Bicyclic peptide ligands specific for CD137

Abstract

The present invention relates to polypeptides covalently attached to a molecular scaffold such that two or more peptide loops are intercalated between the attachment points to the scaffold. In particular, the present invention describes peptides that are high-affinity binders of CD137. The present invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and the use of the peptide ligands and drug conjugates in the prevention, suppression, or treatment of diseases or disorders mediated by CD137.

Description

[Technical field]

[0001] The present invention relates to a polypeptide covalently attached to a molecular scaffold such that two or more peptide loops are intercalated between the attachment points to the scaffold.In particular, the present invention describes peptides that are high affinity binders of CD137.The present invention also includes drug conjugates comprising said peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising said peptide ligands and drug conjugates, and the use of said peptide ligands and drug conjugates in the prevention, inhibition or treatment of diseases or disorders mediated by CD137. [Background technology]

[0002] Cyclic peptides are able to bind with high affinity and target specificity to protein targets and are therefore an attractive class of molecules for therapeutic development. Indeed, several cyclic peptides have already been successfully used in the clinic, for example the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The superior binding properties are due to the reduced conformational flexibility of the cyclic structures together with the relatively large interaction surface formed between the peptide and the target. Typically, macrocycles are used in the development of macrocycles, for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ;Wu et al. (2007), Science 330, 1066-71), a cyclic peptide (355 Å) with an Arg-Gly-Asp motif that binds to integrin αVb3 2 ) (Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 (603 Å) that binds to urokinase-type plasminogen activator. 2;Zhao et al. (2007), J Struct Biol 160(1), 1-10), which bind to surfaces measuring several hundred square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to the target, resulting in higher binding affinity. The reduced flexibility also leads to a target-specific conformational fixation, increasing binding specificity, compared to linear peptides. Such an effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity over other MMPs when its ring is opened (Cherney et al. (1998), J Med Chem 41(11), 1749-51). The favorable binding properties achieved by macrocyclization are even more evident in multicyclic peptides with more than one peptide ring, as in, for example, vancomycin, nisin and actinomycin.

[0004] Different research teams have previously tethered polypeptides bearing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers have used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds, where the compounds arise by linking cysteine-containing polypeptides to molecular scaffolds, e.g., tris(bromomethyl)benzene, are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] A phage display-based combinatorial approach has been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, three cysteine ​​residues and two regions of six random amino acids (Cys-(Xaa) 6 -Cys-(Xaa) 6 A combinatorial library of linear peptides containing -Cys was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the Invention

[0006] According to a first aspect of the present invention there is provided a peptide ligand specific for CD137 comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences such that at least two polypeptide loops are formed on the molecular scaffold, and the molecular scaffold forms covalent bonds with the reactive groups of the polypeptide.

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

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

[0009] According to a further aspect of the invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by CD137. [Brief description of the drawings]

[0010] [Figure 1] 1 shows the results of a CD137 cell activation assay using the bicyclic peptide BCY592. [Diagram 2] Competition of CD137L or the monoclonal antibody utomirumab with the fluorescently labeled peptide BCY640 for binding to CD137 as measured by fluorescence polarization. [Diagram 3] Competition of monoclonal antibodies utomirumab or urelumab with fluorescently labeled peptide BCY640 for binding to CD137 as measured by fluorescence polarization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention According to one particular aspect of the invention that may be mentioned, a peptide ligand specific for CD137 is provided, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, such that at least two polypeptide loops are formed on the molecular scaffold, and the molecular scaffold forms covalent bonds with the cysteine ​​residues of the polypeptide.

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

[0013] In a further embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, both of six amino acids.

[0014] In a further embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, one of five amino acids and the other of six amino acids.

[0015] In one embodiment, the peptide ligand is: C i -IEEGQYC ii -X 1-X 2 -DX 3 -Y / Q / MX 4 -C iii (SEQ ID NO:20); C i -DIGPPYC ii -YR / ADM / PYMC iii (SEQ ID NO:21); C i -DEWGLF / YC ii -I / FP / AHS / PDC iii (SEQ ID NO:22); and C i IEPGPFC ii YADPYMC iii (SEQ ID NO:19); (In the formula, X 1 ~X 4 represents any amino acid residue, C i , C ii and C iii represent a first, second and third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0016] Alanine scanning experiments were performed on selected peptides of the present invention. Alanine scanning is used to predict which amino acid positions are most amenable to substitution and further optimization of affinity and / or other desired properties. Based on affinity to the parent peptide sequence (BCY7151; SEQ ID NO: 92), the alanine scanning peptides were characterized into three categories: 1. no loss of affinity, 2. 2-10 fold weaker affinity, 3. >10 fold weaker affinity. Peptides in category 1 and category 2 can undergo extensive SAR testing with alternative amino acid substitutions. Peptides in category 3 were kept fixed or substituted only with highly similar amino acids. The results of the alanine scanning are shown in Table 2, and show that the aspartic acid (D) amino acid residue at position 9 is most important for binding, since substitution of this amino acid residue with an alanine residue abolished binding activity.

[0017] D-alanine scanning experiments were also performed on selected peptides of the present invention. Since the default preparation of all bicyclic peptides is in the L-configuration, D-alanine scanning indicates which amino acid positions are amenable to D-amino acid substitution. The results of D-alanine scanning are shown in Table 2, and show that substituting D-Ala for glycine (G) at position 4 improved affinity to the reference peptide. This means that the D-Ala4 peptide (BCY7297; SEQ ID NO: 106) is important because it provides improved affinity and other benefits associated with unnatural D-isomer amino acids.

[0018] In one embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, both of six amino acids, the peptide ligand comprising: C i -IEEGQYC ii -X 1 -X 2 -DX 3 -Y / Q / MX 4 -C iii (SEQ ID NO:20); C i -DIGPPYC ii -YR / ADM / PYMC iii (SEQ ID NO:21); and C i IEPGPFC ii YADPYMC iii (SEQ ID NO:19); (In the formula, X 1 ~X 4 represents any amino acid residue, C i , C ii and C iii represent a first, second and third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0019] In one embodiment, X 1 is selected from Y, F, and H.

[0020] In one embodiment, X 2 is selected from R, A, and S.

[0021] In one embodiment, X 3 is selected from M, P, and H.

[0022] In one embodiment, X 4 is selected from M, Y, L, and F.

[0023] In one embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence of six amino acids and a second loop sequence of five amino acids, the peptide ligand comprising: C i -DEWGLF / YC ii -I / FP / AHS / PDC iii (SEQ ID NO:22); (In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively, or a pharma- ceutically acceptable salt thereof.

[0024] In a further embodiment, C i -IEEGQYC ii -X 1 -X 2 -DX 3 -Y / Q / MX 4 -C iii The peptide ligand (SEQ ID NO: 20) is represented by SEQ ID NOs: 1 to 14: C i IEEGQYC ii YRDMYMC iii (SEQ ID NO:1); C i IEEGQYC ii YADPYMC iii (SEQ ID NO:2); C i IEEGQYC ii YADPYYC iii (SEQ ID NO:3); C i IEEGQYC ii YSDPYYC iii (SEQ ID NO:4); C i IEEGQYC ii FADPYMC iii (SEQ ID NO:5); C i IEEGQYC ii YADHQLC iii (SEQ ID NO:6); C i IEEGQYC ii HADPYYC iii (SEQ ID NO:7); C i IEEGQYC ii HADPYFC iii (SEQ ID NO:8); C i IEEGQYC ii YADHYMC iii (SEQ ID NO:9); C i IEEGQYC ii YADPYLC iii (SEQ ID NO:10); C i IEEGQYC ii YSDPYLC iii (SEQ ID NO:11); C i IEEGQYC ii FADPYLC iii (SEQ ID NO:12); C i IEEGQYC ii HADPYMC iii (SEQ ID NO: 13); and C i IEEGQYC ii HADPQMC iii (SEQ ID NO:14); (In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively), or a pharma- ceutically acceptable salt thereof.

[0025] In a further embodiment, C i -IEEGQYC ii -X 1 -X 2 -DX 3 -Y / Q / MX 4 -C iii The peptide ligand of (SEQ ID NO:20) is: A-(SEQ ID NO:1)-A (referred to herein as 74-01-00-N004); A-(SEQ ID NO:2)-A (referred to herein as 74-01-01-N001); A-(SEQ ID NO:3)-A (referred to herein as 74-01-02-N001); A-(SEQ ID NO:4)-A (referred to herein as 74-01-03-N001); A-(SEQ ID NO:5)-A (referred to herein as 74-01-04-N001); A-(SEQ ID NO:6)-A (referred to herein as 74-01-05-N001); A-(SEQ ID NO:7)-A (referred to herein as 74-01-06-N001); A-(SEQ ID NO:8)-A (referred to herein as 74-01-07-N001); A-(SEQ ID NO:9)-A (referred to herein as 74-01-08-N001); A-(SEQ ID NO:10)-A (referred to herein as 74-01-09-N001); A-(SEQ ID NO:10)-SVG (referred to herein as 74-01-09-T03-N002); A-(SEQ ID NO:11)-A (referred to herein as 74-01-10-N001); A-(SEQ ID NO:12)-A (referred to herein as 74-01-11-N001); A-(SEQ ID NO:13)-A (referred to herein as 74-01-13-N001); and A-(SEQ ID NO:14)-A (referred to herein as 74-01-14-N001) The amino acid sequence is selected from the group consisting of:

[0026] In a further embodiment, C i -DIGPPYC ii -YR / ADM / PYMC iii The peptide ligand of (SEQ ID NO:21) is: C i DIGPPYC ii YRDMYMC iii (SEQ ID NO:15); and C i DIGPPYC ii YADPYMC iii (SEQ ID NO:16); (In the formula, C i , C ii and C iii represent a first, second and third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0027] In a further embodiment, C i -DIGPPYC ii -YR / ADM / PYMC iii The peptide ligand of (SEQ ID NO:21) is: A-(SEQ ID NO:15)-A (referred to herein as 74-01-16-N001); and A-(SEQ ID NO:16)-A (referred to herein as 74-01-17-N001) The amino acid sequence is selected from the group consisting of:

[0028] In a further embodiment, C i -DEWGLF / YC ii -I / FP / AHS / PDC iii The peptide ligand of (SEQ ID NO:22) is: C i DEWGLFC ii IPHSDC iii (SEQ ID NO:17); and C i DEWGLYC ii FAHPDC iii (SEQ ID NO:18); (In the formula, C i , C iiand C iii represent a first, second and third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0029] In a further embodiment, C i -DEWGLF / YC ii -I / FP / AHS / PDC iii The peptide ligand of (SEQ ID NO:22) is: Ac-A-(SEQ ID NO:17)-A (referred to herein as 74-02-00-N004); and A-(SEQ ID NO:18)-A (referred to herein as 74-02-01-N001) The amino acid sequence is selected from the group consisting of:

[0030] In one embodiment, C i IEPGPFC ii YADPYMC iii The peptide ligand of (SEQ ID NO: 19) is: A-(SEQ ID NO:19)-NRV (referred to herein as 74-19-00-T01-N002) The amino acid sequence of

[0031] In one embodiment, the molecular scaffold is selected from 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and the peptide ligand is: A-(SEQ ID NO:1)-A (referred to herein as 74-01-00-N004); A-(SEQ ID NO:2)-A (referred to herein as 74-01-01-N001); A-(SEQ ID NO:3)-A (referred to herein as 74-01-02-N001); A-(SEQ ID NO:4)-A (referred to herein as 74-01-03-N001); A-(SEQ ID NO:5)-A (referred to herein as 74-01-04-N001); A-(SEQ ID NO:6)-A (referred to herein as 74-01-05-N001); A-(SEQ ID NO:7)-A (referred to herein as 74-01-06-N001); A-(SEQ ID NO:8)-A (referred to herein as 74-01-07-N001); A-(SEQ ID NO:9)-A (referred to herein as 74-01-08-N001); A-(SEQ ID NO:10)-A (referred to herein as 74-01-09-N001); A-(SEQ ID NO:10)-SVG (referred to herein as 74-01-09-T03-N002); A-(SEQ ID NO:11)-A (referred to herein as 74-01-10-N001); A-(SEQ ID NO:12)-A (referred to herein as 74-01-11-N001); A-(SEQ ID NO:13)-A (referred to herein as 74-01-13-N001); A-(SEQ ID NO:14)-A (referred to herein as 74-01-14-N001); A-(SEQ ID NO:15)-A (referred to herein as 74-01-16-N001); A-(SEQ ID NO:16)-A (referred to herein as 74-01-17-N001); Ac-A-(SEQ ID NO:17)-A (referred to herein as 74-02-00-N004); and A-(SEQ ID NO:18)-A (referred to herein as 74-02-01-N001) The amino acid sequence is selected from the group consisting of:

[0032] The scaffold / peptide ligand of this embodiment demonstrated excellent CD137 competitive binding, as shown in Table 1 herein.

[0033] In a further embodiment, the peptide ligand is: C i IEEGQYC ii FADPY(Nle)C iii (SEQ ID NO:23); C i IKEGQYC iiFADPY(Nle)C iii (SEQ ID NO:24); C i IEKGQYC ii FADPY(Nle)C iii (SEQ ID NO:25); C i IEE(DK)QYC ii FADPY(Nle)C iii (SEQ ID NO:26); C i IEEGKYC ii FADPY(Nle)C iii (SEQ ID NO:27); C i IEEGQYC ii KADPY(Nle)C iii (SEQ ID NO:28); C i IEEGQYC ii FADKY(Nle)C iii (SEQ ID NO:29); and C i IEEGQYC ii FADPYKC iii (SEQ ID NO:30); (In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively), or a pharma- ceutically acceptable salt thereof.

[0034] In a further embodiment, the peptide ligand comprises an N-terminal and a C-terminal modification, AC i IEEGQYC ii FADPY(Nle)C iii -A (SEQ ID NO: 31; BCY3814); Ac-AC i IEEGQYC ii FADPY(Nle)C iii -Dap(SEQ ID NO:32; BCY7732); Ac-AC i IKEGQYC ii FADPY(Nle)C iii -A (SEQ ID NO: 33; BCY7733); Ac-AC i IEKGQYC ii FADPY(Nle)C iii -A (SEQ ID NO: 34; BCY7734); Ac-AC i IEE(DK)QYC ii FADPY(Nle)C iii -A (SEQ ID NO: 35; BCY7735); Ac-AC i IEEGKYC ii FADPY(Nle)C iii -A (SEQ ID NO: 36; BCY7736); Ac-AC i IEEGQYC ii KADPY(Nle)C iii -A (SEQ ID NO: 37; BCY7737); Ac-AC i IEEGQYC ii FADKY(Nle)C iii -A (SEQ ID NO: 38; BCY7738); and Ac-AC i IEEGQYC ii FADPYKC iii -A (SEQ ID NO: 39; BCY7739); (In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively; Ac represents an N-terminal acetyl group; and Dap represents diaminopropionic acid), or a pharma- ceutically acceptable salt thereof.

[0035] In an alternative embodiment, the peptide ligand is: C i LPPGQYC ii FPDLLLC iii (SEQ ID NO: 40; 74-22-00) (In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively.

[0036] In an alternative embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, both of six amino acids, the peptide ligand comprising: C i -I / L / M / VE / D / P / SP / E / AGP / QY / FC ii -YADPY / MM / L / YC iii (SEQ ID NO:41); (In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively.

[0037] In a further embodiment, the amino acid sequence is selected from the peptide sequences listed in Table 1. In a further embodiment, the amino acid sequence is selected from the peptide sequences listed in Table 1, excluding the following peptides: BCY7238, BCY7241, BCY7243, and BCY7246. The peptides of this embodiment were tested in a direct CD137 binding assay and demonstrated good levels of binding.

[0038] In a further embodiment, the amino acid sequence is selected from the peptide sequences listed in Table 3. The peptides of this embodiment were tested in a direct CD137 binding assay and demonstrated good levels of binding.

[0039] In a further embodiment, the amino acid sequence is selected from the peptide sequences listed in Tables 4 and 5. The peptides of this embodiment were tested in an SPR assay for CD137 and demonstrated good levels of binding.

[0040] In a further embodiment, the amino acid sequence is selected from BCY592. Data are presented herein in Figure 1, which shows that the bicyclic peptide BCY592 inhibited CD137L activity in a cell-based assay.

[0041] In an alternative embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, both of six amino acids, the peptide ligand comprising: C i -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -C ii -X 11 -X 12 -DX 13 -X 14 -X 15 -C iii (SEQ ID NO:266); (In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively; X 5 represents Ile, tBuAla, or Chg; X 6 represents Glu, Pro, Asp, Lys, Aad, HyP, or Oxa; X 7 represents Glu, Lys, or Aad; X 8 represents Gly, D-Lys, D-Ala, L-Ala, D-Phe, D-Glu, D-Gln, D-Leu, D-Ser, or D-Trp; X 9 represents Gln, Lys, Ala, Pro, 5,5-dmP, Oic, Oxa, HyP, Aib, or Ac5c; X 10 represents Tyr, Phe, 3MePhe, 4MePhe, 4FPhe, 2Nal, 4MeOPhe, or 4,4-BPA; X 11 represents Phe, Lys, 4MePhe, 2FPhe, 4FPhe, 4Pal, 4,4-BPA, 4tBuPhe, NO2Phe, or 4BrPhe; X 12 represents Ala or Lys; X13 represents Pro or Lys; X 14 represents Tyr or Lys; X 15 stands for Met, Lys, Nle, HLeu, or Ahp) The amino acid sequence is

[0042] In one embodiment, the peptide ligand of SEQ ID NO: 266 is the following peptide: BCY7239, BCY7240, BCY7242, BCY7244, BCY7245, BCY7247, BCY7248, BCY7249, BCY7416, BCY7287, BCY7297, BCY7154, BCY7156, BCY7157, BCY7158, BCY7162, BCY7 165, BCY7166, BCY7167, BCY7168, BCY7169, BCY7170, BCY7174, BCY7175, BCY717 7, BCY7178, BCY7179, BCY7183, BCY7185, BCY7195, BCY7198, BCY7211, BCY7311, BCY7768, BCY7770, BCY7772, BCY7773, BCY7774, BCY7775, BCY7776, BCY7796, BCY7798, BCY7801, BCY7802, BCY7936, BCY7941, BCY7942, BCY7944, BCY7950, BCY7954, BCY7958, BCY7959, BCY7960, BCY7952, BCY7961, BCY8656, BCY8659, BCY8663, BCY8668, BCY8669, BCY8674, BCY8675, BCY9273, BCY3814, BCY7527 and BCY7965 C i ~C iii The sequences are selected from the following (i.e., having no N- or C-terminal additions):

[0043] In a further embodiment, the peptide ligand of SEQ ID NO: 266 is the following peptide: BCY7239, BCY7240, BCY7242, BCY7244, BCY7245, BCY7247, BCY7248, BCY7249, BCY7416, BCY7287, BCY7297, BCY7154, BCY7156, BCY7157, BCY7158, BCY7162, BCY7 165, BCY7166, BCY7167, BCY7168, BCY7169, BCY7170, BCY7174, BCY7175, BCY717 7, BCY7178, BCY7179, BCY7183, BCY7185, BCY7195, BCY7198, BCY7211, BCY7311, BCY7768, BCY7770, BCY7772, BCY7773, BCY7774, BCY7775, BCY7776, BCY7796, BCY7798, BCY7801, BCY7802, BCY7936, BCY7941, BCY7942, BCY7944, BCY7950, BCY7954, BCY7958, BCY7959, BCY7960, BCY7952, BCY7961, BCY8656, BCY8659, BCY8663, BCY8668, BCY8669, BCY8674, BCY8675, BCY9273, BCY3814, BCY7527 and BCY7965 is selected from the complete sequence (i.e., including all N- and C-terminal additions).

[0044] All of these peptides demonstrated good levels of binding in the direct binding or SPR assays described herein, or represented peptides that tolerated substitution with Lys residues.

[0045] In one embodiment, X 5 represents Ile or tBuAla.

[0046] In one embodiment, X 6 represents Lys, Glu, or Pro.

[0047] In one embodiment, X 7 represents Glu or D-Lys.

[0048] In one embodiment, X8 represents Gly, D-Lys, D-Phe, or D-Ala.

[0049] In one embodiment, X 9 represents Gln, Lys, or Pro.

[0050] In one embodiment, X 10 represents Tyr or 4MePhe.

[0051] In one embodiment, X 11 represents Phe or 4FPhe.

[0052] In one embodiment, X 12 stands for Ala.

[0053] In one embodiment, X 13 stands for Pro.

[0054] In one embodiment, X 14 represents Tyr.

[0055] In one embodiment, X 15 represents Met or Nle.

[0056] In a further embodiment, the peptide ligand comprises three cysteine ​​residues separated by two loop sequences, both of six amino acids, and the peptide ligand comprises: C i -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -C ii -X 11 -ADPYX 15 -C iii (SEQ ID NO:267); (In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively; X 5stands for Ile or tBuAla; X 6 represents Lys, Glu, or Pro; X 7 represents Glu or D-Lys; X 8 represents Gly, D-Lys, D-Phe, or D-Ala; X 9 represents Gln, Lys, or Pro; X 10 represents Tyr or 4MePhe; X 11 represents Phe or 4FPhe; X 15 represents Met or Nle).

[0057] In one embodiment, the peptide ligand of SEQ ID NO: 267 is the following peptide: BCY7239, BCY7240, BCY7242, BCY7416, BCY7156, BCY7166, BCY7174, BCY7774, BCY9273, BCY3814, BCY7527 and BCY7965 C i ~C iii The sequences are selected from the following (i.e., having no N- or C-terminal additions):

[0058] In a further embodiment, the peptide ligand of SEQ ID NO: 267 is the following peptide: BCY7239, BCY7240, BCY7242, BCY7416, BCY7156, BCY7166, BCY7174, BCY7774, BCY9273, BCY3814, BCY7527 and BCY7965 is selected from the complete sequence (i.e., including all N- and C-terminal additions).

[0059] All of these peptides demonstrated excellent levels of binding in the direct binding or SPR assays described herein or represented peptides that tolerated substitution with Lys residues.

[0060] In one embodiment, the peptide ligand of SEQ ID NO:267 is the following peptide: BCY7239, BCY7240, BCY7242 and BCY7416 C i ~C iii The sequences are selected from the following (i.e., having no N- or C-terminal additions):

[0061] In a further embodiment, the peptide ligand of SEQ ID NO:267 is the following peptide: BCY7239, BCY7240, BCY7242 and BCY7416 is selected from the complete sequence (i.e., including all N- and C-terminal additions).

[0062] These peptides represented peptides that tolerated substitution with Lys residues.

[0063] In one embodiment, the peptide ligand of SEQ ID NO:267 is the following peptide: BCY9273, BCY3814, BCY7527, and BCY7965 C i ~C iii The sequences are selected from the following (i.e., having no N- or C-terminal additions):

[0064] In a further embodiment, the peptide ligand of SEQ ID NO:267 is the following peptide: BCY9273, BCY3814, BCY7527 and BCY7965 is selected from the complete sequence (i.e., including all N- and C-terminal additions).

[0065] All of these peptides demonstrated good levels of binding in the direct binding assays or SPR assays described herein.

[0066] In one embodiment, the peptide ligand of SEQ ID NO:267 is the following peptide: BCY7156, BCY7166, BCY7174 and BCY7774 C i ~C iii The sequences are selected from the following (i.e., having no N- or C-terminal additions):

[0067] In a further embodiment, the peptide ligand of SEQ ID NO:267 is the following peptide: BCY7156, BCY7166, BCY7174 and BCY7774 is selected from the complete sequence (i.e., including all N- and C-terminal additions).

[0068] All of these peptides demonstrated excellent levels of binding in the direct binding or SPR assays described herein.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the art, e.g., peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques for molecular biology, genetic and biochemical methods are used (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4, incorporated herein by reference). th ed., John Wiley & Sons, Inc.).

[0070] [Nomenclature] Numbering When referring to the position of an amino acid residue in a compound of formula (I), a cysteine ​​residue (C i , C ii and C iii) are excluded from the numbering since they are invariant. Thus, the numbering of amino acid residues in compounds of formula (I) is referred to as follows: -C i -I 1 -E 2 -E 3 -G 4 -Q 5 -Y 6 -C ii -Y 7 -R 8 -D 9 -M 10 -Y 11 -M 12 -C iii - (SEQ ID NO: 1)

[0071] For the purposes of this description, all bicyclic peptides are considered to be cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) or 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) to yield trisubstituted structures. Cyclization with TBMB and TATA is i , C ii and C iii Happens on top.

[0072] Molecular Format Extensions N- or C-terminal to the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail is βAla-Sar10-A-(SEQ ID NO:X) It is shown as:

[0073] [Peptide Ligand] A peptide ligand, as referred to herein, refers to a peptide that is covalently attached to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond to the scaffold, and a sequence interposed between the reactive groups, referred to as a loop sequence, to form a loop when the peptide is attached to the scaffold. In this case, the peptide comprises at least three cysteine ​​residues (referred to herein as C i , C ii and C iii ) which form at least two loops on the scaffold.

[0074] [Advantages of peptide ligands] The particular bicyclic peptides of the present invention have several advantageous properties that allow them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include: -Species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic assessments; - Protease stability. Bicyclic peptide ligands should ideally exhibit stability against plasma proteases, epithelial ("membrane anchor") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained between different species so that bicyclic lead candidates can be developed in animal models and reliably administered to humans; - a desirable solubility profile, which is a function of charge and the ratio of hydrophobic to hydrophilic residues and intra- / inter-molecular hydrogen bonds, which are important for formulation and absorption purposes; and - Optimal plasma half-life in circulation. Clinical indications and treatment regimens may necessitate the development of bicyclic peptides for short exposure in acute disease management settings, or bicyclic peptides with enhanced retention in the circulation, and therefore optimal for the management of more chronic disease conditions. Another factor driving a desirable plasma half-life is the need for sustained exposure for maximum therapeutic efficacy versus the associated toxicology of sustained exposure of the drug. -Selectivity. Certain peptide ligands of the invention demonstrate better selectivity over other CDs.

[0075] [Pharmaceutically acceptable salts] It will be appreciated that salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of said ligand.

[0076] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or free base forms of these compounds with the appropriate base or acid in water or an organic solvent, or a mixture of the two.

[0077] Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts are acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane ... laminic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid The mono- or di-salts formed with acids selected from the group consisting of pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as with acylated amino acids and cation exchange resins.

[0078] One particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0079] The compound is anionic or has a functional group which may be anionic (e.g., -COOH is -COO - In the case of cations such as cations of 1 to 5 carbon atoms, salts may be formed with organic or inorganic bases to give suitable cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions, such as Li + , Na + and K. + , alkaline earth metal cations, e.g., Ca 2+ and Mg 2+ , and other cations, e.g., Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , N.H. 2 R 2 + , N.H.R. 3 + , N.R. 4 + ). Some examples of suitable substituted ammonium ions include methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 )4 + It is.

[0080] When the compounds of formula (I) contain amine functions, they may be formed into quaternary ammonium salts by methods well known to those skilled in the art, for example, by reaction with alkylating agents. Such quaternary ammonium compounds are within the scope of formula (I).

[0081] [Modified derivatives] It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; bicyclic peptide ligands, and the like. The modifications include one or more modifications selected from N-alkylation of one or more amide bonds in the tide ligand; replacement of one or more peptide bonds with alternative bonds; modification of the peptide backbone length; replacement of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids, e.g., cysteine, lysine, glutamic acid / aspartic acid and tyrosine, with appropriate amine-, thiol-, carboxylic acid- and phenol-reactive reagents to functionalize said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivity suitable for functionalization, e.g., introduction or replacement of azide- or alkyne-group-bearing amino acids that allow functionalization with alkyne- or azide-bearing moieties, respectively.

[0082] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, the modified derivative comprises an N-terminal modification using appropriate amino-reactive chemistry and / or a C-terminal modification using appropriate carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator, or a chromophore.

[0083] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, an N-terminal cysteine ​​group (referred to herein as C i The N-terminal end of the cyclic peptide (the group referred to as "N-terminus acetylation") is capped with acetic anhydride or other suitable reagent during peptide synthesis, resulting in a molecule that is N-terminally acetylated. This embodiment offers the advantage of eliminating potential recognition points for aminopeptidases, avoiding the possibility of degradation of the bicyclic peptide.

[0084] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of an effector group and retention of potency of the bicyclic peptide against its target.

[0085] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine ​​group (referred to herein as C iii The C-terminal end of the bicyclic peptide (the group referred to as C-terminal end) is synthesized as an amide during peptide synthesis, resulting in a molecule that is C-terminally amidated. This embodiment offers the advantage of eliminating a potential recognition point for carboxypeptidases, reducing the likelihood for proteolysis of the bicyclic peptide.

[0086] In one embodiment, the modified derivative comprises the substitution of one or more amino acid residues with one or more unnatural amino acid residues. In this embodiment, unnatural amino acids can be selected that have isosteric / isoelectronic side chains that are not recognized by degradative proteases and do not have adverse effects on targeting efficacy.

[0087] Alternatively, unnatural amino acids with constrained amino acid side chains can be used such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, these involve proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids, the simple derivative amino-cyclopropyl carboxylic acid.

[0088] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of an N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii ) including the addition of a spacer group to

[0089] In one embodiment, the modified derivative comprises the substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises the substitution of tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0090] In one embodiment, the modified derivative comprises the substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. In an alternative embodiment, the modified derivative comprises the substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of hydrophobic amino acid residues to charged amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with phospholipid membranes on cell surfaces. The two in combination may affect the half-life, distribution volume and exposure of the peptide drug and can be adjusted depending on the clinical endpoint. Furthermore, the correct combination and number of hydrophobic amino acid residues to charged amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0091] In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues, which is believed to increase proteolytic stability through steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

[0092] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine. This embodiment offers the advantage of removing potential proteolytic attack sites.

[0093] It should be noted that each of the above modifications serves to purposefully improve the potency or stability of the peptide. Further potency improvements based on the modifications may be achieved by the following mechanisms: - taking advantage of the hydrophobic effect and incorporating hydrophobic moieties resulting in lower off rates so that higher affinity is achieved; - incorporating charged groups that take advantage of long-range ionic interactions, resulting in faster on rates and higher affinity (see, e.g., Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - Incorporating further constraints into the peptide, for example by precisely constraining the side chains of amino acids so that the loss in entropy is minimal on target binding, constraining the torsion angles of the backbone so that the loss in entropy is minimal upon target binding, and introducing further cyclization into the molecule for the same reasons. (For reviews, see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

[0094] [Isotope variations] The present invention includes all pharma- ceutically acceptable (radio)isotopically labeled peptide ligands of the invention in which one or more atoms have been replaced by an atom having the same atomic number, but an atom having an atomic mass or mass number different from that normally found in nature, peptide ligands of the invention to which a metal chelating group capable of bearing a relevant (radio)isotope is attached (termed an "effector"), and peptide ligands of the invention in which a particular functional group has been covalently replaced with a relevant (radio)isotope, or with an isotopically labeled functional group.

[0095] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, e.g., 2 H(D) and 3 H(T), an isotope of carbon, e.g.11 C. 13 C, and 14 C, an isotope of chlorine, e.g. 36 Cl, isotopes of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I, and 131 I, isotopes of nitrogen, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O, and 18 O, isotopes of phosphorus, e.g. 32 P, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y, an isotope of lutetium, e.g. 177 Lu, and bismuth isotopes, e.g. 213 Contains Bi.

[0096] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating radioisotopes, are useful for clinically evaluating the presence and / or absence of CD137 targets on diseased tissues in drug and / or substrate tissue distribution studies. The peptide ligands of the invention may further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may use compounds labeled with labeling agents, e.g., radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase), and the like. Radioisotopes such as tritium, i.e., 3 H(T), and carbon-14, i.e. 14 C is particularly useful for this purpose in view of its ease of incorporation and ready means of detection.

[0097] Heavier isotopes, such as deuterium, i.e. 2 Substitution with H(D) may offer certain therapeutic advantages resulting from greater metabolic stability, for example, increased half-life in vivo or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0098] Positron-emitting isotopes, e.g. 11 C. 18 F, 15 O and 13 Substitution with N may be useful in positron emission tomography (PET) studies to examine target occupancy.

[0099] Isotopically labeled compounds of the peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art, or by processes similar to those described in the accompanying Examples, using appropriate isotopically labeled reagents in place of previously used non-labeled reagents.

[0100] [Molecular scaffolds] Molecular scaffolds are described, for example, in WO 2009 / 098450 and the references cited therein, in particular WO 2004 / 077062 and WO 2006 / 078161.

[0101] As noted in the above references, the molecular scaffold may be a small molecule, for example a small organic molecule.

[0102] In one embodiment, the molecular scaffold can be a macromolecule, hi one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides, or carbohydrates.

[0103] In one embodiment, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0104] The molecular scaffold may contain chemical groups that form links with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0105] In one embodiment, the molecular scaffold may comprise or consist of a hexahydro-1,3,5-triazine, in particular 1,3,5-triacryloylhexahydro-1,3,5-triazine ("TATA"), or a derivative thereof.

[0106] In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene (TBMB), but contains three additional methyl groups attached to the benzene ring. This has the advantage that the additional methyl groups can form additional contacts with the polypeptide and thus impose additional structural constraints.

[0107] The molecular scaffolds of the invention contain chemical groups that allow functional groups of the polypeptides of the encoded libraries of the invention to form covalent links with the molecular scaffold, said chemical groups being selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

[0108] The scaffold reactive groups used on the molecular scaffold that can react with the thiol group of cysteine ​​are alkyl halides (also called halogenoalkanes or haloalkanes).

[0109] Specific examples include bromomethylbenzene or iodoacetamide. Other scaffold reactive groups that can be used to selectively couple compounds to cysteines in proteins are maleimides, αβ-unsaturated carbonyl-containing compounds, and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of an α-halomethyl carbonyl-containing compound is N,N',N"-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a natural amino acid that has similar reactivity to cysteine ​​and can be used for the same reactions. Thus, whenever cysteine ​​is mentioned, it is typically permissible to substitute selenocysteine ​​unless the context suggests otherwise.

[0110] [Reactive group] The molecular scaffolds of the invention may be attached to the polypeptide via functional or reactive groups on the polypeptide (i.e., covalently). Such groups are typically formed from the side chains of certain amino acids found within the polypeptide polymer. Such reactive groups may be cysteine ​​side chains, lysine side chains, or N-terminal amine groups, or any other suitable reactive groups. Details can be found in WO 2009 / 098450.

[0111] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenol group of tyrosine, or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups, including azide groups, ketocarbonyl groups, alkyne groups, vinyl groups, or aryl halide groups. The amino and carboxyl groups at the ends of polypeptides can also function as reactive groups to form covalent bonds to molecular scaffolds / cores.

[0112] In one embodiment, the reactive group comprises a cysteine ​​residue.In an alternative embodiment, the reactive group comprises penicillamine.

[0113] The polypeptides of the invention contain at least three reactive groups. They can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed within the molecular scaffold.

[0114] In a preferred embodiment, a polypeptide having three reactive groups is generated. A single product isomer is generated by the reaction of said polypeptide with a molecular scaffold / molecular core having three-fold rotational symmetry. The generation of a single product isomer is advantageous for several reasons. The nucleic acid of the compound library only codes for the primary sequence of the polypeptide, not the isomeric state of the molecule formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is unambiguously defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer isolated in the screening or selection process. The formation of a single product isomer is also advantageous if a specific member of the library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold produces a single product isomer, not a mixture of isomers.

[0115] In another embodiment of the present invention, a polypeptide having four reactive groups is generated. The reaction of said polypeptide with a molecular scaffold / core having tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomers can be determined by chemically synthesizing both isomers, separating the two isomers, and testing both isomers for binding to a target ligand.

[0116] In one embodiment of the present invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of an orthogonal reactive group allows the orthogonal reactive group to be directed to a specific site on the molecular core. A linking strategy involving an orthogonal reactive group may be used to limit the number of product isomers formed. In other words, by selecting a reactive group for one or more of the at least three bonds that is separate or different from the reactive groups selected for the remaining of the at least three bonds, a specific order of binding or directing a specific reactive group of the polypeptide to a specific location on the molecular scaffold can be advantageously achieved.

[0117] In another embodiment, the reactive group of the polypeptide of the invention can react with a molecular linker, which can react with the molecular scaffold so that it is interposed between the molecular scaffold and the polypeptide in the final binding state. In some embodiments, the amino acids of the members of the library or set of polypeptides can be replaced by any amino acid, natural or unnatural. Excluded from these exchangeable amino acids are those that carry a functional group that crosslinks the polypeptide to the molecular core such that the loop sequence is solely exchangeable. Exchangeable polypeptide sequences have either random sequences, invariant sequences, or sequences with random and invariant amino acids. The amino acid with the reactive group is positioned within a defined position in the polypeptide, since the position of the amino acid determines the loop size.

[0118] Alternatives to thiol-mediated conjugation can be used to attach molecular scaffolds to peptides via covalent interactions. Alternatively, the technique can be used to modify or attach additional moieties (e.g., small molecules of interest, different from the molecular scaffold) to the polypeptide after it has been selected or isolated according to the invention. In this embodiment, the attachment obviously does not need to be covalent, but can include non-covalent attachment. The method can be used instead of (or in combination with) the thiol-mediated method by generating phage that display proteins and peptides with unnatural amino acids bearing the required chemical reactive group in combination with small molecules bearing complementary reactive groups, or by incorporating unnatural amino acids into chemically or recombinantly synthesized polypeptides, if the molecules are to be produced after the selection / isolation phase. Further details can be found in WO 2009 / 098450 or Heinis et al., Nat Chem Biol 2009,5(7),502-7.

[0119] [Effectors and functional groups] According to a further aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0120] The effectors and / or functional groups can be attached, for example, to the N-terminus and / or C-terminus of the polypeptide, to amino acids within the polypeptide, or to a molecular scaffold.

[0121] Suitable effector groups include antibodies, and portions or fragments thereof. For example, effector groups can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to one or more constant region domains. Effector groups can also include an antibody hinge region (such as that normally found between the CH1 and CH2 domains of an IgG molecule).

[0122] In a further embodiment of this aspect of the invention, the effector group according to the invention is an Fc region of an IgG molecule. Advantageously, a peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion with a tβ half-life of 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Most advantageously, a peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion with a tβ half-life of 1 day or more.

[0123] Functional groups generally include linking groups, reactive groups for attachment of drugs or other entities, functional groups that aid in uptake of the macrocyclic peptide into cells, and the like.

[0124] The ability of peptides to enter cells makes them effective against intracellular targets. Targets that can be accessed by peptides with the ability to enter cells include transcription factors, intracellular signaling molecules, such as tyrosine kinases and molecules involved in the pathway of apoptosis. Functional groups that allow cell entry include peptides or chemical groups added to either the peptide or the molecular scaffold. Peptides such as VP22, HIV-Tat, and Drosophila homeobox protein (antennapedia) are described, for example, in Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examples of short peptides shown to be effective in translocation through the plasma membrane include the 16 amino acid invasion peptide from the Drosophila antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 amino acid "amphiphilic peptide model" (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127), and the arginine-rich region of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimetics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies that add guanidinium groups to molecules also enhance cell invasion (Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585). Small molecular weight molecules such as steroids may be added to the molecular scaffold to enhance uptake into cells.

[0125] One class of functional groups that can be attached to the peptide ligands includes antibodies and binding fragments thereof, such as Fab, Fv, or single domain fragments. In particular, antibodies that bind to proteins that can increase the half-life of the peptide ligand in vivo may be used.

[0126] In one embodiment, a peptide ligand-effector group according to the invention has a tβ half-life selected from the group consisting of 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, or 20 days or more. Advantageously, a peptide ligand-effector group or composition according to the invention will have a tβ half-life in the range of 12 to 60 hours. In a further embodiment, it will have a tβ half-life of 1 day or more. In a further embodiment, it will be in the range of 12 to 26 hours.

[0127] In one particular embodiment of the invention, the functional group is selected from metal chelators, which are suitable for complexing medicamentously relevant metal radioisotopes.

[0128] Possible effector groups also include enzymes, such as carboxypeptidase G2, which is used in enzyme / prodrug therapy, where the peptide ligand replaces the antibody in ADEPT.

[0129] In a particular embodiment of the present invention, functional group is selected from drugs, for example cytotoxic agents for cancer treatment.Suitable examples include alkylating agents, such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites (including purine analogs azathioprine and mercaptopurine or pyrimidine analogs); plant alkaloids and terpenoids (including vinca alkaloids, such as vincristine, vinblastine, vinorelbine, and vindesine); podophyllotoxin, and its derivatives etoposide and teniposide; taxanes (including paclitaxel, originally known as taxol); topoisomerase inhibitors (including camptothecins: irinotecan and topotecan), and type II inhibitors (including amsacrine, etoposide, etoposide phosphate, and teniposide). Additional drugs can include antitumor antibiotics, which include the immunosuppressants dactinomycin (used in kidney transplants), doxorubicin, epirubicin, bleomycin, calicheamicin, and the like.

[0130] In one further particular embodiment of the invention, the cytotoxic agent is selected from a maytansinoid (eg, DM1) or a monomethylauristatin (eg, MMAE).

[0131] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the structure [ka] has.

[0132] Monomethylauristatin E (MMAE) is a synthetic antineoplastic agent having the following structure: [ka] has.

[0133] In a further particular embodiment of the invention, the cytotoxic agent is selected from maytansinoids (eg, DM1).

[0134] In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulfide bond or a protease-sensitive bond, hi a further embodiment, the groups adjacent to the disulfide bond are modified to control disruption of the disulfide bond, thereby controlling the rate of cleavage and the concomitant release of the cytotoxic agent.

[0135] Published studies have established the possibility of modifying disulfide bonds to make them less sensitive by introducing steric hindrance on either side of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione as well as extracellular (systemic) reducing agents, and consequently reduces the ease with which the toxin is released both inside and outside the cell. Thus, selection of optimal conditions for disulfide stability in circulation (minimizing undesirable side effects of the toxin) versus efficient release within the intracellular environment (maximizing therapeutic efficacy) can be achieved by careful selection of the degree of hindrance on either side of the disulfide bond.

[0136] The lesions on either side of the disulfide bond are adjusted by introducing one or more methyl groups onto the targeting entity (here, the bicyclic peptide) or onto the toxin side of the molecular construct.

[0137] In one embodiment, the cytotoxic agents and linkers are selected from any combination of those described in WO 2016 / 067035, which are incorporated herein by reference.

[0138] [Synthesis] The peptides of the present invention can be synthetically produced by standard techniques followed by reaction with molecular scaffolds in vitro. When this is done, standard chemistry can be used. This allows for rapid and large-scale preparation of soluble material for further downstream experimentation or validation. Such methods can be achieved using conventional chemistry, for example, as disclosed in Timmerman et al. (supra).

[0139] Thus, the present invention also relates to the production of a polypeptide or conjugate selected as indicated herein, which production comprises optional further steps as exemplified below, which in one embodiment are carried out on the final product, the polypeptide / conjugate, produced by chemical synthesis.

[0140] Optionally, amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

[0141] The peptides can also be extended, for example, to incorporate additional loops and thus introduce multiple specificities.

[0142] To extend a peptide, it can simply be chemically extended at its N- or C-terminus using orthogonally protected lysines (and analogues) or within loops using standard solid- or solution-phase chemistries. Activated or activatable N- or C-termini can be introduced using standard (bio)conjugation techniques. Alternatively, the addition can be performed by fragment condensation or native chemical ligation, e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or enzymatically using a subtiligase, e.g. as described in (Chang et al Proc Natl Acad Sci US A. 1994 Dec 20;91(26):12544-8 or Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0143] Alternatively, peptides can be extended or modified by further conjugation via disulfide bonds. This has the additional advantage that once in the reducing environment of cells, the first and second peptides can dissociate from each other. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups. Then, an additional cysteine ​​or thiol can be added to the N- or C-terminus of the first peptide, so that this cysteine ​​or thiol only reacts with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0144] Similar techniques apply equally to the synthesis / coupling of two bicyclic bispecific macrocycles, potentially giving rise to a tetraspecific molecule.

[0145] Additionally, addition of other functional or effector groups can be accomplished in the same manner using appropriate chemistry, coupling at the N- or C-terminus, or via a side chain, In one embodiment, coupling is performed in a manner that does not block the activity of either entity.

[0146] [Pharmaceutical composition] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0147] Generally, the peptide ligand will be utilized in purified form together with pharmacologically appropriate additives or carriers.Typically, these additives or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's.Suitable physiologically acceptable adjuvants, if necessary to keep the polypeptide complex in suspension, may be selected from viscosity enhancing agents, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0148] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0149] The peptide ligands of the invention can be used as compositions administered separately or in conjunction with other drugs. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin or cisplatin and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other drugs in conjunction with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities, for example polypeptides selected using different targeting ligands, whether or not pooled prior to administration.

[0150] The route of administration of the pharmaceutical composition according to the invention may be any one generally known to those skilled in the art. For treatment, the peptide ligands of the invention may be administered to any patient by standard techniques. Administration may be by any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or by direct injection through a suitable catheter. Preferably, the pharmaceutical composition according to the invention will be administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, the concomitant administration of other drugs, counterindications and other parameters considered by the clinician.

[0151] The peptide ligand of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use.This technique has been shown to be effective, and known lyophilization and reconstitution techniques can be used.Those skilled in the art will recognize that lyophilization and reconstitution can result in varying degrees of loss of activity, and the level may have to be adjusted upward to compensate.

[0152] Compositions containing the peptide ligands or cocktails thereof can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, an appropriate amount that achieves at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a population of selected cells is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease and the overall status of the patient's own immune system, but generally ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof may also be administered at similar or slightly lower doses.

[0153] Compositions containing peptide ligands according to the invention may be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or elimination of selected target cell populations in a mammal. Additionally, the peptide ligands described herein may be selectively used ex vivo or in vitro to kill, deplete or otherwise effectively remove target cell populations from a heterogeneous collection of cells. Blood from a mammal may be combined ex vivo with the selected peptide ligand, whereby undesirable cells are killed or otherwise removed from the blood for return to the mammal by standard techniques.

[0154] Therapeutic Use The bicyclic peptides of the invention have particular utility as CD137 binding agents.

[0155] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its alternative name is tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-IBB, and is induced by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but to a greater extent on CD8+ than on CD4+ T cells. CD137 expression is also found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells of the blood vessel wall at sites of inflammation. One of the characterized activities of CD137 is its costimulatory activity for activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In addition, it can enhance immune activity that eliminates tumors in mice.

[0156] CD137 is a T cell costimulatory receptor that is induced immediately after TCR activation (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu. Rev, Immunol., 23:23-68 (2005)). In addition to expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described on antigen-presenting cells, including B cells, monocytes / macrophages, and dendritic cells (Watts et al. Annu. Rev. Immunol, 23:23-68 (2005)). Upon interaction with its ligand, CD137 enhances TCR-induced T cell proliferation, cytokine production, functional maturation, and long-term CD8+ T cell survival (Nam et al, Curr. Cancer Drug Targets, 5:357-363 (2005); Watts et al., Annu. Rev. Immunol, 23:23-68 (2005)).

[0157] Signaling through CD137 by CD137L or agonistic monoclonal antibodies (mAbs) to CD137 enhances TCR-induced T cell proliferation, cytokine production, functional maturation, and long-term CD8+ T cell survival. These effects are due to (1) activation of NF-κB, c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways and (2) regulation of the expression of antiapoptosis- and cell cycle-related genes.

[0158] Experiments performed in both CD137- and CD137L-deficient mice additionally demonstrated the importance of CD137 costimulation in generating fully competitive T cell responses.

[0159] IL-2 and IL-15 activated NK cells express CD137, and ligation of CD137 by agonistic mAbs stimulates NK cell proliferation and IFN-γ secretion, but not cytolytic activity.

[0160] Furthermore, CD137-stimulated NK cells promote the in vitro expansion of activated T cells.

[0161] In accordance with their costimulatory function, agonistic mAbs against CD137 have been shown to promote the rejection of cardiac and skin allografts, eradicate established tumors, broaden primary antiviral CD8+ T cell responses, and increase T cell cytolytic capacity. These studies support the view that CD137 signaling promotes T cell functions that can enhance immunity against tumors and infections.

[0162] Polypeptide ligands selected according to the methods of the invention may be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, etc. Ligands with a selected level of specificity are useful in applications involving testing in non-human animals, where cross-reactivity is desirable, or in diagnostic applications, where cross-reactivity with homologs or paralogs must be carefully controlled. In some applications, e.g., vaccine applications, the ability to elicit an immune response against a range of antigens can be exploited to tailor vaccines to specific diseases and pathogens.

[0163] Substantially pure peptide ligands of at least 90-95% homogeneity are preferred for administration to mammals, with 98-99% or greater homogeneity being most preferred for pharmaceutical uses, particularly when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptide may be used diagnostically or therapeutically (including in vitro), or in developing and performing assay procedures, immunofluorescence staining, and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

[0164] According to a further aspect of the invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by CD137.

[0165] According to a further aspect of the present invention there is provided a method for preventing, inhibiting or treating a disease or disorder mediated by CD137 comprising administering to a patient in need thereof a drug conjugate of an effector group and a peptide ligand as defined herein.

[0166] In one embodiment, the CD137 is a mammalian CD137. In a further embodiment, the mammalian CD137 is human CD137 (hCD137).

[0167] In one embodiment, the disease or disorder mediated by CD137 is selected from cancer, infection, and inflammation. In a further embodiment, the disorder or disease mediated by CD137 is selected from cancer.

[0168] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), such as tumors of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and carcinomas of the ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., thyroid follicular adenocarcinoma), adrenal gland, prostate, skin and adnexa (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies, and associated conditions of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma, lymphomas and leukemias, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), as well as hematological malignancies and associated conditions of myeloid cell lineages (e.g., acute myeloid leukemia [AML], chronic myeloid 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 hematological malignancies (i.e., leukemias, lymphomas), including leukemias, as well as premalignant hematological disorders and borderline malignant disorders; tumors of mesenchymal origin, such as sarcomas of soft tissue, bone or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor and Schwannoma);These include endocrine tumors (e.g., pituitary, adrenal, islet cell, parathyroid, carcinoid, and medullary carcinoma of the thyroid); eye and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas); and childhood and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors); or congenital or other syndromes that predispose a patient to malignancies (e.g., xeroderma pigmentosum).

[0169] In further embodiments, the cancer is selected from hematopoietic malignancies, e.g., non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

[0170] References herein to the term "prevention" involve administration of the protective composition prior to induction of the disease. "Suppression" refers to administration of the composition after an inductive event but prior to clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become evident.

[0171] Animal model systems are available that can be used to screen the effectiveness of peptide ligands in protecting against or treating disease. The use of animal model systems is facilitated by the present invention, which allows for the development of polypeptide ligands that can cross-react with human and animal targets, allowing the use of animal models.

[0172] The invention is further described below with reference to the following examples. EXAMPLES

[0173] [material and method] <Peptide synthesis> Peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer by MultiSynTech. Standard Fmoc-amino acids with appropriate side chain protection groups were used (Sigma, Merck). Where applicable, standard coupling conditions were used in each case followed by deprotection using standard methodologies. Peptides were purified using HPLC and, after isolation, modified with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma). For this, linear peptides were chromatographed with 50:50 MeCN:H 2 Dilute to approximately 35 mL with HO, add approximately 500 µL of 100 mM TATA in acetonitrile, and incubate in HO. 2 5 mL of 1 M NH in HO 4 HCO 3 The reaction was initiated with H. The reaction was allowed to proceed at RT for approximately 30-60 min and, once complete (as determined by MALDI), was lyophilized. 2 One ml of 1 M L-cysteine ​​hydrochloride monohydrate (Sigma) in O was added to the reaction for approximately 60 min at RT to quench excess TATA.

[0174] Following lyophilization, the modified peptide was purified as above, except that the Luna C8 was replaced with a Gemini C18 column (Phenomenex) and the acid was changed to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA-modified material were pooled, lyophilized, and kept at -20°C for storage.

[0175] All amino acids were used in the L-configuration unless otherwise stated.

[0176] In some cases, the peptide was converted to an activated disulfide prior to coupling with a free thiol group of the toxin using the following method: A solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol equiv.) was added to a solution of peptide (20 mM) in dry DMSO (1 mol equiv.). The reaction was mixed thoroughly and DIPEA (20 mol equiv.) was added. The reaction was monitored by LC / MS until completion.

[0177] <abbreviation> Aad 2-aminoadipic acid Abu 2-aminobutyric acid Ac5c Aminocyclopentanecarboxylic acid Ahp aminoheptanoic acid Aib Aminoisobutyric Acid Me-Ala Methylalanine NMe-Ala N-methylalanine tBuAla t-Butylalanine Api aminopimelic acid Aze Azetidine 4,4-BPA 4,4-Biphenylalanine CF3G Trifluoromethyl-alanine Cha 3-Cyclohexylalanine Chg L-Cyclohexylglycine Cit Citrulline H-Cys homocysteine Dap Diaminopimelic acid Fl Fluorescein NMeGlu N-methylglutamic acid HGln Homoglutamine HyP Hydroxyproline Hleu homoleucine Nle Norleucine Nal Naphthylalanine NMelle N-Methyl-Isoleucine Oic Octahydroindole carboxylic acid Oxa Oxazolidine-4-carboxylic acid Pal Pyridylalanine Pen Penicillamine pCaPhe para-carbamoyl-phenylalanine pCoPhe para-carboxy-phenylalanine Phg Phenylglycine HPhe homophenylalanine FPhe Fluorophenylalanine MePhe Methylphenylalanine MeOPhe Methoxyphenylalanine tBuPhe t-Butylphenylalanine NO2Phe Nitrophenylalanine BrPhe Bromophenylalanine Pip Pipecolic acid 5,5-dmP 5,5-Dimethyl-L-proline Sar Sarcosine HSe(me) Homoserine (Me) TetraZ Tetrazole Alanine NMeTyr N-methyl tyrosine

[0178] [Biological data] <1. CD137 direct binding assay> The affinity of the peptides of the invention for human CD137 (Ki) was determined using a fluorescence polarization assay according to the method disclosed in WO 2016 / 067035. The peptides of the invention were labeled with a fluorescent tag (fluorescein, Fl) and diluted to 2.5 nM in 50 mM HEPES with 100 mM NaCl and 0.05% tween (pH 7.5). CD137 protein was titrated starting at 3 μM in the same assay buffer as the peptide in a black wall and bottom low volume 384 well plate to assay 1 nM peptide in a total volume of 25 μL. Typically, the assay was set up by adding 5 μL assay buffer, 10 μL CD137 protein, followed by 10 μL fluorescent peptide. The concentration of CD137 protein was a half-step dilution to give 12 different concentrations starting at 3 μM. Measurements were performed on a BMG PHERAstar FS equipped with a FP 485 520 520 light module at 25°C with 200 flashes per well and a positioning delay of 0.1 seconds. Alternatively, measurements were performed using an Envision (PerkinElmer) equipped with a FITC FP Dual Enh mirror set at 30 flashes. Each well was measured every 5 minutes for 60 minutes. The gain used in the analysis was determined for each tracer after the end of the 60 minutes when no protein was in the well. The mP was fitted to a standard 1:1 binding model of a quadratic equation to generate Kd values. Selected peptides of the invention were tested in the assay described above. The results are shown in Tables 1-3.

[0179] [Table 1A] [Table 1B] [Table 1C]

[0180] [Table 2A] [Table 2B]

[0181] [Table 3A] [Table 3B] [Table 3C] [Table 3D]

[0182] <2. CD137 Biacore experiment> (a) Description of the amine-coupled CD137 target assay Perform a Biacore experiment to identify peptides that bind to human CD137 (AcroBiosystems) protein. a (M -1 s -1 ), k d (s -1 ), K D Values ​​(nM) were determined. CD137 protein was diluted and immobilized on chip CM5 (#BR-1005-30) using standard amine coupling procedures. CD137 protein was diluted to 10 μg / ml in NaAc pH 5.5 and used for coupling. Ethanolamine was then injected to inactivate remaining active esters.

[0183] CD137 protein was immobilized at 180 RU of CD137 protein to generate a maximum theoretical binding response of approximately 25 RU for a 2500 MW peptide. A blank immobilization of a reference flow cell (Fc1 or Fc3) was performed in the case of amine coupling following exactly the same procedure except that no protein target was injected. Peptides were tested at starting concentrations of 300-450 nM and diluted in 1 / 2 serial dilutions. The DMSO concentration was adjusted to remain constant.

[0184] Peptide binding kinetics analysis was performed at a flow rate of 50 μl / min, 200 s association, 600 s dissociation, and 60 s stabilization as follows: Bicyclic peptides were fitted using the 1:1 model Biacore T200 Evaluation software.

[0185] Selected peptides of the present invention were tested in the assay described above and the results are shown in Table 4.

[0186] [Table 4]

[0187] (b) Description of the biotinylated CD137 target assay Perform a Biacore experiment to identify the k of peptides that bind to human CD137 protein. a (M -1 s -1 ), k d (s -1 ), K D Values ​​(nM) were determined. Recombinant human CD137 homotrimers (R&D systems) were resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the manufacturer's suggested protocol. Proteins were desalted and uncoupled biotin was removed into PBS using spin columns.

[0188] For binding analysis, a Biacore T200 instrument was used utilizing a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyldextran surface was activated by injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min for 7 min. For streptavidin capture, the protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl over the activated chip surface. Remaining activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5) to capture biotinylated CD137 to levels of 800-1800 RU. The buffer was changed to PBS / 0.05% Tween 20 and serial dilutions of peptides were prepared in buffer with a final DMSO concentration of 0.5%. Top peptide concentrations were 500 nM or 10 μM, with 6-fold, further 3-fold (500 nM), or 2-fold (10 μM) dilutions in PBS / 0.05% Tween 20. SPR analysis was run at 25 °C with a flow rate of 90 μl / min, with 60 s of association and 100-600 s of dissociation. A regeneration step (10 μl of 10 mM glycine (pH 2)) was used after each cycle. Data were corrected for DMSO excluded volume effect. All data were double-referenced to a blank injection and a reference surface using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a mass transfer model that allowed for mass transfer effects where necessary.

[0189] Selected peptides of the present invention were tested in the assays described above, and the results are shown in Table 5.

[0190] [Table 5A] [Table 5B] [Table 5C] [Table 5D]

[0191] <3. CD137 cell activity> The biological activity of CD137-specific peptides was tested using the Cellular CD137 Luciferase Reporter Assay Kit (Promega). The cells in this commercially available kit express luciferase, which is activated downstream of CD137. This assay can be used to evaluate agonism (exemplified by CD137 ligand, CD137L) and antagonism (exemplified by bicyclic peptide 74-01-04-N002).

[0192] The Promega CD137 cell activity assay uses NF-κB luciferase luminescence as a readout for CD137 activation in Jurkat cells. Briefly, experiments were performed by thawing FBS and preparing media by adding 1% FBS to RPMI-1640 (Promega kit CS196005). Agonists were diluted at concentrations that gave a final concentration of agonist CD137L (R&D systems 2295-4L / CF) diluted to 100 nM in RPMI-1640 media in the assay. Once diluted, bicyclic peptides were titrated down in sterile 96-well plates. The suggested starting concentration for the bicyclic peptides is 10 μM, a 100-fold excess over agonist CD137L. Enough reagent was prepared for replicate samples and then 1 / 3 serial dilutions were performed. Only the positive control CD137L and bicyclic peptide were included. CD137-Jurkat cells were thawed in a water bath and then 500 μl of cells were added to 9.5 ml pre-warmed RPMI-1640 medium with 1% FBS. 50 μl cells / well were added to a white cell culture plate. 12.5 μl of bicyclic peptide (at 6× final concentration) was added to the cells. Then 12.5 μl of agonist (at 6× final concentration) was added as replicate samples or RPMI-1640 with 1% FBS alone was added as background control.

[0193] Cells were incubated with CD137L agonists and bicyclic peptides at 37°C, 5% CO 2 The plates were co-incubated at 4°C for 6 hours. After 6 hours, Bio-Glo™ was thawed and the assay was allowed to proceed at room temperature. 75 μl Bio-Glo™ was added per well and incubated for 5-10 minutes. Luminescence signals were read on a Pherastar plate reader LUM plus model with a gain of 3600 using MARS software. Data were analyzed by calculating the percentage inhibition compared to CD137L alone. Data were transformed to x=log(X) and then plotted as log(inhibitor) against the response variable slope (4 parameters) to determine IC 50 The value was calculated.

[0194] The antagonistic effect of peptide BCY592 (74-01-04-N002; SEQ ID NO: 189) in inhibiting natural ligand CD137L induction was determined using the Promega CD137 cell reporter assay (product number CS196008). CD137 assay cells were co-incubated with trimeric CD137L (R&D systems) + BCY592 peptide. CD137 reporter activity was determined as NF-κB promoter-driven luciferase activity. The effect of peptide BCY592 was plotted as % inhibition relative to baseline CD137L activity in the assay and used to determine IC50 values.

[0195] The results are shown in Figure 1. Here, it is found that the bicyclic peptide BCY592 specific for CD137 can act as an antagonist to inhibit CD137L activity. This result indicates that this peptide can be used in situations where it is desired to block CD137 biological activity. It is known that CD137 activity can cause liver damage due to inflammation driven by local immune cells. Therefore, it is believed that the bicyclic peptide BCY592 (and by inference, other bicyclic CD137 peptides of the present invention) can reduce CD137-CD137L-driven inflammation, which will reduce the liver toxicity of CD137 agonists.

[0196] 4. Fluorescence polarization competitive binding assay The binding site of the hCD137-specific Bicycle peptide was determined by a competition experiment between a fluorescently labeled CD137-binding peptide and the natural ligand CD137L, and the agonistic antibodies urelumab and utomirumab. The urelumab antibody binds to a unique binding site, while both CD137L and utomirumab bind to a site called the ligand-binding site.

[0197] Competitor agonists CD137L (R&D systems), urelumab, and utomilumab were diluted in assay buffer (20 mM HEPES, 150 mM NaCl, 0.05% P20, pH 7.5) to a top concentration of 500-1000 nM. Human CD137 protein (AcroBiosystems) was diluted in the assay to a final concentration of 500 nM. Finally, the fluorescent tracer peptide BCY640 (74-01-04-N001) was added at 1 nM. Typically, the assay was set up by adding 5 μL of agonist competitor, 10 μL of CD137 protein, followed by 10 μL of fluorescent peptide. A total volume of 25 μL was prepared in a black wall and bottom low binding low volume 384-well plate. Measurements were performed on a BMG PHERAstar FS equipped with a FP 485 520 520 light module at 25°C with 200 flashes per well and a positioning delay of 0.1 seconds. Each well was measured every 5 minutes for 60 minutes. The gain was set in wells containing tracer without target protein. The mP values ​​at the end of the 60 minute lead were plotted against the concentration of agonist. A decrease in the mP value indicates competition between a known agonist and the tracer peptide.

[0198] The results are shown in Figures 2 and 3, where it can be seen that CD137-binding Bicycle (BCY640) binds to a physiologically relevant epitope shared with both the natural CD137 ligand (CD137L) and the CD137 antibody (utomirumab).

Claims

1. 1. A peptide ligand specific for CD137 comprising a polypeptide comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences such that at least two polypeptide loops are formed on the molecular scaffold, and a molecular scaffold that forms a covalent bond with said reactive groups of said polypeptide: C ｉ IEPGPFC ｉｉ YADPYMC ｉｉｉ (SEQ ID NO: 19); C ｉ -I-E-E-G-Q-Y-C ｉｉ -X １ -X ２ -D-X ３ -Y / Q-X ４ -C ｉｉｉ (SEQ ID NO:20); C ｉ -D-I-G-P-PY-C ｉｉ -Y-R / A-D-M / P-Y-M-C ｉｉｉ (SEQ ID NO:21); C ｉ -D-E-W-G-L-F / Y-C ｉｉ -I / F-P / A-HS / P-DC ｉｉｉ (SEQ ID NO:22); C ｉ IEEGQYC ｉｉ FADPY(Nle)C ｉｉｉ (SEQ ID NO:23); C ｉ IKEGQYC ｉｉ FADPY(Nle)C ｉｉｉ (SEQ ID NO:24); C ｉ I.E.K.G.Q.Y.C. ｉｉ FADPY(Nle)C ｉｉｉ (SEQ ID NO:25); C ｉ IEE(D-K)QYC ｉｉ FADPY(Nle)C ｉｉｉ (SEQ ID NO:26); C ｉ IEEGKYC ｉｉ FADPY(Nle)C ｉｉｉ (SEQ ID NO:27); C ｉ IEEGQYC ｉｉ KADPY(Nle)C ｉｉｉ (SEQ ID NO:28); C ｉ IEEGQYC ｉｉ FADKY(Nle)C ｉｉｉ (SEQ ID NO:29); C ｉ IEEGQYC ｉｉ FADPYKC ｉｉｉ (SEQ ID NO:30); C ｉ LPP GQYC ｉｉ FPDLLLC ｉｉｉ (SEQ ID NO: 40; 74-22-00); C ｉ -I / L / V-E / D / P-P / E / A-GP / Q-Y / F-C ｉｉ -Y-A-D-P-Y-M / L / Y-C ｉｉｉ (SEQ ID NO:41); and C ｉ -X ５ -X ６ -X ７ -X ８ -X ９ -X １０ -C ｉｉ -X １１ -X １２ -D-X １３ -X １４ -X １５ -C ｉｉｉ (SEQ ID NO: 266) (In the formula, C ｉ , C ｉｉ , and C ｉｉｉ represent the first, second, and third cysteine ​​residues, respectively, and are the three reactive groups that form covalent bonds with the molecular scaffold; X 1 to X 4 represent any amino acid residue; X ５ represents Ile, tBuAla, or Chg; X ６ represents Glu, Pro, Asp, Lys, Aad, HyP, or Oxa; X ７ represents Glu, Lys, or Aad; X ８ represents Gly, D-Lys, D-Ala, L-Ala, D-Phe, D-Glu, D-Gln, D-Leu, D-Ser, or D-Trp; X ９ represents Gln, Lys, Ala, Pro, 5,5-dmP, Oic, Oxa, HyP, Aib, or Ac5c; X １０ represents Tyr, Phe, 3MePhe, 4MePhe, 4FPhe, 2Nal, 4MeOPhe, or 4,4-BPA; X １１ represents Phe, Lys, 4MePhe, 2FPhe, 4FPhe, 4Pal, 4,4-BPA, 4tBuPhe, NO2Phe, or 4BrPhe; X １２ represents Ala or Lys; X １３ represents Pro or Lys; X １４ represents Tyr or Lys; X １５ represents Met, Lys, Nle, HLeu, or Ahp) or a pharma- ceutically acceptable salt thereof.

2. Both contain three cysteine ​​residues separated by two loop sequences of six amino acids, the peptide ligands being: C ｉ -I-E-E-G-Q-Y-C ｉｉ -X １ -X ２ -D-X ３ -Y / Q-X ４ -C ｉｉｉ (SEQ ID NO:20); C ｉ -D-I-G-P-PY-C ｉｉ -Y-R / A-D-M / P-Y-M-C ｉｉｉ (SEQ ID NO:21); and C ｉ IEPGPFC ｉｉ YADPYMC ｉｉｉ (SEQ ID NO: 19); (In the formula, X １ ~X ４ represents any amino acid residue, C ｉ , C ｉｉ and C ｉｉｉ 2. The peptide ligand of claim 1, comprising an amino acid sequence selected from the group consisting of:

3. The peptide ligand comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence of six amino acids and a second loop sequence of five amino acids, the peptide ligand comprising: C ｉ -D-E-W-G-L-F / Y-C ｉｉ -I / F-P / A-HS / P-DC ｉｉｉ (SEQ ID NO:22); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ 2. The peptide ligand of claim 1, comprising an amino acid sequence in which:

4. C ｉ -I-E-E-G-Q-Y-C ｉｉ -X １ -X ２ -D-X ３ -Y / Q-X ４ -C ｉｉｉ The peptide ligand of (SEQ ID NO:20) is selected from SEQ ID NOs:1-14: C ｉ IEEGQYC ｉｉ YRDMYMC ｉｉｉ (SEQ ID NO: 1); C ｉ IEEGQYC ｉｉ YADPYMC ｉｉｉ (SEQ ID NO:2); C ｉ IEEGQYC ｉｉ YADPYYC ｉｉｉ (SEQ ID NO:3); C ｉ IEEGQYC ｉｉ YSDPYYC ｉｉｉ (SEQ ID NO:4); C ｉ IEEGQYC ｉｉ FADPYMC ｉｉｉ (SEQ ID NO:5); C ｉ IEEGQYC ｉｉ YADHQLC ｉｉｉ (SEQ ID NO:6); C ｉ IEEGQYC ｉｉ HADPPYYC ｉｉｉ (SEQ ID NO:7); C ｉ IEEGQYC ｉｉ HADPYFC ｉｉｉ (SEQ ID NO:8); C ｉ IEEGQYC ｉｉ YADHYMC ｉｉｉ (SEQ ID NO:9); C ｉ IEEGQYC ｉｉ YADPYLC ｉｉｉ (SEQ ID NO: 10); C ｉ IEEGQYC ｉｉ YSDPYLC ｉｉｉ (SEQ ID NO: 11); C ｉ IEEGQYC ｉｉ FADPILC ｉｉｉ (SEQ ID NO: 12); C ｉ IEEGQYC ｉｉ HADPYMC ｉｉｉ (SEQ ID NO: 13); and C ｉ IEEGQYC ｉｉ H.A.D.P.Q.M.C. ｉｉｉ (SEQ ID NO: 14); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively, or a pharma- ceutically acceptable salt thereof; for example, A-(SEQ ID NO:1)-A (referred to herein as 74-01-00-N004); A-(SEQ ID NO:2)-A (referred to herein as 74-01-01-N001); A-(SEQ ID NO:3)-A (referred to herein as 74-01-02-N001); A-(SEQ ID NO:4)-A (referred to herein as 74-01-03-N001); A-(SEQ ID NO:5)-A (referred to herein as 74-01-04-N001); A-(SEQ ID NO:6)-A (referred to herein as 74-01-05-N001); A-(SEQ ID NO:7)-A (referred to herein as 74-01-06-N001); A-(SEQ ID NO:8)-A (referred to herein as 74-01-07-N001); A-(SEQ ID NO:9)-A (referred to herein as 74-01-08-N001); A-(SEQ ID NO:10)-A (referred to herein as 74-01-09-N001); A-(SEQ ID NO:10)-SVG (referred to herein as 74-01-09-T03-N002); A-(SEQ ID NO:11)-A (referred to herein as 74-01-10-N001); A-(SEQ ID NO:12)-A (referred to herein as 74-01-11-N001); A-(SEQ ID NO:13)-A (referred to herein as 74-01-13-N001); and A-(SEQ ID NO:14)-A (referred to herein as 74-01-14-N001) 3. The peptide ligand of claim 1 or 2, comprising an amino acid sequence selected from:

5. C ｉ -D-I-G-P-PY-C ｉｉ -Y-R / A-D-M / P-Y-M-C ｉｉｉ The peptide ligand of (SEQ ID NO:21) is: C ｉ DIGPPYC ｉｉ YRDMYMC ｉｉｉ (SEQ ID NO: 15); and C ｉ DIGPPYC ｉｉ YADPYMC ｉｉｉ (SEQ ID NO: 16); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively, or a pharma- ceutically acceptable salt thereof; for example, A-(SEQ ID NO:15)-A (referred to herein as 74-01-16-N001); and A-(SEQ ID NO:16)-A (referred to herein as 74-01-17-N001) 3. The peptide ligand of claim 1 or 2, comprising an amino acid sequence selected from:

6. C ｉ -D-E-W-G-L-F / Y-C ｉｉ -I / F-P / A-HS / P-DC ｉｉｉ The peptide ligand of (SEQ ID NO:22) is: C ｉ DEWGLFC ｉｉ IPHSDC ｉｉｉ (SEQ ID NO: 17); and C ｉ DEWGLYC ｉｉ F.A.H.P.D.C. ｉｉｉ (SEQ ID NO: 18); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively, or a pharma- ceutically acceptable salt thereof; for example, Ac-A-(SEQ ID NO:17)-A (referred to herein as 74-02-00-N004); and A-(SEQ ID NO:18)-A (referred to herein as 74-02-01-N001) 4. The peptide ligand of claim 1 or 3, comprising an amino acid sequence selected from:

7. C ｉ IEPGPFC ｉｉ YADPYMC ｉｉｉ The peptide ligand of (SEQ ID NO:19) is: A-(SEQ ID NO:19)-NRV (referred to herein as 74-19-00-T01-N002) 3. The peptide ligand of claim 1 or 2, comprising the amino acid sequence:

8. including N-terminal and C-terminal modifications, A-C ｉ IEEGQYC ｉｉ FADPY(Nle)C ｉｉｉ -A (SEQ ID NO: 31; BCY3814); A-C ｉ IEEGQYC ｉｉ FADPY(Nle)C ｉｉｉ - Dap (SEQ ID NO: 32; BCY7732); A-C ｉ IKEGQYC ｉｉ FADPY(Nle)C ｉｉｉ -A (SEQ ID NO: 33; BCY7733); A-C ｉ I.E.K.G.Q.Y.C. ｉｉ FADPY(Nle)C ｉｉｉ -A (SEQ ID NO: 34; BCY7734); A-C ｉ IEE(D-K)QYC ｉｉ FADPY(Nle)C ｉｉｉ -A (SEQ ID NO: 35; BCY7735); A-C ｉ IEEGKYC ｉｉ FADPY(Nle)C ｉｉｉ -A (SEQ ID NO: 36; BCY7736); A-C ｉ IEEGQYC ｉｉ KADPY(Nle)C ｉｉｉ -A (SEQ ID NO: 37; BCY7737); A-C ｉ IEEGQYC ｉｉ FADKY(Nle)C ｉｉｉ -A (SEQ ID NO: 38; BCY7738); and A-C ｉ IEEGQYC ｉｉ FADPYKC ｉｉｉ -A (SEQ ID NO: 39; BCY7739); (In the formula, C ｉ , C ｉｉ and C ｉｉｉ 2. The peptide ligand of claim 1, comprising an amino acid sequence selected from: wherein R represents the first, second and third cysteine ​​residues, respectively; Ac represents an N-terminal acetyl group; and Dap represents diaminopropionic acid, or a pharma- ceutically acceptable salt thereof.

9. The amino acid sequences listed in the table below, excluding peptides BCY7238, BCY7241, BCY7243, and BCY7246: 【Table 1】 【Table 2】 【Table 3】 【Table 4】 A peptide ligand specific for CD137, comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences such that at least two polypeptide loops are formed on the molecular scaffold, and a molecular scaffold that forms a covalent bond with the reactive groups of the polypeptide, wherein the reactive groups are cysteine ​​residues, homocysteine ​​residues or penicillamine residues.

10. The peptide ligand of SEQ ID NO:266 is the following peptide: Ac-CIK(Peg12)EGQYCFADPYMC (SEQ ID NO:52; BCY7239), Ac-CIEK(Peg12)GQYCFADPYMC (SEQ ID NO:53; BCY7240), Ac-CIEEGK(Peg12)YCFADPYMC (SEQ ID NO:55; BCY7242), Ac-CIEEGQYCK(Peg12)ADPYMC (SEQ ID NO:57; BCY7244), Ac-CIEEGQYCFK(Peg12)DPYMC (SEQ ID NO:58; BCY7245), Ac-CIEEGQYCFADK(Peg12)YMC (SEQ ID NO:60; BCY7247), Ac-CIEEGQYCFADPK(Peg12)MC (SEQ ID NO:61; BCY7248), Ac-CIEEGQYCFADPYK(Peg12)C (SEQ ID NO:62; BCY7249), [Ac]CIEE[dK(PEG12Fl)]QYCFADPY[Nle]C (SEQ ID NO: 63; BCY7416), [PEG3]-ACIEEGAYCFADPY(Nle)CA (SEQ ID NO:97; BCY7287), [PEG3]-ACIEaQYCFADPY(Nle)CA (SEQ ID NO: 106; BCY7297), [PEG3]-AC-Chg-EEGQYCFADPY[Nle]CA (SEQ ID NO: 117; BCY7154), [PEG3]-ACIPEGQYCFADPY[Nle]CA (SEQ ID NO: 119; BCY7156), [PEG3]-ACIDEGQYCFADPY[Nle]CA (SEQ ID NO: 120; BCY7157), [PEG3]-ACI-Aad-EGQYCFADPY[Nle]CA (SEQ ID NO: 121; BCY7158), [PEG3]-ACIE-Aad-GQYCFADPY[Nle]CA (SEQ ID NO: 125; BCY7162), [PEG3]-ACIEE-DLys-QYCFADPY[Nle]CA (SEQ ID NO: 128; BCY7165), [PEG3]-ACIEE-DPhe-QYCFADPY[Nle]CA (SEQ ID NO: 129; BCY7166), [PEG3]-ACIEE-DGlu-QYCFADPY[Nle]CA (SEQ ID NO: 130); BCY7167), [PEG3]-ACIEE-DGln-QYCFADPY[Nle]CA (SEQ ID NO: 131; BCY7168), [PEG3]-ACIEE-DLeu-QYCFADPY[Nle]CA (SEQ ID NO: 132; BCY7169), [PEG3]-ACIEE-DSer-QYCFADPY[Nle]CA (SEQ ID NO: 133; BCY7170), [PEG3]-ACIEGPYCFADPY[Nle]CA (SEQ ID NO: 136; BCY7174), [PEG3]-ACIEEGQFCFADPY[Nle]CA (SEQ ID NO: 137; BCY7175), [PEG3]-ACIEEGQ-3MeF-CFADPY[Nle]CA (SEQ ID NO: 139; BCY7177), [PEG3]-ACIEEGQ-4MeF-CFADPY[Nle]CA (SEQ ID NO: 140; BCY7178), [PEG3]-ACIEEGQ-4FF-CFADPY[Nle]CA (SEQ ID NO: 141; BCY7179), [PEG3]-ACIEEGQYC-4MeF-ADPY[Nle]CA (SEQ ID NO: 145; BCY7183), [PEG3]-ACIEEGQYC-4FF-ADPY[Nle]CA (SEQ ID NO: 147; BCY7185), [PEG3]AC[tBuAla]EEGQYCFADPY[Nle]CA (SEQ ID NO: 156; BCY7195), [PEG3]ACIEEGQYC[2FPhe]ADPY[Nle]CA (SEQ ID NO: 159; BCY7198), [PEG3]ACIEEGQYCFADPY[HLeu]CA (SEQ ID NO: 170; BCY7211), [PEG3]-ACIEEGQYCFADPY[Ahp]CA (SEQ ID NO: 188; BCY7311), [PEG3]ACIPE[dF]QYCFADPY[Nle]CA (SEQ ID NO: 195; BCY7768), [PEG3]ACIPE[dF]PYCFADPY[Nle]CA (SEQ ID NO: 196; BCY7770), [PEG3]ACIEEE[dF]PYCFADPY[Nle]CA (SEQ ID NO: 197; BCY7772), [PEG3]ACIPEGPYCFADPY[Nle]CA (SEQ ID NO: 198; BCY7773), [PEG3]AC[tBuAla]PE[dF]P[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 199; BCY7774), [PEG3]AC[tBuAla]PE[dF]Q[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 200; BCY7775), [PEG3]AC[tBuAla]EE[dF]P[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 201; BCY7776), [PEG3]ACI[HyP]EGQYCFADPY[Nle]CA (SEQ ID NO: 203; BCY7796), [PEG3]ACIE[dW]QYCFADPY[Nle]CA (SEQ ID NO: 204; BCY7798), [PEG3]ACIEEGQ[2Nal]CFADPY[Nle]CA (SEQ ID NO: 207; BCY7801), [PEG3]ACIEEGQ[4MeoPhe]CFADPY[Nle]CA (SEQ ID NO: 208; BCY7802), [Ac]ACIEEGQ[44BPA]CFADPY[Nle]CA (SEQ ID NO: 223; BCY7936), [Ac]ACIEEGQYC[4Pal]ADPY[Nle]CA (SEQ ID NO: 227; BCY7941), [Ac]ACIEEGQYC[44BPA]ADPY[Nle]CA (SEQ ID NO: 228; BCY7942), [Ac]ACIEEGQYC[4tBuPhe]ADPY[Nle]CA (SEQ ID NO: 230; BCY7944), [Ac]ACIEEG[55DMP]YCFADPY[Nle]CA (SEQ ID NO: 232; BCY7950), [Ac]ACIEEG[Oic]YCFADPY[Nle]CA (SEQ ID NO: 234; BCY7954), [Ac]ACI[Oxa]EGQYCFADPY[Nle]CA (SEQ ID NO: 238; BCY7958), [Ac]ACIEEG[Oxa]YCFADPY[Nle]CA (SEQ ID NO: 239; BCY7959), [Ac]ACIPEGPYCFADPY[Nle]CA (SEQ ID NO: 240; BCY7960), [Ac]ACIEEG[HyP]YCFADPY[Nle]CA (SEQ ID NO: 241; BCY7952), [Ac]ACIPE[dA]PYCFADPY[Nle]CA (SEQ ID NO: 242; BCY7961), [Ac]AC[tBuAla]EEGQYCFADPY[Nle]CA (SEQ ID NO: 245; BCY8656), [Ac]ACIPEGQYCFADPY[Nle]CA (SEQ ID NO: 248; BCY8659), [Ac]ACIEE[dF]QYCFADPY[Nle]CA (SEQ ID NO: 252; BCY8663), [Ac]ACIEEG[Aib]YCFADPY[Nle]CA (SEQ ID NO: 256; BCY8668), [Ac]ACIEEG[AC5C]YCFADPY[Nle]CA (SEQ ID NO: 257; BCY8669), [Ac]ACIEEGQYC[NO2Phe]ADPY[Nle]CA (SEQ ID NO: 261; BCY8674), [Ac]ACIEEGQYC[4BrPhe]ADPY[Nle]CA (SEQ ID NO: 262; BCY8675), [Ac]ACIEEGQYCFADPYMCA (SEQ ID NO: 265; BCY9273), ACIEEGQYCFADPY(Nle)CA (SEQ ID NO:31; BCY3814), [Ac]CIEEGQYCFADPY[Nle]C[Dap] (SEQ ID NO: 194; BCY7527) and [Ac]AC[tBuAla]PE[dA]PYCFADPY[Nle]CA (SEQ ID NO: 243; BCY7965) or the Ci to Ciii sequences of The following peptides: Ac-CIK(Peg12)EGQYCFADPYMC (SEQ ID NO:52; BCY7239), Ac-CIEK(Peg12)GQYCFADPYMC (SEQ ID NO:53; BCY7240), Ac-CIEEGK(Peg12)YCFADPYMC (SEQ ID NO:55; BCY7242), Ac-CIEEGQYCK(Peg12)ADPYMC (SEQ ID NO:57; BCY7244), Ac-CIEEGQYCFK(Peg12)DPYMC (SEQ ID NO:58; BCY7245), Ac-CIEEGQYCFADK(Peg12)YMC (SEQ ID NO:60; BCY7247), Ac-CIEEGQYCFADPK(Peg12)MC (SEQ ID NO:61; BCY7248), Ac-CIEEGQYCFADPYK(Peg12)C (SEQ ID NO:62; BCY7249), [Ac]CIEE[dK(PEG12Fl)]QYCFADPY[Nle]C (SEQ ID NO: 63; BCY7416), [PEG3]-ACIEEGAYCFADPY(Nle)CA (SEQ ID NO:97; BCY7287), [PEG3]-ACIEaQYCFADPY(Nle)CA (SEQ ID NO: 106; BCY7297), [PEG3]-AC-Chg-EEGQYCFADPY[Nle]CA (SEQ ID NO: 117; BCY7154), [PEG3]-ACIPEGQYCFADPY[Nle]CA (SEQ ID NO: 119; BCY7156), [PEG3]-ACIDEGQYCFADPY[Nle]CA (SEQ ID NO: 120; BCY7157), [PEG3]-ACI-Aad-EGQYCFADPY[Nle]CA (SEQ ID NO: 121; BCY7158), [PEG3]-ACIE-Aad-GQYCFADPY[Nle]CA (SEQ ID NO: 125; BCY7162), [PEG3]-ACIEE-DLys-QYCFADPY[Nle]CA (SEQ ID NO: 128; BCY7165), [PEG3]-ACIEE-DPhe-QYCFADPY[Nle]CA (SEQ ID NO: 129; BCY7166), [PEG3]-ACIEE-DGlu-QYCFADPY[Nle]CA (SEQ ID NO: 130; BCY7167), [PEG3]-ACIEE-DGln-QYCFADPY[Nle]CA (SEQ ID NO: 131; BCY7168), [PEG3]-ACIEE-DLeu-QYCFADPY[Nle]CA (SEQ ID NO: 132; BCY7169), [PEG3]-ACIEE-DSer-QYCFADPY[Nle]CA (SEQ ID NO: 133; BCY7170), [PEG3]-ACIEGPYCFADPY[Nle]CA (SEQ ID NO: 136; BCY7174), [PEG3]-ACIEEGQFCFADPY[Nle]CA (SEQ ID NO: 137; BCY7175), [PEG3]-ACIEEGQ-3MeF-CFADPY[Nle]CA (SEQ ID NO: 139; BCY7177), [PEG3]-ACIEEGQ-4MeF-CFADPY[Nle]CA (SEQ ID NO: 140; BCY7178), [PEG3]-ACIEEGQ-4FF-CFADPY[Nle]CA (SEQ ID NO: 141; BCY7179), [PEG3]-ACIEEGQYC-4MeF-ADPY[Nle]CA (SEQ ID NO: 145; BCY7183), [PEG3]-ACIEEGQYC-4FF-ADPY[Nle]CA (SEQ ID NO: 147; BCY7185), [PEG3]AC[tBuAla]EEGQYCFADPY[Nle]CA (SEQ ID NO: 156; BCY7195), [PEG3]ACIEEGQYC[2FPhe]ADPY[Nle]CA (SEQ ID NO: 159; BCY7198), [PEG3]ACIEEGQYCFADPY[HLeu]CA (SEQ ID NO: 170; BCY7211), [PEG3]-ACIEEGQYCFADPY[Ahp]CA (SEQ ID NO: 188; BCY7311), [PEG3]ACIPE[dF]QYCFADPY[Nle]CA (SEQ ID NO: 195; BCY7768), [PEG3]ACIPE[dF]PYCFADPY[Nle]CA (SEQ ID NO: 196; BCY7770), [PEG3]ACIEEE[dF]PYCFADPY[Nle]CA (SEQ ID NO: 197; BCY7772), [PEG3]ACIPEGPYCFADPY[Nle]CA (SEQ ID NO: 198; BCY7773), [PEG3]AC[tBuAla]PE[dF]P[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 199; BCY7774), [PEG3]AC[tBuAla]PE[dF]Q[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 200; BCY7775), [PEG3]AC[tBuAla]EE[dF]P[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 201; BCY7776), [PEG3]ACI[HyP]EGQYCFADPY[Nle]CA (SEQ ID NO: 203; BCY7796), [PEG3]ACIE[dW]QYCFADPY[Nle]CA (SEQ ID NO: 204; BCY7798), [PEG3]ACIEEGQ[2Nal]CFADPY[Nle]CA (SEQ ID NO: 207; BCY7801), [PEG3]ACIEEGQ[4MeoPhe]CFADPY[Nle]CA (SEQ ID NO: 208; BCY7802), [Ac]ACIEEGQ[44BPA]CFADPY[Nle]CA (SEQ ID NO: 223; BCY7936), [Ac]ACIEEGQYC[4Pal]ADPY[Nle]CA (SEQ ID NO: 227; BCY7941), [Ac]ACIEEGQYC[44BPA]ADPY[Nle]CA (SEQ ID NO: 228; BCY7942), [Ac]ACIEEGQYC[4tBuPhe]ADPY[Nle]CA (SEQ ID NO: 230; BCY7944), [Ac]ACIEEG[55DMP]YCFADPY[Nle]CA (SEQ ID NO: 232; BCY7950), [Ac]ACIEEG[Oic]YCFADPY[Nle]CA (SEQ ID NO: 234; BCY7954), [Ac]ACI[Oxa]EGQYCFADPY[Nle]CA (SEQ ID NO: 238; BCY7958), [Ac]ACIEEG[Oxa]YCFADPY[Nle]CA (SEQ ID NO: 239; BCY7959), [Ac]ACIPEGPYCFADPY[Nle]CA (SEQ ID NO: 240; BCY7960), [Ac]ACIEEG[HyP]YCFADPY[Nle]CA (SEQ ID NO: 241; BCY7952), [Ac]ACIPE[dA]PYCFADPY[Nle]CA (SEQ ID NO: 242; BCY7961), [Ac]AC[tBuAla]EEGQYCFADPY[Nle]CA (SEQ ID NO: 245; BCY8656), [Ac]ACIPEGQYCFADPY[Nle]CA (SEQ ID NO: 248; BCY8659), [Ac]ACIEE[dF]QYCFADPY[Nle]CA (SEQ ID NO: 252; BCY8663), [Ac]ACIEEG[Aib]YCFADPY[Nle]CA (SEQ ID NO: 256; BCY8668), [Ac]ACIEEG[AC5C]YCFADPY[Nle]CA (SEQ ID NO: 257; BCY8669), [Ac]ACIEEGQYC[NO2Phe]ADPY[Nle]CA (SEQ ID NO: 261; BCY8674), [Ac]ACIEEGQYC[4BrPhe]ADPY[Nle]CA (SEQ ID NO: 262; BCY8675), [Ac]ACIEEGQYCFADPYMCA (SEQ ID NO: 265; BCY9273), ACIEEGQYCFADPY(Nle)CA (SEQ ID NO:31; BCY3814), [Ac]CIEEGQYCFADPY[Nle]C[Dap] (SEQ ID NO: 194; BCY7527) and [Ac]AC[tBuAla]PE[dA]PYCFADPY[Nle]CA (SEQ ID NO: 243; BCY7965) 2. The peptide ligand of claim 1, wherein the peptide ligand is selected from the complete sequence (i.e., including all N- and C-terminal additions) of:

11. Both contain three cysteine ​​residues separated by two loop sequences of six amino acids, the peptide ligands being: C ｉ -X ５ -X ６ -X ７ -X ８ -X ９ -X １０ -C ｉｉ -X １１ -A-D-P-Y-X １５ -C ｉｉｉ (SEQ ID NO: 267); (In the formula, C ｉ , C ｉｉ , and C ｉｉｉ represent the first, second, and third cysteine ​​residues, respectively; X ５ represents Ile or tBuAla; X ６ represents Lys, Glu, or Pro; X ７ represents Glu or Lys; X ８ represents Gly, D-Lys, D-Phe, or D-Ala; X ９ represents Gln, Lys, or Pro; X １０ represents Tyr or 4MePhe; X １１ represents Phe or 4FPhe; X １５ The peptide ligand of claim 1 , comprising an amino acid sequence selected from the group consisting of:

12. The peptide ligand of SEQ ID NO:267 is the following peptide: Ac-CIK(Peg12)EGQYCFADPYMC (SEQ ID NO:52; BCY7239), Ac-CIEK(Peg12)GQYCFADPYMC (SEQ ID NO:53; BCY7240), Ac-CIEEGK(Peg12)YCFADPYMC (SEQ ID NO:55; BCY7242), [Ac]CIEE[dK(PEG12Fl)]QYCFADPY[Nle]C (SEQ ID NO: 63; BCY7416), [PEG3]-ACIPEGQYCFADPY[Nle]CA (SEQ ID NO: 119; BCY7156), [PEG3]-ACIEE-DPhe-QYCFADPY[Nle]CA (SEQ ID NO: 129; BCY7166), [PEG3]-ACIEGPYCFADPY[Nle]CA (SEQ ID NO: 136; BCY7174), [PEG3]AC[tBuAla]PE[dF]P[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 199; BCY7774), [Ac]ACIEEGQYCFADPYMCA (SEQ ID NO: 265; BCY9273), ACIEEGQYCFADPY(Nle)CA (SEQ ID NO:31; BCY3814), [Ac]CIEEGQYCFADPY[Nle]C[Dap] (SEQ ID NO: 194; BCY7527) and [Ac]AC[tBuAla]PE[dA]PYCFADPY[Nle]CA (SEQ ID NO: 243; BCY7965) C ｉ ~C ｉｉｉ the sequence (i.e., without any N- or C-terminal additions); or The following peptides: Ac-CIK(Peg12)EGQYCFADPYMC (SEQ ID NO:52; BCY7239), Ac-CIEK(Peg12)GQYCFADPYMC (SEQ ID NO:53; BCY7240), Ac-CIEEGK(Peg12)YCFADPYMC (SEQ ID NO:55; BCY7242), [Ac]CIEE[dK(PEG12Fl)]QYCFADPY[Nle]C (SEQ ID NO: 63; BCY7416), [PEG3]-ACIPEGQYCFADPY[Nle]CA (SEQ ID NO: 119; BCY7156), [PEG3]-ACIEE-DPhe-QYCFADPY[Nle]CA (SEQ ID NO: 129; BCY7166), [PEG3]-ACIEGPYCFADPY[Nle]CA (SEQ ID NO: 136; BCY7174), [PEG3]AC[tBuAla]PE[dF]P[4MePhe]C[4FPhe]ADPY[Nle]CA (SEQ ID NO: 199; BCY7774), [Ac]ACIEEGQYCFADPYMCA (SEQ ID NO: 265; BCY9273), ACIEEGQYCFADPY(Nle)CA (SEQ ID NO:31; BCY3814), [Ac]CIEEGQYCFADPY[Nle]C[Dap] (SEQ ID NO: 194; BCY7527) and [Ac]AC[tBuAla]PE[dA]PYCFADPY[Nle]CA (SEQ ID NO: 243; BCY7965) 12. The peptide ligand of claim 11, selected from the complete sequence (i.e., including all N- and C-terminal additions) of:

13. 13. The peptide ligand of any one of claims 1 to 12, wherein the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

14. The peptide ligand of any one of claims 1 to 13, wherein the pharma- ceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt.

15. The peptide ligand according to any one of claims 1 to 14, wherein the CD137 is human CD137.

16. A drug conjugate comprising a peptide ligand according to any one of claims 1 to 15 conjugated to one or more effectors and / or functional groups.

17. A drug conjugate comprising a peptide ligand according to any one of claims 1 to 15 conjugated to one or more cytotoxic agents.

18. A pharmaceutical composition comprising a peptide ligand according to any one of claims 1 to 15 or a drug conjugate according to claim 16 or 17 in combination with one or more pharma- ceutically acceptable excipients.

19. A peptide ligand according to any one of claims 1 to 15 or a drug conjugate according to claim 16 or 17 for use in the prevention, inhibition or treatment of a disease or disorder mediated by CD137.

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