Bicyclic peptide ligands specific for PD-l1

Abstract

To provide polypeptides which are covalently bound to non-aromatic molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffolds.SOLUTION: A peptide ligand specific for PD-L1 is provided which comprises: a polypeptide comprising at least three reactive groups separated by at least two loop sequences; and a non-aromatic molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide, so that at least two polypeptide loops are formed on the molecular scaffold.SELECTED DRAWING: Figure 1

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 polypeptides covalently attached to non-aromatic molecular scaffolds. The invention describes peptides that are high affinity binders of PD-L1. A drug conjugate comprising the peptide conjugated to an effector and / or functional group. and a pharmaceutical composition comprising said peptide ligand and drug conjugate, The peptide ligands and This also includes the use of 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, macrocycles are used as cyclic peptide CXCR4 antagonists, such as CVX15 (400 Å 2 (Wu et al., 2007), Science 330, 1066-71), Arg-Gly that binds to integrin αVb3 -Asp motif cyclic peptide (355 Å 2 )(Xiong et al. (2002), Science 296(5565), 151-5), or cyclic peptide inhibitors that bind to urokinase-type plasminogen activator Upain-1 (603Å 2 (Zhao et al., 2007), J Struct Biol 160(1), 1-10), It bonds to a surface of square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides. This results in a 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, such as actinomycin and cyclopentadiene, 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 capable of converting a cysteine-containing polypeptide, e.g., 1,1',1''-(1,3,5- Molecular scaffolding such as triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) The method for the preparation of the 1,2-diaminodiphenyl ether (1,2-diphenyl ether) is described in Heinis et al. (2014) Angewandt et al. e Chemie, International Edition 53(6) 1602-1606).

[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 reactive groups and capable of forming a covalent bond with the reactive groups of the polypeptide. A non-aromatic molecular scaffold, which results in at least two polypeptide loops. A peptide ligand specific for PD-L1 is provided, wherein the peptide ligand is formed on the molecular scaffold. can be.

[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, the present invention relates to a method for preventing or inhibiting a disease or disorder mediated by PD-L1. or a peptide ligand or drug conjugate as defined herein for use in therapy. Jugate is provided. [Brief description of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: PD-1 / PD-L1 blockade bioassay results with BCY10467, BCY10939, and BCY10959. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention According to one particular embodiment of the invention, at least two loop sequences are provided. A polypeptide comprising at least three cysteine ​​residues spaced apart and a cysteine ​​fragment of said polypeptide. It contains a non-aromatic molecular scaffold that forms a covalent bond with the stain residue, resulting in At least two polypeptide loops are formed on the molecular scaffold, which is specific for PD-L1. Different peptide ligands are provided.

[0012] In one embodiment, the loop sequence comprises 3, 5, 6, 7, or 9 amino acids.

[0013] In a further embodiment, the loop sequences, the first of which consists of 7 amino acids. The second one has three nucleotides separated by two loop sequences of five amino acids each. Contains a reactive group.

[0014] In a further embodiment, the loop sequences, the first of which consists of 7 amino acids. The second one has three nucleotides separated by two loop sequences of five amino acids each. It contains reactive groups (i.e., two Cys residues and one Pen residue).

[0015] In a further embodiment, the loop sequences, the first of which consists of 7 amino acids. The second one has three nucleotides separated by two loop sequences of five amino acids each. Contains a cysteine ​​residue.

[0016] In a further embodiment, the loop sequences, the first of which consists of 5 amino acids. The second one has three loops separated by two loops of six amino acids. Contains a reactive group.

[0017] In a further embodiment, the loop sequences, the first of which consists of 5 amino acids. The second one has three nucleotides separated by two loop sequences of six amino acids. Contains a cysteine ​​residue.

[0018] In a further embodiment, the loop sequences, the first of which consists of three amino acids. The second one has three nucleotides separated by two loop sequences of nine amino acids. Contains a reactive group.

[0019] In a further embodiment, the loop sequences, the first of which consists of three amino acids. The second one has three nucleotides separated by two loop sequences of nine amino acids. Contains a cysteine ​​residue.

[0020] In a further embodiment, the loop sequences, the first of which consists of 7 amino acids. The second one has three repeats separated by two loop sequences of four amino acids. Includes reaction bases.

[0021] In a further embodiment, the loop sequences, the first of which consists of 7 amino acids. The second one has three sequences separated by two loop sequences of four amino acids. Contains stain residues.

[0022] In one embodiment, the peptide ligand is [ka] TIFF2025020121000003.tif245170TIFF2025020121000004.tif246170TIFF2025020121000005.tif15170(where C i , [Pen] i , C ii [Pen] ii , C iii , and [Pen] iii are the first, second, and and the third reactive group, HArg represents homoarginine, HSer represents homoserine, HP he represents homophenylalanine, Aib represents 2-aminoisobutyric acid, and Abu represents 2-amino butyric acid, 2Nal represents 3-(2-naphthyl)-L-alanine, and Chg represents cyclohexylglycine. Nva stands for norvaline, 7-AzaW stands for 7-azatryptophan, and Aad stands for 2-aminoadipic acid, Cpa represents β-cyclopropylalanine, and Dab represents 2,4-diaminoadipic acid. 3HyV stands for 2-amino-3-hydroxy-3-methyl-butyric acid, Nle stands for nor leucine, Pen stands for penicillamine, and PYA stands for 4-pentynoic acid). or a pharma- ceutically acceptable salt thereof.

[0023] In a further embodiment, the peptide ligand is (B-Ala)-Sar5-A-(SEQ ID NO:1)-A (hereinafter referred to as 73-07-00-N001); A-(SEQ ID NO:1)-A (referred to herein as 73-07-00-N002 or BCY519); A-(SEQ ID NO:2)-A (referred to herein as 73-08-00-N002 or BCY521); A-(SEQ ID NO:3)-A (referred to herein as 73-09-00-N002 or BCY522); A-(SEQ ID NO:4)-A (referred to herein as 73-10-00-N002 or BCY523); A-(SEQ ID NO:5)-A (referred to herein as 73-13-00-N002 or BCY526) A-(SEQ ID NO:6)-A (referred to herein as 73-14-00-N002 or BCY527); A-(SEQ ID NO:7)-A (referred to herein as 73-14-01-N001 or BCY528); A-(SEQ ID NO:8)-A (referred to herein as 73-14-02-N001 or BCY529); A-(SEQ ID NO:9)-A (referred to herein as 73-14-03-N001 or BCY530); A-(SEQ ID NO:10)-A (referred to herein as 73-14-04-N001 or BCY531); A-(SEQ ID NO:11)-A (referred to herein as 73-14-05-N001 or BCY532); A-(SEQ ID NO:12)-A (referred to herein as 73-14-06-N001 or BCY533); A-(SEQ ID NO:13)-A (referred to herein as 73-14-07-N001 or BCY534); A-(SEQ ID NO:14)-A (referred to herein as 73-14-08-N001 or BCY535); A-(SEQ ID NO:15)-A (referred to herein as 73-14-10-N001 or BCY537); A-(SEQ ID NO:16)-A (referred to herein as 73-15-00-N002 or BCY538); A-(SEQ ID NO:17)-A (referred to herein as 73-16-00-N002 or BCY539); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:18) (hereinafter referred to as BCY8938); SDK-(SEQ ID NO:19)-A (hereafter referred to as BCY3835); [PYA]-[B-Ala]-[Sar 10 ]-SDK-(SEQ ID NO:19) (hereinafter referred to as BCY10043); NH2-SDK-(SEQ ID NO:19)-[Sar 10 ]-[K(PYA)] (hereafter referred to as BCY10044); [Ac][dS]DK-(SEQ ID NO:19)-A (hereafter referred to as BCY10051); SD[HArg]-(SEQ ID NO:19)-A (hereafter referred to as BCY10052); SD[HSer]-(SEQ ID NO:19)-A (hereafter referred to as BCY10053); [Ac]SDK-(SEQ ID NO:19)-A (hereafter referred to as BCY10075); [Ac]SDR-(SEQ ID NO:19) (hereafter referred to as BCY10076); [Ac]SDK-(SEQ ID NO:19)-PSH (hereafter referred to as BCY10943); [Ac]SEK-(SEQ ID NO:19) (hereinafter referred to as BCY10951); [Ac]-SDK-(SEQ ID NO:19) (hereafter referred to as BCY11832); [Ac]-A-(SEQ ID NO:19)-PSH (hereafter referred to as BCY11833); SDK-(SEQ ID NO:19)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY566); [Ac]D[HArg]-(SEQ ID NO:19) (hereafter referred to as BCY11865); NH2-SDK-(SEQ ID NO:20) (hereinafter referred to as BCY10045); Ac-SDK-(SEQ ID NO:20)-PSH (hereafter referred to as BCY10861); Ac-SDK-(SEQ ID NO:20) (hereafter referred to as BCY11013); SDK-(SEQ ID NO:21)-A (hereafter referred to as BCY10054); SDK-(SEQ ID NO:22)-A (hereafter referred to as BCY10055); SDK-(SEQ ID NO:23)-A (hereafter referred to as BCY10057); SDK-(SEQ ID NO:24)-A (hereafter referred to as BCY10058); SDK-(SEQ ID NO:25)-A (hereinafter referred to as BCY10060); SDK-(SEQ ID NO:26)-A (hereafter referred to as BCY10061); SDK-(SEQ ID NO:27)-A (hereafter referred to as BCY10062); SDK-(SEQ ID NO:28)-A (hereafter referred to as BCY10064); SDK-(SEQ ID NO:29)-A (hereafter referred to as BCY10065); SDK-(SEQ ID NO:30)-A (hereafter referred to as BCY10066); SDK-(SEQ ID NO:31)-A (hereafter referred to as BCY10067); [Ac]-SDK-(SEQ ID NO:31)-PSH (hereafter referred to as BCY11830); SDK-(SEQ ID NO:32)-A (hereafter referred to as BCY10068); SDK-(SEQ ID NO:33)-A (hereafter referred to as BCY10069); SDK-(SEQ ID NO:34)-A (hereafter referred to as BCY10071); SDK-(SEQ ID NO:35)-A (hereafter referred to as BCY10072); SDK-(SEQ ID NO:36)-A (hereafter referred to as BCY10073); SDK-(SEQ ID NO:37)-A (hereafter referred to as BCY10074); [Ac]SDK-(SEQ ID NO:38) (hereinafter referred to as BCY10078); SDK-(SEQ ID NO:39)-A (hereafter referred to as BCY10083); SDK-(SEQ ID NO:40)-A (hereafter referred to as BCY10084); SDK-(SEQ ID NO:41)-A (hereafter referred to as BCY10085); [Ac]SDK-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10467); [Ac]SDK-(SEQ ID NO:42)-PS (hereafter referred to as BCY10934); [Ac]SDK-(SEQ ID NO:42)-P (hereafter referred to as BCY10935); [Ac]SDK-(SEQ ID NO:42)-PS-COOH (hereinafter referred to as BCY10936); [Ac]SDK-(SEQ ID NO:42)-P-COOH (hereafter referred to as BCY10937); [Ac]SDK-(SEQ ID NO:42)-COOH (hereinafter referred to as BCY10938); [Ac]SDK-(SEQ ID NO:42)-PSH-COOH (hereinafter referred to as BCY10939); [Ac]DK-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10940); [Ac]K-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10941); [Ac]-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10942); [Ac]SD[HArg]-(SEQ ID NO:43)-PSH (hereafter referred to as BCY10944); [Ac]SDK-(SEQ ID NO:44) (hereinafter referred to as BCY10945); [Ac]SDK-(SEQ ID NO:45) (hereinafter referred to as BCY10946); [Ac]SDK-(SEQ ID NO:46) (hereinafter referred to as BCY10947); [Ac]SDK-(SEQ ID NO:47) (hereinafter referred to as BCY10948); [Ac]SDK-(SEQ ID NO:48) (hereinafter referred to as BCY10949); [Ac]SDK-(SEQ ID NO:49) (hereinafter referred to as BCY10950); [Ac]SDK-(SEQ ID NO:50) (hereinafter referred to as BCY10952); [Ac]SDK-(SEQ ID NO:51) (hereinafter referred to as BCY10954); [Ac]SDK-(SEQ ID NO:52) (hereinafter referred to as BCY10956); [Ac]SD[HArg]-(SEQ ID NO:53)-PSH (hereafter referred to as BCY10959); [Ac]SDK-(SEQ ID NO:54) (hereinafter referred to as BCY10960); [Ac]SDK-(SEQ ID NO:55) (hereinafter referred to as BCY10961); [B-Ala][Sar]5A-(SEQ ID NO:56)-A (the fluoresced derivative thereof is hereafter referred to as BCY562); VER-(SEQ ID NO:57)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY567); REN-(SEQ ID NO:58)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY568); QQE-(SEQ ID NO:59)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY569); SGK-(SEQ ID NO:59)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY577); AGS-(SEQ ID NO:60)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY570); AQT-(SEQ ID NO:61)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY571); APV-(SEQ ID NO:62)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY572); ADV-(SEQ ID NO:63)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY573); GNK-(SEQ ID NO:64)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY574); KPK-(SEQ ID NO:64)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY580); ERV-(SEQ ID NO:65)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY575); AER-(SEQ ID NO:66)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY576); KEL-(SEQ ID NO:67)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY578); G[Sar]5KEL-(SEQ ID NO:67)-A (the fluoresced derivative thereof is hereafter referred to as BCY3811); KEL-(SEQ ID NO:68)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY579); A-(SEQ ID NO:69)-PSH[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY581); A-(SEQ ID NO:70)-DHEN[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY582); REE-(SEQ ID NO:71)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY3812); QAEK-(SEQ ID NO:72)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY8210); A-(SEQ ID NO:73)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7126); A-(SEQ ID NO:74)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7374); A-(SEQ ID NO:75)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7375); A-(SEQ ID NO:76)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7376); A-(SEQ ID NO:77)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7377); A-(SEQ ID NO:78)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7384); A-(SEQ ID NO:79)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7385); A-(SEQ ID NO:80)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7386); A-(SEQ ID NO:81)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7387); A-(SEQ ID NO:82)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7388); A-(SEQ ID NO:83)-A[Sar]6 (the fluoresced derivative of which is hereafter referred to as BCY7389); Ac-DK-(SEQ ID NO:84) (hereafter referred to as BCY11818); Ac-K-(SEQ ID NO:84) (hereafter referred to as BCY11819); Ac-SEK-(SEQ ID NO:84) (hereafter referred to as BCY11820); Ac-EK-(SEQ ID NO:84) (hereafter referred to as BCY11821); Ac-DK-(SEQ ID NO:84)-PSH-COOH (hereinafter referred to as BCY11853); Ac-SDK-(SEQ ID NO:85) (hereafter referred to as BCY11822); Ac-DK-(SEQ ID NO:85) (hereafter referred to as BCY11823); Ac-K-(SEQ ID NO:85) (hereafter referred to as BCY11824); Ac-SEK-(SEQ ID NO:85) (hereinafter referred to as BCY11825); Ac-EK-(SEQ ID NO:85) (hereafter referred to as BCY11826); Ac-[HArg]-(SEQ ID NO:85) (hereinafter referred to as BCY11827); Ac-SDK-(SEQ ID NO:85)-A (hereafter referred to as BCY11828); Ac-SEK-(SEQ ID NO:85)-PSH (hereafter referred to as BCY11831); Ac-SDK-(SEQ ID NO:86) (hereafter referred to as BCY11829); Ac-D[HArg]-(SEQ ID NO:87)-PSH (hereafter referred to as BCY11866); Ac-D[HArg]-(SEQ ID NO:88)-PSH (hereafter referred to as BCY11867); Ac-D[HArg]-(SEQ ID NO:89)-PSH (hereafter referred to as BCY11868); Ac-D[HArg]-(SEQ ID NO:90)-PSH (hereafter referred to as BCY11869); Ac-SD[HArg]-(SEQ ID NO:91)-PSHK (hereafter referred to as BCY12479); Ac-SD[HArg]-(SEQ ID NO:92)-PSHK (hereinafter referred to as BCY12477); Ac-[HArg]-(SEQ ID NO:93)- PSH (hereafter referred to as BCY12640); Ac-[HArg]-(SEQ ID NO:94)-PSH (hereafter referred to as BCY12641); Ac-[HArg]-(SEQ ID NO:95)-PSH (hereafter referred to as BCY12642); Ac-[HArg]-(SEQ ID NO:96)-PSH (hereinafter referred to as BCY12643); and Ac-[HArg]-(SEQ ID NO:97)-PSH (referred to herein as BCY12644); (wherein PYA stands for 4-pentynoic acid, B-Ala stands for β-alanine, and Sar stands for sarcosyl HSe represents homoserine, and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof.

[0024] In one embodiment, the peptide ligand is any of: BCY10467, BCY10939, and BCY10959. The peptide sequences are selected from the following: In Table 1 and Table 6, the PD-L1-binding bicyclic peptides bind to PD-1-expressing T cells and stably express PD-L1. It was shown that the antibody showed the ability to block PD-1 / PD-L1 interaction between CHO-K1 and the PD-1 / PD-L1-expressing can.

[0025] In one embodiment, the peptide ligand is BCY11818, BCY11819, BCY11820, BCY11821 , BCY11822, BCY11823, BCY11824, BCY11825, BCY11826, BCY11827, BCY11828, BCY11829 , BCY11830, BCY11831, BCY11832, BCY11833, and / or BCY11853: The peptide sequences are selected from peptide sequences other than the number

[0026] In one embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5 -triyl)triprop-2-en-1-one (TATA).

[0027] In one embodiment, the peptide ligand is a peptide listed in any of Tables 1-5. is selected from the

[0028] 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. Standard practice has the same meaning as commonly understood by practitioners in the field of science and technology. 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).

[0029] (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 -S1-W2-S3-W4-L5-T6-M7-C ii -Q8-K9-L 10 -H 11 -L 12 -C iii - (SEQ ID NO: 1) .

[0030] For the purposes of this description, all bicyclic peptides are represented as 1,1',1''-(1,3,5-triazinane-1,3,5- It is believed that the cyclization with TATA (triyl)triprop-2-en-1-one results in a trisubstituted structure. Cyclization by TATA is i , C ii , and C iii This occurs above.

[0031] (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:

[0032] (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).

[0033] (Peptide Ligand) The peptide ligands referred to herein are covalently attached to a molecular scaffold. Typically, such peptides are covalently bonded to a scaffold. and two or more reactive groups (i.e., cysteine ​​residues and / or Pen residues) capable of forming Since the peptide forms a loop when it binds to the scaffold, it is called a loop sequence. In this case, the peptide comprises at least three sequences, and an internal sequence between the reactive groups. Stain residues (herein referred to as C i , C ii , and C iii (called a penicillin) or one and two cysteine ​​residues, and at least one cysteine ​​residue on the scaffold. also forms two loops.

[0034] (Reactive group) The molecular scaffolds of the present invention can be attached to polypeptides via functional or reactive groups on the polypeptide. These are typically found in polypeptide polymers. Such reactive groups are formed from the side chains of certain amino acids that are The side chain, or N-terminal amino group, or any other suitable reactive group, e.g., in penicillamine Details of suitable reactive groups can be found in WO 2009 / 098450.

[0035] Examples of reactive groups in natural amino acids are the thiol group of cysteine, the amino group of lysine, and the amino group of asparagine. Carboxyl group of ginic acid or glutamic acid, guanidinium group of arginine, tyrosine The phenolic group of phenylalanine or the hydroxyl group of serine. -Provides a wide range of reactive groups including carbonyl, alkyne, vinyl, or aryl halide groups The amino and carboxyl groups at the ends of the polypeptide can also be used as molecular scaffolds. The aryl group can act as a reactive group to form a covalent bond with the ring / molecular core.

[0036] The polypeptides of the present invention contain at least three reactive groups. The more reactive groups used, the more The loops can be formed in the molecular scaffold.

[0037] In a preferred embodiment, a polypeptide having three reactive groups is produced. Reaction of peptides with molecular scaffolds / cores with three-fold symmetry results in the formation of single Production of a single product isomer is preferred for several reasons. The nucleic acids of the compound library only encode the primary sequence of the polypeptide, but It does not code for the isomeric state of the molecule that is formed upon reaction of the base with the molecular core. In cases where isomers can be formed, the assignment of nucleic acids to product isomers is clearly defined. If multiple product isomers are formed, the nucleic acid may be useful in screening or selection processes. No information can be given about the nature of the isolated product isomers. Single product isomers This information is also advantageous when specific members of the libraries of the invention are synthesized. In the case of Instead, a single product isomer is produced.

[0038] In another embodiment, a polypeptide having four reactive groups is produced. The reaction of tides with molecular scaffolds / molecular cores with tetrahedral symmetry gives two products: Isomers are produced. When two different product isomers are encoded by the same nucleic acid, However, it is possible to chemically synthesize both isomers, separate the two isomers, and then target both isomers with the target ligand. The nature of the isolated isomers can be determined by testing for binding to the isomers. do.

[0039] In one embodiment of the invention, at least one of the reactive groups of the polypeptide is The use of an orthogonal reactive group allows the orthogonal reactive group to be attached to a specific portion of the molecular core. Using a linking strategy involving orthogonal reactive groups, the product formed can be In other words, the number of product isomers can be limited by at least three bonds. Attach at least three reactive groups separate or different from the reactive groups selected for the remaining ones. By selecting for one or more of the following, a specific position on the molecular scaffold can be obtained. This effectively achieves a specific order of attachment or orientation of specific reactive groups on a polypeptide to the corresponding positions. This can be done.

[0040] In another embodiment, the reactive group of the polypeptide of the invention is reacted with a molecular linker, In this case, the linker is a molecular scaffold and a polypeptide in a final conjugated state. The peptides can react with the molecular scaffold so as to intercalate between the peptides.

[0041] In some embodiments, the identification of members of a library or set of polypeptides The amino acids can be replaced with any natural or unnatural amino acid. Only the loop sequence is replaced. those with functional groups for cross-linking the polypeptide to the molecular core so that it can be exchanged; These replaceable amino acids are excluded. The replaceable polypeptide sequences are random. Either a regular sequence, a fixed sequence, or a sequence with random and fixed amino acids The position of these amino acids determines the loop size, so the amino acids with reactive groups Each of the acids is at a defined position within the polypeptide.

[0042] In one embodiment, the polypeptide having three reactive groups has the sequence (X) l Y(X) m Y(X) nY(X) o where Y represents an amino acid having a reactive group and X represents a random amino acid. , m and n are the intervening polypeptide segments (which may be the same or different); represents a number from 3 to 6 that defines the length of the adjacent polypeptide segments, Represents a number from 0 to 20 that specifies the length.

[0043] An alternative to thiol-mediated conjugation is via covalent interactions Alternatively, these techniques can be used to attach the molecular scaffold to the peptide. In accordance with the present invention, a further moiety (e.g., a small molecule of interest different from the molecular scaffold) is included. Thus, after selection or isolation, the further moiety can be modified or attached to the polypeptide. can be used - in this embodiment, obviously, the bond is covalent. These methods involve the use of complementary reactive groups. Proteins with unnatural amino acids having the required chemically reactive groups in combination with small molecules and By producing phages displaying the peptides or by selecting molecules after a selection / isolation step. Unnatural amino acids are added to chemically or recombinantly synthesized polysaccharides as they are made. Incorporation into peptides can be used instead of (or in combination with) thiol-mediated methods. Further details can be found in WO 2009 / 098450 or in Heinis et al., Nat Ch em Biol 2009, 5(7), 502-7.

[0044] In one embodiment, the reactive groups are cysteine ​​and / or penicillamine: [ka] is selected from.

[0045] In one embodiment, each of the reactive groups comprises a cysteine. wherein one of the reactive groups comprises penicillamine and the remaining (i.e., two) reactive groups comprise Contains cysteine.

[0046] (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 fixed, short-term exposure or to develop bicyclic peptides with enhanced retention in the circulation. It may be necessary to develop novel peptides that are best suited to managing more chronic disease states. Other factors driving new plasma half-lives are the need for sustained exposure for maximum therapeutic efficacy and the need for drug Concomitant toxicity with sustained exposure; and -Selectivity. The specific peptide ligands of the present invention have better selectivity than other transmembrane proteins. Indicates gender.

[0047] (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

[0048] 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.

[0049] 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., For example, 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] (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 examples of suitable 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:

[0054] 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.

[0055] 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.

[0056] 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. .

[0057] 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.

[0058] 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.

[0059] Alternatively, proteolytic hydrolysis of nearby peptide bonds may result in conformational and steric Non-natural amino acids with constrained amino acid side chains may be used so as to be hindered. 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.

[0060] 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.

[0061] 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 aryloxy group residue with a naphthylalanine or alanine residue. Offers the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligands do.

[0062] 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. The correct balance of amino acid residues is an important feature of bicyclic peptide ligands. For example, Hydrophobic amino acid residues affect the degree of plasma protein binding and therefore the available free radicals in plasma. The concentration of the isolated fraction is affected, 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 correct combination and number of residues reduces irritation at the injection site (if the peptide drug is administered subcutaneously). It can be reduced.

[0063] 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 . 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).

[0064] 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.

[0065] 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, amino acid side chains should be correctly oriented so that entropy loss is minimized upon target binding. The torsion angles of the backbone should be tightly constrained so that entropy loss is minimized upon target binding. By restricting the degree of cyclization and for the same reason by 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).

[0066] (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.

[0067] 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.

[0068] Certain isotopically labeled peptide ligands of the invention, e.g., incorporate a radioisotope. These include drug and / or substrate tissue distribution studies and the presence of PD-L1 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, luminescence 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.

[0069] 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.

[0070] 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

[0071] 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.

[0072] (Non-aromatic molecular scaffolds) Reference herein to the term "non-aromatic molecular scaffold" refers to an aromatic ( i.e., any of the ring systems defined herein that do not contain (unsaturated) carbocyclic or heterocyclic ring systems. This refers to the molecular scaffold of

[0073] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chem. ie, International Edition 53(6) 1602-1606.

[0074] As described in the aforementioned documents, molecular scaffolds are small molecules, such as small organic molecules. It may be a child.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In one embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5 -triyl)triprop-2-en-1-one (TATA), or a derivative thereof, It may consist of these.

[0079] An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1''-(1,3,5-triazinane-1,3,5-triphenylphosphine) (yl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition, on(2014), 53(6), 1602-1606).

[0080] (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. .

[0081] 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.

[0082] 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

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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).

[0092] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure: do: [ka] .

[0093] Monomethylauristatin E (MMAE) is a synthetic anti-neoplastic agent having the following structure: [ka] .

[0094] In yet a further specific embodiment of the invention, the cytotoxic agent is maytansinoic acid. The DM1 is selected from the group consisting of

[0095] 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.

[0096] Published studies have demonstrated that the introduction of steric hindrance on either side of the disulfide bond results in 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 lead to 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. , versus optimizing efficient release in the intracellular environment, which maximizes therapeutic efficacy. Disulfide stability in circulation, which minimizes unwanted side effects of the toxin The selection of optimal conditions involves careful selection of the degree of disorder on either side of the disulfide bond. This can be achieved by:

[0097] 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.

[0098] 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).

[0099] (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

[0100] 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: The procedure is performed on the final polypeptide conjugate produced by the method of the present invention.

[0101] Optionally, the amino acid residues in the polypeptide of interest are linked to form a conjugate or complex. They may be replaced when manufacturing.

[0102] The peptide can be extended, for example, to incorporate additional loops and thus introduce multiple specificities. You can also enter.

[0103] 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, or by other methods such as those described in, for example, 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).

[0104] 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 The cysteine ​​residues are added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups. then an additional cysteine ​​or thiol can be added to the N- or C-terminus of the first peptide; A cysteine ​​or thiol can be added to the second peptide so that the cysteine ​​or thiol can be released from the second peptide. It reacts only with cysteine ​​or thiol of the peptide to form a disulfide-bonded bicyclic peptide. A tide conjugate was formed.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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 combinations of 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.

[0111] 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.

[0112] The peptide ligands of the present invention may be lyophilized prior to storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and is compatible with freezing methods known in the art. Freeze-drying and reconstitution techniques are available. 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.

[0113] 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 / day, with doses of 0.05 to 2.0 mg / kg / day being more commonly used. In addition, compositions containing the peptide ligands or cocktails thereof also have similar or slightly Small dosages may be administered.

[0114] 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 Additionally, the peptide ligands described herein can be selected ex vivo or in vitro. selectively used to selectively kill or deplete a target cell population from a heterogeneous collection of cells. The blood from the mammal can be filtered or otherwise effectively removed. The peptide ligand can then be combined in vitro and then expressed in mammalian cells according to standard techniques. Killing or otherwise removing undesirable cells from the blood for return to the animal .

[0115] (therapeutic use) The bicyclic peptides of the invention have particular utility as PD-L1 binding agents.

[0116] Programmed cell death 1 ligand 1 (PD-L1) is located on mouse chromosome 19 and human chromosome 9. It is a type I transmembrane protein of 290 amino acids encoded by the CD274 gene. Chronic infections, such as chronic viral infections (including, for example, HIV, HBV, HCV, and HTLV, among others). chronic bacterial infections (e.g., including, among others, Helicobacter pylori) as well as chronic parasitic infections (including Schistosoma mansoni). PD-L1 expression is involved in evading immune responses associated with tumors. Several types of cells, including hematopoietic cells, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, muscle cells, and placenta. It has been detected in several tissues and cell types.

[0117] PD-L1 expression is also involved in suppressing antitumor immune activity. Tumors are recognized by host T cells and Although most tumors express antigens that can be recognized, immunological clearance of the tumors is rare. Part of the defect is due to immunosuppression by the tumor microenvironment. PD-L1 expression is a component of this suppressive environment and acts in concert with other immunosuppressive signals. PD-L1 expression is seen in breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, and nerve. It has been shown to be effective in a wide variety of solid tumors in situ, including glioma, thyroid, thymic epithelium, and head and neck. (Brown JA et al., 2003 Immunol. 170:1257-66; Dong H et al., 2003 Immunol. 170:1257-66) 2002 Nat. Med. 8:793-800; Hamanishi J et al., 2007 Proc. Natl. Acad. Sci. USA 1 04:3360-65; Strome SE et al., 2003 Cancer Res. 63:6501-5; Inman BA et al., 200 7 Cancer 109:1499-505; Konishi J et al., 2004 Clin. Cancer Res. 10:5094-100; Na Kanishi 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. S ci. USA 101: 17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). In addition, PD Expression of programmed cell death protein 1 (also known as PD-1 and CD279), the receptor for IL-1 This expression is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C (2003 Immunol. 171:4574-81). Most importantly, tumor PD-L1 expression was correlated with disease progression. Studies linking PD-L1 expression to outcome have shown that PD-L1 expression is associated with renal, ovarian, bladder, breast, gastric, and It has been shown to be strongly correlated with poor prognosis in pancreatic cancer (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al., 2007 Cancer 1 09:1499-505; 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 Proc. Natl. Acad. Sci. USA 1 01:17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). Furthermore, these studies Therefore, higher levels of PD-L1 expression in tumors are associated with advanced tumor stages and deeper tissue structures. It has been suggested that this may facilitate invasion of the tissue.

[0118] The PD-1 pathway may also play a role in hematological malignancies. PD-L1 is involved in a number of bone marrow It is expressed in tumor cells but not in normal plasma cells (Liu J et al., 2007 Blood 1 10:296-304). PD-L1 is expressed in some primary T-cell lymphomas, particularly anaplastic large cell T-lymphoma. (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is an angioimmunoblastic phospholipase A (AN-IMB) kinase inhibitor. PD-L1 is highly expressed on T cells in lymphomas, and PD-L1 is expressed in the associated follicular dendritic cell network. (Dorfman DM et al., 2006 Am. J. Surg. Pathol. 30:802-10). In Hodgkin lymphoma, T cells associated with lymphocytes or histiocytes (L&H) cells express PD-1. Microarray analysis using readout of genes induced by PD-1 ligation Ray analysis reveals that tumor-associated T cells express PD-1 signaling in situ in Hodgkin lymphoma (Chemnitz JM et al., 2007 Blood 110:3226-33). D-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma (S Himauchi T et al., 2007 Int. J. Cancer 121: 2585-90). These tumor cells express TCR signaling. It has low response to signals.

[0119] Studies in animal models have shown that PD-L1 on tumors blocks T cell activation and tumor cell lysis. It has been shown that T cell proliferation and proliferation in tumors can be inhibited by T cells, and in some cases, can result in 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 regulate antitumor T cell responses. PD-L1 expression on a population of tumor-associated myeloid DCs is upregulated by factors in the tumor environment. (Curiel TJ et al., 2003 Nat. Med. 9:562-67). Intranodal plasmacytoid dendritic cells (DCs) express IDO, which potently activates the suppressive activity of regulatory T cells The suppressive activity of IDO-treated regulatory T cells required cell contact with IDO-expressing DCs ( Sharma MD et al., 2007 Clin. Invest. 117:2570-82).

[0120] Thus, infectious diseases, e.g., chronic intracellular infectious diseases, e.g., viral diseases, For example, hepatitis infection, or bacterial infection, for example, tuberculosis infection; and cancer, for example, liver cancer, for example There is a need in the art for effective treatments for PD-L1 associated diseases, such as hepatocellular carcinoma.

[0121] 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.

[0122] 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).

[0123] According to a further aspect of the present invention, a method for preventing, inhibiting, or preventing a disease or disorder mediated by PD-L1 is provided. or a peptide ligand or drug conjugate as defined herein for use in therapy. Jugate is provided.

[0124] According to a further aspect of the present invention, a method for preventing, inhibiting, or preventing a disease or disorder mediated by PD-L1 is provided. or a method of treatment comprising administering to a patient in need thereof a peptide as defined herein. Methods are provided that include administering an effector group of a ligand and a drug conjugate. can be.

[0125] In one embodiment, the PD-L1 is a mammalian PD-L1. Mammalian PD-L1 may be human PD-L1 (hPD-L1) or murine PD-L1 (mPD-L1). All peptide sequences identified in this study were identical to the human PD-L1 binding sequence, except for BCY8938, which binds to mouse PD-L1. In a further embodiment, the mammalian PD-L1 is human PD-L1 (hPD-L1).

[0126] In one embodiment, the disease or disorder mediated by PD-L1 is a chronic infection or disease, The tumor is selected from a solid tumor, and a hematological malignancy.

[0127] In one embodiment, the chronic infection or disease mediated by PD-L1 is a chronic viral infection. The infection is selected from chronic bacterial infections, chronic parasitic infections, hepatitis infections, and viral diseases.

[0128] In one embodiment, the solid tumor mediated by PD-L1 is breast, lung, colon, ovarian, Melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymic epithelium, head and neck tumors is selected from.

[0129] In a further embodiment, the disease or disorder mediated by PD-L1 is selected from cancer. can be.

[0130] 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:

[0131] In further embodiments, the cancer is renal, ovarian, bladder, breast, stomach, and pancreatic cancer. .

[0132] In yet further embodiments, the cancer is selected from liver cancer, e.g., hepatocellular carcinoma and hepatocellular carcinoma. can be.

[0133] In further embodiments, the cancer is, for example, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BLL), or Myeloma (BL), multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (TCL) Leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), chronic myeloid leukemia (CML), and nodular lymphocyte-predominant Hodgkin's lymphoma malignant tumors of the hematopoietic system selected from lymphomas.

[0134] 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.

[0135] 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.

[0136] The invention will now be further described with reference to the following examples. EXAMPLES

[0137] (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 was converted to 1,3,5-triacryloylhexahydro-1 To this end, the linear peptide was reacted with 50:50 MeCN: Dilute to approximately 35 mL with HO, add approximately 500 μL of 100 mM TATA in acetonitrile, and add 5 mM The reaction was initiated with L of 1M NH4HCO3. The reaction was allowed to proceed at RT for approximately 30 to 60 min and then diluted with (MALD When the reaction was complete (as judged by I), it was lyophilized. Cysteine ​​hydrochloride monohydrate (Sigma) was added to the reaction mixture for approximately 60 min at RT to quench excess TATA. After lyophilization, the modified peptide was purified as described above, while Luna C8 was The column was replaced with a 0.1% C18 column (Phenomenex) and the acid was changed to 0.1% trifluoroacetic acid. Pure fractions containing TA-modified material were pooled, lyophilized, and kept at -20 °C for storage. did.

[0138] Unless otherwise noted, all amino acids were used in the L-configuration.

[0139] In some cases, the peptide is converted to an activated disulfide and then purified using the following method: Coupling to the free thiol group of the toxin; 4-methyl(succinimidyl) 4-(2-pyridinyl A solution of the peptide (20 mM) in anhydrous DMSO (1.25 mol equivalents) was dissolved in anhydrous DMSO. The reaction mixture was mixed well and DIPEA (20 mol equiv.) was added. The reaction was monitored by LC / MS until completion.

[0140] (Biological Data) (1. PD-L1 Direct Binding Assay) Human PD-L1 was detected using a fluorescence polarization assay according to the method disclosed in WO2016 / 067035. The affinity (Ki) of the peptide of the present invention for the fluorescent tag (fluorescein, SIGMA or The peptide of the present invention having Alexa Fluor488 (trademark, Fisher Scientific) was used for 0. PBS containing 0.1% Tween 20 or 50 mM HEPES pH 7.4 containing 100 mM NaCl and 0.01% Tween (both The peptides were diluted to 2.5 nM in the same assay buffer as the peptides. In conjunction with titration of proteins in buffer, a low-binding, low-volume 384-well plate with black walls and bottom is Typically, 5 μL of assay buffer, 10 μL of protein, and then 10 μL of A total volume of 25 μL of fluorescent peptide gave rise to 1 nM peptide. A 1:2 serial dilution was used: Maximum concentrations range from 500 nM for known high affinity binders to 100 nM for low affinity binders and selectivity enhancers. Twelve different concentrations were produced, ranging from 10 μM for the assay. Measurements were performed with excitation at 485 nm and excitation at 520 nm. BMG equipped with the optical module "FP 485 520 520" for parallel and perpendicular emission detection at 1000 nm The experiment was performed using a PHERAstar FS. The PHERAstar FS was set at 25°C and 200 flashes were performed per well. Each well was measured for 60 minutes at 5-10 minute intervals with a 0.1 second positioning delay. The gain used in the analysis was calculated based on the number of tracers present in the well. The absence was determined at the end of 60 min. Data were analyzed using Systat Sigmaplot version 12.0. The mP values ​​were fitted to a user-defined quadratic equation to obtain Kd values: f = ymin + (ym ax-ymin) / Lig*((x+Lig+Kd) / 2-sqrt((((x+Lig+Kd) / 2)^2)-(Lig*x))). "Lig" is the The concentration of the tracer was the specified value.

[0141] The following selected bicyclic peptide ligands of the invention were assayed in the PD-L1 direct binding assay described above: The results are shown in Tables 1 and 2: Table 1: Human direct binding assay data for selected bicyclic peptide ligands of the invention [Table 1] TIFF2025020121000010.tif235170TIFF2025020121000011.tif228170TIFF2025020121000012.tif87170Table 2: Mouse direct binding assay data for selected bicyclic peptide ligands of the invention [Table 2]

[0142] (2. PD-L1 Competitive Binding Assay) The peptide without a fluorescent tag was Fl-G-Sar5-ACPDPHNICHLWCA (Kd=68.75 nM - see above protocol). The peptides were tested in competition with DMSO up to 5%. Dilute to the appropriate concentration in assay buffer as described in the indirect binding assay. This was followed by a 1:2 serial dilution. 5 μL of the diluted peptide was added to the plate, followed by 10 μL of Human PD-L1 was then added, followed by 10 μL of fluorescent peptide. The measurements were performed, with the gain determined before the first measurement. Data analysis was performed using Systat Sigma. ot version 12.0, where the mP values ​​were fitted to a user-defined cubic equation to obtain the Ki I got the value: f=ymin+(ymax-ymin) / Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-P rot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-P rot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+K lig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*((((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kc omp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+C omp-Prot*c))) / ((3*Klig)+((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c )+Klig*(Comp-Prot*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c )^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*K comp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*((((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*( Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp-P rot*c)))). "Lig", "KLig", and "Prot" are all respectively: fluorescent peptide concentration, fluorescent peptide These were the defined values ​​for Kd and PD-L1 concentration.

[0143] The following selected bicyclic peptide ligands of the invention were assayed in the PD-L1 competitive binding assay described above: The results are shown in Table 3: Table 3: Competitive binding assay data for selected bicyclic peptide ligands of the invention. [Table 3]

[0144] (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 D (nM) value To determine the binding of PD-L1 to recombinant human PD-L1 (Sino Biologicals or R&D system), a Biacore experiment was performed. tems) or mouse PD-L1 (R&D systems) were resuspended in PBS and incubated as per the manufacturer's suggested protocol. Biotin was then synthesized using EZ-Link™ Sulfo-NHS-LC-LC-Biotin Reagent (Thermo Fisher). The protein was desalted and uncoupled biotin was removed using a spin column. For binding analysis, a CM5 sensor chip (GE Healthcare) was used. A Biacore 3000 instrument was used. HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) was used as the running buffer. Streptavidin was coupled to the chiral marker using standard amine coupling chemistry at 25 °C. The carboxymethyl dextran surface was immobilized on the tip in a 1:1 ratio at a flow rate of 10 μl / min. 0.4M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1M N-Hydroxyethyl The antibody was activated with a 12-minute injection of hydroxysuccinimide (NHS). For capture, protein was diluted to 0.1 mg / ml in 10 mM sodium acetate (pH 4.5) and 70 μl was added to The remaining activated groups were captured by injecting them onto the activated chip surface. The cells were blocked with a 7-minute injection of 1:100 PBS (pH 8.5). Biotinylated PD-L1 stock was diluted 1:100 in HBS-N. The buffer was diluted and captured on one flow cell at 5 μl / min to a level of 1000-1300 RU. The solution was replaced with PBS / 0.05% Tween 20 and a dilution series of the peptides was added to this buffer to a final D The MSO concentrations were adjusted. The maximum concentration was 200 nM or 500 nM with six 2-fold dilutions. SPR analysis was performed. The assay was run at 25° C. with a flow rate of 50 μl / min, with an association time of 60 s and a dissociation time of 400 s. Data were analyzed using a DMS All data were processed using standard processing procedures. The injection and reference planes were double-referenced, and data processing and kinetic fitting were performed using a Scrutinized The analysis was performed using bber software, version 2.0c (BioLogic Software). A mass transfer model was used for fitting, taking into account mass transfer effects where appropriate.

[0145] The following selected bicyclic peptide ligands of the invention were tested in the SPR binding assay described above: The results are shown in Tables 4 and 5: Table 4: Human SPR binding assay data for selected bicyclic peptide ligands of the invention. [Table 4] TIFF2025020121000016.tif242170TIFF2025020121000017.tif243170TIFF2025020121000018.tif243170TIFF2025020121000019.tif242170TIFF2025020121000020.tif36170NB: No binding at 500nM Table 5: Mouse SPR binding assay data for selected bicyclic peptide ligands of the invention. [Table 5]

[0146] 3. PD-1 / PD-L1 Blockade Bioassay Overexpressing human PD-1 and expressing the luciferase gene under the NFAT response element Effector Jurkat cells modified as described above were purchased from Promega. Jurkat T cell receptor Human PD-L1 and engineered cell surface proteins designed to activate the immune system in an antigen-independent manner Target CHO-K1 cells overexpressing the protein were also purchased from Promega. The day before the assay, CHO-K1 cells were transfected with 100% ethanol. The cells were plated in Ham's F-12 medium + 10% FBS and allowed to adhere to the plate. On the day of the assay, the medium was removed and Jurkat cells were cultured in a dose-response test in RPMI 1640 medium containing 1% FBS. Plated with test article (total volume of 80 μL per well). PD-L1 monoclonal An antibody (PD-L1 mAb) was included as a positive control (clone MIH1, Invitrogen catalog #16598382) After 6 hours of incubation at 37°C and 5% CO2, 80 μL of Bio-Glo reagent (Promega) was added to each well. 10 wells and allowed to equilibrate at room temperature for 10 minutes. Luminescence was measured using a Clariostar plate reader. The luminescence signal was measured using a background well (both cell lines were Fold induction was calculated by dividing the mean of wells (wells containing test article and wells without test article) by the mean of wells containing test article and wells without test article. Data were graphed in Prism and fitted to a 4-parameter logistic curve.

[0147] The results are shown in Figure 1 and Table 6. In Figure 1 and Table 6, the PD-L1 binding bicyclic peptides The 1444T cells expressing PD-1 and 1444T cells stably expressing PD-L1 were treated with 1444T cells (BCY10467, BCY10939, and BCY10959). It can be seen that the results showed the ability to block PD-1 / PD-L1 interaction with O-K1. Table 6: PD-1 / PD-L1 Blockade Bioassay Results [Table 6]

Claims

1. A polypeptide containing at least three reactive groups separated by at least two loop sequences. and a non-aromatic molecular scaffold that forms a covalent bond with the reactive group of the polypeptide. so that at least two polypeptide loops are formed on the molecular scaffold. A peptide ligand specific for PD-L1.

2. 2. The peptide of claim 1, wherein the loop sequence comprises 3, 5, 6, 7, or 9 amino acids. Gund.

3. 1 or claim 1 , wherein the reactive groups are selected from cysteine ​​and / or penicillamine residues. Item 3. A peptide ligand according to item 2. 【Request 4】 【Chemical 1】 【change】 【change】 【change】 (Here, C i , [Pen] i , C ii [Pen] ii , C iii , and [Pen] iii are the first, second, and and the third reactive group, HArg represents homoarginine, HSer represents homoserine, HP he represents homophenylalanine, Aib represents 2-aminoisobutyric acid, and Abu represents 2-amino butyric acid, 2Nal represents 3-(2-naphthyl)-L-alanine, and Chg represents cyclohexylglycine. Nva stands for norvaline, 7-AzaW stands for 7-azatryptophan, and Aad stands for 2-aminoadipic acid, Cpa represents β-cyclopropylalanine, and Dab represents 2,4-diaminoadipic acid. 3HyV stands for 2-amino-3-hydroxy-3-methyl-butyric acid, Nle stands for nor leucine, Pen stands for penicillamine, and PYA stands for 4-pentynoic acid). or a pharma- ceutical acceptable salt thereof. A peptide ligand according to any one of claims 1 to 4.

5. (B-Ala)-Sar5-A-(SEQ ID NO:1)-A (referred to herein as 73-07-00-N001); A-(SEQ ID NO:1)-A (referred to herein as 73-07-00-N002 or BCY519); A-(SEQ ID NO:2)-A (referred to herein as 73-08-00-N002 or BCY521); A-(SEQ ID NO:3)-A (referred to herein as 73-09-00-N002 or BCY522); A-(SEQ ID NO:4)-A (referred to herein as 73-10-00-N002 or BCY523); A-(SEQ ID NO:5)-A (referred to herein as 73-13-00-N002 or BCY526); A-(SEQ ID NO:6)-A (referred to herein as 73-14-00-N002 or BCY527); A-(SEQ ID NO:7)-A (referred to herein as 73-14-01-N001 or BCY528); A-(SEQ ID NO:8)-A (referred to herein as 73-14-02-N001 or BCY529); A-(SEQ ID NO:9)-A (referred to herein as 73-14-03-N001 or BCY530); A-(SEQ ID NO:10)-A (referred to herein as 73-14-04-N001 or BCY531); A-(SEQ ID NO:11)-A (referred to herein as 73-14-05-N001 or BCY532); A-(SEQ ID NO:12)-A (referred to herein as 73-14-06-N001 or BCY533); A-(SEQ ID NO:13)-A (referred to herein as 73-14-07-N001 or BCY534); A-(SEQ ID NO:14)-A (referred to herein as 73-14-08-N001 or BCY535); A-(SEQ ID NO:15)-A (referred to herein as 73-14-10-N001 or BCY537); A-(SEQ ID NO:16)-A (referred to herein as 73-15-00-N002 or BCY538); A-(SEQ ID NO:17)-A (referred to herein as 73-16-00-N002 or BCY539); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:18) (hereinafter referred to as BCY8938); SDK-(SEQ ID NO:19)-A (hereafter referred to as BCY3835); [PYA]-[B-Ala]-[Sar 10 ]-SDK-(SEQ ID NO:19) (hereinafter referred to as BCY10043); NH 2 -SDK-(SEQ ID NO:19)-[Sar 10 ]-[K(PYA)] (hereafter referred to as BCY10044); [Ac][dS]DK-(SEQ ID NO:19)-A (hereafter referred to as BCY10051); SD[HArg]-(SEQ ID NO:19)-A (hereafter referred to as BCY10052); SD[HSer]-(SEQ ID NO:19)-A (hereafter referred to as BCY10053); [Ac]SDK-(SEQ ID NO:19)-A (hereafter referred to as BCY10075); [Ac]SDR-(SEQ ID NO:19) (hereafter referred to as BCY10076); [Ac]SDK-(SEQ ID NO:19)-PSH (hereafter referred to as BCY10943); [Ac]SEK-(SEQ ID NO:19) (hereinafter referred to as BCY10951); [Ac]-SDK-(SEQ ID NO:19) (hereafter referred to as BCY11832); [Ac]-A-(SEQ ID NO:19)-PSH (hereafter referred to as BCY11833); SDK-(SEQ ID NO:19)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY566); [Ac]D[HArg]-(SEQ ID NO:19) (hereafter referred to as BCY11865); NH 2 -SDK- (SEQ ID NO: 20) (hereinafter referred to as BCY10045); Ac-SDK-(SEQ ID NO:20)-PSH (hereafter referred to as BCY10861); Ac-SDK-(SEQ ID NO:20) (hereafter referred to as BCY11013); SDK-(SEQ ID NO:21)-A (hereafter referred to as BCY10054); SDK-(SEQ ID NO:22)-A (hereafter referred to as BCY10055); SDK-(SEQ ID NO:23)-A (hereafter referred to as BCY10057); SDK-(SEQ ID NO:24)-A (hereafter referred to as BCY10058); SDK-(SEQ ID NO:25)-A (hereinafter referred to as BCY10060); SDK-(SEQ ID NO:26)-A (hereafter referred to as BCY10061); SDK-(SEQ ID NO:27)-A (hereafter referred to as BCY10062); SDK-(SEQ ID NO:28)-A (hereafter referred to as BCY10064); SDK-(SEQ ID NO:29)-A (hereafter referred to as BCY10065); SDK-(SEQ ID NO:30)-A (hereafter referred to as BCY10066); SDK-(SEQ ID NO:31)-A (hereafter referred to as BCY10067); [Ac]-SDK-(SEQ ID NO:31)-PSH (hereafter referred to as BCY11830); SDK-(SEQ ID NO:32)-A (hereafter referred to as BCY10068); SDK-(SEQ ID NO:33)-A (hereafter referred to as BCY10069); SDK-(SEQ ID NO:34)-A (hereafter referred to as BCY10071); SDK-(SEQ ID NO:35)-A (hereafter referred to as BCY10072); SDK-(SEQ ID NO:36)-A (hereafter referred to as BCY10073); SDK-(SEQ ID NO:37)-A (hereafter referred to as BCY10074); [Ac]SDK-(SEQ ID NO:38) (hereinafter referred to as BCY10078); SDK-(SEQ ID NO:39)-A (hereafter referred to as BCY10083); SDK-(SEQ ID NO:40)-A (hereafter referred to as BCY10084); SDK-(SEQ ID NO:41)-A (hereafter referred to as BCY10085); [Ac]SDK-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10467); [Ac]SDK-(SEQ ID NO:42)-PS (hereafter referred to as BCY10934); [Ac]SDK-(SEQ ID NO:42)-P (hereafter referred to as BCY10935); [Ac]SDK-(SEQ ID NO:42)-PS-COOH (hereinafter referred to as BCY10936); [Ac]SDK-(SEQ ID NO:42)-P-COOH (hereafter referred to as BCY10937); [Ac]SDK-(SEQ ID NO:42)-COOH (hereinafter referred to as BCY10938); [Ac]SDK-(SEQ ID NO:42)-PSH-COOH (hereinafter referred to as BCY10939); [Ac]DK-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10940); [Ac]K-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10941); [Ac]-(SEQ ID NO:42)-PSH (hereafter referred to as BCY10942); [Ac]SD[HArg]-(SEQ ID NO:43)-PSH (hereafter referred to as BCY10944); [Ac]SDK-(SEQ ID NO:44) (hereinafter referred to as BCY10945); [Ac]SDK-(SEQ ID NO:45) (hereinafter referred to as BCY10946); [Ac]SDK-(SEQ ID NO:46) (hereinafter referred to as BCY10947); [Ac]SDK-(SEQ ID NO:47) (hereinafter referred to as BCY10948); [Ac]SDK-(SEQ ID NO:48) (hereinafter referred to as BCY10949); [Ac]SDK-(SEQ ID NO:49) (hereinafter referred to as BCY10950); [Ac]SDK-(SEQ ID NO:50) (hereinafter referred to as BCY10952); [Ac]SDK-(SEQ ID NO:51) (hereinafter referred to as BCY10954); [Ac]SDK-(SEQ ID NO:52) (hereinafter referred to as BCY10956); [Ac]SD[HArg]-(SEQ ID NO:53)-PSH (hereafter referred to as BCY10959); [Ac]SDK-(SEQ ID NO:54) (hereinafter referred to as BCY10960); [Ac]SDK-(SEQ ID NO:55) (hereinafter referred to as BCY10961); [B-Ala][Sar] 5 A-(SEQ ID NO:56)-A (the fluoresced derivative thereof is hereafter referred to as BCY562); VER-(SEQ ID NO:57)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY567); REN-(SEQ ID NO:58)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY568); QQE-(SEQ ID NO:59)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY569); SGK-(SEQ ID NO:59)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY577); AGS-(SEQ ID NO:60)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY570); AQT-(SEQ ID NO:61)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY571); APV-(SEQ ID NO:62)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY572); ADV-(SEQ ID NO:63)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY573); GNK-(SEQ ID NO:64)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY574); KPK-(SEQ ID NO:64)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY580); ERV-(SEQ ID NO:65)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY575); AER-(SEQ ID NO:66)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY576); KEL-(SEQ ID NO:67)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY578); G[Sar] 5 KEL-(SEQ ID NO:67)-A (the fluoresced derivative of which is hereafter referred to as BCY3811); KEL-(SEQ ID NO:68)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY579); A-(SEQ ID NO:69)-PSH[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY581); A-(SEQ ID NO:70)-DHEN[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY582); REE-(SEQ ID NO:71)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY3812); QAEK-(SEQ ID NO:72)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY8210); A-(SEQ ID NO:73)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7126); A-(SEQ ID NO:74)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7374); A-(SEQ ID NO:75)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7375); A-(SEQ ID NO:76)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7376); A-(SEQ ID NO:77)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7377); A-(SEQ ID NO:78)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7384); A-(SEQ ID NO:79)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7385); A-(SEQ ID NO:80)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7386); A-(SEQ ID NO:81)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7387); A-(SEQ ID NO:82)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7388); A-(SEQ ID NO:83)-A[Sar] 6 (The fluorescent derivative is hereafter referred to as BCY7389); Ac-DK-(SEQ ID NO:84) (hereafter referred to as BCY11818); Ac-K-(SEQ ID NO:84) (hereafter referred to as BCY11819); Ac-SEK-(SEQ ID NO:84) (hereafter referred to as BCY11820); Ac-EK-(SEQ ID NO:84) (hereafter referred to as BCY11821); Ac-DK-(SEQ ID NO:84)-PSH-COOH (hereinafter referred to as BCY11853); Ac-SDK-(SEQ ID NO:85) (hereafter referred to as BCY11822); Ac-DK-(SEQ ID NO:85) (hereafter referred to as BCY11823); Ac-K-(SEQ ID NO:85) (hereafter referred to as BCY11824); Ac-SEK-(SEQ ID NO:85) (hereinafter referred to as BCY11825); Ac-EK-(SEQ ID NO:85) (hereafter referred to as BCY11826); Ac-[HArg]-(SEQ ID NO:85) (hereinafter referred to as BCY11827); Ac-SDK-(SEQ ID NO:85)-A (hereafter referred to as BCY11828); Ac-SEK-(SEQ ID NO:85)-PSH (hereafter referred to as BCY11831); Ac-SDK-(SEQ ID NO:86) (hereafter referred to as BCY11829); Ac-D[HArg]-(SEQ ID NO:87)-PSH (hereafter referred to as BCY11866); Ac-D[HArg]-(SEQ ID NO:88)-PSH (hereafter referred to as BCY11867); Ac-D[HArg]-(SEQ ID NO:89)-PSH (hereafter referred to as BCY11868); Ac-D[HArg]-(SEQ ID NO:90)-PSH (hereafter referred to as BCY11869); Ac-SD[HArg]-(SEQ ID NO:91)-PSHK (hereafter referred to as BCY12479); Ac-SD[HArg]-(SEQ ID NO:92)-PSHK (hereafter referred to as BCY12477); Ac-[HArg]-(SEQ ID NO:93)-PSH (hereafter referred to as BCY12640); Ac-[HArg]-(SEQ ID NO:94)-PSH (hereafter referred to as BCY12641); Ac-[HArg]-(SEQ ID NO:95)-PSH (hereafter referred to as BCY12642); Ac-[HArg]-(SEQ ID NO:96)-PSH (hereinafter referred to as BCY12643); and Ac-[HArg]-(SEQ ID NO:97)-PSH (hereinafter referred to as BCY12644); (wherein PYA stands for 4-pentynoic acid, B-Ala stands for β-alanine, and Sar stands for sarcosyl HSe represents homoserine, and HArg represents homoarginine. or a pharma- ceutical acceptable salt thereof. Peptide ligands.

6. The peptide according to any one of claims 1 to 3, which is listed in any one of Tables 1 to 5. Peptide ligands.

7. The molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropane. The peptide ligase according to any one of claims 1 to 6, selected from the group consisting of per-2-en-1-one (TATA). And.

8. The pharma- ceutical acceptable salts may be either the free acid or sodium, potassium, calcium, ammonium, 8. The peptide ligand of claim 1, wherein the peptide ligand is selected from the group consisting of ammonium salts.

9. 9. Any of claims 1 to 8, wherein the PD-L1 is human or mouse PD-L1, e.g., human PD-L1. A peptide ligand according to claim 1.

10. Any of claims 1 to 9 conjugated to one or more effectors and / or functional groups. A drug conjugate comprising the peptide ligand according to claim 1.

11. The peptide of any one of claims 1 to 9 conjugated to one or more cytotoxic agents. A drug conjugate comprising a doligand.

12. A peptide ligand according to any one of claims 1 to 9 or claim 10 or claim 11. A pharmaceutical composition comprising the drug conjugate of the present invention in combination with one or more pharma- ceutical acceptable excipients. Composition.

13. For use in the prevention, inhibition, or treatment of a disease or disorder mediated by PD-L1 The peptide ligand according to any one of claims 1 to 9 or the peptide ligand according to claim 10 or claim 11. The drug conjugates described above.

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