Bicyclic peptide ligand specific for NK cells

Abstract

The present invention relates to a polypeptide covalently attached to a molecular scaffold, with two or more peptide loops sandwiched between the attachment points to the scaffold. In particular, the present invention describes peptides that bind to natural killer (NK) cells. The present invention also relates to a multimeric binding complex comprising at least two of said bicyclic peptide ligands. The present invention also relates to pharmaceutical compositions comprising the peptide ligands or multimeric binding complexes, and the use of said peptide ligands, multimeric binding complexes, and pharmaceutical compositions in the prevention, inhibition, or treatment of natural killer (NK) cell-mediated diseases or disorders, such as inflammatory disorders, autoimmune diseases, and cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a polypeptide covalently attached to a molecular scaffold, with two or more peptide loops sandwiched between the attachment points to the scaffold. In particular, the present invention describes peptides that bind to natural killer (NK) cells. The present invention also relates to a multimeric binding complex comprising at least two of said bicyclic peptide ligands. The present invention also relates to pharmaceutical compositions comprising the peptide ligands or multimeric binding complexes, and the use of said peptide ligands, multimeric binding complexes, and pharmaceutical compositions in the prevention, inhibition, or treatment of natural killer (NK) cell-mediated diseases or disorders, such as inflammatory disorders, autoimmune diseases, and cancer. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are able to bind protein targets with high affinity and specificity and are therefore an attractive class of molecules for the development of therapeutic drugs. Indeed, some cyclic peptides have already been successfully used in the clinic, such as, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al., (2008), Nat. Rev. Drug. Discov. 7(7), 608-24). The excellent binding properties are due not only to the relatively large interaction surface formed between the peptide and the target, but also to the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are used with a 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 and experience less entropy loss when binding to targets, resulting in higher binding affinity. The reduced flexibility also results in a target-specific conformational fixation, increasing binding specificity compared to linear peptides. This effect is exemplified by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity against other MMPs when its ring is opened (Cherney et al., (1998), J. Med. Chem. 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with multiple peptide rings, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously tethered polypeptides with cysteine ​​residues into 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 generating candidate drug compounds have been disclosed by linking cysteine-containing polypeptides to molecular scaffolds such as 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Heinis et al., (2014) Angewandte Chemie, International Edition 53(6)1602-1606).

[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 WO2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine ​​residues and two random six amino acid regions (Cys-(Xaa)6-Cys-(Xaa)6-Cys) is displayed on phage and cyclized by covalently linking the cysteine ​​side chains to a small molecule scaffold. Summary of the Invention

[0006] (Summary of the invention) In a first aspect of the present invention, there is provided a peptide ligand specific for natural killer (NK) cells, comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, wherein at least two polypeptide loops are formed on the molecular scaffold.

[0007] In a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0008] In a further aspect of the invention there is provided a peptide ligand or pharmaceutical composition as defined herein for use in the prevention, inhibition or treatment of a natural killer (NK) cell-mediated disease or disorder. [Brief description of the drawings]

[0009] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1 shows the binding of Alexa Fluor® 488-labeled NKp46 bicyclic peptide dimers to CHO-K1 / NKp46 or CHO-K1 NKp46-less parental cell lines. (A) Alexa Fluor® 488-tagged NKp46 bicyclic peptide dimers bind to NKp46-expressing cells in a dose-dependent manner. No binding is observed in the NKp46-less CHO-K1 parental cell line (B). Several AF488-NKp46 dimers were evaluated and are shown in Table 4. BCY15665 is a non-linked bicyclic peptide dimer composed of all D-amino acids. [Diagram 2] Figure 2 shows the binding of Alexa Fluor® 488-labeled NKp46 bicyclic peptide dimers to CD56+ cells (NK cells) or CD56- cells from human PBMCs. (A) shows the binding of Alexa Fluor® 488-labeled NKp46 bicyclic peptide dimers to a PBMC population expressing CD56 (NK cell population). (B) shows no binding to cells of the PBMC population (T cell population) that do not express CD56 and are CD3+. Data (n=4) from one representative PBMC donor. BCY15665 is a non-binding bicyclic peptide dimer composed of all D-amino acids. [Diagram 3] FIG. 3 shows an IgG competition assay of C-terminally biotinylated human CD16a F176 variant (F variant) using BCY15914 (A), BCY15915 (B), and BCY15916 (C). [Figure 4] FIG. 4 shows an IgG competition assay of N-terminally biotinylated human CD16a V176 variant (V variant) using BCY15914 (A), BCY15915 (B), and BCY15916 (C). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention In a first aspect of the present invention, there is provided a peptide ligand specific for natural killer (NK) cells, comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, wherein at least two polypeptide loops are formed on the molecular scaffold.

[0011] Natural killer (NK) cells are members of the innate immune system and represent a small fraction of peripheral blood mononuclear cells. As frontline responders, these immune cells detect and eliminate unhealthy cells, bridging the gap between innate and adaptive immune responses. Their unique properties make NK cells excellent candidates to enhance therapeutic approaches in tumor immunity and autoimmunity.

[0012] NK cells play a role in immune surveillance through various inhibitory and activating receptors. These activating and inhibitory receptors on the surface of NK cells are a complex means through which NK cell activity is balanced in healthy individuals. NK cells recognize MHC class I molecules on the surface of healthy cells and are inhibited through inhibitory receptors so as not to eliminate these healthy cells. During times of stress, infection, or transformation, NK cells recognize unhealthy cells that have lost MHC class I on their cell surface and triggered NK cell receptor ligands that bind to the activating receptors. Recognition of non-self by NK cells triggers a cytotoxic response, resulting in the release of cytokines and cytotoxic molecules to eliminate the unhealthy cells.

[0013] NK cell activity is based on a complex mechanism involving both activating and inhibitory signals. Several studies have provided evidence for the central role of NK cell receptors in natural cytotoxicity and their usefulness in cancer therapy. An open challenge is to further understand and enhance NK cell-mediated tumor cell recognition and killing. Reports suggest that tumor cells use many mechanisms to reduce NK activity and that the presence and availability of NK cells correlates with a favorable patient prognosis (Pasero et al. (2015) Oncotarget 6(16), 14360-14373; Stringaris et al. (2014) Haematologica 99(5), 836-847). Therapeutic interventions could exploit the potential of NK cells to mediate immune responses to combat cancer and autoimmune diseases.

[0014] (NKp46-binding bicyclic peptide) In one embodiment, the bicyclic peptide is specific for (i.e., binds to) a natural cytotoxicity receptor present on the surface of NK cells. In a further embodiment, the bicyclic peptide is specific for (i.e., binds to) a natural cytotoxicity receptor selected from NKp30, NKp44, and NKp46. In an even further embodiment, the bicyclic peptide is specific for (i.e., binds to) NKp46.

[0015] Natural cytotoxicity receptors (NCRs) are a family of stimulatory receptors expressed on the surface of NK cells that induce NK activation and NK cell-mediated cytotoxicity. The NCR family consists of three members: NKp30, NKp44, and NKp46. Although the cellular ligand for NKp46 is unknown, a role for NKp46 in antitumor immunity has been shown. NK cell activation of NKp46 via viral antigens leads to tumor rejection (Chinnery et al., 2012). The NKp46 receptor, upon interaction with its ligand, primes NK cells to direct cytotoxicity, as shown by the use of anti-NKp46 blocking antibodies that inhibit the ability of NK cells to lyse targets (Arnon et al., 2004). The amount of NCR expression on the surface of NK cells also increases NK cytotoxicity. A strong correlation has been identified between the density of NCR expression and the ability of NK cells to kill target cells, including a wide variety of tumor cells (Moretta et al., 2006). In AML and cervical cancer and its precursor lesions, tumor cells are resistant to NK cytotoxicity due to insufficient amounts of NCR or NCR ligands (Costello et al., 2002; Garcia-Iglesias et al., 2009). In many solid tumors, NK cells are downregulated by the tumor microenvironment, including tumors that shed NCR ligands and immunoedit to impede the ability of NK cells to recognize, infiltrate, and kill tumor cells (Nayyar, 2019; Stojanovic et al., 2011; Sordo-Bahamonde et al., 2020; Watanabe et al., 2010; Izawa et al., 2011; Koo et al., 2013; Sun et al., 2015; Hasmim et al., 2015; Han et al., 2018). Stringaris et al. (2014) reported downregulation of NKp46, upregulation of the NK cell inhibitory receptor NKG2A, and reduced cytotoxicity of NK cells from AML patients.Furthermore, in solid tumors such as prostate cancer, a decrease in the expression of several activating receptors (CD16, NKp30, NKp46, NKG2D, and DNAM-1) and an increase in the inhibitory receptor CD85j have been reported (Pesaro et al., 2016). In contrast, Gautheir et al. (2019) identified NKp46 as a potential candidate for targeting activating receptors on NK cells in cancer and demonstrated no statistically significant downregulation of NKp46 in the periphery in patients with SCCHN, breast cancer, liver cancer, lung cancer, kidney cancer, and metastatic melanoma. Furthermore, persistent expression of NKp46 in multiple solid tumors associated with downregulation of other activating receptors, e.g., NKG2D, NKp30, and NKp44, as well as low expression of CD16 on tumor-infiltrating lymphocytes, has been reported in cancers such as acute myeloid leukemia, breast cancer, and lung cancer (Fauriat et al., 2007; Mamessier et al., 2011; Platonova et al., 2011; Levi et al., 2015; Kim et al., 2010; MacFarlane et al., 2017). Thus, NKp46 has been shown to be a specific NK surface marker suitable for therapeutic applications, allowing NK cells to identify and target tumors.

[0016] In a further embodiment, the loop sequence comprises 4, 5, 6, or 7 amino acids.

[0017] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 4 amino acids.

[0018] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, and the bicyclic peptide ligand comprises: [ka] (In the formula, C i , Cii , and C iii represent a first, second, and third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0019] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:1)-A-Sar6-KFl (referred to herein as BCY14654); A-(SEQ ID NO:1)-A (referred to herein as BCY14456); A-(SEQ ID NO:2)-A-Sar6-KFl (referred to herein as BCY15125); A-(SEQ ID NO:2)-A (referred to herein as BCY15102); and A-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY18004), wherein PYA represents pentynic acid, Sar represents sarcosine, and Fl represents fluorescein.

[0020] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and: A-(SEQ ID NO:1)-A-Sar6-KFl (referred to herein as BCY14654); and A-(SEQ ID NO:2)-A-Sar6-KFl (referred to herein as BCY15125), wherein Sar represents sarcosine and Fl represents fluorescein.

[0021] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 6 amino acids.

[0022] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 6 amino acids, and the bicyclic peptide ligand comprises: [ka] TIFF2024502351000004.tif196170 (in the formula, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively; Cba represents cyclobutylalanine; tBuAla represents β-t-butyl-L-alanine; Sar represents sarcosine; Cis-HyP represents L-4-cis-hydroxyproline; HyP represents hydroxyproline; NMeAla represents N-methylalanine; Nle represents norleucine; dA represents D-alanine; Agb represents norarginine; and tBuGly represents tert-butylglycine. , Cha represents 3-cyclohexyl-L-alanine, Chg represents cyclohexylglycine, Cbg represents cyclobutylglycine, HArg represents homoarginie, Aze represents azetidine-2-carboxylic acid, Pip represents pipecolic acid, Nva represents norvaline, AlloIle represents L-allo-isoleucine, and EPA represents diethyl-L-alanine), or a pharma- ceutical acceptable salt thereof.

[0023] In an even further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 6 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0024] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 6 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY15687); A-(SEQ ID NO:3)-A (referred to herein as BCY15098); A-(SEQ ID NO:4)-A-Sar6-KFl (referred to herein as BCY15115); A-(SEQ ID NO:4)-A (referred to herein as BCY15099); Ac-(SEQ ID NO:4) (referred to herein as BCY18293); Ac-A-(SEQ ID NO:4)-A (referred to herein as BCY18294); (SEQ ID NO:4)-A (referred to herein as BCY19694); K-(SEQ ID NO:4)-A (referred to herein as BCY19695); dK-(SEQ ID NO:4)-A (referred to herein as BCY19696); Dap-(SEQ ID NO:4)-A (referred to herein as BCY19697); Dab-(SEQ ID NO:4)-A (referred to herein as BCY19698); GABA-(SEQ ID NO:4)-A (referred to herein as BCY19699); A-(SEQ ID NO:63)-A (referred to herein as BCY14454); A-(SEQ ID NO:64)-A (referred to herein as BCY18299); A-(SEQ ID NO:65)-A (referred to herein as BCY18301); A-(SEQ ID NO:66)-A (referred to herein as BCY18302); A-(SEQ ID NO:67)-A (referred to herein as BCY18303); A-(SEQ ID NO:68)-A (referred to herein as BCY18304); A-(SEQ ID NO:69)-A (referred to herein as BCY18305); A-(SEQ ID NO:70)-A (referred to herein as BCY18307); A-(SEQ ID NO:71)-A (referred to herein as BCY18309); A-(SEQ ID NO:72)-A (referred to herein as BCY18310); A-(SEQ ID NO:73)-A (referred to herein as BCY18311); A-(SEQ ID NO:74)-A (referred to herein as BCY18312); A-(SEQ ID NO:75)-A (referred to herein as BCY18313); A-(SEQ ID NO:76)-A (referred to herein as BCY18314); A-(SEQ ID NO:77)-A (referred to herein as BCY18315); A-(SEQ ID NO:78)-A (referred to herein as BCY18316); A-(SEQ ID NO:79)-A (referred to herein as BCY18317); A-(SEQ ID NO:80)-A (referred to herein as BCY18318); A-(SEQ ID NO:81)-A (referred to herein as BCY18319); A-(SEQ ID NO:82)-A (referred to herein as BCY18320); A-(SEQ ID NO:83)-A (referred to herein as BCY18321); A-(SEQ ID NO:84)-A (referred to herein as BCY18322); A-(SEQ ID NO:85)-A (referred to herein as BCY18323); A-(SEQ ID NO:86)-A (referred to herein as BCY18324); A-(SEQ ID NO:87)-A (referred to herein as BCY18325); A-(SEQ ID NO:88)-A (referred to herein as BCY18326); A-(SEQ ID NO:89)-A (referred to herein as BCY18327); A-(SEQ ID NO:90)-A (referred to herein as BCY18328); A-(SEQ ID NO:91)-A (referred to herein as BCY19700); A-(SEQ ID NO:92)-A (referred to herein as BCY19701); A-(SEQ ID NO:93)-A (referred to herein as BCY19702); A-(SEQ ID NO:94)-A (referred to herein as BCY19703); A-(SEQ ID NO:95)-A (referred to herein as BCY19704); A-(SEQ ID NO:96)-A (referred to herein as BCY19706); A-(SEQ ID NO:97)-A (referred to herein as BCY19707); A-(SEQ ID NO:98)-A (referred to herein as BCY19708); A-(SEQ ID NO:99)-A (referred to herein as BCY19710); A-(SEQ ID NO:100)-A (referred to herein as BCY19711); A-(SEQ ID NO:101)-A (referred to herein as BCY19712); and A-(SEQ ID NO:102)-A (referred to herein as BCY19713), wherein PYA represents pentynic acid, Sar represents sarcosine, Fl represents fluorescein, dK represents D-lysine, Dap represents L-2,3-diaminopropionic acid, Dab represents L-2,4-diaminobutyric acid, and GABA represents γ-aminobutyric acid.

[0025] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 6 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY15687); and A-(SEQ ID NO:4)-A-Sar6-KFl (referred to herein as BCY15115), wherein PYA represents pentynic acid, Sar represents sarcosine, and Fl represents fluorescein.

[0026] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 7 amino acids.

[0027] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 7 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0028] In an even further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 7 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0029] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 7 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:5)-A (referred to herein as BCY15103); A-(SEQ ID NO:5)-A-[K(PYA)] (referred to herein as BCY18005); A-(SEQ ID NO:5)-A-Sar6-KFl (referred to herein as BCY15118); A-(SEQ ID NO:6)-A (referred to herein as BCY15104); A-(SEQ ID NO:6)-A-Sar6-KFl (referred to herein as BCY15119); A-(SEQ ID NO:7)-A (referred to herein as BCY15105); A-(SEQ ID NO:7)-A-Sar6-KFl (referred to herein as BCY15120); A-(SEQ ID NO:8)-A (referred to herein as BCY15106); A-(SEQ ID NO:8)-A-Sar6-KFl (referred to herein as BCY15121); and A-(SEQ ID NO:103)-A (referred to herein as BCY14457), wherein PYA represents pentynic acid, Sar represents sarcosine, and Fl represents fluorescein.

[0030] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 7 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:5)-A-Sar6-KFl (referred to herein as BCY15118); A-(SEQ ID NO:6)-A-Sar6-KFl (referred to herein as BCY15119); A-(SEQ ID NO:7)-A-Sar6-KFl (referred to herein as BCY15120); and A-(SEQ ID NO:8)-A-Sar6-KFl (referred to herein as BCY15121); wherein Sar represents sarcosine and Fl represents fluorescein.

[0031] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 6 amino acids and the second loop sequence consisting of 4 amino acids.

[0032] In a further embodiment, the loop sequence comprises three cysteine ​​or penicillamine residues separated by two loop sequences, a first loop sequence consisting of six amino acids and a second loop sequence consisting of four amino acids, and the bicyclic peptide ligand comprises: [ka] TIFF2024502351000009.tif247170TIFF2024502351000010.tif248170TIFF2024502351000011.tif61170 (in the formula, C i , Pen i , C ii , Pen ii , C iii and Pen iiirepresent the first, second, and third reactive groups, respectively; 2,6DiMeTyr represents 2,6-dimethyl-tyrosine; tBuAla represents t-butyl-alanine; Cba represents β-cyclobutylalanine; HyP represents hydroxyproline; Aze represents azetidine; Aib represents aminoisobutyric acid; 1Nal represents 1-naphthylalanine; 2Nal represents 2-naphthylalanine; 3Pal represents 3-pyridylalanine; C5A represents β-cyclopentyl-L-alanine; Cha represents 3- cyclohexyl-L-alanine, 4FPhe stands for 4-fluoro-L-phenylalanine, 4MeOPhe stands for 4-methoxy-phenylalanine, NO2Phe stands for 4-nitro-phenylalanine, 4MePhe stands for 4-methyl-phenylalanine, pCoPhe stands for p-carboxy-phenylalanine, pCaPhe stands for p-carboxamido-phenylalanine, 4tBuPhe stands for 4-tert-butyl-phenylalanine, CF3Phe stands for 4-trifluoromethyl-phenylalanine, tBuTyr stands for 3-tert-butyl tyrosine, 4CNPhe stands for 4-cyano-phenylalanine, 4ClPhe stands for 4-chloro-phenylalanine, HPhe stands for homophenylalanine, 3tBuTyr stands for 3-tert-butyl tyrosine, His3Me stands for 3-methylhistidine, 2Pal stands for 2-pyridylalanine, His1Me stands for 1-methylhistidine, 4ThiAz stands for β-4-thiazol-L-ylalanine, Trp(Me) stands for 1-methyltryptophan, t BuGly stands for Lt-butylglycine, Chg stands for cyclohexylglycine, HSer stands for homoserine, NMeAla stands for N-methylalanine, 4Pal stands for 4-pyridylalanine, Cis-HyP stands for 4-cis-hydroxyproline, dA stands for D-alanine, dV stands for D-valine, dNva stands for D-norvaline, dNle stands for D-norleucine, dF stands for D-phenylalanine, dCha stands for 3-cyclohexyl-D-alanine, and DOPA stands for 3,4-dihydroxyphenylalanine, 2FTyr represents 2-fluorotyrosine, Nle represents norleucine, dC represents D-cysteine, AzaTrp represents azatryptophan, 2MeTrp represents 2-methyltryptophan, 6MeTrp represents 6-methyltryptophan, 6FTrp represents 6-fluorotryptophan, 5FTrp represents 5-fluorotryptophan, Dht represents 2,3-dihydrotryptophan, 2FuAla represents 2-furylalanine, and AlloIle represents L-allo-isoleucine), or a pharma- ceutical acceptable salt thereof.

[0033] In an even further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 6 amino acids and a second loop sequence consisting of 4 amino acids, and the bicyclic peptide ligand comprises: [ka] TIFF2024502351000013.tif114170 (in the formula, C i , C ii , and C iiirepresent the first, second, and third cysteine ​​residues, respectively; 2,6DiMeTyr represents 2,6-dimethyl-tyrosine; tBuAla represents t-butyl-alanine; Cba represents β-cyclobutylalanine; HyP represents hydroxyproline; Aze represents azetidine; Aib represents aminoisobutyric acid; 1Nal represents 1-naphthylalanine; 2Nal represents 2-naphthylalanine; 3Pal represents 3-pyridylalanine; C5A represents β-cyclopentyl-L-alanine; Cha represents 3-cyclohexyl-L-alanine; 4FPhe represents 4-fluoro-L-phenylalanine; 4MeOPhe represents 4-methyl-L-alanine; wherein NO2Phe represents 4-nitro-phenylalanine, 4MePhe represents 4-methyl-phenylalanine, pCoPhe represents p-carboxy-phenylalanine, pCaPhe represents p-carboxamido-phenylalanine, 4tBuPhe represents 4-tert-butyl-phenylalanine, CF3Phe represents 4-trifluoromethyl-phenylalanine, 4CNPhe represents 4-cyano-phenylalanine, 4ClPhe represents 4-chloro-phenylalanine, and HPhe represents homophenylalanine), or a pharma- ceutically acceptable salt thereof.

[0034] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of six amino acids and a second loop sequence consisting of four amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:9)-A-[dK(PYA)] (referred to herein as BCY15013); A-(SEQ ID NO:10)-A (referred to herein as BCY18470); A-(SEQ ID NO:10)-A-[dK(PYA)] (referred to herein as BCY15452); A-(SEQ ID NO:11)-A (referred to herein as BCY15107); Ac-(SEQ ID NO:11) (referred to herein as BCY18260); Ac-A-(SEQ ID NO:11)-A (referred to herein as BCY18261); A-(SEQ ID NO:11)-[Aib] (referred to herein as BCY19717); (SEQ ID NO:11)-A (referred to herein as BCY19718); A-(SEQ ID NO:11)-A-[dK(PYA)] (referred to herein as BCY19719); A-(SEQ ID NO:11)-A-[K(PYA)] (referred to herein as BCY15686); A-(SEQ ID NO:12)-A (referred to herein as BCY15100); Ac-(SEQ ID NO:12) (referred to herein as BCY15634); Ac-A-(SEQ ID NO:12)-A (referred to herein as BCY15635); dA-(SEQ ID NO:12) (referred to herein as BCY16134); A-(SEQ ID NO:12)-A-Sar6-KFl (referred to herein as BCY15116); A-(SEQ ID NO:13)-A (referred to herein as BCY14455); (SEQ ID NO:13)-Sar6-KFl (referred to herein as BCY15023); Ac-(SEQ ID NO:13)-Sar6-KFl (referred to herein as BCY15024); A-(SEQ ID NO:13)-A-Sar6-KFl (referred to herein as BCY14652); A-(SEQ ID NO:14)-A (referred to herein as BCY15636); A-(SEQ ID NO:15)-A (referred to herein as BCY15638); A-(SEQ ID NO:16)-A (referred to herein as BCY15639); A-(SEQ ID NO:17)-A (referred to herein as BCY15640); A-(SEQ ID NO:18)-A (referred to herein as BCY15641); A-(SEQ ID NO:19)-A (referred to herein as BCY15642); A-(SEQ ID NO:20)-A (referred to herein as BCY15643); A-(SEQ ID NO:21)-A (referred to herein as BCY15644); A-(SEQ ID NO:22)-A (referred to herein as BCY15645); A-(SEQ ID NO:23)-A (referred to herein as BCY15646); A-(SEQ ID NO:24)-A (referred to herein as BCY15648); A-(SEQ ID NO:25)-A (referred to herein as BCY15649); A-(SEQ ID NO:26)-A (referred to herein as BCY15650); A-(SEQ ID NO:27)-A (referred to herein as BCY15651); A-(SEQ ID NO:28)-A (referred to herein as BCY15653); A-(SEQ ID NO:29)-A (referred to herein as BCY15654); A-(SEQ ID NO:30)-A (referred to herein as BCY15655); A-(SEQ ID NO:31)-A (referred to herein as BCY15656); A-(SEQ ID NO:32)-A (referred to herein as BCY15657); A-(SEQ ID NO:33)-A (referred to herein as BCY15658); A-(SEQ ID NO:34)-A (referred to herein as BCY15659); A-(SEQ ID NO:35)-A (referred to herein as BCY15660); A-(SEQ ID NO:36)-A (referred to herein as BCY15661); A-(SEQ ID NO:37)-A (referred to herein as BCY15662); A-(SEQ ID NO:38)-A (referred to herein as BCY16130); A-(SEQ ID NO:39)-A (referred to herein as BCY16131); A-(SEQ ID NO:40)-A (referred to herein as BCY16132); Ac-(SEQ ID NO:40) (referred to herein as BCY16133); Ac-(SEQ ID NO:40)-[K(PYA)] (referred to herein as BCY17224); A-(SEQ ID NO:41)-A (referred to herein as BCY16135); A-(SEQ ID NO:42)-A (referred to herein as BCY16136); A-(SEQ ID NO:43)-A (referred to herein as BCY16137); A-(SEQ ID NO:44)-A (referred to herein as BCY16138); A-(SEQ ID NO:45)-A (referred to herein as BCY16139); A-(SEQ ID NO:46)-A (referred to herein as BCY16140); A-(SEQ ID NO:47)-A (referred to herein as BCY16141); A-(SEQ ID NO:48)-A (referred to herein as BCY16142); A-(SEQ ID NO:49)-A (referred to herein as BCY16143); A-(SEQ ID NO:50)-A (referred to herein as BCY16144); A-(SEQ ID NO:51)-A (referred to herein as BCY16145); A-(SEQ ID NO:52)-A-Sar6-KFl (referred to herein as BCY15025); A-(SEQ ID NO:53)-A-Sar6-KFl (referred to herein as BCY15028); A-(SEQ ID NO:54)-A-Sar6-KFl (referred to herein as BCY15030); A-(SEQ ID NO:55)-A-Sar6-KFl (referred to herein as BCY15035); A-(SEQ ID NO:56)-A-Sar6-KFl (referred to herein as BCY15117); A-(SEQ ID NO:57)-A-Sar6-KFl (referred to herein as BCY15122); A-(SEQ ID NO:58)-A (referred to herein as BCY15108); A-(SEQ ID NO:58)-A-Sar6-KFl (referred to herein as BCY15123); A-(SEQ ID NO:59)-A-Sar6-KFl (referred to herein as BCY15124); A-(SEQ ID NO:104)-A (referred to herein as BCY14458); A-(SEQ ID NO:105)-A (referred to herein as BCY15109); A-(SEQ ID NO:106)-A (referred to herein as BCY15568); A-(SEQ ID NO:107)-A (referred to herein as BCY15637); A-(SEQ ID NO:108)-A (referred to herein as BCY15647); A-(SEQ ID NO:109)-A (referred to herein as BCY15652); A-(SEQ ID NO:110)-A (referred to herein as BCY16834); A-(SEQ ID NO:111)-A (referred to herein as BCY16837); A-(SEQ ID NO:112)-A (referred to herein as BCY16840); A-(SEQ ID NO:113)-A (referred to herein as BCY16842); A-(SEQ ID NO:114)-A (referred to herein as BCY16843); A-(SEQ ID NO:115)-A (referred to herein as BCY17662); A-(SEQ ID NO:116)-A (referred to herein as BCY18263); A-(SEQ ID NO:117)-A (referred to herein as BCY18265); A-(SEQ ID NO:118)-A (referred to herein as BCY18266); A-(SEQ ID NO:119)-A (referred to herein as BCY18269); A-(SEQ ID NO:120)-A (referred to herein as BCY18272); A-(SEQ ID NO:121)-A (referred to herein as BCY18273); A-(SEQ ID NO:122)-A (referred to herein as BCY18277); A-(SEQ ID NO:123)-A (referred to herein as BCY18278); A-(SEQ ID NO:124)-A (referred to herein as BCY18279); A-(SEQ ID NO:125)-A (referred to herein as BCY18280); A-(SEQ ID NO:126)-A (referred to herein as BCY18281); A-(SEQ ID NO:127)-A (referred to herein as BCY18282); A-(SEQ ID NO:128)-A (referred to herein as BCY18285); A-(SEQ ID NO:129)-A (referred to herein as BCY18286); A-(SEQ ID NO:130)-A (referred to herein as BCY18287); A-(SEQ ID NO:131)-A (referred to herein as BCY18288); A-(SEQ ID NO:132)-A (referred to herein as BCY18289); A-(SEQ ID NO:133)-A (referred to herein as BCY18290); A-(SEQ ID NO:134)-A (referred to herein as BCY18291); A-(SEQ ID NO:135)-A (referred to herein as BCY18292); Ac-(SEQ ID NO:136) (referred to herein as BCY18433); Ac-(SEQ ID NO:137) (referred to herein as BCY18434); Ac-(SEQ ID NO:138) (referred to herein as BCY18435); Ac-(SEQ ID NO:139) (referred to herein as BCY18437); Ac-(SEQ ID NO:140) (referred to herein as BCY18439); Ac- (SEQ ID NO: 141) (referred to herein as BCY18440); Ac- (SEQ ID NO: 142) (referred to herein as BCY18441); Ac- (SEQ ID NO: 143) (referred to herein as BCY18442); Ac-(SEQ ID NO:144) (referred to herein as BCY18443); Ac-(SEQ ID NO:145) (referred to herein as BCY18444); Ac-(SEQ ID NO:146) (referred to herein as BCY19682); Ac-(SEQ ID NO:147) (referred to herein as BCY19683); Ac-(SEQ ID NO:148) (referred to herein as BCY19684); Ac-(SEQ ID NO:149) (referred to herein as BCY19685); Ac-(SEQ ID NO:150) (referred to herein as BCY19686); Ac-(SEQ ID NO:151) (referred to herein as BCY19687); Ac-(SEQ ID NO:152) (referred to herein as BCY19688); Ac-(SEQ ID NO:153) (referred to herein as BCY19689); Ac-(SEQ ID NO:154) (referred to herein as BCY19690); Ac-(SEQ ID NO:155) (referred to herein as BCY19691); Ac-(SEQ ID NO:156) (referred to herein as BCY19692); Ac-(SEQ ID NO:157) (referred to herein as BCY19693); A-(SEQ ID NO:158)-A (referred to herein as BCY19720); A-(SEQ ID NO:159)-A (referred to herein as BCY19721); A-(SEQ ID NO:160)-A (referred to herein as BCY19722); A-(SEQ ID NO:161)-A (referred to herein as BCY19723); A-(SEQ ID NO:162)-A (referred to herein as BCY19724); A-(SEQ ID NO:163)-A (referred to herein as BCY19725); A-(SEQ ID NO:164)-A (referred to herein as BCY19730); A-(SEQ ID NO:165)-A (referred to herein as BCY19732); A-(SEQ ID NO:166)-A (referred to herein as BCY19733); A-(SEQ ID NO:167)-A (referred to herein as BCY19734); and A-(SEQ ID NO:168)-A (referred to herein as BCY19735), wherein Aib represents aminoisobutyric acid, PYA represents pentanoic acid, Sar represents sarcosine, and Fl represents fluorescein.

[0035] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 6 amino acids and a second loop sequence consisting of 4 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and: A-(SEQ ID NO:9)-A-[dK(PYA)] (referred to herein as BCY15013); A-(SEQ ID NO:10)-A-[dK(PYA)] (referred to herein as BCY15452); A-(SEQ ID NO:11)-A-[K(PYA)] (referred to herein as BCY15686); A-(SEQ ID NO:12)-A (referred to herein as BCY15100); (SEQ ID NO:12) (referred to herein as BCY15633); Ac-(SEQ ID NO:12) (referred to herein as BCY15634); Ac-A-(SEQ ID NO:12)-A (referred to herein as BCY15635); dA-(SEQ ID NO:12) (referred to herein as BCY16134); A-(SEQ ID NO:12)-A-Sar6-KFl (referred to herein as BCY15116); A-(SEQ ID NO:13)-A (referred to herein as BCY14455); (SEQ ID NO:13)-Sar6-KFl (referred to herein as BCY15023); Ac-(SEQ ID NO:13)-Sar6-KFl (referred to herein as BCY15024); A-(SEQ ID NO:13)-A-Sar6-KFl (referred to herein as BCY14652); A-(SEQ ID NO:14)-A (referred to herein as BCY15636); A-(SEQ ID NO:15)-A (referred to herein as BCY15638); A-(SEQ ID NO:16)-A (referred to herein as BCY15639); A-(SEQ ID NO:17)-A (referred to herein as BCY15640); A-(SEQ ID NO:18)-A (referred to herein as BCY15641); A-(SEQ ID NO:19)-A (referred to herein as BCY15642); A-(SEQ ID NO:20)-A (referred to herein as BCY15643); A-(SEQ ID NO:21)-A (referred to herein as BCY15644); A-(SEQ ID NO:22)-A (referred to herein as BCY15645); A-(SEQ ID NO:23)-A (referred to herein as BCY15646); A-(SEQ ID NO:24)-A (referred to herein as BCY15648); A-(SEQ ID NO:25)-A (referred to herein as BCY15649); A-(SEQ ID NO:26)-A (referred to herein as BCY15650); A-(SEQ ID NO:27)-A (referred to herein as BCY15651); A-(SEQ ID NO:28)-A (referred to herein as BCY15653); A-(SEQ ID NO:29)-A (referred to herein as BCY15654); A-(SEQ ID NO:30)-A (referred to herein as BCY15655); A-(SEQ ID NO:31)-A (referred to herein as BCY15656); A-(SEQ ID NO:32)-A (referred to herein as BCY15657); A-(SEQ ID NO:33)-A (referred to herein as BCY15658); A-(SEQ ID NO:34)-A (referred to herein as BCY15659); A-(SEQ ID NO:35)-A (referred to herein as BCY15660); A-(SEQ ID NO:36)-A (referred to herein as BCY15661); A-(SEQ ID NO:37)-A (referred to herein as BCY15662); A-(SEQ ID NO:38)-A (referred to herein as BCY16130); A-(SEQ ID NO:39)-A (referred to herein as BCY16131); A-(SEQ ID NO:40)-A (referred to herein as BCY16132); Ac-(SEQ ID NO:40) (referred to herein as BCY16133); Ac-(SEQ ID NO:40)-[K(PYA)] (referred to herein as BCY17224); A-(SEQ ID NO:41)-A (referred to herein as BCY16135); A-(SEQ ID NO:42)-A (referred to herein as BCY16136); A-(SEQ ID NO:43)-A (referred to herein as BCY16137); A-(SEQ ID NO:44)-A (referred to herein as BCY16138); A-(SEQ ID NO:45)-A (referred to herein as BCY16139); A-(SEQ ID NO:46)-A (referred to herein as BCY16140); A-(SEQ ID NO:47)-A (referred to herein as BCY16141); A-(SEQ ID NO:48)-A (referred to herein as BCY16142); A-(SEQ ID NO:49)-A (referred to herein as BCY16143); A-(SEQ ID NO:50)-A (referred to herein as BCY16144); A-(SEQ ID NO:51)-A (referred to herein as BCY16145); A-(SEQ ID NO:52)-A-Sar6-KFl (referred to herein as BCY15025); A-(SEQ ID NO:53)-A-Sar6-KFl (referred to herein as BCY15028); A-(SEQ ID NO:54)-A-Sar6-KFl (referred to herein as BCY15030); A-(SEQ ID NO:55)-A-Sar6-KFl (referred to herein as BCY15035); A-(SEQ ID NO:56)-A-Sar6-KFl (referred to herein as BCY15117); A-(SEQ ID NO:57)-A-Sar6-KFl (referred to herein as BCY15122); A-(SEQ ID NO:58)-A-Sar6-KFl (referred to herein as BCY15123); and A-(SEQ ID NO:59)-A-Sar6-KFl (referred to herein as BCY15124), wherein PYA represents pentynic acid, Sar represents sarcosine, and Fl represents fluorescein.

[0036] (CD16a-binding bicyclic peptide) In one embodiment, the bicyclic peptide is specific for (i.e., binds to) an Fc receptor present on the surface of NK cells. In a further embodiment, the bicyclic peptide is specific for (i.e., binds to) a low affinity Fcγ receptor (FcγR) selected from FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB. In an even further embodiment, the bicyclic peptide is specific for (i.e., binds to) FcγRIIIA (also known as CD16a).

[0037] Fc receptors are expressed on the surface of many leukocytes. In humans, five classical low affinity Fcγ receptors (FcγR) (FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB) bind to the Fc portion of immunoglobulin G (IgG) and are mediators of inflammation through activation and suppression of immune cells (Muta et al., 1994; Ravetch et al., 2001). FcγRIIIA (CD16a) is activated by engagement with the Fc portion of IgG molecules and is crucial for the antibody-dependent cellular cytotoxicity (ADCC) process. ADCC is a mechanism by which antigen-specific antibodies direct NK cells to kill cancer cells expressing the antigen (Arnould et al., 2006). Given its important role in the antitumor effects of IgG1 mAbs, several studies have shown that the human IgG1 anti-human epidermal growth factor receptor 2 (HER-2) antibody trastuzumab owes its antitumor effect partly to ADCC, as does the EGFR antibody cetuximab in metastatic colorectal patients (Zhang et al., 2007, 2020; Wu et al., 2003). Similar results have been reported for rituximab, a chimeric IgG1 mAb against the B cell differentiation antigen CD20 (Manches et al., 2003; Clynes et al., 2000). The utility of CD16 expression in inducing immune cells to promote tumor cell death was shown in overexpression studies. Expression of IgFc on the surface of B16 melanoma cells through retroviral transduction of Ig-Fc results in tumor killing in vivo (Riddle et al., 2005). The role of FcγR engagement in targeting NK cells to tumors was also demonstrated in studies whereby chimeric antigen receptor T cells expressing CD16 scFv targeted antibody-coated tumor cells. In vivo model transfer of CD16-CarT into EGFR or CD20 tumor-bearing mice treated with cetuximab or rituximab enhanced immune cell targeting and ADCC killing, thereby abolishing tumor immune evasion (Rataj et al., 2019; Caratelli et al., 2017).

[0038] The contribution of FcγR genes to autoimmune diseases has attracted much attention, and functional FcγR polymorphisms have been reported to play an important role in the pathogenesis of autoimmune diseases (Salmon et al., 2001; Morgan et al., 2003; Wu et al., 1997). FcγR knockout mouse models show that both activating and inhibitory FcγRs affect the development of autoimmune diseases (Nabbe et al., 2003; Kleinau et al., 2000; Bolland et al., 2000). Changes in FcγR expression greatly affect the signal threshold mediated by IgG immune complexes. In particular, proinflammatory and anti-inflammatory cytokines can regulate the expression levels of activating and inhibitory FcγRs and affect the threshold of immune cell responses to IgG immune complexes (Pricop et al., 2001; Boruchov et al., 2005). FcγRIIIA and FcγRIIIB copy number variations were associated with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in Taiwanese patients (Chen et al., 2014). Elevated copy number of FcγRIIIA was demonstrated to be a risk factor for SLE. High frequencies of cytokine-producing FcγRIIIA-positive dendritic cells (DCs) were observed in SLE patients, especially in those with active disease, suggesting that FcγRIIIA-mediated proinflammatory cytokine production in DCs may contribute to disease pathogenesis (Henriques et al., 2012). High density of activating FcγRIIIA on the surface of immune cells (NK cells, monocytes, DCs, macrophages, and a subset of T cells) may tip the delicate balance of the immune response toward intense inflammation, resulting in the development of SLE (Chen et al., 2014). A correlation between low copy numbers of FcγRIIIA and low expression of CD16a in NK cells has been demonstrated, suggesting that the copy number of FcγRIIIA has physiological significance in NK cell function. Most importantly, FcγRIIIA deficiency has been associated with two different autoimmune diseases (SLE and RA), suggesting that functional defects of FcγRIIIA may be a common risk factor for various autoimmune diseases.This disease association suggests that modulation of FcγRIIIA function may be an important therapeutic target for lupus nephritis (Chen et al., 2014).

[0039] Therapeutic innovation as a means to block inflammation in autoimmune diseases through FcγR interactions offers some hope. Previous studies support the potential of FcγR blockade as a therapeutic strategy to suppress immune complex-mediated events in autoimmune diseases (Clarkson et al., 1986; Flaherty et al., 2012). IgG Fc fragments by themselves have demonstrated good efficacy in animal models of RA and in humans with thrombocytopenia (ITP) or Kawasaki disease (Anthony et al., 2008; Debre M et al., 1993; Hsu et al., 1993). Multivalent Fc constructs have been shown to suppress immune complex processes in murine models of thrombocytopenia and arthritis (Ortiz et al., 2016). Furthermore, infusion of soluble FcγR3a and 2a can suppress immune complex-driven inflammation in a murine lupus model (Li et al., 2014). Blocking immune complex formation on NK cells is an avenue to be explored to reduce inflammatory activation and therefore autoimmune disease.

[0040] In further embodiments, the loop sequence comprises 3, 4, 5, 6, 7, or 8 amino acids. In even further embodiments, the loop sequence comprises 3, 4, 5, 7, or 8 amino acids.

[0041] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of four amino acids and the second loop sequence consisting of eight amino acids.

[0042] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 4 amino acids and a second loop sequence consisting of 8 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0043] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of four amino acids and a second loop sequence consisting of eight amino acids, the molecular scaffold is TATB, and the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition: A-(SEQ ID NO:265)-A (referred to herein as BCY15044); and A-(SEQ ID NO:265)-AGAAAE (referred to herein as BCY19416), The amino acid sequence is selected from the group consisting of:

[0044] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 4 amino acids.

[0045] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0046] In an even further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0047] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, the molecular scaffold is TATA, and the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition: A-(SEQ ID NO:60)-A (referred to herein as BCY11808); A-(SEQ ID NO:60)-A-[K(PYA)] (referred to herein as BCY15685); A-(SEQ ID NO:60)-A-[Sar6]-[KBiot] (referred to herein as BCY12621); A-(SEQ ID NO:169)-A (referred to herein as BCY11673); A-(SEQ ID NO:170)-A (referred to herein as BCY11809); A-(SEQ ID NO:171)-A (referred to herein as BCY11810); A-(SEQ ID NO:172)-A (referred to herein as BCY11811); A-(SEQ ID NO:173)-A (referred to herein as BCY11812); and A-(SEQ ID NO:174)-A-[Sar6]-[KBiot] (referred to herein as BCY12622), (wherein Sar represents sarcosine and Biot represents biotin).

[0048] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 5 amino acids and a second loop sequence consisting of 4 amino acids, the molecular scaffold is TATA, and the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition: A-(SEQ ID NO:60)-A-[K(PYA)] (referred to herein as BCY15685) The amino acid sequence is

[0049] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 6 amino acids and the second loop sequence consisting of 3 amino acids.

[0050] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 6 amino acids and a second loop sequence consisting of 3 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0051] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of six amino acids and a second loop sequence consisting of three amino acids, the molecular scaffold is TATB, and the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition: A-(SEQ ID NO:266)-A (referred to herein as BCY15045); and A-(SEQ ID NO:266)-AGAAAE (referred to herein as BCY19417) The amino acid sequence is selected from the group consisting of:

[0052] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 7 amino acids and the second loop sequence consisting of 3 amino acids.

[0053] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 7 amino acids and a second loop sequence consisting of 3 amino acids, and the bicyclic peptide ligand comprises: [ka] TIFF2024502351000019.tif248170TIFF2024502351000020.tif47170 (in the formula, C i , C ii , and C iiirepresent the first, second, and third cysteine ​​residues, respectively; Gla represents γ-carboxyglutamic acid; Aze represents azetidine-2-carboxylic acid; 2MeTrp represents 2-methyltryptophan; 6MeTrp represents 6-methyltryptophan; 6FTrp represents 6-fluorotryptophan; 5FTrp represents 5-fluorotryptophan; 6ClTrp represents 6-chloro-L-tryptophan; 5MeoTrp represents 6-methyltryptophan; p represents 5-methoxy-L-tryptophan, Trp(S) represents 3-(3-benzothienyl)-L-alanine, AzaTrp represents azatryptophan, 4MeoTrp represents 4-methoxy-L-tryptophan, DOPA represents 3,4-dihydroxyphenylalanine, His1Me represents 1-methylhistidine, His3Me represents 3-methylhistidine, and 4MeoPhe represents 4-methoxyphenylalanine. , 26DiMeTyr represents 2,6-dimethyl-L-tyrosine, 3tBuTyr represents 3-tert-butyltyrosine, 4FPhe represents 4-fluoro-L-phenylalanine, 4MePhe represents 4-methyl-phenylalanine, HyP represents hydroxyproline, 4tBuPhe represents 4-t-butyl-phenylalanine, HPhe represents homophenylalanine, and 4PhePro represents (2S,4S)-4-phenyl wherein HGlu represents L-proline, HSer represents homoserine, HGlu represents L-2-aminoadipic acid, dA represents D-alanine, HArg represents homoarginine, tBuGly represents tert-butylglycine, Cis-HyP represents 4-cis-hydroxyproline, and 4BnPro represents (2S,4R)-4-benzyl-pyrrolidine-2-carboxylic acid, or a pharma- ceutical acceptable salt thereof.

[0054] In an even further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 7 amino acids and a second loop sequence consisting of 3 amino acids, and the bicyclic peptide ligand comprises: [ka] (In the formula, Ci , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively, or a pharma- ceutically acceptable salt thereof.

[0055] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of seven amino acids and a second loop sequence consisting of three amino acids, the molecular scaffold is TBMT, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and: A-(SEQ ID NO:61)-A (referred to herein as BCY14950); Ac-A-(SEQ ID NO:61)-A (referred to herein as BCY16599); A-(SEQ ID NO:61)-AGAAAE (referred to herein as BCY19040); A-(SEQ ID NO:61)-A-[K(PYA)] (referred to herein as BCY13883); Ac-A-(SEQ ID NO:61)-A-[K(PYA)] (referred to herein as BCY19596); A-(SEQ ID NO:175)-A (referred to herein as BCY14953); A-(SEQ ID NO:176)-A (referred to herein as BCY14954); [PYA]-A-(SEQ ID NO:177) (referred to herein as BCY14955); A-(SEQ ID NO:178)-A (referred to herein as BCY14956); A-(SEQ ID NO:179)-A (referred to herein as BCY16608); A-(SEQ ID NO:180)-A (referred to herein as BCY16610); A-(SEQ ID NO:181)-A (referred to herein as BCY19032); A-(SEQ ID NO:182)-A (referred to herein as BCY19033); A-(SEQ ID NO:183)-A (referred to herein as BCY19034); A-(SEQ ID NO:184)-A (referred to herein as BCY19035); A-(SEQ ID NO:185)-A (referred to herein as BCY19036); A-(SEQ ID NO:186)-A (referred to herein as BCY19037); A-(SEQ ID NO:187)-AGAAAE (referred to herein as BCY19038); A-(SEQ ID NO:188)-AGAAAE (referred to herein as BCY19039); A-(SEQ ID NO:189)-AGAAAE (referred to herein as BCY19041); A-(SEQ ID NO:190)-AGAAAE (referred to herein as BCY19042); A-(SEQ ID NO:191)-AGAAAE (referred to herein as BCY19043); A-(SEQ ID NO:192)-AGAAAE (referred to herein as BCY19044); A-(SEQ ID NO:193)-A (referred to herein as BCY19045); A-(SEQ ID NO:194)-A (referred to herein as BCY19046); A-(SEQ ID NO:195)-A (referred to herein as BCY19048); A-(SEQ ID NO:196)-A (referred to herein as BCY19049); A-(SEQ ID NO:197)-A (referred to herein as BCY19050); A-(SEQ ID NO:198)-A (referred to herein as BCY19053); A-(SEQ ID NO:199)-A (referred to herein as BCY19055); A-(SEQ ID NO:200)-A (referred to herein as BCY19057); A-(SEQ ID NO:201)-A (referred to herein as BCY19060); A-(SEQ ID NO:202)-A (referred to herein as BCY19061); A-(SEQ ID NO:203)-A (referred to herein as BCY19063); A-(SEQ ID NO:204)-A (referred to herein as BCY19064); A-(SEQ ID NO:205)-A (referred to herein as BCY19065); A-(SEQ ID NO:206)-A (referred to herein as BCY19068); A-(SEQ ID NO:207)-A-[K(PYA)] (referred to herein as BCY20360); A-(SEQ ID NO:208)-A-[K(PYA)] (referred to herein as BCY20361); A-(SEQ ID NO:209)-A (referred to herein as BCY20363); A-(SEQ ID NO:210)-A (referred to herein as BCY20364); A-(SEQ ID NO:211)-A (referred to herein as BCY20365); A-(SEQ ID NO:212)-A (referred to herein as BCY20366); A-(SEQ ID NO:213)-A (referred to herein as BCY20367); A-(SEQ ID NO:214)-A (referred to herein as BCY20368); A-(SEQ ID NO:215)-A (referred to herein as BCY20369); A-(SEQ ID NO:216)-A (referred to herein as BCY20370); A-(SEQ ID NO:217)-A (referred to herein as BCY20371); A-(SEQ ID NO:218)-A (referred to herein as BCY20372); A-(SEQ ID NO:219)-A (referred to herein as BCY20373); A-(SEQ ID NO:220)-A (referred to herein as BCY20374); A-(SEQ ID NO:221)-A (referred to herein as BCY20375); A-(SEQ ID NO:222)-A (referred to herein as BCY20376); A-(SEQ ID NO:223)-A (referred to herein as BCY20377); A-(SEQ ID NO:224)-A (referred to herein as BCY20378); A-(SEQ ID NO:225)-A (referred to herein as BCY20379); A-(SEQ ID NO:226)-A (referred to herein as BCY20380); A-(SEQ ID NO:227)-A (referred to herein as BCY20382); A-(SEQ ID NO:228)-A (referred to herein as BCY20383); A-(SEQ ID NO:229)-A (referred to herein as BCY20384); A-(SEQ ID NO:230)-A (referred to herein as BCY20385); (SEQ ID NO:230)-A (referred to herein as BCY21690); Ac-(SEQ ID NO:230)-A (referred to herein as BCY21691); A-(SEQ ID NO:230) (referred to herein as BCY21692); Ac- (SEQ ID NO: 230) (referred to herein as BCY21694); [Nle]-(SEQ ID NO:230)-A (referred to herein as BCY21695); Ac-[Nle]-(SEQ ID NO:230)-A (referred to herein as BCY21696); Ac-A-(SEQ ID NO:230)-A (referred to herein as BCY21697); A-(SEQ ID NO:231)-A (referred to herein as BCY20386); A-(SEQ ID NO:232)-A (referred to herein as BCY20388); A-(SEQ ID NO:233)-A (referred to herein as BCY20389); A-(SEQ ID NO:234)-A (referred to herein as BCY20390); A-(SEQ ID NO:235)-A (referred to herein as BCY20392); A-(SEQ ID NO:236)-A (referred to herein as BCY20394); A-(SEQ ID NO:237)-A (referred to herein as BCY20395); A-(SEQ ID NO:238)-A (referred to herein as BCY20396); A-(SEQ ID NO:239)-A (referred to herein as BCY20397); A-(SEQ ID NO:240)-A (referred to herein as BCY20526); Ac-A-(SEQ ID NO:241)-A (referred to herein as BCY20527); [Aib]-(SEQ ID NO:242)-A (referred to herein as BCY20528); A-(SEQ ID NO:243)-[Aib] (referred to herein as BCY20529); A-(SEQ ID NO:267)-A (referred to herein as BCY20387); and A-(SEQ ID NO:268)-A (referred to herein as BCY21689), wherein PYA represents pentynic acid and Aib represents aminoisobutryic acid.

[0056] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of seven amino acids and a second loop sequence consisting of three amino acids, the molecular scaffold is TBMT, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and: A-(SEQ ID NO:61)-A-[K(PYA)] (referred to herein as BCY13883), wherein PYA represents pentynic acid.

[0057] In another embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 8 amino acids and the second loop sequence consisting of 3 amino acids.

[0058] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 8 amino acids and a second loop sequence consisting of 3 amino acids, and the bicyclic peptide ligand comprises: [ka] (In the formula, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively; dA represents D-alanine; tBuAla represents β-t-butyl-L-alanine; Cba represents cyclobutylalanine; 1Nal represents 1-napthylalanine; 2Nal represents 2-naphthylalanine; Cha represents 3-cyclohexyl-D-alanine; Nle represents norleucine; Gla represents γ-carboxyglutamic acid; Aze represents azetidine-2-carboxylic acid; HLeu represents homoleucine; and HGlu represents L-2-aminoadipic acid), or a pharma- ceutically acceptable salt thereof.

[0059] In an even further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of 8 amino acids, a second loop sequence consisting of 3 amino acids, and the bicyclic peptide ligand comprises: [ka] (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.

[0060] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 8 amino acids and a second loop sequence consisting of 3 amino acids, the molecular scaffold is TBMT, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and: A-(SEQ ID NO:62)-A (referred to herein as BCY16493); Ac-(SEQ ID NO:62) (referred to herein as BCY16495); A-(SEQ ID NO:62)-A-[K(PYA)] (referred to herein as BCY13886); A-(SEQ ID NO:244)-A-[K(PYA)] (referred to herein as BCY16165); A-(SEQ ID NO:245)-A (referred to herein as BCY16498); A-(SEQ ID NO:246)-A (referred to herein as BCY16499); A-(SEQ ID NO:247)-A (referred to herein as BCY16501); A-(SEQ ID NO:248)-A (referred to herein as BCY16502); A-(SEQ ID NO:249)-A (referred to herein as BCY16504); A-(SEQ ID NO:250)-A (referred to herein as BCY16505); A-(SEQ ID NO:251)-A (referred to herein as BCY16506); A-(SEQ ID NO:252)-A (referred to herein as BCY16507); A-(SEQ ID NO:253)-A (referred to herein as BCY16508); A-(SEQ ID NO:254)-A (referred to herein as BCY16509); A-(SEQ ID NO:255)-A (referred to herein as BCY16510); A-(SEQ ID NO:256)-A (referred to herein as BCY16511); A-(SEQ ID NO:257)-A (referred to herein as BCY16512); A-(SEQ ID NO:258)-A (referred to herein as BCY16520); A-(SEQ ID NO:259)-A (referred to herein as BCY16522); A-(SEQ ID NO:260)-A (referred to herein as BCY16523); A-(SEQ ID NO:261)-A (referred to herein as BCY16524); A-(SEQ ID NO:262)-A (referred to herein as BCY16527); A-(SEQ ID NO:263)-A (referred to herein as BCY16528); and A-(SEQ ID NO:264)-A (referred to herein as BCY16529), wherein PYA represents pentynic acid.

[0061] In yet a further embodiment, the loop sequence comprises three reactive groups separated by two loop sequences, a first loop sequence consisting of 8 amino acids and a second loop sequence consisting of 3 amino acids, the molecular scaffold is TBMT, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and: A-(SEQ ID NO:62)-A-[K(PYA)] (referred to herein as BCY13886), wherein PYA represents pentynic acid.

[0062] In a further embodiment, the pharma- ceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, or ammonium salt.

[0063] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, for example, peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetics, and biochemistry methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th ed., John Wiley & Sons, Inc., incorporated herein by reference).

[0064] (Numbering) When referring to amino acid residue positions within the peptides of the present invention, the reactive group, i.e., a cysteine ​​residue or a penicillamine residue (C i , Pen i , C ii , Pen ii , and C iii and Pen iii ) are omitted from the numbering since they are invariant. The numbering of amino acid residues within the peptides of the invention is therefore represented as follows: -C i -N1-L2-Q3-A4-P5-C ii -M6-Q7-T8-G9-K 10 -V 11 -C iii -(Sequence number: 1).

[0065] For the purposes of this description, all bicyclic peptides are considered to be cyclized with TATA, TATB, or TBMT, resulting in a trisubstituted structure. Cyclization with TATA, TATB, or TBMT removes the first, second, and third reactive groups (i.e., C i , C ii , C iii ) occurs on

[0066] (Molecular format) N- or C-terminal extensions to the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal biotin-G-Sar5 tail is: [Biot]-G-[Sar5]-A-(SEQ ID NO:X) It is symbolized as follows.

[0067] (Reverse peptide sequence) In view of the disclosure in Nair et al., (2003) J. Immunol. 170(3), 1362-1373, it is contemplated that the peptide sequences disclosed herein will also find utility in their retro-inverso forms, for example where the sequence is reversed (i.e., N-terminus becomes C-terminus and vice versa) and the stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids and vice versa).

[0068] (Definition of Peptide Ligand) As used herein, a peptide ligand refers to a peptide, peptidic agent, or peptidomimetic that is covalently attached to a molecular scaffold. Typically, these peptides, peptidic agents, or peptidomimetics include peptides that have a natural or unnatural amino acid, two or more reactive groups (i.e., cysteine ​​or penicillamine residues) that can form covalent bonds with the scaffold, and a sequence between the reactive groups (referred to as a loop sequence because the peptide, peptidic agent, or peptidomimetic forms a loop when bound to the scaffold). In this case, the peptide, peptidic agent, or peptidomimetic has at least three cysteine ​​or penicillamine residues (referred to herein as a loop sequence). i , Pen i , C ii , Pen ii , and C iii and Pen iii ) forming at least two loops on the scaffold.

[0069] (Advantages of peptide ligands) The particular bicyclic peptides of the present invention have many advantageous properties that allow them to be considered suitable drug-like molecules for injection, inhalation, nasal, ophthalmic, oral or topical administration. Such advantageous properties include: - species cross-reactivity (which is usually required for preclinical pharmacodynamic and pharmacokinetic evaluation); - Protease stability (bicyclic peptide ligands should in most circumstances demonstrate stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic peptide lead candidates can not only be developed in animal models but also be safely administered to humans); - a desirable solubility profile (which is a function of the ratio of "charged and hydrophilic residues" to "hydrophobic residues" and intra- / inter-molecular hydrogen bonds, which is important for formulation and absorption purposes); and - Optimal plasma half-life within the circulation (depending on the clinical indication and treatment regimen, there will be a desire to develop bicyclic peptides for short or long in vivo exposure times in the management setting of either chronic or acute disease stages. The optimal exposure time will be governed by the need for sustained exposure (for maximum therapeutic efficacy) versus short exposure times to minimize toxic effects resulting from sustained exposure of the drug).

[0070] (Pharmaceutical acceptable salts) It will be understood that salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of that ligand.

[0071] The salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods, such as those described in "Pharmaceutical Salts: Properties, Selection, and Use", P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. Typically, such salts can be prepared by reacting the free acid or free base form of these compounds with the appropriate base or acid, in water or in an organic solvent, or in a mixture of the two.

[0072] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, 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, etc.), glutamic acid (e.g., L-glutamic acid, etc.), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, salts acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, Examples of suitable salts include mono- or di-salts formed with acids selected from the group consisting of acetic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0073] A particular group of salts consists of the 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. Another particular salt is the acetate.

[0074] If the compound is anionic or has a functional group which may be anionic (e.g., -COOH becomes -COO - Salts can be formed with organic or inorganic bases to provide suitable cations. Examples of suitable inorganic cations include, but are not limited to, Li + , Na + , and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NH3R + , NH2R2 + , NHR3 + , NR4 + Some suitable examples of substituted ammonium ions include those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0075] When the peptides of the invention contain amine groups, they may form quaternary ammonium salts, for example, by reaction with alkylating agents by methods well known to those of skill in the art. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

[0076] (multimeric complex) In a further aspect of the invention there is provided a multimeric binding complex comprising at least two bicyclic peptide ligands as defined herein, which may be the same or different.

[0077] In one embodiment, the multimeric binding complex comprises more than one identical bicyclic peptide (i.e., is a homomultimer). In another embodiment, the multimeric binding complex comprises different bicyclic peptides (i.e., is a heteromultimer).

[0078] In one embodiment, the multimeric binding complex further comprises a fluorophore.

[0079] In one embodiment, the multimeric binding complex comprises two identical bicyclic peptides (i.e., is a homomultimer). Examples of suitable dimeric NKp46 homodimers are shown in Table A below.

[0080] Table A: Dimeric NKp46 multimer binding complexes of the present invention [Table 1]

[0081] Examples of suitable dimeric CD16a homodimers are shown in Table B below.

[0082] Table B: Dimeric CD16a multimer binding complexes of the invention [Table 2]

[0083] (Modified derivative) It will be appreciated that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of suitable modified derivatives include N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more sterically equivalent or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-naturally occurring sterically equivalent or isoelectronic amino acids); the addition of a spacer group; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with one or more substituted amino acids, such as alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; bicyclic peptide ligands. replacement of one or more peptide bonds with an alternative bond; alteration 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 such as cysteine, lysine, glutamate / aspartate and tyrosine with amine-, thiol-, carboxylic acid- and phenol-reactive reagents suitable for functionalizing these amino acids, and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, e.g., introduction or substitution of amino acids bearing an azide or alkyne group, which allows functionalization of an alkyne- or azide-containing moiety, respectively.

[0084] In one embodiment, the modified derivatives include N-terminal and / or C-terminal modifications. In a further embodiment, the modified derivatives include N-terminal modifications using suitable amino-reactive chemistry and / or C-terminal modifications using suitable carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modifications include the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator, or a chromophore.

[0085] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal residue is capped with acetic anhydride or other suitable reagent during peptide synthesis, resulting in an N-terminally acetylated molecule. This embodiment offers the advantage of removing an aminopeptidase recognition point, avoiding potential degradation of the bicyclic peptide.

[0086] In another embodiment, the N-terminal modification involves the addition of a molecular spacer group that facilitates conjugation of an effector group and promotes retention of efficacy of the bicyclic peptide against its target.

[0087] 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 residue is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment provides the advantage of removing a carboxypeptidase recognition point, reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0088] In one embodiment, the modified derivatives include the substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids can be selected that have sterically equivalent / isoelectronic side chains that are not recognized by degradative proteases and do not adversely affect targeting efficacy in any way.

[0089] Alternatively, unnatural amino acids with constrained amino acid side chains may be used, such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. These in particular relate to proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids (a simple derivative is amino-cyclopropyl carboxylic acid).

[0090] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises an N-terminal cysteine ​​or penicillamine (C i Or Pen i ) and / or C-terminal cysteine ​​or penicillamine (C iii Or Pen iii ) including the addition of a spacer group to

[0091] In one embodiment, the modified derivative comprises the substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises the substitution of a tryptophan residue with a naphthylalanine residue or an alanine residue. This embodiment provides the advantage that the pharmaceutical stability profile of the resulting bicyclic peptide ligand is improved.

[0092] In one embodiment, the modified derivative comprises the substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In another 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 "charged amino acid residues" versus "hydrophobic amino acid residues" is an important property of bicyclic peptide ligands. For example, hydrophobic amino acid residues will 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) will affect the interaction of the peptide with the phospholipid membrane of the cell surface. The combination of the two will affect the half-life, volume of distribution, and exposure of the peptide drug and can be tailored to the clinical evaluation endpoint. Furthermore, the correct combination and number of "charged amino acid residues" versus "hydrophobic amino acid residues" will reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

[0093] 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 enhance proteolytic stability by steric hindrance and by the properties of D-amino acids to stabilize the β-turn conformation (Tugyi et al., (2005) PNAS, 102(2), 413-418).

[0094] In one embodiment, the modified derivative comprises the removal of any amino acid residue and substitution with alanine, e.g., D-alanine. This embodiment offers the advantage of identifying critical binding residues and removing potential site(s) of proteolytic attack.

[0095] It should be noted that each of the above modifications serves to deliberately improve the potency or stability of the peptide. Further potency improvements based on the modifications could be achieved by the following mechanisms: - Higher affinity is achieved by incorporating hydrophobic moieties that exploit the hydrophobic effect to reduce the dissociation rate; - the incorporation of charged groups that take advantage of long-range ionic interactions, increasing the rate of association and resulting in higher affinity (see, for example, Schreiber et al., "Rapid, electrostatically assisted association of proteins," (1996), Nature Struct. Biol. 3, 427-31); and - Incorporating additional constraints into the peptide, for example by precisely constraining the amino acid side chains so as to minimize entropic loss upon target binding, by restricting the torsion angles of the backbone so as to minimize entropic loss upon target binding, and by introducing additional cyclizations into the molecule for the same reasons.

[0096] (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418.)

[0097] (isotopic variants) The present invention includes all pharma- ceutically acceptable (radio)isotopically labeled peptide ligands of the invention in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, as well as peptide ligands of the invention in which a metal chelating group (called an "effector") capable of bearing a relevant (radio)isotope is conjugated, and peptide ligands of the invention in which a particular functional group is covalently substituted with a relevant (radio)isotope or an isotopically labeled functional group.

[0098] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include hydrogen isotopes, e.g. 2 H(D) and 3 H(T), carbon isotopes, e.g. 11 C. 13 C and 14 C, chlorine isotopes, e.g. 36 Cl, fluorine isotopes, e.g. 18 F, iodine isotopes, e.g. 123 I, 125 I and 131 I, nitrogen isotopes, e.g. 13 N and 15 N, oxygen isotopes, e.g. 15 O. 17 O and 18 O, phosphorus isotopes, e.g. 32 P, sulfur isotopes, e.g. 35 S, copper isotopes, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y, as well as lutetium isotopes, e.g. 177Lu, as well as bismuth isotopes, e.g. 213 Contains Bi.

[0099] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies and for clinically assessing the presence and / or absence of targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compound and other molecules, peptides, proteins, enzymes, or receptors. This detection or identification method may employ compounds that are labeled with labeling agents, such as, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). The radioisotope tritium, i.e. 3 H(T), and carbon-14, i.e. 14 C is particularly useful for this purpose given its ease of incorporation and the means of detection available.

[0100] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), may be preferable in some circumstances because it may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0101] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, can be utilized in Positron Emission Topography (PET) studies for examining target occupancy.

[0102] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the Examples herein, substituting an appropriate isotopically labeled reagent for the non-labeled reagent already utilized.

[0103] (Molecular scaffolds) In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. Reference herein to a "non-aromatic molecular scaffold" refers to any molecular scaffold, as defined herein, that does not contain an aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring system.

[0104] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6)1602-1606.

[0105] As mentioned in said document, the molecular scaffold may be a small molecule, for example a small organic molecule.

[0106] In one embodiment, the molecular scaffold may be a polymer. In one embodiment, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates.

[0107] In one embodiment, the molecular scaffold comprises a reactive group that can react with a functional group(s) of a polypeptide to form a covalent bond.

[0108] The molecular scaffold can contain chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0109] In one embodiment, the molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA)): [ka] .

[0110] In that case, the C of the bicyclic peptide of the present invention i , Pen i , C ii , Pen ii , C iii and Pen iii After cyclization on the cysteine ​​or penicillamine residues, the molecular scaffold forms a trisubstituted 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)tripropan-1-one derivative of TATA having the following structure: [ka] (where * indicates the attachment points of the three cysteine ​​residues).

[0111] In another embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)-s-triazine (TBMT): [ka] .

[0112] In that case, the C of the bicyclic peptide of the present invention i , Pen i , C ii , Pen ii , C iii and Pen iii After cyclization on the cysteine ​​or penicillamine residues, the molecular scaffold forms a trisubstituted TBMT derivative having the following structure: [ka] (where * indicates the attachment points of the three cysteine ​​residues).

[0113] In another embodiment, the molecular scaffold is 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine (TATB): [ka] .

[0114] In that case, the C of the bicyclic peptide of the present invention i , Pen i , C ii , Pen ii , C iii and Pen iii After cyclization on the cysteine ​​or penicillamine residues, the molecular scaffold forms a trisubstituted 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine derivative of TATB having the following structure: [ka] (where * indicates the attachment points of the three cysteine ​​residues).

[0115] (synthesis) The peptides of the present invention may be synthetically produced by standard techniques and then reacted with the molecular scaffold in vitro. Standard chemical methods can be used to carry this out. This allows for rapid large-scale production of soluble material for further downstream experimentation or validation. Such methods can be accomplished using conventional chemical methods such as those disclosed in Timmerman et al. (supra).

[0116] The present invention therefore also relates to the production of a polypeptide or conjugate selected as described herein, which production comprises any further steps as explained below. In one embodiment, these steps are carried out on the final product polypeptide / conjugate produced by chemical synthesis.

[0117] Optionally, amino acid residues in the polypeptide of interest may be substituted during preparation of the conjugate or complex.

[0118] The peptides can also be extended to incorporate, for example, additional loops, thereby introducing multiple specificities.

[0119] To elongate a peptide, it may simply be chemically extended at its N- or C-terminus or within a loop with orthogonally protected lysines (and analogues) using standard solid- or solution-phase chemistries. Activated or activatable N- or C-termini may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be by fragment condensation or by native chemical ligation as described, for example, in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation, Science 266:776-779) or enzymatically using subtiligase as described, for example, in (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20;91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, 15 Nov. 2008, pp. 6000-6003).

[0120] Alternatively, peptides may be extended or modified by further conjugation via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA, TATB, or TBMT) is added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; then, an additional cysteine ​​or thiol is added to the N-terminus or C-terminus of the first peptide, so that this cysteine ​​or thiol can only react with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0121] Furthermore, the addition of other functional or effector groups may be achieved similarly using appropriate chemical methods, coupling at the N-terminus or C-terminus or via a side chain, In one embodiment, the coupling is carried out in such a manner as not to block the activity of either entity.

[0122] Pharmaceutical Composition In a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0123] Typically, the peptide ligands may be used in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous liquids, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride solution, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants may be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, as necessary to keep the polypeptide complex in suspension.

[0124] Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives can also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack, (1982), Remington's Pharmaceutical Sciences, 16th Edition).

[0125] The peptide ligands of the invention can be used as a composition administered separately or in combination with other drugs. These can include antibodies, antibody fragments, and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin, or cisplatin, and immunotoxins. Further examples of other drugs that can be administered separately or in combination with the peptide ligands of the invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic factors, and antithrombotic factors. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other drugs in combination with the protein ligands of the invention, or even combinations of selected polypeptides of the invention with different specificities, such as, for example, polypeptides selected with different targeting ligands, whether pooled or not prior to administration.

[0126] The route of administration of the pharmaceutical composition of the present invention may be any of those generally known to those skilled in the art. For therapy, the peptide ligand of the present invention may be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or, also appropriately, by direct injection using a catheter. Preferably, the pharmaceutical composition of the present invention will be administered intravenously. The dosage and frequency of administration will depend on the age, sex and condition of the patient, the concomitant administration of other drugs, contraindications, and other parameters taken into account by the clinician.

[0127] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be utilized. Those skilled in the art will recognize that lyophilization and reconstitution can result in varying degrees of activity loss, and that concentration levels may need to be adjusted upward to compensate for this loss.

[0128] Compositions containing the peptide ligands or cocktails thereof can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, an amount adequate to achieve at least partial inhibition, pacification, modulation, killing, or any other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dosage will depend on the severity of the disease, but will generally range from 0.005-5.0 mg of the selected peptide ligand per kilogram of body weight, with doses in the range of 0.05-2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof may also be administered at similar or slightly lower doses.

[0129] Compositions containing the peptide ligands of the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in a mammal. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove target cell populations from heterogeneous populations of cells. Blood from a mammal can be combined ex vivo with the selected peptide ligand, thereby killing or otherwise removing undesirable cells from the blood according to standard techniques, and then returned to the mammal.

[0130] (therapeutic use) Polypeptide ligands selected by 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 for applications involving testing in non-human animals where cross-reactivity is desirable, or for diagnostic applications where cross-reactivity with homologs or paralogs must be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response against a range of antigens can be exploited to tailor the vaccine to specific diseases and pathogens.

[0131] Substantially pure peptide ligands having at least 90-95% homogeneity are preferred for administration to mammals, with 98-99% or more homogeneity being most preferred for pharmaceutical uses, particularly where the mammal is a human. Selected polypeptides can be purified, partially or to the desired degree of homogeneity, and used diagnostically or therapeutically (including in vitro), or in the development and implementation of assay procedures, immunofluorescence staining, and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Vols. I and II, Academic Press, New York).

[0132] In a further aspect of the invention there is provided a peptide ligand or pharmaceutical conjugate as defined herein for use in the prevention, inhibition or treatment of an NK cell mediated disease or disorder.

[0133] In a further aspect of the invention, there is provided a method for preventing, inhibiting or treating an NK cell-mediated disease or disorder comprising administering to a patient in need thereof a peptide ligand as defined herein.

[0134] In one embodiment, the NK cell mediated disease or disorder is selected from inflammatory disorders, autoimmune diseases, and cancer. In a further embodiment, the NK cell mediated disease or disorder is selected from rheumatoid arthritis (RA), bone erosion, intraperitoneal abscess, inflammatory bowel disease, allograft rejection, psoriasis, angiogenesis, atherosclerosis, asthma, multiple sclerosis, systemic lupus erythematosus (SLE), ocular surface disorders (such as dry eye), ankylosing spondylitis, psoriatic arthritis, cancer (such as multiple myeloma and breast cancer).

[0135] In a further embodiment, the NK cell mediated disease or disorder is selected from cancer.

[0136] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and various types of cancers, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other cancers), such as bladder and urinary tract cancer, breast cancer, carcinomas of the digestive tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, carcinomas of the lung (e.g., lung adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck cancers (e.g., tongue, oral cavity, larynx, pharynx, and thyroid cancer), and tumors of the thyroid gland, thyroid gland, and thyroid gland. , nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses); ovarian, fallopian tube, peritoneal, vaginal, vulvar, penile, cervical, myometrial, endometrial, thyroid (e.g., follicular thyroid carcinoma), adrenal, prostate, skin, and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and premalignant hematological disorders and borderline malignant disorders, e.g., hematological malignancies and related disorders of the lymphatic system (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], and B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and T-cell leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and related disorders of the myeloid system (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, polycythemia vera) myeloproliferative disorders such as myelofibrosis, essential thrombocythemia and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia; tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytoma, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma);These include endocrine tumors (e.g., pituitary, adrenal, islet cell, parathyroid, carcinoid, and medullary carcinoma of the thyroid); ophthalmic and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas), as well as pediatric and embryonal tumors (e.g., medulloblastomas, neuroblastomas, Wilms' tumors, and primitive neuroectodermal tumors) or syndromes, whether congenital or not, that predispose a patient to malignancies (e.g., xeroderma pigmentosum);

[0137] References herein to the term "prevention" include administration of the protective composition prior to the induction of disease. "Suppression" refers to administration of the composition after an inductive event but prior to the clinical appearance of disease. "Treatment" includes administration of the protective composition after disease symptoms are manifest.

[0138] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease. The present invention facilitates the use of animal model systems and enables the development of polypeptide ligands that can cross-react with human and animal targets, thereby making the use of animal models possible.

[0139] The invention will now be further described with reference to the following examples. EXAMPLES

[0140] (Example) Materials and Methods (Preparation of Bicyclic Peptide Ligands (General Method)) Bicyclic peptides were synthesized on Rink amide resin using standard Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis methods, either by manual coupling (for large scale) or using a Biotage Syro II automated peptide synthesizer (for small scale). After TFA-based cleavage from the resin, the peptide was precipitated with diethyl ether and dissolved in 50:50 acetonitrile / water. The crude peptide (approximately 1 mM concentration) was then cyclized with 1.3 equivalents of the scaffold using ammonium bicarbonate (100 mM) as the base. Completion of the cyclization was determined by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) or LC-MS. Once complete, the cyclization reaction was quenched using N-acetylcysteine ​​(10 equivalents relative to peptide) and the solution was lyophilized. The residue was dissolved in an appropriate solvent and purified by RP-HPLC. Peptide fractions of sufficient purity and correct molecular weight (verified by MALDI-TOF and either HPLC or LC-MS) were pooled and lyophilized. Concentrations based on Trp / Tyr content were determined by UV absorption using the extinction coefficient at 280 nm.

[0141] Unless otherwise stated, all amino acids were used in the L-configuration.

[0142] (Example) In general, some of the fluorescent bicyclic peptide dimer conjugates of the present invention can be prepared according to the following general method: [ka] .

[0143] A mixture of BP24369 (1.0 equiv.) and AF488-NHS (1) (1.5 equiv.) is dissolved in DMF. The solution is adjusted to pH 8 by dropwise addition of DIEA (3.0 equiv.). The reaction mixture is stirred at 25 °C for 1 h and then a sample is taken for analysis. If the reaction is incomplete, additional AF488-NHS is added and the mixture is continued to stir for an additional hour. Once the reaction is complete, the reaction mixture is filtered to remove undissolved residues and the crude product is purified by preparative HPLC to give intermediate 2.

[0144] A mixture of intermediate 2 (1.0 equiv.), bicyclic (2.1 equiv.), and THPTA (2.2 equiv.) is dissolved in degassed t-BuOH / H2O (1:1), then an aqueous solution of CuSO4 (0.4 M, 2.2 equiv.) and VcNa (4.0 equiv.) is added. Finally, 0.2 M NH4HCO3 is added to adjust the pH to 8. The reaction mixture is stirred under N2 atmosphere for up to 16 h. The reaction mixture is directly purified by preparative HPLC.

[0145] Fluorescently labeled bicyclic peptide dimer conjugates prepared using this method are listed below: [Table 3] .

[0146] More detailed experiments on selected bicyclic peptide multimeric complexes of the invention are provided herein below.

[0147] (Synthesis of intermediate AF488-BP24369) [ka] A mixture of compound 1 (10.0 mg, 18.2 μmol, 1.0 equiv.), compound 2 (17.0 mg, 27.3 μmol, 1.5 equiv.) was dissolved in DMF (0.4 mL). The solution was adjusted to pH 8 by dropwise addition of DIEA (15.8 μL, 181.60 μmol, 3.0 equiv.). The reaction mixture was stirred at 25° C. for 1 h. LC-MS showed that BP24369 was not completely consumed, so additional compound 2 (17.0 mg, 27.24 μmol, 1.5 equiv.) was added to the reaction mixture and stirred at 25° C. for 1 h. LC-MS showed one main peak with the desired m / z. The reaction mixture was filtered to remove undissolved residue. The crude product was purified by preparative HPLC to give AF488-BP24369 (10.0 mg, 8.90 μmol, 49.02% yield, 95.0% purity) as a red solid. Calculated MW: 1067.1, observed m / z: 1068.3 ([M+H] + ), 534.7([M+2H] 2+ ).

[0148] (Synthesis of BCY15663) [ka] A mixture of compound 1 (3.0 mg, 2.81 μmol, 1.0 equiv.), compound 2 (14.0 mg, 6.47 μmol, 2.3 equiv.), and THPTA (1.2 mg, 2.81 μmol, 1.0 equiv.) was dissolved in degassed and N2-purged t-BuOH / H2O (1:1, 0.5 mL), then an aqueous solution of CuSO4 (0.4 M, 7.0 μL, 1.0 equiv.) and Vc (2.0 mg, 11.25 μmol, 4.0 equiv.) was added under N2. The solution was adjusted to pH 8, and the solution turned bright yellow. The reaction mixture was stirred at 25 °C for 1 h under N2 atmosphere. LC-MS showed that AF488-BP24369 was completely consumed, some BCY15452 remained, and one main peak with the desired m / z was detected. The reaction mixture was filtered to remove undissolved residues. The crude product was purified by preparative HPLC to give BCY15663 (9.4 mg, 1.66 μmol, 59.06% yield, 96.4% purity) as a white solid. Calculated MW: 5377.9, observed m / z: 1345.5 ([M+4H] 4+ ).

[0149] BCY15921 [ka] . BCY15922 [ka] . BCY15455 [ka] . BCY18043 [ka] .

[0150] (Synthesis of BCY15916) [ka] A mixture of compound 1 (2.0 mg, 1.87 μmol, 1.0 equiv.), compound 2 (7.5 mg, 3.94 μmol, 2.1 equiv.), and THPTA (1.8 mg, 4.12 μmol, 2.2 equiv.) was dissolved in t-BuOH / H2O (1:1, 0.5 mL, degassed and purged with N2), then an aqueous solution of CuSO4 (0.4 M, 10.3 μL, 2.2 equiv.) and VcNa (1.5 mg, 7.5 μmol, 4.0 equiv.) was added under N2. The solution was adjusted to pH 8 by dropwise addition of 0.2 M NH4HCO3 (1:1 in t-BuOH / H2O), turning the solution bright yellow. The reaction mixture was stirred at 25-30 °C for 1 h under N2 atmosphere. LC-MS showed that compound 1 was completely consumed and one main peak with the desired m / z was detected. The reaction mixture was filtered to remove undissolved residues. The crude product was purified by preparative HPLC to give BCY15916 (1.6 mg, 0.30 μmol, 16.3% yield, 93.3% purity) as a white solid. Calculated MW: 4875.5, observed m / z: 1219.6 ([M+4H] 4+ ).

[0151] BCY15914 [ka] . BCY15915 [ka] .

[0152] (Biological Data) 1. NKp46 Fluorescence Polarization (FP) Direct Binding Assay The affinity of peptides of the invention for human NKp46 (Acro Biosystems, NC1-H5257 or NC1-H52H4) was determined using a fluorescence polarization assay. Peptides of the invention were labeled with a fluorescent tag (fluorescein) and diluted to 2.5 nM in 25 mM HEPES containing 100 mM NaCl and 0.005% P20, pH 7.4. NKp46 protein was titrated starting at 0.2-5 μM in the same assay buffer as the peptide, and 1 nM, 25 μL total peptide was assayed in a black-walled, low-binding, small-volume 384-well plate. The assay was typically set up by adding 5 μL assay buffer, 10 μL NKp46 protein, then 10 μL fluorescent peptide. NKp46 protein concentrations were either 2-fold or 3-fold dilutions, with 12 concentrations starting at 0.2-5 μM. Measurements were performed at 25 °C on a BMG PHERAstar FS equipped with an FP 485 520 520 optical module with 200 flashes per well and a transfer delay of 0.1 seconds. Each well was measured every 5 minutes for 60 minutes. The gain used in the analysis was determined for each tracer at the end of 60 minutes, when no protein was present in the well. The mP was fitted to a standard 1:1 binding model with a quadratic equation, K D Selected peptides of the present invention were tested in the above assay and the results are shown in Table 1.

[0153] (FP Direct Binding Assay Materials) Human NKp46 Fc tag (Acro Biosystems, NC1-H5257) Human NKp46 His tag (Acro Biosystems, NC1-H52H4)

[0154] Table 1: Fluorescence polarization-direct binding assay data for selected bicyclic peptides of the invention. [Table 4]

[0155] 2. NKp46 Fluorescence Polarization (FP) Competitive Binding Assay The affinity of the peptides of the invention was determined in a fluorescence polarization (FP) competition assay using the fluorescently labeled peptide BCY15035 as a tracer to compete with the untagged peptide.

[0156] Untagged competitor peptides were diluted in assay buffer 25 mM HEPES, 100 mM NaCl, 0.01% P20, pH 7.4 to a maximum concentration of 5000 nM, with 11 two-fold dilutions. Human NKp46-His tagged protein (Acro Biosystems) was diluted to a final concentration of 40 nM in the assay. Finally, the fluorescent tracer peptide BCY15035 was added at 1 nM. The assay was typically set up by adding 5 μL of competitor, 10 μL of NKp46 protein, then 10 μL of fluorescent peptide. A total volume of 25 μL was prepared in a black-walled flat-bottom low-binding small-volume 384-well plate. Measurements were performed using an Envision (PerkinElmer) with excitation filter FITC FP 480, emission filter FITC FP P-pol 535 and second emission filter FITC FP S-pol 535 at a 30 flash setting. Gain was set in wells containing only tracer without target protein. The mP values ​​of the final readings after 60 min were converted to percentage inhibition and used to calculate Ki values ​​(Kcomp) in the software Sigmaplot (Systat Software Inc.) using the following formula:

number

[0157] Selected peptides of the invention were tested in the above assays and the results are shown in Table 2.

[0158] Table 2: Competitive fluorescence polarization assay data for selected bicyclic peptides of the invention. [Table 5] TIFF2024502351000046.tif243170TIFF2024502351000047.tif17170

[0159] 3a. NKp46 SPR Assay (Method 1) SPR experiments were performed to measure the kA activity of various bicyclic peptides against immobilized NKp46. a (M -1 s -1 ) value, k d (s -1 ) value, K D Measurements were taken of (nM) values ​​(when available).

[0160] For analysis of peptide binding to NKp46, a Biacore 3000 instrument was used with a Cytiva streptavidin chip. Protein (recombinant human NKp46-Fc fusion biotinylated via Avi-tag) capture was performed at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as running buffer, and biotinylated NKp46 was captured to a level of 400-800 RU using protein dilution buffer up to 0.2 μM. The buffer was then changed to PBS / 0.05% Tween 20, and a dilution series of peptide was prepared in this buffer with a final DMSO concentration of 0.5%, a maximum peptide concentration of 100 nM to 5000 nM, and six further 2- or 3-fold dilutions. SPR analysis was performed at 25°C, with a flow rate of 50 μL / min, an association time of 60 s, and a dissociation time of 200-400 s. Data were corrected for DMSO excluded volume effects. 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, Inc.). Data were fitted using a simple 1:1 binding model allowing for mass transport effects where applicable.

[0161] Selected peptides of the invention were tested in the above assays and the results are shown in Table 3A.

[0162] Table 3A: SPR assay data for selected bicyclic peptides of the invention. [Table 6]

[0163] (3b. NKp46 SPR Assay (Method 2)) Peptides were tested on Fc-tagged human NKp46 (ACROBiosystems, Cat. No. NC1-H5257).

[0164] For peptide binding analysis, a Biacore 8k+ instrument was used with a CM5 chip (Cytiva). All experiments were performed at 25°C. Anti-human IgG (Fc) antibody (Cytiva) was immobilized on all flow cells by standard amine coupling chemistry. The running buffer was HBS-N (Cytiva, 10 mM HEPES, 0.15 M NaCl, pH 7.4) with a flow rate of 10 μL / min. The carboxymethyldextran surface was activated with a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) (Cytiva) for 420 s. Anti-human IgG (Fc) antibody was diluted to 12.5 μg / mL in 10 mM sodium acetate, pH 5.0, and injected over the chip surface for 360 s. Remaining activated groups were blocked by injection of 1 M ethanolamine (pH 8.5) for 420 s. Final surface density was 3000–5000 RU. Fc-tagged human NKp46 was diluted to 0.1 μM in HBS-N and captured to 500–1000 RU only on flow cell 2 of the sensor chip at a flow rate of 10 μL / min. The buffer was then changed to PBS-P+ (Cytiva, supplemented with 2% DMSO). Peptide dilutions were prepared in this buffer to a final concentration of 2% DMSO. A flow rate of 50 μL / min was used with an association time of 60 s and a dissociation time of 400 s. Data were corrected for DMSO excluded volume effects. All data were double-referenced to a blank injection and a reference surface reaction using standard processing procedures. Data processing and kinetic fitting were performed using Biacore Insight Evaluation software and Extended Screening and Characterization Extension (Cytiva). The data was fitted using a steady-state affinity 1:1 binding model to obtain K D The data were fitted with a kinetic 1:1 binding model to determine reaction rate constants, where applicable.

[0165] Selected peptides of the invention were tested by Method 1 and / or Method 2 and the results are shown in Table 3B.

[0166] Table 3B: SPR assay data for selected bicyclic peptides of the invention. [Table 7] TIFF2024502351000050.tif245170TIFF2024502351000051.tif244170TIFF2024502351000052.tif244170TIFF2024502351000053.tif232170

[0167] 4. Binding to CHO-K1 / NKp46 cell line The affinity of NKp46 bicyclic peptide dimers conjugated with Alexa Fluor® 488 for commercially available NKp46-overexpressing CHO-K1 cells was determined using a flow cytometry assay.

[0168] Commercially available CHO-K1 cells stably transfected with human natural activating cell receptor 46 (NKp46, also known as natural cytotoxicity inducing receptor 1 (NCR1) or CD355) (referred to herein as CHO-K1 / NKp46 cells) were procured from Abeomics. CHO-K1 / NKp46 cells were cultured in DMEM medium (Corning® 15-017 CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), 1% penicillin-streptomycin (Corning™ 30-002-CI), and 10 μg / mL blasticidin (Gibco™ A1113902). Culture maintenance and subculture conditions were in accordance with the manufacturer's recommendations.

[0169] On the day of the experiment, CHO-K1 / NKp46 were detached from the culture vessel using trypsin / EDTA and washed once with 15 mL of pre-warmed working medium (defined as follows: DMEM medium (Corning® 15-017 CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI)) at 500 rpm for 5 min. The cell pellet was then diluted with 3 × 10 5 The cells were resuspended in working medium at a concentration of 100 cells / mL. 100 μL of the cell suspension was then plated into a 96-well V-bottom polypropylene plate (Greiner Bio-One 651201).

[0170] NKp46 bicyclic peptide dimers conjugated to Alexa Fluor® 488 were diluted in working medium and added to the plate at a starting concentration of 1 μM and added dropwise in a 1:4 dilution series to perform an 11-point serial dilution. The plate was then incubated for 1 hour at room temperature in the dark. After incubation, the plate was centrifuged at 500 rpm for 5 minutes and the supernatant was removed. The samples were then washed twice with 200 μL of 1× phosphate buffered saline (PBS; Gibco™ 10-010-023) at 500 rpm for 5 minutes. The cells were resuspended in 100 μL of PBS and transferred to a new 96-well V-bottom polypropylene plate (Greiner Bio-One 651201). The plate was centrifuged at 500 rpm for 5 minutes and the supernatant was removed.

[0171] Sample preparation for flow cytometry: Zombie Violet™ Fixable Viability Dye (BioLegend® 423113) was prepared by diluting 1:1000 in PBS and 100 μL of Viability Dye was added to each well and incubated at 4° C. in the dark for 30 min. To ensure NKp46 expression detection, untreated CHO-K1 / NKp46 cells were stained with either anti-human NKp46 or isotype control. Antibody cocktails were prepared by diluting 1.5 μL of either PE anti-human NKp46 (BioLegend® 331908; clone 9E2) or PE isotype control IgG1, kappa (BioLegend® 400112) per 100 μL of staining buffer (1×PBS supplemented with 2% FBS). Cells were resuspended in master mix cocktail (100 μL) and incubated at 4° C. for 30 min in the dark. The cells were then washed three times with 100 μL staining buffer at 500 rpm for 5 min, the supernatant was removed, and the cells were resuspended in 200 μL staining buffer and kept in the dark at 4° C. until read on a BD FACSCelesta™ flow cytometer.

[0172] Flow cytometry results were analyzed using FlowJo™ software with a gating strategy to exclude debris, gate on single and live cells, and determine the geometric mean of the Alexa Fluor® 488+ / - population. Binding affinities of the bicyclic peptides were calculated using 4-parameter logistic regression with Prism GraphPad software.

[0173] The data presented in FIG. 1 and Table 4 demonstrate that Alexa Fluor® 488-tagged NKp46 bicyclic peptide dimers bind to NKp46-expressing cells in a dose-dependent manner.

[0174] Table 4: Binding affinity (K) of Alexa Fluor® 488 NKp46 bicyclic peptide dimer binding to CHO-K1 / NKp46 cells d,app )) [Table 8]

[0175] 5. Binding to PBMCs The affinity of NKp46 bicyclic peptide dimers conjugated to Alexa Fluor® 488 for healthy primary peripheral blood mononuclear cells (PBMCs) was determined using a flow cytometry assay.

[0176] On the day of the experiment, medium was prepared by supplementing RPMI-1640 (Gibco™ 11875-093; with L-glutamine) with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI), referred to herein as working medium. Human peripheral blood mononuclear cells (PBMCs) previously isolated from whole blood were rapidly thawed in a water bath and washed once with 10 mL of pre-warmed working medium at 500 rpm for 5 min. The PBMC pellet was then diluted with 5 × 10 6 The cells were resuspended in working medium to a concentration of 100 cells / mL and allowed to stand horizontally overnight (12 to 18 hours) in a coated flask for tissue culture (T-183; CELLTREAT Scientific, Inc. 229351).

[0177] After overnight incubation, the PBMCs were removed from the flasks and washed once with 10 mL of pre-warmed working medium at 500 rpm for 5 min. The PBMC pellet was then diluted with 1 × 10 5Cells were resuspended in working medium at a concentration of 100 μL cells / mL. 100 μL of the cell suspension was then plated into a 96-well V-bottom polypropylene plate (Greiner Bio-One 651201). Bicyclic peptides were diluted in working medium and added to the corresponding cell plate at a starting concentration of 300 nM, dropwise in a 1:4 dilution series, to perform a 12-point serial dilution. The plates were then incubated for 1 hour at 37° C. and 5% CO2. After incubation, the plates were centrifuged at 500 rpm for 5 minutes and the supernatant was removed. Samples were then washed once with 200 μL 1× phosphate buffered saline (PBS; Gibco™ 10-010-023) at 500 rpm for 5 minutes and the supernatant was removed.

[0178] Sample preparation for flow cytometry: Zombie Aqua™ Fixable Viability Dye (BioLegend® 423102) was prepared by diluting 1:1000 in PBS, and 100 μL of Viability Dye was added to each well and incubated for 30 min at 4° C. in the dark. The wells were then washed with 100 μL of PBS at 500 rpm for 5 min and the supernatant was removed. Next, human TruStain FcX™ block (BioLegend® 422302) was prepared by diluting 1.5 μL of FcX in 25 μL of staining buffer (1×PBS, supplemented with 2% FBS). Fc block solution (25 μL / well) was incubated for 10 min at room temperature (RT) in the dark. An antibody master mix cocktail was prepared by diluting 1.5 μL of the following antibodies per 100 μL of staining buffer: Brilliant Violet 605™ anti-human CD3 (BioLegend® 344836; clone SK7), and PE / Cyanine 7 anti-human CD56 (BioLegend® 362510; clone 5.1H11). Cells were resuspended in the master mix cocktail (100 μL) and incubated at 4° C. for 30 min in the dark. Cells were then washed three times with 100 μL of staining buffer at 500 rpm for 5 min and the supernatant was removed. Cells were resuspended in 200 μL of staining buffer and kept in the dark at 4° C. until reading on a BD FACSCelesta™ flow cytometer.

[0179] Flow cytometry results were analyzed using FlowJo™ software with a gating strategy to assess lymphocyte populations, then exclude debris, gate on single and live cells, and determine the geometric mean of Alexa Fluor® 488 for CD3+ and CD3- / CD56+ populations. Binding affinities were calculated using 4-parameter logistic regression with Prism Graphpad™ 8.0.2 software.

[0180] Table 5: Binding affinity (K) of Alexa Fluor® 488 NKp46 dimers binding to subpopulations of PBMC cellsd,app )) [Table 9]

[0181] The data presented in Table 5 and Figure 2 demonstrate that Alexa Fluor® 488-tagged NKp46 BCY15663 induces dose-dependent binding exclusively to NK cells (CD56+ population), whereas Alexa Fluor® 488-tagged non-binding NKp46 BCY15665 induces no binding to the PBMC population.

[0182] 6a. Description of FcγRIII (CD16a) SPR Experiment (Method A) SPR experiments were performed to measure the kappa binding of various bicyclic peptides to immobilized human FcγRIII protein. a (M -1 s -1 ) value, k d (s -1 ) value, K D Measurements were taken of (nM) values ​​(when available).

[0183] For peptide binding analysis, a Biacore 3000 or T200 instrument was used with a CM5 chip (GE Healthcare). Streptavidin was immobilized on the chip by standard amine coupling chemistry at 25° C. in HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as running buffer. Briefly, the carboxymethyldextran surface was activated by injecting 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. To capture streptavidin, 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) and the levels of captured biotinylated FcγRIII ranged from 400 to 1400 RU. The buffer was changed to PBS / 0.05% Tween 20 and peptide dilution series were prepared in this buffer to a final concentration of 0.5% DMSO. The maximum concentration of peptide ranged from 200 nM to 20 μM (depending on predicted affinity) with an additional 6 2- or 3-fold dilutions. SPR analysis was performed at 25 °C with a flow rate of 50 μL / min, with association times of 60 s and dissociation times between 60 and 200 s. Data were corrected for DMSO excluded volume effects. 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, Inc.). Data were fitted using a simple 1:1 binding model allowing for mass transport effects where applicable.

[0184] Selected peptides of the invention were tested in the above assay and the results are shown in Table 6A.

[0185] Table 6A: SPR assay data for selected bicyclic peptides of the invention. [Table 10]

[0186] (6b. Description of FcγRIII (CD16a) SPR Experiment (Method B)) For peptide binding analysis, a Biacore 8k+ instrument was used with a CM5 chip (Cytiva). All experiments were performed at 25°C. Streptavidin was immobilized on all flow cells by standard amine coupling chemistry. The running buffer was HBS-N (Cytiva, 10 mM HEPES, 0.15 M NaCl, pH 7.4) at a flow rate of 10 μL / min. The carboxymethyldextran surface was activated with a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) (Cytiva) for 420 seconds. Streptavidin (ProSpec, Cat. No. PRO-791) was diluted to 0.2 mg / mL in 10 mM sodium acetate, pH 4.5, and dispensed onto the chip surface for 600 seconds. Remaining activated groups were blocked by injection of 1 M ethanolamine (pH 8.5) for 420 seconds. Final surface density was 2000-3000 RU. Biotinylated CD16a was diluted to 5 μg / mL in HBS-N and captured to 1500-2500 RU only on flow cell 2 of the streptavidin chip at a flow rate of 10 μL / min. The buffer was then changed to PBS-P+ (Cytiva, supplemented with 0.5-2% DMSO). Peptide dilutions were prepared in this buffer to a final concentration of 0.5-2% DMSO. Flow rates were 30 μL / min with association times of 100 seconds and dissociation times of 400 seconds. Data were corrected for DMSO excluded volume effects. All data were double referenced to blank injections and reference surface reactions using standard processing procedures. Data processing and kinetic fitting were performed using Biacore Insight Evaluation software and Extended Screening and Characterization Extension (Cytiva). Data were fitted using a steady-state affinity 1:1 binding model to obtain K D The data were fitted with a kinetic 1:1 binding model to determine reaction rate constants, where applicable.

[0187] Selected peptides of the invention were tested by Method A and / or Method B and the results are shown in Table 6B.

[0188] Table 6B: SPR assay data for selected bicyclic peptides of the invention. [Table 11] TIFF2024502351000058.tif244170TIFF2024502351000059.tif244170TIFF2024502351000060.tif89170

[0189] 7. IgG Competition Assay The bicyclic peptides were evaluated in a CD16a-IgG competition assay using PerkinElmer AlphaScreen™ technology. Acceptor beads (PerkinElmer 6762003) were chemically conjugated with mouse IgG2a mAb by reductive amination reaction according to the manufacturer's protocol, linking via the free amine. Briefly, beads were centrifuged and resuspended in 130 mM sodium phosphate buffer pH 8.0 containing 0.2 mg mAb, 20 mM NaBH3CN (Sigma 296945) and 0.06% P20 surfactant (Cytiva Life Sciences BR-1000-54) and incubated for 22 hours at 44°C on a rotary shaker. Unbound sites were then blocked by adding carboxymethoxylamine (CMO) (Sigma C13408) at a final concentration of 3 mg / mL, after which the shaking incubation was continued for another hour. The beads were washed by centrifugation and resuspended in 100 mM TRIS-HCl pH 8.0 (Sigma T2694), then finally resuspended at 5 mg / mL in PBS (Gibco 14190) and 0.05% Proclin 300 (Sigma 48912-U) and stored at 4 °C until use.

[0190] The assay was performed in white 384-well plates (PerkinElmer 6007290) using assay buffer 25 mM HEPES (Sigma-Aldrich H0887), 100 mM NaCl (Sigma-Aldrich S5150), 0.5% BSA (Sigma-Aldrich A3294), 0.05% P20 (Cytiva Life Sciences BR-1000-54), pH 7.4 for all dilutions. The bicyclic peptides were incubated dropwise with C-terminally biotinylated human CD16a F176 (ACRO Biosystems CDA-H82E8) or N-terminally biotinylated V176 variant (R&D Systems 4325-Fc, biotinylated in-house) at a final concentration of 10 nM and incubated for 30 min at room temperature. Acceptor beads conjugated with mouse IgG2a as described above were added to a final concentration of 20 ug / mL and incubated at room temperature for 30 minutes under subdued lighting. Streptavidin-coated donor beads (PerkinElmer, 6760002) were added to a final concentration of 20 ug / mL and incubated at room temperature for 60 minutes under subdued lighting. Excitation emission at 570 nm was read using a BMG Labtech PHERAstar FS plate reader. Data were normalized to a high control containing all assay components except the bicyclic peptide and fitted to a four-parameter nonlinear regression using Graphpad Prism 8.0.2 to obtain IC 50 Created a value.

[0191] Selected peptides of the invention were tested in the above assays and the results are shown in Table 7 and Figures 3 and 4. The data presented in Table 7 and Figures 3 and 4 demonstrate that peptides of the invention bind to a biologically relevant epitope on FcγRIIIA (F and V variants) and can compete with IgG for binding to this epitope.

[0192] Table 7: IgG competition assay data for selected bicyclic peptides of the invention. [Table 12]

[0193] (8.CD16a / FCgRIIIa HTRF Assay) Binding to cellular CD16a / FCgRIIIa (F158 variant) was determined by competition with IgG for binding to the CD16a receptor expressed on HEK 293 cells. The assay is based on fluorescence resonance energy transfer (FRET), whereby a flash of light induces FRET between the CD16a terbium donor dye and the IgG-d2 acceptor, generating a fluorescent signal when the donor-acceptor pair is in close proximity. Unlabeled antibodies or bicyclic peptides competing with the IgG-d2 acceptor reduce the overall signal in a dose-dependent manner, thus determining the IC 50 The value can be determined.

[0194] The experimental procedure followed the manufacturer's instructions detailed in the product information for the CD16 (FCγRIIIA) kit (Cisbio 6FR3APAG). Two human IgG antibodies were included as controls (Cisbio 6FR3APAG, max concentration 267 nM, and R&D Systems 1-001-A, max concentration 3000 nM). Tag-lite buffer was prepared to generate the IgG-d2 acceptor solution. The bicyclic peptide was dispensed at a max concentration of 20 μM using an Echo compound handler. Competing unlabeled antibodies and bicyclic peptides were added to a PerkinElmer ProxiPlate-384 Plus in a 3-fold dilution series in Tag-lite buffer.

[0195] HEK 293 cells expressing terbium-labeled CD16a were thawed, washed with PBS, and added to the wells of a PerkinElmer ProxiPlate-384 Plus containing competitor compounds. Finally, acceptor IgG-D2 reagent was added and the plate was incubated for 2 hours at room temperature (RT). FRET signals were read on an Envision plate reader using Ex 340 nM and Em 655 / 620 nM settings. Data were plotted in Graphpad Prism to obtain IC 50 Values ​​were determined that indicated competition of unlabeled antibody or bicyclic peptide for cellular binding to the CD16a receptor.

[0196] Selected peptides of the invention were tested in the above assay and the results are shown in Table 8.

[0197] Table 8: IgG competition assay data for selected bicyclic peptides of the invention (HTRF) [Table 13] TIFF2024502351000063.tif143170

[0198] (List of References) [Table 14] TIFF2024502351000065.tif247170TIFF2024502351000066.tif247170TIFF2024502351000067.tif176170

Claims

1. A peptide ligand specific for natural killer (NK) cells, comprising: a polypeptide comprising at least three reactive groups separated by at least two loop sequences; and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, wherein at least two polypeptide loops are formed on the molecular scaffold.

2. The peptide ligand of claim 1 , wherein the reactive group comprises a cysteine ​​residue or a penicillamine residue.

3. 3. The peptide ligand of claim 1 or claim 2, wherein the bicyclic peptide is specific for natural cytotoxicity receptors present on the surface of NK cells, such as NKp30, NKp44 and NKp46, in particular NKp46.

4. The peptide ligand of claim 3 , wherein the loop sequence comprises 4, 5, 6, or 7 amino acids.

5. 5. A peptide ligand according to claim 3 or claim 4, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 4 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 1】 (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, in particular The molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:1)-A-Sar 6 -KFl (referred to herein as BCY14654); A-(SEQ ID NO:1)-A (referred to herein as BCY14456); A-(SEQ ID NO:2)-A-Sar 6 -KFl (referred to herein as BCY15125); A-(SEQ ID NO:2)-A (referred to herein as BCY15102); and A-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY18004), wherein PYA represents pentynic acid, Sar represents sarcosine, and Fl represents fluorescein.

6. 5. A peptide ligand according to claim 3 or claim 4, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 6 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 2】 【change】 (In the formula, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively; Cba represents cyclobutylalanine; tBuAla represents β-t-butyl-L-alanine; Sar represents sarcosine; Cis-HyP represents L-4-cis-hydroxyproline; HyP represents hydroxyproline; NMeAla represents N-methylalanine; Nle represents norleucine; dA represents D-alanine; Agb represents norarginine; tBuGly represents tert-butylglycine; C ha represents 3-cyclohexyl-L-alanine, Chg represents cyclohexylglycine, Cbg represents cyclobutylglycine, HArg represents homoarginie, Aze represents azetidine-2-carboxylic acid, Pip represents pipecolic acid, Nva represents norvaline, AlloIle represents L-allo-isoleucine, and EPA represents diethyl-L-alanine), or a pharma- ceutically acceptable salt thereof, in particular: The molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY15687); A-(SEQ ID NO:3)-A (referred to herein as BCY15098); A-(SEQ ID NO:4)-A-Sar 6 -KFl (referred to herein as BCY15115); A-(SEQ ID NO:4)-A (referred to herein as BCY15099); Ac-(SEQ ID NO:4) (referred to herein as BCY18293); Ac-A-(SEQ ID NO:4)-A (referred to herein as BCY18294); (SEQ ID NO:4)-A (referred to herein as BCY19694); K-(SEQ ID NO:4)-A (referred to herein as BCY19695); dK-(SEQ ID NO:4)-A (referred to herein as BCY19696); Dap-(SEQ ID NO:4)-A (referred to herein as BCY19697); Dab-(SEQ ID NO:4)-A (referred to herein as BCY19698); GABA-(SEQ ID NO:4)-A (referred to herein as BCY19699); A-(SEQ ID NO:63)-A (referred to herein as BCY14454); A-(SEQ ID NO:64)-A (referred to herein as BCY18299); A-(SEQ ID NO:65)-A (referred to herein as BCY18301); A-(SEQ ID NO:66)-A (referred to herein as BCY18302); A-(SEQ ID NO:67)-A (referred to herein as BCY18303); A-(SEQ ID NO:68)-A (referred to herein as BCY18304); A-(SEQ ID NO:69)-A (referred to herein as BCY18305); A-(SEQ ID NO:70)-A (referred to herein as BCY18307); A-(SEQ ID NO:71)-A (referred to herein as BCY18309); A-(SEQ ID NO:72)-A (referred to herein as BCY18310); A-(SEQ ID NO:73)-A (referred to herein as BCY18311); A-(SEQ ID NO:74)-A (referred to herein as BCY18312); A-(SEQ ID NO:75)-A (referred to herein as BCY18313); A-(SEQ ID NO:76)-A (referred to herein as BCY18314); A-(SEQ ID NO:77)-A (referred to herein as BCY18315); A-(SEQ ID NO:78)-A (referred to herein as BCY18316); A-(SEQ ID NO:79)-A (referred to herein as BCY18317); A-(SEQ ID NO:80)-A (referred to herein as BCY18318); A-(SEQ ID NO:81)-A (referred to herein as BCY18319); A-(SEQ ID NO:82)-A (referred to herein as BCY18320); A-(SEQ ID NO:83)-A (referred to herein as BCY18321); A-(SEQ ID NO:84)-A (referred to herein as BCY18322); A-(SEQ ID NO:85)-A (referred to herein as BCY18323); A-(SEQ ID NO:86)-A (referred to herein as BCY18324); A-(SEQ ID NO:87)-A (referred to herein as BCY18325); A-(SEQ ID NO:88)-A (referred to herein as BCY18326); A-(SEQ ID NO:89)-A (referred to herein as BCY18327); A-(SEQ ID NO:90)-A (referred to herein as BCY18328); A-(SEQ ID NO:91)-A (referred to herein as BCY19700); A-(SEQ ID NO:92)-A (referred to herein as BCY19701); A-(SEQ ID NO:93)-A (referred to herein as BCY19702); A-(SEQ ID NO:94)-A (referred to herein as BCY19703); A-(SEQ ID NO:95)-A (referred to herein as BCY19704); A-(SEQ ID NO:96)-A (referred to herein as BCY19706); A-(SEQ ID NO:97)-A (referred to herein as BCY19707); A-(SEQ ID NO:98)-A (referred to herein as BCY19708); A-(SEQ ID NO:99)-A (referred to herein as BCY19710); A-(SEQ ID NO:100)-A (referred to herein as BCY19711); A-(SEQ ID NO:101)-A (referred to herein as BCY19712); and A-(SEQ ID NO:102)-A (referred to herein as BCY19713), wherein PYA represents pentynic acid, Sar represents sarcosine, Fl represents fluorescein, dK represents D-lysine, Dap represents L-2,3-diaminopropionic acid, Dab represents L-2,4-diaminobutyric acid, and GABA represents γ-aminobutyric acid.

7. 5. A peptide ligand according to claim 3 or claim 4, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 7 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 3】 (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, in particular The molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:5)-A (referred to herein as BCY15103); A-(SEQ ID NO:5)-A-[K(PYA)] (referred to herein as BCY18005); A-(SEQ ID NO:5)-A-Sar 6 -KFl (referred to herein as BCY15118); A-(SEQ ID NO:6)-A (referred to herein as BCY15104); A-(SEQ ID NO:6)-A-Sar 6 -KFl (referred to herein as BCY15119); A-(SEQ ID NO:7)-A (referred to herein as BCY15105); A-(SEQ ID NO:7)-A-Sar 6 -KFl (referred to herein as BCY15120); A-(SEQ ID NO:8)-A (referred to herein as BCY15106); A-(SEQ ID NO:8)-A-Sar 6 -KFl (herein referred to as BCY15121); and A-(SEQ ID NO:103)-A (referred to herein as BCY14457), wherein PYA represents pentynic acid, Sar represents sarcosine, and Fl represents fluorescein.

8. 5. A peptide ligand according to claim 3 or claim 4, wherein the loop sequence comprises three cysteine ​​or penicillamine residues separated by two loop sequences, the first loop sequence consisting of 6 amino acids and the second loop sequence consisting of 4 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 4】 【change】 【change】 【change】 (In the formula, C i , Pen i , C ii , Pen ii , C iii and Pen iii represent the first, second, and third reactive groups, respectively; 2,6DiMeTyr represents 2,6-dimethyl-tyrosine; tBuAla represents t-butyl-alanine; Cba represents β-cyclobutylalanine; HyP represents hydroxyproline; Aze represents azetidine; Aib represents aminoisobutyric acid; 1Nal represents 1-naphthylalanine; 2Nal represents 2-naphthylalanine; 3Pal represents 3-pyridylalanine; C5A represents β-cyclopentyl-L-alanine; Cha represents 3- cyclohexyl-L-alanine, 4FPhe stands for 4-fluoro-L-phenylalanine, 4MeOPhe stands for 4-methoxy-phenylalanine, NO2Phe stands for 4-nitro-phenylalanine, 4MePhe stands for 4-methyl-phenylalanine, pCoPhe stands for p-carboxy-phenylalanine, pCaPhe stands for p-carboxamido-phenylalanine, 4tBuPhe stands for 4-tert-butyl-phenylalanine, CF3Phe stands for 4-trifluoromethyl-phenylalanine, tBuTyr stands for 3-tert-butyl tyrosine, 4CNPhe stands for 4-cyano-phenylalanine, 4ClPhe stands for 4-chloro-phenylalanine, HPhe stands for homophenylalanine, 3tBuTyr stands for 3-tert-butyl tyrosine, His3Me stands for 3-methylhistidine, 2Pal stands for 2-pyridylalanine, His1Me stands for 1-methylhistidine, 4ThiAz stands for β-4-thiazol-L-ylalanine, Trp(Me) stands for 1-methyltryptophan, t BuGly stands for Lt-butylglycine, Chg stands for cyclohexylglycine, HSer stands for homoserine, NMeAla stands for N-methylalanine, 4Pal stands for 4-pyridylalanine, Cis-HyP stands for 4-cis-hydroxyproline, dA stands for D-alanine, dV stands for D-valine, dNva stands for D-norvaline, dNle stands for D-norleucine, dF stands for D-phenylalanine, dCha stands for 3-cyclohexyl-D-alanine, and DOPA stands for 3,4-dihydroxyphenylalanine, 2FTyr represents 2-fluorotyrosine, Nle represents norleucine, dC represents D-cysteine, AzaTrp represents azatryptophan, 2MeTrp represents 2-methyltryptophan, 6MeTrp represents 6-methyltryptophan, 6FTrp represents 6-fluorotryptophan, 5FTrp represents 5-fluorotryptophan, Dht represents 2,3-dihydrotryptophan, 2FuAla represents 2-furylalanine, and AlloIle represents L-allo-isoleucine), or a pharma- ceutically acceptable salt thereof, in particular: The molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:9)-A-[dK(PYA)] (referred to herein as BCY15013); A-(SEQ ID NO:10)-A (referred to herein as BCY18470); A-(SEQ ID NO:10)-A-[dK(PYA)] (referred to herein as BCY15452); A-(SEQ ID NO:11)-A (referred to herein as BCY15107); Ac-(SEQ ID NO:11) (referred to herein as BCY18260); Ac-A-(SEQ ID NO:11)-A (referred to herein as BCY18261); A-(SEQ ID NO:11)-[Aib] (referred to herein as BCY19717); (SEQ ID NO:11)-A (referred to herein as BCY19718); A-(SEQ ID NO:11)-A-[dK(PYA)] (referred to herein as BCY19719); A-(SEQ ID NO:11)-A-[K(PYA)] (referred to herein as BCY15686); A-(SEQ ID NO:12)-A (referred to herein as BCY15100); Ac-(SEQ ID NO:12) (referred to herein as BCY15634); Ac-A-(SEQ ID NO:12)-A (referred to herein as BCY15635); dA-(SEQ ID NO:12) (referred to herein as BCY16134); A-(SEQ ID NO:12)-A-Sar 6 -KFl (referred to herein as BCY15116); A-(SEQ ID NO:13)-A (referred to herein as BCY14455); SEQ ID NO:13 -Sar 6 -KFl (referred to herein as BCY15023); Ac-(SEQ ID NO: 13)-Sar 6 -KFl (referred to herein as BCY15024); A-(SEQ ID NO:13)-A-Sar 6 -KFl (referred to herein as BCY14652); A-(SEQ ID NO:14)-A (referred to herein as BCY15636); A-(SEQ ID NO:15)-A (referred to herein as BCY15638); A-(SEQ ID NO:16)-A (referred to herein as BCY15639); A-(SEQ ID NO:17)-A (referred to herein as BCY15640); A-(SEQ ID NO:18)-A (referred to herein as BCY15641); A-(SEQ ID NO:19)-A (referred to herein as BCY15642); A-(SEQ ID NO:20)-A-(referred to herein as BCY15643); A-(SEQ ID NO:21)-A (referred to herein as BCY15644); A-(SEQ ID NO:22)-A (referred to herein as BCY15645); A-(SEQ ID NO:23)-A (referred to herein as BCY15646); A-(SEQ ID NO:24)-A (referred to herein as BCY15648); A-(SEQ ID NO:25)-A (referred to herein as BCY15649); A-(SEQ ID NO:26)-A (referred to herein as BCY15650); A-(SEQ ID NO:27)-A (referred to herein as BCY15651); A-(SEQ ID NO:28)-A (referred to herein as BCY15653); A-(SEQ ID NO:29)-A (referred to herein as BCY15654); A-(SEQ ID NO:30)-A (referred to herein as BCY15655); A-(SEQ ID NO:31)-A (referred to herein as BCY15656); A-(SEQ ID NO:32)-A (referred to herein as BCY15657); A-(SEQ ID NO:33)-A (referred to herein as BCY15658); A-(SEQ ID NO:34)-A (referred to herein as BCY15659); A-(SEQ ID NO:35)-A (referred to herein as BCY15660); A-(SEQ ID NO:36)-A (referred to herein as BCY15661); A-(SEQ ID NO:37)-A (referred to herein as BCY15662); A-(SEQ ID NO:38)-A (referred to herein as BCY16130); A-(SEQ ID NO:39)-A (referred to herein as BCY16131); A-(SEQ ID NO:40)-A (referred to herein as BCY16132); Ac- (SEQ ID NO: 40) (referred to herein as BCY16133); Ac-(SEQ ID NO:40)-[K(PYA)] (referred to herein as BCY17224); A-(SEQ ID NO:41)-A (referred to herein as BCY16135); A-(SEQ ID NO:42)-A (referred to herein as BCY16136); A-(SEQ ID NO:43)-A (referred to herein as BCY16137); A-(SEQ ID NO:44)-A (referred to herein as BCY16138); A-(SEQ ID NO:45)-A (referred to herein as BCY16139); A-(SEQ ID NO:46)-A (referred to herein as BCY16140); A-(SEQ ID NO:47)-A (referred to herein as BCY16141); A-(SEQ ID NO:48)-A (referred to herein as BCY16142); A-(SEQ ID NO:49)-A (referred to herein as BCY16143); A-(SEQ ID NO:50)-A (referred to herein as BCY16144); A-(SEQ ID NO:51)-A (referred to herein as BCY16145); A-(SEQ ID NO:52)-A-Sar 6 -KFl (referred to herein as BCY15025); A-(SEQ ID NO:53)-A-Sar 6 -KFl (referred to herein as BCY15028); A-(SEQ ID NO:54)-A-Sar 6 -KFl (referred to herein as BCY15030); A-(SEQ ID NO:55)-A-Sar 6 -KFl (referred to herein as BCY15035); A-(SEQ ID NO:56)-A-Sar 6 -KFl (referred to herein as BCY15117); A-(SEQ ID NO:57)-A-Sar 6 -KFl (referred to herein as BCY15122); A-(SEQ ID NO:58)-A (referred to herein as BCY15108); A-(SEQ ID NO:58)-A-Sar 6 -KFl (referred to herein as BCY15123); A-(SEQ ID NO:59)-A-Sar 6 -KFl (referred to herein as BCY15124); A-(SEQ ID NO:104)-A (referred to herein as BCY14458); A-(SEQ ID NO:105)-A (referred to herein as BCY15109); A-(SEQ ID NO:106)-A (referred to herein as BCY15568); A-(SEQ ID NO:107)-A (referred to herein as BCY15637); A-(SEQ ID NO:108)-A (referred to herein as BCY15647); A-(SEQ ID NO:109)-A (referred to herein as BCY15652); A-(SEQ ID NO:110)-A (referred to herein as BCY16834); A-(SEQ ID NO:111)-A (referred to herein as BCY16837); A-(SEQ ID NO:112)-A (referred to herein as BCY16840); A-(SEQ ID NO:113)-A (referred to herein as BCY16842); A-(SEQ ID NO:114)-A (referred to herein as BCY16843); A-(SEQ ID NO:115)-A (referred to herein as BCY17662); A-(SEQ ID NO:116)-A (referred to herein as BCY18263); A-(SEQ ID NO:117)-A (referred to herein as BCY18265); A-(SEQ ID NO:118)-A (referred to herein as BCY18266); A-(SEQ ID NO:119)-A (referred to herein as BCY18269); A-(SEQ ID NO:120)-A (referred to herein as BCY18272); A-(SEQ ID NO:121)-A (referred to herein as BCY18273); A-(SEQ ID NO:122)-A (referred to herein as BCY18277); A-(SEQ ID NO:123)-A (referred to herein as BCY18278); A-(SEQ ID NO:124)-A (referred to herein as BCY18279); A-(SEQ ID NO:125)-A (referred to herein as BCY18280); A-(SEQ ID NO:126)-A (referred to herein as BCY18281); A-(SEQ ID NO:127)-A (referred to herein as BCY18282); A-(SEQ ID NO:128)-A (referred to herein as BCY18285); A-(SEQ ID NO:129)-A (referred to herein as BCY18286); A-(SEQ ID NO:130)-A (referred to herein as BCY18287); A-(SEQ ID NO:131)-A (referred to herein as BCY18288); A-(SEQ ID NO:132)-A (referred to herein as BCY18289); A-(SEQ ID NO:133)-A (referred to herein as BCY18290); A-(SEQ ID NO:134)-A (referred to herein as BCY18291); A-(SEQ ID NO:135)-A (referred to herein as BCY18292); Ac- (SEQ ID NO: 136) (referred to herein as BCY18433); Ac- (SEQ ID NO: 137) (referred to herein as BCY18434); Ac- (SEQ ID NO: 138) (referred to herein as BCY18435); Ac- (SEQ ID NO: 139) (referred to herein as BCY18437); Ac- (SEQ ID NO: 140) (referred to herein as BCY18439); Ac- (SEQ ID NO: 141) (referred to herein as BCY18440); Ac- (SEQ ID NO: 142) (referred to herein as BCY18441); Ac- (SEQ ID NO: 143) (referred to herein as BCY18442); Ac- (SEQ ID NO: 144) (referred to herein as BCY18443); Ac- (SEQ ID NO: 145) (referred to herein as BCY18444); Ac- (SEQ ID NO: 146) (referred to herein as BCY19682); Ac- (SEQ ID NO: 147) (referred to herein as BCY19683); Ac- (SEQ ID NO: 148) (referred to herein as BCY19684); Ac- (SEQ ID NO: 149) (referred to herein as BCY19685); Ac- (SEQ ID NO: 150) (referred to herein as BCY19686); Ac- (SEQ ID NO: 151) (referred to herein as BCY19687); Ac- (SEQ ID NO: 152) (referred to herein as BCY19688); Ac- (SEQ ID NO: 153) (referred to herein as BCY19689); Ac- (SEQ ID NO: 154) (referred to herein as BCY19690); Ac- (SEQ ID NO: 155) (referred to herein as BCY19691); Ac- (SEQ ID NO: 156) (referred to herein as BCY19692); Ac- (SEQ ID NO: 157) (referred to herein as BCY19693); A-(SEQ ID NO:158)-A (referred to herein as BCY19720); A-(SEQ ID NO:159)-A (referred to herein as BCY19721); A-(SEQ ID NO:160)-A (referred to herein as BCY19722); A-(SEQ ID NO:161)-A (referred to herein as BCY19723); A-(SEQ ID NO:162)-A (referred to herein as BCY19724); A-(SEQ ID NO:163)-A (referred to herein as BCY19725); A-(SEQ ID NO:164)-A (referred to herein as BCY19730); A-(SEQ ID NO:165)-A (referred to herein as BCY19732); A-(SEQ ID NO:166)-A (referred to herein as BCY19733); A-(SEQ ID NO:167)-A (referred to herein as BCY19734); and A-(SEQ ID NO:168)-A (referred to herein as BCY19735), wherein Aib represents aminoisobutyric acid, PYA represents pentanoic acid, Sar represents sarcosine, and Fl represents fluorescein.

9. 3. The peptide ligand of claim 1 or claim 2, wherein the bicyclic peptide is specific for an Fc receptor present on the surface of NK cells, such as a low affinity Fcγ receptor (FcγR) selected from FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB, in particular FcγRIIIA (also known as CD16a).

10. 10. The peptide ligand of claim 9, wherein the loop sequence comprises 3, 4, 5, 6, 7, or 8 amino acids.

11. 11. A peptide ligand according to claim 9 or claim 10, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of four amino acids and the second loop sequence consisting of eight amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 5】 (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, in particular The molecular scaffold is TATB, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:265)-A (referred to herein as BCY15044); and A-(SEQ ID NO:265)-AGAAAE (referred to herein as BCY19416), The peptide ligand comprises an amino acid sequence selected from:

12. 11. A peptide ligand according to claim 9 or claim 10, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 5 amino acids and the second loop sequence consisting of 4 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 6】 (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, in particular: The molecular scaffold is TATA, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:60)-A (referred to herein as BCY11808); A-(SEQ ID NO:60)-A-[K(PYA)] (referred to herein as BCY15685); A-(SEQ ID NO:60)-A-[Sar 6 ]-[KBiot] (referred to herein as BCY12621); A-(SEQ ID NO:169)-A (referred to herein as BCY11673); A-(SEQ ID NO:170)-A (referred to herein as BCY11809); A-(SEQ ID NO:171)-A (referred to herein as BCY11810); A-(SEQ ID NO:172)-A (referred to herein as BCY11811); A-(SEQ ID NO:173)-A (referred to herein as BCY11812); and A-(SEQ ID NO: 174)-A-[Sar 6 ]-[KBiot] (referred to herein as BCY12622), (wherein Sar represents sarcosine and Biot represents biotin),

13. 11. A peptide ligand according to claim 9 or claim 10, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 6 amino acids and the second loop sequence consisting of 3 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 7】 (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, in particular The molecular scaffold is TATB, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:266)-A (referred to herein as BCY15045); and A-(SEQ ID NO:266)-AGAAAE (referred to herein as BCY19417), The peptide ligand comprises an amino acid sequence selected from:

14. 11. A peptide ligand according to claim 9 or claim 10, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 7 amino acids and the second loop sequence consisting of 3 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 8】 【change】 【change】 (In the formula, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively; Gla represents γ-carboxyglutamic acid; Aze represents azetidine-2-carboxylic acid; 2MeTrp represents 2-methyltryptophan; 6MeTrp represents 6-methyltryptophan; 6FTrp represents 6-fluorotryptophan; 5FTrp represents 5-fluorotryptophan; 6ClTrp represents 6-chloro-L-tryptophan; 5MeoTrp represents 6-methyltryptophan; represents 5-methoxy-L-tryptophan, Trp(S) represents 3-(3-benzothienyl)-L-alanine, AzaTrp represents azatryptophan, 4MeoTrp represents 4-methoxy-L-tryptophan, DOPA represents 3,4-dihydroxyphenylalanine, His1Me represents 1-methylhistidine, His3Me represents 3-methylhistidine, 4MeoPhe represents 4-methoxyphenylalanine, 26DiMeTyr represents 2,6-dimethyl-L-tyrosine, 3tBuTyr represents 3-tert-butyltyrosine, 4FPhe represents 4-fluoro-L-phenylalanine, 4MePhe represents 4-methyl-phenylalanine, HyP represents hydroxyproline, 4tBuPhe represents 4-t-butyl-phenylalanine, HPhe represents homophenylalanine, and 4PhePro represents (2S,4S)-4-phenylproline. wherein R represents 5-aminophosphorus, HSer represents homoserine, HGlu represents L-2-aminoadipic acid, dA represents D-alanine, HArg represents homoarginine, tBuGly represents tert-butylglycine, Cis-HyP represents 4-cis-hydroxyproline, and 4BnPro represents (2S,4R)-4-benzyl-pyrrolidine-2-carboxylic acid, or a pharma- ceutically acceptable salt thereof, in particular: The molecular scaffold is TBMT, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:61)-A (referred to herein as BCY14950); Ac-A-(SEQ ID NO:61)-A (referred to herein as BCY16599); A-(SEQ ID NO:61)-AGAAAE (referred to herein as BCY19040); A-(SEQ ID NO:61)-A-[K(PYA)] (referred to herein as BCY13883); Ac-A-(SEQ ID NO:61)-A-[K(PYA)] (referred to herein as BCY19596); A-(SEQ ID NO:175)-A (referred to herein as BCY14953); A-(SEQ ID NO:176)-A (referred to herein as BCY14954); [PYA]-A-(SEQ ID NO:177) (referred to herein as BCY14955); A-(SEQ ID NO:178)-A (referred to herein as BCY14956); A-(SEQ ID NO:179)-A (referred to herein as BCY16608); A-(SEQ ID NO:180)-A (referred to herein as BCY16610); A-(SEQ ID NO:181)-A (referred to herein as BCY19032); A-(SEQ ID NO:182)-A (referred to herein as BCY19033); A-(SEQ ID NO:183)-A (referred to herein as BCY19034); A-(SEQ ID NO:184)-A (referred to herein as BCY19035); A-(SEQ ID NO:185)-A (referred to herein as BCY19036); A-(SEQ ID NO:186)-A (referred to herein as BCY19037); A-(SEQ ID NO:187)-AGAAAE (referred to herein as BCY19038); A-(SEQ ID NO:188)-AGAAAE (referred to herein as BCY19039); A-(SEQ ID NO:189)-AGAAAE (referred to herein as BCY19041); A-(SEQ ID NO:190)-AGAAAE (referred to herein as BCY19042); A-(SEQ ID NO:191)-AGAAAE (referred to herein as BCY19043); A-(SEQ ID NO:192)-AGAAAE (referred to herein as BCY19044); A-(SEQ ID NO:193)-A (referred to herein as BCY19045); A-(SEQ ID NO:194)-A (referred to herein as BCY19046); A-(SEQ ID NO:195)-A (referred to herein as BCY19048); A-(SEQ ID NO:196)-A (referred to herein as BCY19049); A-(SEQ ID NO:197)-A (referred to herein as BCY19050); A-(SEQ ID NO:198)-A (referred to herein as BCY19053); A-(SEQ ID NO:199)-A (referred to herein as BCY19055); A-(SEQ ID NO:200)-A (referred to herein as BCY19057); A-(SEQ ID NO:201)-A (referred to herein as BCY19060); A-(SEQ ID NO:202)-A (referred to herein as BCY19061); A-(SEQ ID NO:203)-A (referred to herein as BCY19063); A-(SEQ ID NO:204)-A (referred to herein as BCY19064); A-(SEQ ID NO:205)-A (referred to herein as BCY19065); A-(SEQ ID NO:206)-A (referred to herein as BCY19068); A-(SEQ ID NO:207)-A-[K(PYA)] (referred to herein as BCY20360); A-(SEQ ID NO:208)-A-[K(PYA)] (referred to herein as BCY20361); A-(SEQ ID NO:209)-A (referred to herein as BCY20363); A-(SEQ ID NO:210)-A (referred to herein as BCY20364); A-(SEQ ID NO:211)-A (referred to herein as BCY20365); A-(SEQ ID NO:212)-A (referred to herein as BCY20366); A-(SEQ ID NO:213)-A (referred to herein as BCY20367); A-(SEQ ID NO:214)-A (referred to herein as BCY20368); A-(SEQ ID NO:215)-A (referred to herein as BCY20369); A-(SEQ ID NO:216)-A (referred to herein as BCY20370); A-(SEQ ID NO:217)-A (referred to herein as BCY20371); A-(SEQ ID NO:218)-A (referred to herein as BCY20372); A-(SEQ ID NO:219)-A (referred to herein as BCY20373); A-(SEQ ID NO:220)-A (referred to herein as BCY20374); A-(SEQ ID NO:221)-A (referred to herein as BCY20375); A-(SEQ ID NO:222)-A (referred to herein as BCY20376); A-(SEQ ID NO:223)-A (referred to herein as BCY20377); A-(SEQ ID NO:224)-A (referred to herein as BCY20378); A-(SEQ ID NO:225)-A (referred to herein as BCY20379); A-(SEQ ID NO:226)-A (referred to herein as BCY20380); A-(SEQ ID NO:227)-A (referred to herein as BCY20382); A-(SEQ ID NO:228)-A (referred to herein as BCY20383); A-(SEQ ID NO:229)-A (referred to herein as BCY20384); A-(SEQ ID NO:230)-A (referred to herein as BCY20385); (SEQ ID NO:230)-A (referred to herein as BCY21690); Ac-(SEQ ID NO:230)-A (referred to herein as BCY21691); A-(SEQ ID NO:230) (referred to herein as BCY21692); Ac- (SEQ ID NO: 230) (referred to herein as BCY21694); [Nle]-(SEQ ID NO:230)-A (referred to herein as BCY21695); Ac-[Nle]-(SEQ ID NO:230)-A (referred to herein as BCY21696); Ac-A-(SEQ ID NO:230)-A (referred to herein as BCY21697); A-(SEQ ID NO:231)-A (referred to herein as BCY20386); A-(SEQ ID NO:232)-A (referred to herein as BCY20388); A-(SEQ ID NO:233)-A (referred to herein as BCY20389); A-(SEQ ID NO:234)-A (referred to herein as BCY20390); A-(SEQ ID NO:235)-A (referred to herein as BCY20392); A-(SEQ ID NO:236)-A (referred to herein as BCY20394); A-(SEQ ID NO:237)-A (referred to herein as BCY20395); A-(SEQ ID NO:238)-A (referred to herein as BCY20396); A-(SEQ ID NO:239)-A (referred to herein as BCY20397); A-(SEQ ID NO:240)-A (referred to herein as BCY20526); Ac-A-(SEQ ID NO:241)-A (referred to herein as BCY20527); [Aib]-(SEQ ID NO:242)-A (referred to herein as BCY20528); A-(SEQ ID NO:243)-[Aib] (referred to herein as BCY20529); A-(SEQ ID NO:267)-A (referred to herein as BCY20387); and A-(SEQ ID NO:268)-A (referred to herein as BCY21689), (wherein PYA represents pentynic acid and Aib represents aminoisobutryic acid).

15. 11. A peptide ligand according to claim 9 or claim 10, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first loop sequence consisting of 8 amino acids and the second loop sequence consisting of 3 amino acids, such as: The bicyclic peptide ligand is: 【Chemistry 9】 (In the formula, C i , C ii , and C iii represent the first, second and third cysteine ​​residues, respectively; dA represents D-alanine; tBuAla represents β-t-butyl-L-alanine; Cba represents cyclobutylalanine; 1Nal represents 1-napthylalanine; 2Nal represents 2-napthylalanine; Cha represents 3-cyclohexyl-D-alanine; Nle represents norleucine; Gla represents γ-carboxyglutamic acid; Aze represents azetidine-2-carboxylic acid; HLeu represents homoleucine; and HGlu represents L-2-aminoadipic acid), or a pharma- ceutically acceptable salt thereof, in particular: The molecular scaffold is TBMT, the bicyclic peptide ligand further comprises an N-terminal addition and / or a C-terminal addition, and A-(SEQ ID NO:62)-A (referred to herein as BCY16493); Ac- (SEQ ID NO:62) (referred to herein as BCY16495); A-(SEQ ID NO:62)-A-[K(PYA)] (referred to herein as BCY13886); A-(SEQ ID NO:244)-A-[K(PYA)] (referred to herein as BCY16165); A-(SEQ ID NO:245)-A (referred to herein as BCY16498); A-(SEQ ID NO:246)-A (referred to herein as BCY16499); A-(SEQ ID NO:247)-A (referred to herein as BCY16501); A-(SEQ ID NO:248)-A (referred to herein as BCY16502); A-(SEQ ID NO:249)-A (referred to herein as BCY16504); A-(SEQ ID NO:250)-A (referred to herein as BCY16505); A-(SEQ ID NO:251)-A (referred to herein as BCY16506); A-(SEQ ID NO:252)-A (referred to herein as BCY16507); A-(SEQ ID NO:253)-A (referred to herein as BCY16508); A-(SEQ ID NO:254)-A (referred to herein as BCY16509); A-(SEQ ID NO:255)-A (referred to herein as BCY16510); A-(SEQ ID NO:256)-A (referred to herein as BCY16511); A-(SEQ ID NO:257)-A (referred to herein as BCY16512); A-(SEQ ID NO:258)-A (referred to herein as BCY16520); A-(SEQ ID NO:259)-A (referred to herein as BCY16522); A-(SEQ ID NO:260)-A (referred to herein as BCY16523); A-(SEQ ID NO:261)-A (referred to herein as BCY16524); A-(SEQ ID NO:262)-A (referred to herein as BCY16527); A-(SEQ ID NO:263)-A (referred to herein as BCY16528); and A-(SEQ ID NO:264)-A (referred to herein as BCY16529), (wherein PYA represents pentynic acid).

16. 16. The peptide ligand of any one of claims 1 to 15, wherein the pharma- ceutically acceptable salt is selected from the free acid or a sodium, potassium, calcium, or ammonium salt.

17. A multimeric binding complex comprising at least two bicyclic peptide ligands according to any one of claims 1 to 16, said peptide ligands being the same or different.

18. 18. The multimeric binding complex of claim 17, comprising an NKp46 homodimer selected from BCY15663, BCY15921, BCY15922, BCY15455, and BCY18043.

19. 18. The multimeric binding complex of claim 17, comprising a CD16a homodimer selected from BCY15914, BCY15915, and BCY15916.

20. 20. A pharmaceutical composition comprising a peptide ligand according to any one of claims 1 to 16, or a multimeric binding complex according to any one of claims 17 to 19, in combination with one or more pharma- ceutically acceptable excipients.

21. A peptide ligand according to any one of claims 1 to 16, or a multimeric binding complex according to any one of claims 17 to 19, or a pharmaceutical composition according to claim 20, for use in the prevention, inhibition or treatment of a natural killer (NK) cell mediated disease or disorder, such as an inflammatory disorder, an autoimmune disease and cancer.

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