivatives include the substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected that have isosteric / isoelectronic side chains that are not recognized by degradative proteases or have any adverse effect on targeting efficacy.

[0043] Alternatively, unnatural amino acids with constrained amino acid side chains may be used such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, these concern proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids that are simple derivatives of amino-cyclopropyl carboxylic acids.

[0044] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of an N-terminal cysteine ​​(C i) and / or a C-terminal cysteine ​​(C iii ) including the addition of a spacer group to

[0045] In one embodiment, the modified derivative comprises the 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 the substitution of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the resulting bicyclic peptide ligand with the advantage of improved pharmaceutical stability profile.

[0046] In one embodiment, the modified derivative comprises the 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 the substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The appropriate balance of charged and hydrophobic 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 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. The combination of the two can affect the half-life, volume of distribution, and exposure of the peptide drug, which can be adjusted depending on the clinical endpoint. Furthermore, the appropriate combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0047] In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to enhance proteolytic stability due to steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0048] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine. This embodiment has the advantage of removing potential sites of proteolytic attack.

[0049] It should be noted that each of the above mentioned modifications serves to purposefully improve the potency or stability of the peptide. Further potency improvements based on the modifications can be achieved by the following mechanisms: - taking advantage of the hydrophobic effect and incorporating hydrophobic sites resulting in lower dissociation rates so that higher affinities are achieved; - taking advantage of long-range ionic interactions, resulting in faster association rates and incorporating charged groups resulting in higher affinity (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - incorporating further constraints into the peptide, for example by appropriately constraining the amino acid side chains so that entropy loss is minimized upon target binding, constraining the backbone torsion angles so that entropy loss is minimized upon target binding, and introducing further cyclization into the molecule for the same reasons. (For reviews see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0050] (Isotopic Variation) The present invention includes all pharma- ceutically acceptable (radio)isotope-labeled peptide ligands of the invention in which one or more atoms have been replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, as well as peptide ligands of the invention to which a metal chelating group capable of bearing a relevant (radio)isotope has been attached (referred to as "effectors"), and peptide ligands of the invention in which a particular functional group has been covalently replaced with a relevant (radio)isotope or an isotopically labeled functional group.

[0051] 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. 36 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, isotopes of phosphorus, e.g. 32 P, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y, as well as isotopes of lutetium, e.g. 177 Lu, as well as isotopes of bismuth, e.g. 213 Contains Bi.

[0052] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies and for clinically assessing the presence and / or absence of PD-L1 targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may employ compounds that are labeled with labeling agents, such as, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). The radioisotope tritium, i.e., 3 H(T) and carbon-14, i.e. 14 C is particularly useful for this purpose given its ease of incorporation and the means of detection available.

[0053] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0054] 11 C. 18 F, 15 O, and 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0055] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the accompanying Examples, substituting appropriate isotopically labeled reagents for previously utilized non-labeled reagents.

[0056] (Aromatic Molecular Scaffolds) References herein to the term "aromatic molecular scaffold" refer to any molecular scaffold as defined herein that contains an aromatic carbocyclic or heterocyclic ring system.

[0057] It will be appreciated that the aromatic molecular scaffold may include aromatic moieties. Examples of suitable aromatic moieties within the aromatic scaffold include biphenylene, terphenylene, naphthalene, or anthracene.

[0058] It will also be appreciated that the aromatic molecular scaffold can include heteroaromatic moieties. Examples of suitable heteroaromatic moieties within an aromatic scaffold include pyridine, pyrimidine, pyrrole, furan, and thiophene.

[0059] It will also be understood that the aromatic molecular scaffold may include a halomethylarene moiety, such as bis(bromomethyl)benzene, tris(bromomethyl)benzene, tetra(bromomethyl)benzene, or derivatives thereof. Non-limiting examples of aromatic molecular scaffolds include: bis-, tris-, or tetra(halomethyl)benzene; bis-, tris-, or tetra(halomethyl)pyridine; bis-, tris-, or tetra(halomethyl)pyridazine; bis-, tris-, or tetra(halomethyl)pyrimidine; bis-, tris-, or tetra(halomethyl)pyrazine; bis-, tris-, or tetra(halomethyl)-1,2,3-triazine; bis-, tris-, or tetra- bis-, tris-, or tetra(halomethyl)pyrroles, -furans, -thiophenes; bis-, tris-, or tetra(halomethyl)imidazoles, -oxazoles, -thiazoles; bis-, tris-, or tetra(halomethyl)-3H-pyrazoles, -isoxazoles, -isothiazoles; bis-, tris-, or tetra(halomethyl)biphenylenes; bis-, tris-, or tetra(halomethyl)terphenylenes; 1,8-bis(halomethyl)naphthalenes; bis-, tris-, or tetra(halomethyl)anthracenes; and bis-, tris-, or tetra(2-halomethylphenyl)methanes.

[0060] More specific examples of aromatic molecular scaffolds include: 1,2-bis(halomethyl)benzenes; 3,4-bis(halomethyl)pyridines; 3,4-bis(halomethyl)pyridazines; 4,5-bis(halomethyl)pyrimidines; 4,5-bis(halomethyl)pyrazines; 4,5-bis(halomethyl)-1,2,3-triazines; 5,6-bis(halomethyl)-1,2,4-triazines; 3,4-bis(halomethyl)pyrroles, -furans, -thiophenes, and other positional isomers; 4,5-bis(halomethyl)imidazoles, -oxazoles, -thiazoles; 4,5-bis(halomethyl)-3H-pyrazoles, -isoxazoles, -isothiazoles; 2,2'-bis(halomethyl)biphenylenes; 2,2''-bis(halomethyl)terphenylenes; 1,8-Bis(halomethyl)naphthalene; 1,10-Bis(halomethyl)anthracene; Bis(2-halomethylphenyl)methane; 1,2,3-Tris(halomethyl)benzene; 2,3,4-Tris(halomethyl)pyridine; 2,3,4-Tris(halomethyl)pyridazine; 3,4,5-Tris(halomethyl)pyrimidine; 4,5,6-Tris(halomethyl)-1,2,3-triazine; 2,3,4-Tris(halomethyl)pyrrole, -furan, -thiophene; 2,4,5-Bis(halomethyl)imidazole, -oxazole, -thiazole; 3,4,5-Bis(halomethyl)-1H-pyrazole, -isoxazole, -isothiazole; 2,4,2'-Tris(halomethyl)biphenylene; 2,3',2''-Tris(halomethyl)terphenylene; 1,3,8-Tris(halomethyl)naphthalene; 1,3,10-Tris(halomethyl)anthracene; Bis(2-halomethylphenyl)methane; 1,2,4,5-Tetra(halomethyl)benzene; 1,2,4,5-Tetra(halomethyl)pyridine; 2,4,5,6-Tetra(halomethyl)pyrimidine; 2,3,4,5-Tetra(halomethyl)pyrrole, -furan, -thiophene; 2,2',6,6'-Tetra(halomethyl)biphenylene; 2,2'',6,6''-Tetra(halomethyl)terphenylene; 2,3,5,6-Tetra(halomethyl)naphthalene, and 2,3,7,8-Tetra(halomethyl)anthracene;and bis(2,4-bis(halomethyl)phenyl)methane;

[0061] As described in the aforementioned documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0062] In one embodiment, the molecular scaffold may be a polymer, hi one embodiment, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates.

[0063] In one embodiment, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0064] The molecular scaffold can include chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0065] In one embodiment, the molecular scaffold may comprise or consist of tris(bromomethyl)benzene, in particular 1,3,5-tris(bromomethyl)benzene ("TBMB"), or a derivative thereof.

[0066] In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene.This molecule is similar to 1,3,5-tris(bromomethyl)benzene, but contains three additional methyl groups attached to the benzene ring.This has the advantage that the additional methyl groups can form additional contacts with polypeptides, thus adding additional structural constraints.

[0067] The molecular scaffolds of the invention contain chemical groups that allow functional groups of the polypeptides of the encoded libraries of the invention to form covalent bonds with the molecular scaffold, the chemical groups being selected from a wide range of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides, and acyl halides.

[0068] Scaffold reactive groups that can be used on molecular scaffolds to react with the thiol groups of cysteine ​​are alkyl halides (also named halogenoalkanes or haloalkanes).

[0069] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups used to selectively couple compounds to cysteines in proteins are maleimides, αβ-unsaturated carbonyl-containing compounds, and α-halomethylcarbonyl-containing compounds. Examples of maleimides that may be used as molecular scaffolds in the present invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an α-halomethylcarbonyl-containing compound is N,N′,N″-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a naturally occurring amino acid with similar reactivity to cysteine ​​and can be used in the same reactions. Thus, whenever cysteine ​​is mentioned, it is generally permissible to use selenocysteine ​​instead, unless the context suggests otherwise.

[0070] (Effectors and Functional Groups) According to a further aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0071] The effectors and / or functional groups can be attached, for example, to the N- and / or C-terminus of the polypeptide, to amino acids within the polypeptide, or to a molecular scaffold.

[0072] Suitable effector groups include antibodies and portions or fragments thereof. For example, an effector group can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to one or more constant region domains. An effector group can include an antibody hinge region (such a region typically found between the CH1 and CH2 domains of an IgG molecule).

[0073] In a further embodiment of this aspect of the invention, the effector group according to the invention is an Fc region of an IgG molecule. Advantageously, a peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion with a tβ half-life of 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Most advantageously, a peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion with a tβ half-life of 1 day or more.

[0074] Functional groups generally include linking groups, reactive groups for attachment of drugs or other entities, functional groups which aid in cellular uptake of the macrocyclic peptide, and the like.

[0075] The ability of peptides to penetrate into cells allows peptides to be effective against intracellular targets. Targets that can be accessed by peptides with the ability to penetrate into cells include intracellular signaling molecules such as transcription factors, tyrosine kinases, and molecules involved in apoptotic pathways. Functional groups that allow cell penetration include chemical groups added to either the peptide or the peptide or molecular scaffold. For example, peptides such as those derived from VP22, HIV-Tat, Drosophila homeobox protein (Antennapedia), etc., as described in Chen and Harrison, Biochemical Society Transactions (2007), Vol. 35, Part 4, p. 821; Gupta et al., Advanced Drug Discovery Reviews (2004), Vol. 57, p. 9637. Examples of short peptides that have been shown to be efficient at translocating through cell membranes include the 16 amino acid penetratin peptide from the Drosophila antennapedia protein (Derossi et al. (1994) J. Biol. Chem. 269:10444), the 18 amino acid "model amphipathic peptide" (Oehlke et al. (1998) Biochim Biophys Acts 1414:127), and the arginine-rich region of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimetics or SMOCs that can be easily attached to biomolecules (Okuyama et al. (2007) Nature Methods 4:153). Another chemical strategy, the addition of guanidinium groups to molecules, also enhances cell penetration (Elson-Scwab et al. (2007) J. Biol. Chem 282:13585). Small molecular weight molecules such as steroids can be added to the molecular scaffold to enhance cellular uptake.

[0076] One class of functional groups that can be attached to the peptide ligands includes antibodies and binding fragments thereof, such as Fab, Fv, or single domain fragments. In particular, antibodies that bind to proteins that can increase the half-life of the peptide ligand in vivo can be used.

[0077] In one embodiment, a peptide ligand effector group according to the invention has a tβ half-life selected from the group consisting of: 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, 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, a peptide ligand-effector group or composition according to the invention has a tβ half-life in the range of 12 hours to 60 hours. In a further embodiment, it has a tβ half-life of 1 day or more. In a still further embodiment, it is in the range of 12 to 26 hours.

[0078] In one particular embodiment of the invention, the functional group is selected from metal chelators that are suitable for complexing medicamentously relevant metal radioisotopes.

[0079] Possible effector groups also include enzymes such as carboxypeptidase G2 for use in enzyme / prodrug therapy, where peptide ligands replace antibodies in ADEPT.

[0080] In one particular embodiment of the present invention, functional group is selected from drugs such as cytotoxic agents for cancer therapy.Suitable examples include: alkylating agents such as cisplatin and carboplatin, and oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, originally known as taxol; topoisomerase inhibitors including camptothecin; irinotecan and topototecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Additional agents may include the immunosuppressant dactinomycin (used in kidney transplants), antitumor antibiotics including doxorubicin, epirubicin, bleomycin, calicheamicin, and the like.

[0081] In one further specific embodiment of the invention, the cytotoxic agent is selected from a maytansinoid (eg, DM1) or a monomethylauristatin (eg, MMAE).

[0082] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure: [ka] .

[0083] Monomethylauristatin E (MMAE) is a synthetic anti-neoplastic agent having the following structure: [ka] .

[0084] In a still further embodiment of the invention, the cytotoxic agent is selected from maytansinoids (eg, DM1).

[0085] In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulfide bond or a protease-sensitive bond, hi a further embodiment, the groups adjacent to the disulfide bond are modified to control the rate of disruption of the disulfide bond and, thereby, cleavage and concomitant release of the cytotoxic agent.

[0086] Published studies have established the possibility of modifying the susceptibility of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also by extracellular (systemic) reducing agents, and consequently reduces the ease with which the toxin is released both inside and outside the cell. Thus, the selection of optimal conditions for disulfide stability in circulation (which minimizes the undesirable side effects of the toxin) versus efficient release in the intracellular environment (which maximizes the therapeutic effect) can be achieved by careful selection of the degree of hindrance on either side of the disulfide bond.

[0087] The disorder on either side of the disulfide bond is adjusted by introducing one or more methyl groups into either the targeting entity (here, the bicyclic peptide) or the toxin side of the molecular construct.

[0088] In one embodiment, the cytotoxic agents and linkers are selected from any combination of those described in WO 2016 / 067035, which cytotoxic agents and linkers thereof are incorporated herein by reference.

[0089] (synthesis) The peptides of the present invention can be synthetically produced by standard techniques and then reacted with molecular scaffolds in vitro. Standard chemistry can be used to carry this out. This allows for rapid large-scale preparation of soluble material for further downstream experimentation or validation. Such methods can be achieved using conventional chemistry such as that disclosed in Timmerman et al. (supra).

[0090] Thus, the present invention also relates to the production of a polypeptide or conjugate selected as described herein, which production comprises any further steps as explained below, in one embodiment these steps are carried out on the final polypeptide / conjugate produced by chemical synthesis.

[0091] Optionally, amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

[0092] The peptide can also be extended, for example, to incorporate additional loops and thus introduce multiple specificities.

[0093] To extend a peptide, it may simply be chemically extended at its N- or C-terminus or within a loop using orthogonally protected lysines (and analogues) using standard solid- or solution-phase chemistries. Activated or activatable N- or C-termini may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be performed enzymatically by fragment condensation or native chemical ligation as described, for example, in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or using subtiligase 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., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, 15 Nov. 2008, pp. 6000-6003).

[0094] Alternatively, peptides 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 in the reducing environment of cells. In this case, a molecular scaffold (e.g., TBMB) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; then, an additional cysteine ​​or thiol can be added to the N- or C-terminus of the first peptide, so that this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0095] Similar techniques apply equally to the synthesis / coupling of two bicyclic bispecific macrocycles, potentially giving rise to a tetraspecific molecule.

[0096] Furthermore, the addition of other functional or effector groups may be achieved in the same manner, coupling at the N- or C-terminus or via a side chain using appropriate chemistry, hi one embodiment, the coupling is carried out in such a way as not to block the activity of either entity.

[0097] Pharmaceutical Composition According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0098] Usually, the peptide ligands are utilized in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including 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, if necessary to keep the polypeptide complex in suspension, may be selected from viscosity-increasing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0099] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose, and preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack, 1982, Remington's Pharmaceutical Sciences, 16th ed.).

[0100] The peptide ligands of the invention may be used as a separately administered composition or in conjunction with other drugs. These may include antibodies, antibody fragments, and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin, or cisplatin, and immunotoxins. Pharmaceutical compositions may include "cocktails" of various cytotoxic or other drugs in conjunction with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities, such as polypeptides selected with different targeting ligands, whether pooled or not prior to administration.

[0101] The route of administration of the pharmaceutical composition according to the invention may be any one generally known to those skilled in the art. For therapy, the peptide ligand of the invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also suitably by direct injection using a catheter. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dosage and frequency of administration will depend on the age, sex, and condition of the patient, the simultaneous administration of other drugs, contraindications, and other parameters taken into account by the clinician.

[0102] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be utilized. It will be understood by those skilled in the art that lyophilization and reconstitution may result in varying degrees of activity loss, and that levels may need to be adjusted upwards to compensate.

[0103] Compositions containing the peptide ligands or cocktails thereof of the present invention can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, modulation, 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 will depend on the severity of the disease and the general state of the patient's own immune system, but generally ranges from 0.005-5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05-2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

[0104] Compositions containing peptide ligands according to the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in a mammal. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove target cell populations from heterogeneous populations of cells. Blood from a mammal can be combined ex vivo with the selected peptide ligand, thereby killing or otherwise removing undesirable cells from the blood for return to the mammal according to standard techniques.

[0105] (therapeutic use) The bicyclic peptides of the invention have particular utility as PD-L1 binding agents.

[0106] Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in evading immune responses involved in chronic infections, such as chronic viral infections (including, among others, HIV, HBV, HCV, and HTLV), chronic bacterial infections (including, among others, Helicobacter pylori), and chronic parasitic infections (including, among others, Schistosoma mansoni). PD-L1 expression has been detected in several tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, muscle cells, and placenta.

[0107] PD-L1 expression has also been implicated in suppressing antitumor immune activity. Although tumors express antigens that can be recognized by host T cells, immunological clearance of tumors is rare. This failure is due in part to immune suppression by the tumor microenvironment. PD-L1 expression in many tumors is a component of this suppressive environment and acts in concert with other immunosuppressive signals. PD-L1 expression has been demonstrated in situ in a wide variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymic epithelium, and head and neck (Brown JA et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003 Cancer Res. 63:6501-5; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi et al. 2004 Clin. Cancer Res. 10:5094-100; J et al., 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al., 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al., 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). In addition, expression of programmed cell death protein 1 (also known as PD-1 and CD279), the receptor for PD-L1, is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al., 2003 Immunol. 171:4574-81).Most importantly, studies relating tumor PD-L1 expression to disease outcome have shown that PD-L1 expression strongly correlates with unfavorable prognosis in renal, ovarian, bladder, breast, gastric, and pancreatic cancers (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 ... 101:17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). Furthermore, these studies suggest that higher levels of PD-L1 expression in tumors may promote tumor stage progression and invasion into deeper tissue structures.

[0108] The PD-1 pathway can also play a role in hematological malignancies. PD-L1 is expressed on many myeloma cells but not on normal plasma cells (Liu J et al., 2007 Blood 110:296-304). PD-L1 is expressed on some primary T-cell lymphomas, particularly anaplastic large cell T-lymphoma (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is highly expressed on T cells in angioimmunoblastic lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al., 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin's lymphoma, T cells associated with lymphocytes or histiocytic (L&H) cells express PD-1. Microarray analysis using the readout of genes induced by PD-1 ligation suggests that tumor-associated T cells respond to PD-1 signals in situ in Hodgkin's lymphoma (Chemnitz JM et al., 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma (Shimauchi T et al., 2007 Int. J. Cancer 121: 2585-90). These tumor cells are hyporesponsive to TCR signals.

[0109] Studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis, and in some cases leads to increased tumor-specific T cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96). Tumor-associated APCs can also exploit the PD-1:PD-L1 pathway to control antitumor T cell responses. PD-L1 expression on a population of tumor-associated bone marrow DCs is upregulated by tumor environmental factors (Curiel TJ et al. 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in tumor-draining lymph nodes of B16 melanoma express IDO, which potently activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cellular contact with IDO-expressing DCs (Sharma MD et al. 2007 Clin. Invest. 117:2570-82).

[0110] Thus, there is a need in the art for effective treatments of PD-L1 associated diseases, such as infectious diseases, e.g., chronic intracellular infectious diseases, e.g., viral diseases, e.g., hepatitis infection, or bacterial infections, e.g., tuberculosis infection; and cancers, e.g., liver cancer, e.g., hepatocellular carcinoma.

[0111] Polypeptide ligands selected by the methods of the invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, etc. Ligands with a selected level of specificity are useful in applications involving testing in non-human animals where cross-reactivity is desirable, or in diagnostic applications where cross-reactivity with homologs or paralogs must be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response against a range of antigens can be exploited to tailor vaccines to specific diseases and pathogens.

[0112] Substantially pure peptide ligands having at least 90-95% homogeneity are preferred for administration to mammals, with 98-99% or greater homogeneity being most preferred for pharmaceutical uses, particularly where the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides can be used diagnostically or therapeutically (including in vitro), or in the development and implementation of assay procedures, immunofluorescence staining, and the like (Lefkovite and Pernis, 1979 and 1981, Immunological Methods, Vols. I and II, Academic Press, NY).

[0113] According to a further aspect of the invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by PD-L1.

[0114] According to a further aspect of the invention there is provided a method for the prevention, inhibition or treatment of a disease or disorder mediated by PD-L1 comprising administering to a patient in need thereof an effector group and drug conjugate of a peptide ligand defined herein.

[0115] In one embodiment, the PD-L1 is mammalian PD-L1. In a further embodiment, the mammalian PD-L1 is human PD-L1 (hPD-L1).

[0116] In one embodiment, the PD-L1 mediated disease or disorder is selected from a chronic infection or disease, a solid tumor, and a hematological malignancy.

[0117] In one embodiment, the chronic infection or disease mediated by PD-L1 is selected from a chronic viral infection, a chronic bacterial infection, a chronic parasitic infection, a hepatitis infection, and a viral disease.

[0118] In one embodiment, the PD-L1 mediated solid tumor is selected from breast, lung, colon, ovarian, melanoma, bladder, liver, salivary gland, gastric, glioma, thyroid, thymic epithelial, head and neck tumors.

[0119] In a further embodiment, the disease or disorder mediated by PD-L1 is selected from cancer.

[0120] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), such as tumors 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 system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, cancers of the nasal cavity, 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 adnexal cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and borderline malignancies including premalignant hematological disorders and hematological malignancies and related disorders of lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [D-cell lymphoma], LBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell 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 disorder), and hematological malignancies and related disorders of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndromes, myeloproliferative disorders, e.g., polycythemia vera, essential thrombocytopenia, myeloma, myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia; tumors of mesenchymal origin, e.g., sarcomas of the soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and Schwannoma);These include, but are not limited to, endocrine tumors (e.g., pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas); and pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors); or congenital or other syndromes that predispose a patient to malignancies (e.g., xeroderma pigmentosum);

[0121] In further embodiments, the cancer is kidney, ovarian, bladder, breast, stomach, and pancreatic cancer.

[0122] In still further embodiments, the cancer is selected from liver cancer, such as hepatocellular carcinoma and hepatocellular carcinoma.

[0123] In further embodiments, the cancer is selected from hematopoietic malignancies, e.g., selected from: non-Hodgkin's lymphoma (NHL), Burkitt's 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's lymphoma (HL), chronic myelogenous leukemia (CML), and nodular lymphocyte-predominant Hodgkin's lymphoma.

[0124] References herein to the term "prevention" include administration of the protective composition prior to the induction of disease. "Suppression" refers to administration of the composition after an inductive event but prior to clinical appearance of the disease. "Treatment" includes administration of the protective composition after disease symptoms are manifest.

[0125] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease, and the use of animal model systems is facilitated by the present invention, which allows for the development of polypeptide ligands that can cross-react with human and animal targets.

[0126] The invention will now be further described with reference to the following examples. EXAMPLES

[0127] (Example) Materials and Methods (Peptide synthesis) Peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer from MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) were used with appropriate side-chain protecting groups: where applicable, standard coupling conditions were used in each case, followed by deprotection using standard methodologies. Peptides were purified using HPLC and, after isolation, were modified with 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma). For this, the linear peptide was diluted to about 35 mL with H2O, about 500 μL of 100 mM TBMB in acetonitrile was added, and the reaction was started with 5 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed for about 30 to 60 min at RT and, once the reaction was completed (as judged by MALDI), it was lyophilized. After lyophilization, the modified peptides were purified as above, except that 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 kept at -20°C for storage.

[0128] Unless otherwise noted, all amino acids were used in the L-configuration.

[0129] In some cases, the peptide was converted to an activated disulfide and then coupled to a free thiol group of the toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in anhydrous DMSO (1.25 mol equiv.) was added to a solution of peptide (20 mM) in anhydrous DMSO (1 mol equiv.). The reaction was mixed well and DIPEA (20 mol equiv.) was added. The reaction was monitored by LC / MS until completion.

[0130] (Biological Data) (1. PD-L1 Direct Binding Assay) The affinity (Ki) of peptides of the invention for human PD-L1 was determined using a fluorescence polarization assay according to the method disclosed in WO2016 / 067035. Peptides of the invention bearing a fluorescent tag (either fluorescein, SIGMA or Alexa Fluor488™, Fisher Scientific) were diluted to 2.5 nM in PBS with 0.01% tween 20 or 50 mM HEPES pH 7.4 with 100 mM NaCl and 0.01% tween (both referred to as assay buffer). This was combined with a titration of protein in the same assay buffer as the peptide to give 1 nM peptide in a total volume of 25 μL, typically 5 μL assay buffer, 10 μL protein, then 10 μL fluorescent peptide in a black walled, low volume 384 well plate with low binding and bottom. A 1:2 serial dilution was used to generate 12 different concentrations ranging from 500 nM for known high affinity binders to 10 μM for low affinity binders and selectivity assays. Measurements were performed on a BMG PHERAstar FS equipped with an optical module "FP 485 520 520" with excitation at 485 nm and detection of parallel and perpendicular emission at 520 nm. The PHERAstar FS was set at 25 °C, with 200 flashes per well and a positioning delay of 0.1 seconds, and each well was measured for 60 minutes at 5-10 minute intervals. The gain used for analysis was determined for each tracer at the end of the 60 minutes when no protein was present in the well. Data were analyzed using Systat Sigmaplot version 12.0. The mP values ​​were fitted to a user-defined quadratic equation to generate Kd values: f = ymin+(ymax-ymin) / Lig*((x+Lig+Kd) / 2-sqrt((((x+Lig+Kd) / 2)^2)-(Lig*x))). "Lig" was the defined concentration value of the tracer used.

[0131] The following selected bicyclic peptide ligands of the invention were tested in the PD-L1 direct binding assay described above, and the results are shown in Table 1: Table 1: Direct binding assay data for selected bicyclic peptide ligands of the invention. [Table 1] ** Represents the average of four experiments. [Table 2]

[0132] (2. PD-L1 Competitive Binding Assay) The peptide without the fluorescent tag was [ka] Peptides were tested in competition with (([Fl]G[Sar]5A-(SEQ ID NO:6)-A); BCY585; Kd=68.75nM - determined using the protocol above). Peptides were diluted to the appropriate concentration in assay buffer as described for the direct binding assay with up to 5% DMSO and then serially diluted 1 to 2. 5μL of diluted peptide was added to the plate, followed by 10μL of human PD-L1, followed by 10μL of fluorescent peptide. Measurements were performed as for the direct binding assay, except that gain was determined prior to the first measurement. Data analysis was performed with Systat Sigmaplot version 12.0, where mP values ​​were fitted to a user-defined cubic equation to generate Ki values: f=ymin+(ymax-ymin) / Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Pro t*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(K comp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*(((Klig+Kcomp+Lig+Com p-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp-P rot*c))) / ((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 ​​for: fluorescent peptide concentration, Kd of fluorescent peptide, and PD-L1 concentration, respectively.

[0133] The following selected bicyclic peptide ligands of the invention were tested in the PD-L1 competitive binding assay described above, and the results are shown in Table 2: Table 2: Competitive binding assay data for selected bicyclic peptide ligands of the invention. [Table 3]

[0134] (3. PD-L1 SPR Binding Assay) k of monomeric peptide binding to human PD-L1 protein a (M -1 s -1 ), k d (s -1 ), K DBiacore experiments were performed to determine (nM) values. Recombinant human PD-L1 (Sino Biologicals or R&D systems) or mouse PD-L1 (R&D systems) were resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin Reagent (Thermo Fisher) following the manufacturer's suggested protocol. Proteins were desalted and uncoupled biotin was removed into PBS using a spin column. For binding analysis, a Biacore 3000 instrument was used, utilizing a CM5 sensor chip (GE Healthcare). Streptavidin was immobilized on the chip using standard amine coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. The carboxymethyldextran surface was activated with a 12 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min. For streptavidin capture, the protein was diluted to 0.1 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 70 μl over the activated chip surface. Remaining activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Biotinylated PD-L1 stock was diluted 1:100 in HBS-N and captured on one flow cell to a level of 1000-1300 RU at 5 μl / min. The buffer was exchanged into PBS / 0.05% Tween 20 and a dilution series of the peptide was prepared in this buffer with a final DMSO concentration of 0.5%. The maximum concentration was 200 nM or 500 nM with six two-fold dilutions. SPR analysis was performed at 25° C. with 60 s association and 400 s dissociation at a flow rate of 50 μl / min. Data were corrected for DMSO excluded volume effects. All data were double-referenced against blank injections and baselines using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software).The data were fitted with a mass transfer model, taking mass transfer effects into account where appropriate.

[0135] The following selected bicyclic peptide ligands of the invention were tested in the SPR binding assay described above, and the results are shown in Table 3: Table 3: PD-L1 SPR binding assay data of selected bicyclic peptide ligands of the invention [Table 4] The present application provides the following aspects of the invention. (Aspect 1) A peptide ligand specific for PD-L1, comprising: a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences; and an aromatic 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. (Aspect 2) 2. The peptide ligand of embodiment 1, wherein the loop sequence comprises 2, 3, or 6 amino acids. (Aspect 3) (chemical 1) TIFF0007658902000018.tif22170 (where X 1 represents any amino acid residue, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. 3. The peptide ligand of embodiment 1 or embodiment 2, comprising an amino acid sequence selected from: (Aspect 4) the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of two amino acids, and the peptide ligand (chemical 2) TIFF0007658902000019.tif7170 (Here, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. 4. The peptide ligand according to any one of aspects 1 to 3, comprising an amino acid sequence selected from: (Aspect 5) the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of three amino acids, and the peptide ligand comprises (C3) TIFF0007658902000020.tif15170 (where X 1 represents any amino acid residue, C i、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. 4. The peptide ligand according to any one of aspects 1 to 3, comprising an amino acid sequence selected from: (Aspect 6) The above (C4) TIFF0007658902000021.tif9170 The peptide ligand is represented by SEQ ID NOs: 1 to 3: (C5) TIFF0007658902000022.tif21170 (Here, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutical acceptable salt thereof; for example: A-(SEQ ID NO:1)-A (referred to herein as BCY507); A-(referred to as SEQ ID NO:1)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY543); A-(referred to as SEQ ID NO:2)-A (referred to herein as BCY508); [B-Ala][Sar] 5 A-(referred to as SEQ ID NO:2)-A (whose fluoresced derivative is hereafter referred to as BCY544); A-(referred to as SEQ ID NO:3)-A (referred to herein as BCY509); [B-Ala][Sar] 5 A-(referred to as SEQ ID NO:3)-A (the fluoresced derivative of which is hereafter referred to as BCY545); and A-(referred to as SEQ ID NO:3)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY546.) 6. The peptide ligand of embodiment 3 or embodiment 5, comprising an amino acid sequence selected from: (Aspect 7) The above (6) TIFF0007658902000023.tif7170 The peptide ligand is represented by SEQ ID NOs: 4 to 6: (C7) TIFF0007658902000024.tif22170 (Here, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutical acceptable salt thereof; for example: A-(SEQ ID NO:4)-A (referred to herein as BCY514); A-(referred to as SEQ ID NO:4)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY550); G[Sar] 5 A-(referred to as SEQ ID NO:4)-A (the fluoresced derivative of which is hereafter referred to as BCY583); A-(referred to as SEQ ID NO:5)-A (referred to herein as BCY515); G[Sar] 5 A-(referred to as SEQ ID NO:5)-A (the fluoresced derivative of which is hereafter referred to as BCY584); A-(referred to as SEQ ID NO:6)-A (referred to herein as BCY516); and G[Sar] 5 A-(referred to as SEQ ID NO:6)-A (whose fluorescent derivative is hereafter referred to as BCY585) 6. The peptide ligand of embodiment 3 or embodiment 5, comprising an amino acid sequence selected from: (Aspect 8) The peptide ligand (8) TIFF0007658902000025.tif8170 but, A-(SEQ ID NO:9)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY551.) 4. The peptide ligand of embodiment 3, comprising an amino acid sequence which is: (Aspect 9) A peptide ligand according to embodiment 1 or embodiment 2, which is a peptide listed in any one of Tables 1 to 3. (Aspect 10) The peptide ligand of any one of embodiments 1 to 9, wherein the molecular scaffold is 1,3,5-tris(bromomethyl)benzene (TBMB). (Aspect 11) 11. The peptide ligand according to any one of aspects 1 to 10, wherein the pharma- ceutical acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt. (Aspect 12) 12. The peptide ligand of any one of aspects 1 to 11, wherein said PD-L1 is human PD-L1. (Aspect 13) 13. A drug conjugate comprising the peptide ligand according to any one of aspects 1 to 12 conjugated to one or more effectors and / or functional groups. (Aspect 14) 13. A drug conjugate comprising the peptide ligand according to any one of embodiments 1 to 12 conjugated to one or more cytotoxic agents. (Aspect 15) A pharmaceutical composition comprising a peptide ligand according to any one of aspects 1 to 12 or one or more drug conjugates according to aspect 13 or aspect 14 in combination with a pharma- ceutical acceptable excipient. (Aspect 16) A peptide ligand according to any one of Aspects 1 to 12 or a drug conjugate according to Aspect 13 or Aspect 14 for use in the prevention, suppression or treatment of a disease or disorder mediated by PD-L1.

Claims

1. 1. A peptide ligand specific for PD-L1, or a pharma- ceutically acceptable salt thereof, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and an aromatic 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, wherein the aromatic molecular scaffold is 1,3,5-tris(bromomethyl)benzene (TBMB), and the peptide ligand is 【Chemistry 1】 (where X 1 represents any amino acid residue, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutical acceptable salt thereof.

2. the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of two amino acids, and the peptide ligand 【Chemistry 2】 (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively.

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

3. the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of six amino acids and the second of which consists of three amino acids, and the peptide ligand comprises 【Chemistry 3】 (where X 1 represents any amino acid residue, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively.

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

4. The peptide ligand is selected from SEQ ID NOs: 1-3: 【Chemistry 4】 (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively.

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

5. The peptide ligand is A-(SEQ ID NO:1)-A (referred to herein as BCY507); A-(SEQ ID NO:1)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY543); A-(SEQ ID NO:2)-A (referred to herein as BCY508); [B-Ala][Sar] 5 A-(SEQ ID NO:2)-A (the fluoresced derivative of which is hereafter referred to as BCY544); A-(SEQ ID NO:3)-A (referred to herein as BCY509); [B-Ala][Sar] 5 A-(SEQ ID NO:3)-A (the fluoresced derivative of which is hereafter referred to as BCY545); and A-(SEQ ID NO:3)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY546.) 5. The peptide ligand of claim 4, or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from:

6. The peptide ligand is selected from SEQ ID NOs: 4-6: 【Chemistry 5】 (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively.

4. The peptide ligand according to claim 3, or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from any one of the following:

7. The peptide ligand is A-(SEQ ID NO:4)-A (referred to herein as BCY514); A-(SEQ ID NO:4)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY550); G[Sar] 5 A-(SEQ ID NO:4)-A (the fluoresced derivative thereof is hereafter referred to as BCY583); A-(SEQ ID NO:5)-A (referred to herein as BCY515); G[Sar] 5 A-(SEQ ID NO:5)-A (the fluoresced derivative of which is hereafter referred to as BCY584); A-(SEQ ID NO:6)-A (referred to herein as BCY516); and G[Sar] 5 A-(SEQ ID NO:6)-A (the fluorescent derivative of which is hereafter referred to as BCY585) 7. The peptide ligand of claim 6, or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from:

8. The peptide ligand is A-(SEQ ID NO:9)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY551.) 4. The peptide ligand of claim 3, comprising an amino acid sequence which is:

9. The peptide ligand or a pharma- ceutically acceptable salt thereof of claim 1, wherein the peptide ligand is selected from BCY507, BCY508, BCY509, BCY514, BCY515, BCY516, BCY543, BCY544, BCY545, BCY546, BCY550, BCY551, BCY583, BCY584, and BCY585.

10. The peptide ligand or a pharma- ceutical acceptable salt thereof according to claim 1, wherein said peptide ligand is BCY509.

11. 11. The peptide ligand or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 10, wherein the pharma- ceutical acceptable salt is selected from sodium, potassium, calcium, or ammonium salts.

12. 12. The peptide ligand or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 11, wherein the PD-L1 is human PD-L1.

13. A drug conjugate comprising the peptide ligand or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 12 conjugated to one or more drugs.

14. 14. A drug conjugate comprising the peptide ligand or a pharma- ceutically acceptable salt thereof according to claim 13, wherein the drug is a cytotoxic agent.

15. A drug conjugate comprising the peptide ligand of any one of claims 1 to 12, or a pharma- ceutical acceptable salt thereof, conjugated to a metal chelating agent.

16. The drug conjugate of claim 15, wherein the metal chelator is complexed to a metal radioisotope.

17. The drug conjugate of claim 16, wherein the metal radioisotope is selected from 64 Cu, 67 Ga, 68 Ga, 177 Lu, 90 Y, and 213 Bi.

18. The drug conjugate of claim 17, wherein the metal radioisotope is 68 Ga.

19. The drug conjugate of claim 15, further incorporating a radioactive isotope, said radioactive isotope being selected from 11 C, 18 F, 15 O and 13 N.

20. The drug conjugate of claim 19, wherein the radioisotope is 18F.

21. A pharmaceutical composition comprising a drug conjugate described in any one of claims 13 to 20.

22. 22. The pharmaceutical composition of claim 21 for preventing, inhibiting, or treating a disease or disorder mediated by PD-L1.

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