Bicyclic peptide ligands specific for NK cells

Abstract

The present invention relates to polypeptides covalently bound to a molecular backbone such that two or more peptide rings are present oppositely between the points of connection to the backbone. In particular, the present invention describes peptides that bind to natural killer (NK) cells. The invention also includes multimeric binding complexes comprising at least two of the bicyclic peptide ligands. The invention also includes a pharmaceutical composition comprising the peptide ligand or the polymer binding complex, and the use of the peptide ligands, multimer binding complexes and pharmaceutical compositions in the detection of diseases and disorders mediated by natural killer (NK) cells, and in the prevention, inhibition or treatment of diseases or disorders mediated by natural killer (NK) cells, such as inflammatory disorders, autoimmune diseases and cancer.

Description

[0001] TECHNICAL FIELD

[0002] The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide loops are present in an antiparallel orientation between the points of attachment to the scaffold. In particular, the present invention describes peptides that bind to natural killer (NK) cells. The present invention also includes multimeric binding complexes comprising at least two of said bicyclic peptide ligands. The present invention also includes pharmaceutical compositions comprising said peptide ligands or said multimeric binding complexes, and the use of said peptide ligands, multimeric binding complexes and pharmaceutical compositions in the detection of, and in the prevention, inhibition or treatment of diseases and disorders mediated by natural killer (NK) cells, such as inflammatory disorders, autoimmune diseases and cancer. BACKGROUND

[0004] Cyclic peptides are able to bind to protein targets with high affinity and specificity and are therefore an attractive class of molecules for the development of therapeutics. In fact, several cyclic peptides have been successfully used in the clinic, e.g. the antibacterial peptide vancomycin, the immunosuppressive drug cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat. Rev. Drug. Discov. 7 Good binding properties result from the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, large rings bind to surfaces of several hundred square angstroms, e.g. the cyclic peptide CXCR4 antagonist CVX15 (400 A 2 ; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide binding to integrin aVb3 with the Arg-Gly-Asp motif (355 A 2 ) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A 2 ; Zhao et al. (2007), J. Struct. Biol. 160(1), 1-10).

[0005] Due to their cyclic conformation, peptides are less flexible than linear peptides, resulting in a smaller entropic loss upon binding to a target, and leading to higher binding affinities. The reduced flexibility also results in locking a target-specific conformation, increasing binding specificity compared to linear peptides. This effect has been exemplified by potent and selective inhibitors of matrix metalloproteinase 8 (MMP-8), which lose their selectivity over other MMPs when their 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 more than one peptide ring, such as in vancomycin, nisin and actinomycin.

[0006] Different research groups have previously tethered polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues have used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds to mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). A method for generating drug candidate compounds, wherein the compounds are generated by linking a cysteine-containing polypeptide to a molecular scaffold (e.g. 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).

[0007] A phage display-based combinatorial approach has been developed to generate and screen large libraries of bicyclic peptides against a target of interest (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine residues and two regions of six random amino acids each (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine side chains to a small molecular scaffold.

[0008] Natural killer (NK) cells are members of the innate immune system. Numerous studies have provided evidence regarding the central role of NK cell receptors in natural cytotoxicity and their potential applications in cancer therapy. New agents capable of binding to NK cells are needed, particularly to guide NK cell activity in the context of mediating immune responses to combat barriers such as cancer, inflammatory diseases, and autoimmune disorders. Invention Overview

[0010] This article provides peptide ligands comprising polypeptides having an amino acid sequence as follows:

[0011] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1);

[0012] or its modified derivatives, wherein

[0013] Where X represents dNva or dA; where dNva represents D-valine, dA represents D-alanine, dC represents D-cysteine, and Cba represents cyclobutylalanine;

[0014] Or its pharmaceutically acceptable salt.

[0015] In some embodiments, the peptide ligand or a pharmaceutically acceptable salt thereof comprises the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the peptide ligand or a pharmaceutically acceptable salt thereof comprises one or more N-terminal and / or C-terminal additions. In some embodiments, the peptide ligand or a pharmaceutically acceptable salt thereof comprises the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the C-terminus is amidated. In some embodiments, the polypeptide of the peptide ligand is linked to a molecular backbone, such as TATA or a derivative thereof as defined herein. In some embodiments, the peptide ligand is specific for natural killer (NK) cells.

[0016] According to another aspect of the invention, a peptide ligand specific to natural killer (NK) cells is provided, comprising an amino acid sequence, said amino acid sequence being:

[0017] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1);

[0018] Where X represents dNva or dA, and Cba represents cyclobutylalanine and Nva represents valine.

[0019] Or its pharmaceutically acceptable salt.

[0020] According to another aspect of the invention, a bicyclic peptide ligand is provided, comprising a peptide ligand and a molecular backbone as defined herein, wherein three cysteine ​​residues of the peptide ligand are covalently bonded to the molecular backbone to form two cyclic sequences.

[0021] According to another aspect of the invention, a multimeric binding complex is provided comprising at least two bicyclic peptide ligands, wherein at least one bicyclic peptide ligand is as defined herein, and wherein the peptide ligands may be the same or different. According to another aspect of the invention, a multimeric binding complex is provided comprising at least two bicyclic peptide ligands as defined herein, wherein the peptide ligands may be the same or different. In some embodiments, the multimeric binding complex further comprises one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores. In one embodiment, the multimeric binding complex comprises a fluorophore.

[0022] According to another aspect of the invention, a heterotandem bicyclic peptide complex is provided, comprising: (a) a first bicyclic peptide ligand capable of binding to a component present on a cancer cell; the first bicyclic peptide ligand being linked to (b) one or more second bicyclic peptide ligands as defined herein, wherein the one or more second bicyclic peptide ligands may be the same or different. According to another aspect of the invention, a heterotandem bicyclic peptide complex is provided, comprising:

[0023] (a) A first bicyclic peptide ligand that binds to a component present on cancer cells; the first bicyclic peptide ligand is conjugated to via a linker.

[0024] (b) One or more second bicyclic peptide ligands that are specific to natural killer (NK) cells as defined herein.

[0025] In some embodiments, the linker is a straight-chain linker or a branched linker. In some embodiments, the linker contains three or four branches and is capable of binding to three or four bicyclic peptide ligands. In some embodiments, the linker contains one or more PEG groups, such as one or more PEGs. nThe linker comprises a group, wherein each n is independently an integer from about 2 to about 25. In some embodiments, the linker comprises one or more azide groups capable of reacting with one or more alkyne groups of one or more peptide ligands; and / or one or more carboxylic acid groups or activated derivatives thereof capable of reacting with one or more amine groups of one or more peptide ligands. In some embodiments, the first bicyclic peptide ligand binds to EphA2, Nectin-4, PD-L1, MT1, or PSMA. In some embodiments, the first bicyclic peptide ligand binds to EphA2 and comprises a polypeptide of SEQ ID NO: 6 or 7. In some embodiments, the polypeptide is linked to a molecular backbone, such as TATA or a derivative thereof as defined herein. In some embodiments, the heterotandem bicyclic peptide complex is BCY27047 or a pharmaceutically acceptable salt thereof, or BCY26129 or a pharmaceutically acceptable salt thereof.

[0026] According to another aspect of the invention, a pharmaceutical composition is provided comprising a combination of a peptide ligand, a bicyclic peptide ligand, a multimeric binding complex or a heterotandem binding complex as defined herein, and one or more pharmaceutically acceptable excipients.

[0027] According to another aspect of the invention, peptide ligands, bicyclic peptide ligands, multimeric binding complexes, heterotandem binding complexes, or pharmaceutical compositions as defined herein are provided for the prevention, inhibition, or treatment of diseases or disorders mediated by natural killer (NK) cells. Brief description of the attached diagram

[0029] Figure 1 : AF647-labeled NKp46 bicyclic dimer (BCY28671) binds to isolated NK cells.

[0030] Figure 2 : AF647-labeled NKp46 bicyclic dimer (BCY28672) binds to isolated NK cells.

[0031] Figure 3 The binding of AF647-labeled NKp46 bicyclic dimer (BCY28671) in PBMC preparation to CD56+ cells.

[0032] Figure 4 The binding of AF647-labeled NKp46 bicyclic dimer (BCY28672) in PBMC preparation to CD56+ cells.

[0033] Figure 5 Results of NK:A431-luc cytotoxicity assay for BCY27047.

[0034] Figure 6Results of NK:A431-luc cytotoxicity assay for BCY26129.

[0035] Figure 7 Results of NK:HT1080-luc cytotoxicity assay for BCY27047.

[0036] Figure 8 Results of NK:HT1080-luc cytotoxicity assay for BCY26129.

[0037] Figure 9 IFN of BCY27047 Secretion test results.

[0038] Figure 10 IFN of BCY26129 Secretion test results.

[0039] Figure 11 Results of TNFα secretion assay for BCY27047.

[0040] Figure 12 Results of TNFα secretion assay for BCY26129. Invention Details

[0042] This invention provides peptides capable of binding to natural killer (NK) cells. The NK cell-binding peptides provided herein can be contained, for example, in peptide ligands, which contain the peptide covalently bound to a molecular backbone (e.g., a molecular backbone as described in more detail herein) such that two or more peptide loops are present facing each other between the linker sites with the backbone. The peptide ligand can be directly or indirectly (e.g., via a linker) linked to another peptide ligand (i.e., two or more peptide ligands, which may be the same or different, can be linked together) to form a complex as described in more detail herein. For example, one or more peptide ligands containing the NK cell-binding peptides described herein can be linked to one or more additional peptide ligands that bind to another biological target, such as EphA2, Nectin-4, PD-L1, MT1, or PSAM (especially EphA2). Peptides, peptide ligands, or complexes containing multiple peptide ligands can be directly or indirectly (e.g., via a linker) linked to one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores.

[0043] Therefore, in one embodiment, this document provides a peptide ligand comprising a polypeptide having an amino acid sequence as follows:

[0044] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1)

[0045] or its modified derivatives, wherein

[0046] X represents dNva or dA;

[0047] Where dNva represents D-valine, dA represents D-alanine, dC represents D-cysteine, and Cba represents cyclobutylalanine;

[0048] Or its pharmaceutically acceptable salt.

[0049] In some implementations, the peptide is able to bind to NK cells. In some implementations, the peptide is specific to NK cells.

[0050] Natural killer (NK) cells are members of the innate immune system, representing a small fraction of peripheral blood mononuclear cells. As frontline responders, these immune cells detect and eliminate unhealthy cells and link innate and adaptive immune responses. Due to their inherent properties, NK cells are excellent candidates for therapeutic tools in enhancing immuno-oncology and autoimmunity. NK cells are responsible for immune surveillance via a variety of inhibitory and activating receptors. These activating and inhibitory receptors on the surface of NK cells form a complex mechanism by which NK cell activity is balanced in healthy individuals. NK cells recognize MHC class I molecules on the surface of healthy cells and are constrained from eliminating these healthy cells by inhibitory receptors. In response to stress, infection, or transformation, NK cells recognize unhealthy cells through the loss of MHC class I molecules on the cell surface and the induction of NK cell receptor ligands that bind to activating receptors. NK cell recognition of non-self cells triggers a cytotoxic response, releasing cytokines and cytotoxic molecules to eliminate unhealthy cells.

[0051] NK cell activity is achieved through a complex mechanism involving both activation and inhibition signals. Numerous reports have provided evidence of the central role of NK cell receptors in natural cytotoxicity and their potential applications in cancer therapy. There is an unmet need for further understanding and enhancement of NK cell-mediated tumor cell recognition and killing. Reports indicate that tumor cells utilize numerous mechanisms to reduce NK cell activity, and that the presence and efficacy of NK cells are associated with favorable patient outcomes (Pasero et al. (2015) Oncotarget 6(16), 14360-14373, Stringaris et al. (2014) Haematologica 99(5), 836-847). Through therapeutic interventions, the potential role of NK cells in mediating immune responses can be utilized to combat cancer and autoimmune diseases.

[0052] NKp46 binding bicyclic peptides

[0053] In one embodiment, the bicyclic peptide is specific to (i.e., binds to) a natural cytotoxic receptor present on the surface of NK cells. Such a receptor may, in some embodiments, comprise or define an epitope capable of being selectively bound by the provided peptide and a ligand containing it. In another embodiment, the bicyclic peptide is specific to (i.e. binds to) a natural cytotoxic receptor selected from NKp30, NKp44, and NKp46. In yet another embodiment, the bicyclic peptide is specific to (i.e. binds to) NKp46.

[0054] Naturally occurring cytotoxic receptors (NCRs) are a family of stimulatory receptors expressed on the surface of NK cells that trigger NK cell activation and cell-mediated cytotoxicity. The NCR family consists of three members: NKp30, NKp44, and NKp46. Although the cellular ligand of NKp46 is unknown, its role in antitumor immunity has been demonstrated. NKp46 activation mediated by viral antigens on NK cells leads to tumor rejection (Chinnery et al., 2012). Upon interaction with its ligand, the NKp46 receptor triggers NK cell-induced targeted cytotoxicity, as illustrated by the ability of NK cells to lyse their targets using anti-NKp46 blocking antibodies (Arnon et al., 2004). The level of NCR expression on the NK cell surface also enhances NK cell cytotoxicity. A strong correlation has been established between NCR expression density and the ability of NK cells to kill target cells, including various tumor cells (Moretta et al., 2006). In AML and cervical cancer and precursor lesions, insufficient NCR or NCR ligand levels confuse tumor cells with NK cell cytotoxicity (Costello et al., 2002; Garcia-Iglesias et al., 2009). In many solid tumors, NK cells are regulated by the tumor microenvironment, including tumor shedding and immune editing of NCR ligands, which prevents NK cells from recognizing, infiltrating, and killing 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 NK cell cytotoxicity from AML patients. Furthermore, in solid cancers such as prostate cancer, decreased expression of several activating receptors (CD16, NKp30, NKp46, NKG2D, and DNAM-1) and increased expression of the inhibitory receptor CD85j have been reported (Pesaro et al., 2016). In contrast, Gautheir et al. (2019) identified NKp46 as a promising candidate for targeting activating receptors on NK cells in cancer, demonstrating that peripheral NKp46 was not statistically significantly downregulated in patients with SCCHN, breast cancer, liver cancer, lung cancer, kidney cancer, and metastatic melanoma.Furthermore, persistent NKp46 expression has been reported in various solid tumors, including cancers such as acute myeloid leukemia, breast cancer, and lung cancer, accompanied by downregulation of other activating receptors such as NKG2D, NKp30, and NKp44, and low CD16 expression on tumor-infiltrating lymphocytes (Fauriat et al. 2007, Mamessier et al. 2011, Platonova et al. 2011, Levi et al. 2015, Kim et al. 2010, MacFarlane et al. 2017). Therefore, NKp46 has been shown to be a specific NK surface marker suitable for therapeutic applications to recognize NK cells and target them to tumors. Accordingly, in some embodiments, the provided peptide and ligands containing such peptides (described in more detail herein) bind to one or more of NKp30, NKp44, and NKp46, such as NKp46. In some embodiments, the provided peptide and ligands containing such peptides (described in more detail herein) are specific for one or more of NKp30, NKp44, and NKp46, such as NKp46.

[0055] As described above, this article provides peptide ligands comprising a polypeptide having an amino acid sequence as follows:

[0056] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1)

[0057] or its modified derivatives,

[0058] Where X represents dNva or dA.

[0059] Where dNva represents D-valine, dA represents D-alanine, dC represents D-cysteine, and Cba represents cyclobutylalanine;

[0060] Or its pharmaceutically acceptable salt.

[0061] In some implementations, X is dNva, and the peptide therefore comprises an amino acid sequence:

[0062] [dC]Y[Cba]PDYLC[dNva]DEYC (SEQ ID NO: 2)

[0063] Or its modified derivatives, or its salts. In some embodiments, X is dA, and the peptide therefore comprises an amino acid sequence.

[0064] [dC]Y[Cba]PDYLC[dA]DEYC (SEQ ID NO: 3)

[0065] Or its modified derivatives, or its salts.

[0066] According to a first aspect of the invention, a peptide ligand specific to natural killer (NK) cells is provided, comprising an amino acid sequence, said amino acid sequence being:

[0067] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1);

[0068] Where X represents dNva or dA, and Cba represents cyclobutylalanine and Nva represents valine.

[0069] Or its pharmaceutically acceptable salt.

[0070] In one implementation, X represents dNva and the peptide ligand contains the sequence:

[0071] [dC]Y[Cba]PDYLC[dNva]DEYC (SEQ ID NO: 2);

[0072] Nva represents valine.

[0073] Or its pharmaceutically acceptable salt.

[0074] In an alternative implementation, X represents [dA] and the peptide ligand comprises the sequence:

[0075] [dC]Y[Cba]PDYLC[dA]DEYC (SEQ ID NO: 3);

[0076] Cba represents cyclobutylalanine.

[0077] Or its pharmaceutically acceptable salt.

[0078] In some embodiments, the peptide comprising an amino acid sequence or a salt thereof is extended at the N-terminus, said amino acid sequence being:

[0079] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1)

[0080] Or its modified derivatives. In some embodiments, the peptide is extended at the N-terminus with one or more (e.g., 1 to 10, 1 to 5, e.g., 1, 2, 3, 4, or 5) additional amino acids or their analogues. In some embodiments, the peptide is extended at the C-terminus. In some embodiments, the peptide is extended at the C-terminus with one or more (e.g., 1 to 10, 1 to 5, e.g., 1, 2, 3, 4, or 5) additional amino acids or their analogues. In some embodiments, the peptide is extended at both the N-terminus and C-terminus. In some embodiments, the peptide is extended at both the N-terminus and C-terminus with one or more (e.g., 1 to 10, 1 to 5, e.g., 1, 2, 3, 4, or 5) additional amino acids or their analogues.

[0081] In some embodiments, the peptide is modified at the N-terminus and / or C-terminus. In some embodiments, the peptide is modified at both the N-terminus and C-terminus. Examples of N-terminal modification include N-terminal acetylation, denoted by "Ac". Examples of C-terminal modification include extending the peptide by one or more amino acids or amino acid analogs such as lysine (K) or variants thereof. An example of a lysine variant is K(PYA), where PYA represents pentynyl acid (e.g., 4-pentynyl acid). In some embodiments, K(PYA) has the following structure (e.g., before being linked to, for example, a linker, such as an azide group contained in the linker):

[0082] .

[0083] An example of C-terminal modification is amidation; that is, the transformation of the C-terminal carboxylic acid group (-C(O)OH or -C(O)O). - It is converted to amide (-C(O)NH2). In some embodiments, the C-terminus of the peptide is amidated.

[0084] An example of a peptide modified at both the N and C ends is SEQ ID NO: 4; and an example of a peptide modified at both the N and C ends is SEQ ID NO: 5.

[0085] In one embodiment, the peptide ligand further comprises an N-terminal and / or C-terminal addition and includes an amino acid sequence selected from:

[0086] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4), and

[0087] Ac-([dC]Y[Cba]PDYLC[dA]DEYC)-[K(PYA)] (SEQ ID NO: 5),

[0088] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0089] Or its pharmaceutically acceptable salt.

[0090] In another embodiment, the peptide ligand further comprises an N-terminal and / or C-terminal addition and contains an amino acid sequence, said amino acid sequence being:

[0091] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4),

[0092] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0093] Or its pharmaceutically acceptable salt.

[0094] In some embodiments, the peptide ligands provided herein comprise a peptide, as provided herein, linked to a molecular backbone. The molecular backbone can be any molecular backbone as described in more detail herein. In some embodiments, the molecular backbone is linked to one or more reactive groups in the peptide. In some embodiments, the molecular backbone is linked to one or more reactive groups in the peptide, such as linked to one or more cysteine ​​groups (e.g., linked to a thiol group contained in a cysteine ​​side chain). In some embodiments, the peptide comprises three cysteine ​​residues (L-cysteine ​​and / or D-cysteine), and the molecular backbone is linked to three cystine groups.

[0095] According to another aspect of the invention, a bicyclic peptide ligand is provided, comprising a peptide ligand and a molecular backbone as defined herein, wherein three cysteine ​​residues of the peptide ligand are covalently bonded to the molecular backbone to form two cyclic sequences.

[0096] An example of a molecule suitable for use as a molecular backbone in peptide ligands as presented herein and in complexes containing them is TATA (1,3,5-triacryloylhexahydro-1,3,5-triazine, available from Sigma Aldrich). TATA has the following structure:

[0097] .

[0098] In some embodiments, TATA reacts with the cysteine-thiol group of a peptide as described herein to form a peptide ligand comprising a backbone, said backbone being a derivative of TATA in the following forms:

[0099]

[0100] Each Cys-S represents a cysteine ​​residue (e.g., L-cysteine ​​or D-cysteine).

[0101] Therefore, in one embodiment, the molecular backbone is a derivative of TATA having the following structure:

[0102] ,

[0103] It can also be described as

[0104]

[0105] in This indicates the junction of three cysteine ​​residues.

[0106] In one embodiment, this document provides a bicyclic peptide ligand comprising a peptide of SEQ ID NO: 1 or a pharmaceutically acceptable salt thereof, said peptide optionally extended at an N-terminus and / or a C-terminus and bonded to a molecular backbone at each of three reactive residues (e.g., at each of three cysteine ​​residues (e.g., L-cysteine ​​or D-cysteine)), said molecular backbone being a derivative of TATA as described herein. In one embodiment, said bicyclic ligand comprises the peptide sequence of SEQ ID NO: 2, 3, 4, or 5. When the bicyclic ligand comprises SEQ ID NO: 4, the ligand may be referred to as BCY26631. When the bicyclic ligand comprises SEQ ID NO: 5, the ligand may be referred to as BCY24188.

[0107] In one embodiment, the bicyclic peptide comprises a molecular backbone that is a derivative of TATA having the following structure:

[0108] ,

[0109] in This indicates a linking point for three cysteine ​​residues, and the peptide ligand comprises an amino acid sequence selected from the following:

[0110] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (its bicyclic peptide is referred to herein as BCY26631), and

[0111] Ac-([dC]Y[Cba]PDYLC[dA]DEYC)-[K(PYA)] (SEQ ID NO: 5) (its bicyclic peptide is referred to in this article as BCY24188),

[0112] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0113] Or its pharmaceutically acceptable salt.

[0114] In another embodiment, the bicyclic peptide comprises a molecular backbone, said molecular backbone being a derivative of TATA having the following structure:

[0115] ,

[0116] in The linker represents a junction of three cysteine ​​residues, and the peptide ligand comprises an amino acid sequence as follows:

[0117] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (its bicyclic peptide is referred to herein as BCY26631), and

[0118] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0119] Or its pharmaceutically acceptable salt.

[0120] In another embodiment, the pharmaceutically acceptable salt is selected from free acids or sodium, potassium, calcium or ammonium salts.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques used in 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.), are incorporated herein by reference.

[0122] Multimer binding complexes

[0123] According to another aspect of the invention, a multimeric binding complex is provided comprising at least two bicyclic peptide ligands (e.g., 2, 3, or 4 bicyclic peptide ligands), said bicyclic peptide ligands may be the same or different, wherein at least one bicyclic peptide ligand is a peptide ligand as defined herein. According to another aspect of the invention, a multimeric binding complex is provided comprising at least two bicyclic peptide ligands as defined herein, said peptide ligands may be the same or different. In some embodiments, the multimeric binding complex comprises, for example, 2, 3, or 4 bicyclic peptide ligands as defined herein, said peptide ligands may be the same or different.

[0124] In one embodiment, the multimeric binding complex comprises more than one identical bicyclic peptide (i.e., homomeric). In an alternative embodiment, the multimeric binding complex comprises different bicyclic peptides (i.e., heteromeric). In one embodiment, the multimeric binding complex comprises at least two identical bicyclic peptide ligands and at least one different bicyclic peptide. In some embodiments, the multimeric binding complex comprises (a) two identical bicyclic peptide ligands and (b) one or two additional bicyclic peptide ligands that may be the same or different, and wherein the two additional bicyclic peptide ligands in (b) may be the same or different from the two bicyclic peptide ligands in (a).

[0125] In one embodiment, the multimeric binding complex further comprises one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores. In one embodiment, the multimeric binding complex further comprises a fluorophore.

[0126] In one embodiment, the multimer binding complex comprises two identical bicyclic peptides (i.e., homodimers).

[0127] Effector groups

[0128] As will be apparent from the discussion herein, in some embodiments, peptide ligands, bicyclic peptide ligands, multimeric binding complexes, or heterotandem binding complexes as described herein may comprise one or more effector groups and / or functional groups, such as one or more cytotoxic agents, chelating agents (e.g., radiochelating agents), chromophores, and / or fluorophores. In some embodiments, peptide ligands, bicyclic peptide ligands, multimeric binding complexes, or heterotandem binding complexes as described herein may comprise one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelating agents, chromophores, and / or fluorophores linked to the ligand or complex via a linker portion. Any suitable linker portion may be used. In some embodiments, the linker for linking one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelating agents, chromophores, and / or fluorophores, may be the linker as described herein.

[0129] In some embodiments, one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores, are directly linked to peptide ligands as described herein. In some embodiments, one or more effector groups and / or functional groups, such as one or more cytotoxic agents, radiochelates, or chromophores, are linked to a complex comprising at least two peptide ligands as described herein, and to a linker that links two or more peptide ligands together.

[0130] In some embodiments, one or more effector groups and / or functional groups include one or more chelating agents, such as one or more metal ion chelating agents. Any suitable metal ion chelating agent can be used. In some embodiments, the chelating agent is a chelating agent for a radioactive isotope (i.e., a radioactive chelating agent).

[0131] In some embodiments, the fluorophore is linked to a peptide ligand, bicyclic peptide ligand, multimeric binding complex, or heterotandem binding complex as described herein. In some embodiments, the fluorophore is a fluorescent dye. In some embodiments, the fluorescent dye comprises a reactive group (e.g., a carboxylic acid group or an activated derivative thereof) for reacting with a peptide ligand, bicyclic peptide ligand, multimeric binding complex, or heterotandem binding complex as described herein; for example, for reacting with a linker contained in the complex described herein. In some implementations, the fluorophore is selected from: Alexa Fluor (AF) dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, and Alexa Fluor 680), AMCA, AMCA-S, and BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 581 / 59 ... 650 / 665), carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, coumarin 343, cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Cy7.5), dansyl sulfonyl, dapoxyl, dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethoxyfluorescein, DM-NERF, eosin, erythrosine, fluorescein, FAM, hydroxycoumarin, IRDyes (IRD40, IRD700, IRD800), JOE, lissaminorhodamine B, MarinaBlue, methoxycoumarin, naphthalenefluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-Tetrabromosulfone-fluorescein, Tetramethyl-Rhodamine (TMR), CarboxytetramethylRhodamine (TAMRA), Texas Red, and Texas Red-X.

[0132] In some embodiments, peptide ligands, bicyclic peptide ligands, multimeric binding complexes, or heterotandem binding complexes as described herein comprise one or more effector groups and / or functional groups, such as fluorophores, as described herein, and can be used to detect diseases and disorders in a subject, such as diseases or disorders mediated by natural killer (NK) cells. Exemplary disorders are described in more detail herein. In some embodiments, the ligand or complex binds to a target, such as NK cells or binding sites thereon, in a subject who has or is suspected of having a disease or disorder as described herein. In some embodiments, the binding of the ligand or complex is detectable, for example, by detecting an effector group (e.g., a fluorophore). In some embodiments, compounds are provided herein that are peptide ligands, bicyclic peptide ligands, multimeric binding complexes, or heterotandem binding complexes as described herein, comprising one or more effector groups and / or functional groups, such as fluorophores, as described herein, for use in methods of diagnosing a subject's pathology; in some embodiments, the pathology is a disease or disorder associated with NK cells (e.g., a disease or disorder as described herein). In some embodiments, the method includes contacting the compound or a composition containing the compound with a sample (e.g., an in vitro, ex vivo, or in vivo sample) and detecting pathology-specific changes in the sample in the presence of the compound or composition. The method may, for example, include detecting the fluorescence of a fluorescently labeled compound as described herein. Therefore, the provided compounds can be used to develop and perform assays, immunofluorescence staining, etc.

[0133] Hetero-serial binding complexes

[0134] According to another aspect of the invention, a heterotandem bicyclic peptide complex is provided herein comprising (i) one or more first bicyclic peptide ligands; said one or more first bicyclic peptide ligands are linked to (ii) one or more second bicyclic peptide ligands, wherein said one or more second bicyclic peptide ligands are one or more bicyclic peptide ligands as provided herein. The second bicyclic peptide ligands provided herein, or each of the second bicyclic peptide ligands provided herein, are generally specific to natural killer (NK) cells. The one or more first bicyclic peptide ligands generally bind to one or more additional biological targets and are generally not specific to NK cells. For example, said one or more first bicyclic peptide ligands may bind to one or more cancer cells (e.g., to one or more components (e.g., epitopes) present on or expressed by one or more cancer cells). When said heterotandem bicyclic peptide complex comprises two or more first bicyclic peptide ligands, said two or more first bicyclic peptide ligands may be the same or different. The two or more first bicyclic peptide ligands may be specific to the same biological target or to different targets.

[0135] Heterotandem bicyclic peptide complexes may include one or more linkers. The linker can link (i) one or more first bicyclic peptide ligands to one or more second bicyclic peptide ligands as provided herein. Any suitable linker can be used. Some exemplary linkers are described in more detail herein. Thus, in some embodiments, heterotandem bicyclic peptide complexes are provided herein comprising (i) one or more first bicyclic peptide ligands; said one or more first bicyclic peptide ligands are linked via linkers to (ii) one or more second bicyclic peptide ligands as provided herein.

[0136] According to another aspect of the present invention, a heterotandem bicyclic peptide complex is provided, comprising:

[0137] (a) A first bicyclic peptide ligand that binds to a component present on cancer cells; the first bicyclic peptide ligand is conjugated to via a linker.

[0138] (b) One or more second bicyclic peptide ligands that are specific to natural killer (NK) cells as defined herein.

[0139] First bicyclic peptide ligand

[0140] The term "cancer cell" as used in this article includes any cell known to be involved in cancer. Cancer cells arise when the genes responsible for regulating cell division are damaged. Carcinogenesis is caused by mutations in the genetic material and epigenetic mutations of normal cells, which disrupt the normal balance between proliferation and cell death. This leads to uncontrolled cell division and the evolution of those cells through natural selection within the body. The uncontrolled and often rapid proliferation of cells can lead to benign or malignant tumors (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors can invade other organs, spread to distant locations (metastases), and become life-threatening.

[0141] In one embodiment, the cancer cells are fibrosarcoma cells, lung cancer cells, ovarian cancer cells, prostate cancer cells, bladder cancer cells, colon cancer cells, or breast cancer cells. In one embodiment, the cancer cells are selected from HT1080, A549, SC-OV-3, PC3, HT1376, NCI-H292, LnCap, MC38, MC38#13, 4T1-D02, H322, HT29, T47D, and RKO tumor cells.

[0142] In one embodiment, the first bicyclic peptide ligand comprises a polypeptide linked to a molecular backbone. In some embodiments, the molecular backbone is a derivative of TATA having the following structure:

[0143]

[0144] in This indicates the connection point with the polypeptide, such as the connection point with three cysteine ​​residues of the polypeptide.

[0145] In one implementation, the component present on the cancer cells is EphA2.

[0146] Eph receptor tyrosine kinase (Eph) belongs to a large group of receptor tyrosine kinases (RTKs)—kinases that phosphorylate proteins at tyrosine residues. Eph and its membrane-bound hepatin ligands (hepatin) control cell localization and tissue structure (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur in a higher ligand oligomerization state (Stein et al. (1998) Genes Dev 12, 667-678).

[0147] In model functions, various Eph and hepatic ligands have been shown to play a role, particularly in vascular development. Knockout of EphB4 and hepatic ligand-B2 results in a lack of ability to remodel capillary beds into blood vessels (Poliakov et al., ibid.) and embryonic lethality. Persistent expression of some Eph receptors and hepatic ligands has also been observed in newly formed adult microvessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0148] The re-emergence of dysregulation of some hepatic glycoproteins and their receptors in adults has also been observed to contribute to tumor invasion, metastasis, and angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., ibid.). Furthermore, some Eph family members have been found to be overexpressed on tumor cells from various human tumors (Brantley-Sieders et al., ibid.; Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0149] EPH receptor A2 (hepatin A receptor 2) is a receptor for liver glycoprotein A in humans. EPHA2 Proteins encoded by genes.

[0150] EphA2 is upregulated in a variety of human cancers and is generally associated with disease progression, metastasis, and poor prognosis, for example: breast (Zelinski et al. (2001) Cancer Res. 61, 2301–2306; Zhuang et al. (2010) Cancer Res. 70, 299–308; Brantley-Sieders et al. (2011) PLoS One 6, e24426), lung (Brannan et al. (2009) Cancer Prev Res (Phila) 2, 1039–1049; Kinch et al. (2003) Clin Cancer Res. 9, 613–618; Guo et al. (2013) J Thorac Oncol. 8, 301–308), stomach (Nakamura et al. (2005) Cancer Sci. 96, 42–47; Yuan et al. (2009) Dig Dis Sci. 96, 42–47). 54, 2410-2417), pancreas (Mudali et al. (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels et al. (1999) Prostate 41, 275-280), liver (Yang et al. (2009) Hepatol Res. 39, 1169-1177), and glioblastoma (Wykosky et al. (2005) Mol Cancer Res. 3, 541–551; Li et al. (2010) Tumor Biol. 31, 477–488). Despite evidence of interactions at many stages of cancer progression, including tumor cell growth, survival, invasion, and angiogenesis, the full role of EphA2 in cancer progression remains unclear. Downregulation of EphA2 expression inhibits tumor cell proliferation (Binda et al. (2012) Cancer Cell 22, 765-780), while EphA2 blockade inhibits VEGF-induced cell migration (Hess et al. (2001) Cancer Res. 61, 3250-3255), budding, and angiogenesis (Cheng et al. (2002) Mol Cancer Res.). 1 , 2 – 11 ； Lin et al. (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al. (2005) FASEB J. 19, 1884– 1886).

[0151] Antibody-drug conjugates for EphA2 have been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al. (2008) Cancer Research 68, 9367-9374), and similar approaches have been attempted in humans, but treatment had to be discontinued due to treatment-related adverse events (Annunziata et al. (2013) Invest New drugs 31, 77-84).

[0152] In one embodiment, the first bicyclic peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

[0153] Suitable examples of EphA2 binding bicyclic peptide ligands are disclosed in WO 2019 / 122860, WO 2019 / 122861 and WO2019 / 122863, the peptides of which are incorporated herein by reference.

[0154] In one embodiment, the EphA2-binding bicyclic peptide ligand comprises an amino acid sequence, said amino acid sequence being:

[0155] C[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 6);

[0156] Or its modified derivatives;

[0157] HyP represents trans-4-hydroxy-L-proline and d1Nal represents D-1-naphthylalanine, or a pharmaceutically acceptable salt thereof. In one embodiment, the EphA2-binding bicyclic peptide ligand comprises an amino acid sequence, said amino acid sequence being:

[0158] C[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 6).

[0159] In some embodiments, the EphA2-binding bicyclic peptide ligand comprises one or more N-terminal and / or C-terminal modifications. In some embodiments, the EphA2-binding ligand comprises:

[0160] A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 7)

[0161] Or its modified derivatives. In some embodiments, the EphA2 binding ligand comprises:

[0162] A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 7).

[0163] In some embodiments, the peptide of the EphA2-binding bicyclic peptide ligand is linked to a molecular backbone as disclosed herein. Thus, in some embodiments, the EphA2-binding bicyclic peptide ligand comprises a peptide linked (e.g., via three cysteine ​​residues) to a molecular backbone of SEQ ID NO: 7 (or a modified derivative thereof), said molecular backbone being a derivative of TATA as defined herein.

[0164] In another embodiment, the molecular backbone is a derivative of TATA having the following structure:

[0165] ,

[0166] in This indicates a junction of three cysteine ​​residues, and the EphA2-binding bicyclic peptide ligand optionally includes N-terminal and / or C-terminal modifications and comprises:

[0167] A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 7) (its bicyclic peptide is referred to as BCY13118 in this article),

[0168] Where HArg represents high arginine, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine.

[0169] Or its pharmaceutically acceptable salt.

[0170] In the alternative implementation, the component present on the cancer cells is Nectin-4.

[0171] Nectin-4 is a surface molecule belonging to the nectin protein family, which includes four members. Nectins are cell adhesion molecules that play crucial roles in various biological processes such as polarity, proliferation, differentiation, and migration of epithelial, endothelial, immune, and neuronal cells during development and adulthood. They are involved in several pathological processes in humans. They are major receptors for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding Nectin-1 (PVRL1) or Nectin-4 (PVRL4) cause ectodermal dysplasia syndrome, accompanied by other abnormalities. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other family members. Nectin-4 is a tumor-associated antigen found in 50%, 49%, and 86% of breast, ovarian, and lung cancers, respectively, primarily in tumors with poor prognosis. Its expression is not detected in the corresponding normal tissues. In breast tumors, Nectin-4 is primarily expressed in triple-negative and ERBB2+ carcinomas. In the serum of patients with these cancers, a soluble form of Nectin-4 was detected and associated with poor prognosis. Serum Nectin-4 levels increased during metastatic progression and decreased after treatment. These results suggest that Nectin-4 could be a reliable target for cancer treatment. Consequently, several anti-Nectin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting Nectin-4 and is currently in clinical trials for the treatment of patients with solid tumors.

[0172] In one embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand.

[0173] Suitable examples of Nectin-4 binding to bicyclic peptide ligands are disclosed in WO 2019 / 243832, the peptide of which is incorporated herein by reference.

[0174] In the alternative implementation, the component present on the cancer cells is PD-L1.

[0175] Programmed cell death 1-ligand 1 (PD-L1) is a 290-amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in immune response escapes related to chronic infections, such as chronic viral infections (including HIV, HBV, HCV, and HTLV), chronic bacterial infections (including Helicobacter pylori), and chronic parasitic infections (including Schistosoma mansoni). PD-L1 expression has been detected in numerous tissue and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, myocytes, and the placenta.

[0176] PD-L1 expression is also involved in the suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immune clearance of tumors is rare. This failure is partly due to the immunosuppression of the tumor microenvironment. PD-L1 expression on many tumors is part of this suppressive environment and works in conjunction with other immunosuppressive signals. PD-L1 expression has been observed in situ in various solid tumors, including the breast, lung, colon, ovary, melanoma, bladder, liver, saliva, stomach, glioma, thyroid, thymic epithelium, head, and neck (Brown JA et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA104:3360-65; Strome SE et al. 2003 Cancer Res. 63:6501-5; Inman BA et al. 2007 Cancer109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother). 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). Furthermore, the expression of the PD-L1 receptor programmed cell death protein 1 (also known as PD-1 and CD279) is upregulated on tumor-infiltrating lymphocytes, and this also contributes to tumor immunosuppression (Blank C et al. 2003 Immunol. 171:4574-81).Most importantly, studies linking PD-L1 expression on tumors with disease outcomes have shown a strong correlation between PD-L1 expression and poor prognosis in renal cell carcinoma, ovarian cancer, bladder cancer, breast cancer, gastric cancer, and pancreatic cancer (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta). Histochem. 108:19-24). Furthermore, these studies indicate that higher levels of PD-L1 expression on tumors can promote tumor staging progression and invasion into deeper tissue structures.

[0177] The PD-1 pathway also plays a role in hematologic malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al., 2007 Blood 110:296-304). PD-L1 is expressed in some primary T-cell lymphomas, particularly anaplastic large cell T-lymphomas (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is highly expressed on T cells in angioimmunoblastic lymphomas, and PD-L1 is expressed on associated follicular dendritic cell networks (Dorfman DM et al., 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, PD-1 is expressed on lymphocyte- or histiocyte-histocyte (L&H)-associated T cells. Microarray analysis using PD-1 ligand binding-induced gene readout showed that tumor-associated T cells respond in situ to PD-1 signaling in Hodgkin lymphoma (Chemnitz JM et al. 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma (Shimauchi T et al. 2007 Int. J. Cancer 121: 2585-90). These tumor cells are poorly responsive to TCR signaling.

[0178] Studies in animal models have demonstrated that PD-L1 on tumors inhibits T cell activation and tumor cell lysis, and in some cases leads to increased tumor-specific T cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96). Tumor-associated APCs can also utilize the PD-1:PD-L1 pathway to control anti-tumor T cell responses. PD-L1 expression in tumor-associated myeloid dendritic cells (DCs) is upregulated by tumor environmental factors (Curiel TJ et al. 2003 Nat. Med. 9:562-67). Plasma-like dendritic cells (DCs) in the tumor-draining lymph nodes of B16 melanoma express IDO, which strongly activates the inhibitory activity of regulatory T cells. The inhibitory activity of IDO-treated regulatory T cells requires contact between cells and DCs expressing IDO (Sharma MD et al. 2007 Clin. Invest. 117:2570-82).

[0179] In one embodiment, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand.

[0180] Suitable examples of PD-L1 binding bicyclic peptide ligands are disclosed in WO 2020 / 128526 and WO 2020 / 128527, the peptides of which are incorporated herein by reference.

[0181] In an alternative embodiment, the component present on cancer cells is membrane-type matrix metallopeptidase 14 (MT1, also known as MMP14). MT1-MMP is a transmembrane metalloproteinase that plays a major role in extracellular matrix remodeling, both directly by degrading several extracellular matrix components and indirectly by activating MMP2 prozymogen. MT1-MMP is crucial for tumor angiogenesis (Sounni et al. (2002) FASEB J. 16(6), 555-564) and is overexpressed in a variety of solid tumors; therefore, the MT1-MMP-binding bicyclic peptide of the present invention has particular utility in the targeted therapy of cancer, particularly solid tumors such as non-small cell lung cancer. In one embodiment, the bicyclic peptide of the present invention is specific to human MT1-MMP. In another embodiment, the bicyclic peptide of the present invention is specific to mouse MT1-MMP. In yet another embodiment, the bicyclic peptide of the present invention is specific to both human and mouse MT1-MMP. In yet another embodiment, the bicyclic peptide of the present invention is specific to human, mouse, and dog MT1-MMP.

[0182] Suitable examples of MT1 binding to bicyclic peptide ligands are disclosed in WO 2016 / 067035, the peptide of which is incorporated herein by reference.

[0183] In the alternative implementation, the component present on the cancer cells is prostate-specific membrane antigen (PSMA).

[0184] Prostate-specific membrane antigen (PSMA) (also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartate-L-glutamate peptidase I (NAALADase I), and NAAG peptidase) is a protein synthesized in humans by... FOLH1 The (folic acid hydrolase 1) gene encodes an enzyme. Human GCPII contains 750 amino acids and has a molecular weight of approximately 84 kDa.

[0185] Human PSMA is highly expressed in the prostate, approximately 100 times higher than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, with levels 8 to 12 times higher than in non-cancerous prostate cells. Due to this high expression, PSMA is being developed as a potential biomarker for the treatment and imaging of some cancers. In human prostate cancer, higher expression in tumors is associated with faster progression and a larger percentage of patients experiencing recurrence.

[0186] In one embodiment, the first peptide ligand comprises a PSMA-binding bicyclic peptide ligand.

[0187] Suitable examples of PSMA binding to bicyclic peptide ligands are disclosed in WO 2019 / 243455 and WO 2020 / 120980, the peptides of which are incorporated herein by reference.

[0188] Second peptide ligand

[0189] It should be understood that in some embodiments, one or more second peptide ligands are required to bind to one or more components present on natural killer (NK) cells. It should also be understood that when more than one second peptide ligand is present, the second peptide ligand can bind to the same or different targets within the NK cell. Therefore, in one embodiment, the second bicyclic peptide ligand is specific to the same target within the NK cell. In another embodiment, the heterotandem bicyclic peptide complex comprises at least two identical second bicyclic peptide ligands. "Identical" means second bicyclic peptides having the same amino acid sequence; most importantly, the same amino acid sequence refers to the binding portion of the second bicyclic peptide (e.g., the sequence linking position can vary). In this embodiment, each of the second bicyclic peptides within the heterotandem bicyclic peptide complex will bind to the exact same epitope on the same target of the NK cell—the resulting target-binding complex will thus produce a homodimer (if the heterotandem bicyclic peptide complex comprises two identical second bicyclic peptides), a homotrimer (if the heterotandem bicyclic peptide complex comprises three identical second bicyclic peptides), or a homotetramer (if the heterotandem bicyclic peptide complex comprises four identical second bicyclic peptides), etc.

[0190] In an alternative embodiment, the heterotandem bicyclic peptide complex comprises at least two distinct second bicyclic peptide ligands. "Distinct" means a second bicyclic peptide with a different amino acid sequence. In this embodiment, the distinct second bicyclic peptide ligands within the heterotandem bicyclic peptide complex will typically bind to distinct epitopes on NK cells—in some embodiments, the resulting target-binding complex will thus produce a double complementary site (if the heterotandem bicyclic peptide complex comprises two distinct second bicyclic peptides), a triple complementary site (if the heterotandem bicyclic peptide complex comprises three distinct second bicyclic peptides), or a quadruple complementary site (if the heterotandem bicyclic peptide complex comprises four distinct second bicyclic peptides), etc.

[0191] Unbound by theory, it is assumed that the resulting heterotandem bicyclic peptide complex can activate receptors by heterocrosslinking different targets, such as different target receptors on NK cells. Therefore, in one embodiment, the second bicyclic peptide ligand is specific to different targets on NK cells. It should be understood that in this embodiment, the heterotandem bicyclic peptide complex contains at least two different second bicyclic peptide ligands (i.e., second bicyclic peptide ligands with different amino acid sequences). In this embodiment, each of the second bicyclic peptides within the heterotandem bicyclic peptide complex will typically bind to a different epitope on the NK cell—the resulting target-binding complex will thus produce bispecific heterotandem bicyclic peptide complexes (if the heterotandem bicyclic peptide complex contains two different second bicyclic peptides), trispecific multimeric binding complexes (if the heterotandem bicyclic peptide complex contains three different second bicyclic peptides), tetraspecific heterotandem bicyclic peptide complexes (if the heterotandem bicyclic peptide complex contains four different second bicyclic peptides), and so on.

[0192] NKp46 binding bicyclic peptides

[0193] Examples of NKp46 binding to bicyclic peptides are described above.

[0194] Linkers

[0195] It should be understood that the first peptide ligand can be conjugated to one or more second peptide ligands via any suitable linker. Typically, the linker is designed such that two or more total bicyclic peptides are presented in such a way that they can bind unhindered to their respective targets, individually or simultaneously to both target receptors. Furthermore, the linker should allow simultaneous binding to two targets while maintaining an appropriate distance between target cells, which will lead to the desired functional outcome. The properties of the linker can be tuned to increase length, rigidity, or solubility to optimize the desired functional outcome. The linker can also be designed to allow more than one bicyclic linker to the same target. Increasing the valence of either binding peptide can be used to increase the affinity of the heterotandem linker for target cells, or it can help induce oligomerization of one or both target receptors.

[0196] In one embodiment, the linker is a straight-chain linker or a branched-chain linker. In some embodiments, the linker is a branched-chain linker and comprises three or four branches. In some embodiments, the linker is capable of binding to three or four bicyclic peptide ligands. In some embodiments, the linker comprises three branches and is capable of binding to three bicyclic peptide ligands. In some embodiments, the linker comprises four branches and is capable of binding to four bicyclic peptide ligands. In some embodiments, the linker comprises one or more repeating monomeric groups.

[0197] In some embodiments, the linker is a bidentate or polydentate group having a length of about 0.3 nm to about 300 nm. In some embodiments, the linker has a length of about 0.5 nm to about 200 nm, such as about 1 nm to about 100 nm, such as about 1.5 nm to about 50 nm, such as about 2 nm to about 20 nm, such as about 3 nm to about 10 nm. In some embodiments, the linker length is a continuous length. In some embodiments, the length is determined when the linker is in an aqueous solution under physiological conditions (e.g., phosphate-buffered saline at pH 7.4 at 37°C), and in some embodiments, it can be determined using atomic force microscopy.

[0198] In some embodiments, the linker comprises one or more linking portions, such as one or more poly(alkylene glycol) groups, such as poly(ethylene glycol) or poly(propylene glycol). In some embodiments, the linker may comprise one or more groups, such as amine groups, amide groups; alkylene groups; urethane groups; ether groups; ester groups; disulfide bonds; hydrazone groups; sulfonamide groups; thioether groups; or cyclic groups, preferably 4-12 membered carbocyclic or heterocyclic groups, 5-12 membered heteroaryl groups, or C6 groups. 6-12 Aryl groups; wherein the alkylene, alkenylene, ynylene, poly(alkylene glycol), amine and cyclic groups are each optionally substituted independently.

[0199] In some embodiments, the linker comprises one or more amino acids or amino acid analogs. In some embodiments, the linker comprises about 1 to about 5 amino acids or amino acid analogs. In some embodiments, the side chains of two amino acids or amino acid analogs in the linker are linked together. In some embodiments, the linker comprises a portion of the following form:

[0200]

[0201] Each R 1 Is it H or C? 1-4 Alkyl; each R 2 Side chains selected from amino acids (e.g., standard amino acids), or C 1-4 Alkyl groups, which can be, for example, OH, SH, SC 1-4 Alkyl, aryl (which can be substituted with OH), heteroaryl, C(O)OH, C(O)NH2, N + H3, NH (C=N) + H2)NH2 substitution, for example, R 2 It may contain arginine or homoarginine side chains; and LINK is a linker, wherein LINK is optionally C. 2-8 A hydrocarbylene (e.g., an alkylene) linker, optionally terminated by or substituted with one or more groups, such as amine groups, amide groups; alkylene groups; urethane groups; ether groups; ester groups; disulfide bonds; hydrazone groups; sulfonamide groups; thioether groups; or cyclic groups, as defined herein; for example, LINK may contain C 3-6 An alkylene group, which is terminated by an amide group or interrupted by an amide group; for example, LINK may contain the form -C 1-4 Alkylene-NHC(O)-C 1-4 The alkylene moiety, such as -C4 alkylene-NHC(O)-C1 alkylene-. In some embodiments, the moiety is linked to a polypeptide contained in a peptide ligand or bicyclic peptide ligand as defined herein, for example at the N or C terminus of the polypeptide contained in a peptide ligand or bicyclic peptide ligand as defined herein.

[0202] In some embodiments, the linker comprises one or more reactive groups for reacting with bicyclic peptide ligands as described herein. Exemplary reactive groups include azide groups (which can react with alkyne groups on bicyclic peptide ligands as described herein, for example, in the presence of suitable conditions, such as in the presence of an azide-alkyne cycloaddition catalyst, thereby forming a 1,2,3-triazole group); carboxylic acids and their activated derivatives (such as NHS-esters) (which can react with amine groups on bicyclic peptide ligands as described herein, for example, in the presence of suitable conditions, thereby forming an amide bond), etc.

[0203] In some embodiments, when the linker comprises a poly(alkylene glycol), the poly(alkylene glycol) is either poly(ethylene glycol)(PEG) or poly(propylene glycol)(PPG). In some embodiments, the linker comprises one or more PEGs. n The linker group, wherein n represents the number of consecutive ethylene glycol units in each PEG group. In some embodiments, n is an integer such as about 2 to about 25, or about 3 to about 10. In some embodiments, the linker comprises one or more branches, such as three branches, and each branch contains PEG. n Group.

[0204] In some embodiments, when the linker comprises an amide group, the amide group has the formula -NHC(O)- or -C(O)NH-. In some embodiments, when the linker comprises an amine group, the amine group has the formula N(R)3, wherein each R may be the same or different. In some embodiments, each R comprises a bicyclic peptide ligand as described herein, for example by comprising one or more PEGs as described herein. n The linker portion of the group is attached to an amine nitrogen. In some embodiments, when the linker contains a cyclic group, the cyclic group is a C6 aryl group. In some embodiments, when the linker contains an alkylene group, the alkylene group is a C6 aryl group. 1-3 Alkylene group.

[0205] In one implementation, the linker is a straight-chain linker. Without being bound by theory, it is considered that straight-chain linkers have the advantage of allowing a first peptide to be present at one end and a second peptide to be present at the other end.

[0206] In some implementations, the linear linker is a group of the following formula:

[0207] N3—(Alk) m —PEG n —(Alk) m —Q

[0208] Each Alk is independently C 1-3Alkylene group; each m is independently 0 or 1; n is an integer from about 2 to about 25; and Q is a carboxylic acid (C(O)OH) or its activated derivative (e.g., NHS-ester group).

[0209] In another embodiment, the linear connector is selected from:

[0210]

[0211] Azide-PEG5-acid; and

[0212]

[0213] Azide-PEG24-acid.

[0214] Those skilled in the art will understand that, when conjugated with a bicyclic peptide ligand as described herein, the azide group will typically react with an alkyne group on one bicyclic peptide ligand (e.g., with an alkyne group contained in the K(PYA) moiety as described herein) and the carboxylic acid or NHS-ester will typically react with an amine group on another bicyclic peptide ligand (e.g., with the N-terminal amino group of the polypeptide as described herein).

[0215] In one implementation, the linker is a branched linker. Without being bound by theory, it is considered that branched linkers have the advantage of allowing a first peptide to be present at one end and two or more second peptides at the other end.

[0216] In some implementations, the branched linker is a group of the following formula:

[0217] [N3—(Alk) m —PEG n —(Alk) m -] x -W-[-(Alk) m —PEG n —(Alk) m —Q] y

[0218] Each Alk is independently C 1-3 An alkylene group, optionally and independently terminated with an amide group or an -O- group; each m is independently 0, 1, or 2; n is an integer from about 2 to about 25; Q is a carboxylic acid or an activated derivative thereof (e.g., an NHS-ester group); W is an N or benzene ring; and x and y are each independently an integer from 0 or 1 to about 4; where x + y equals the number of branches in the branched linker; for example, a linker containing three branches can have x=2 and y=1. To avoid ambiguity, when the linker contains multiple groups [N3—(Alk)]... m —PEG n —(Alk)m -] and / or [-(Alk)] m —PEG n —(Alk) m When —Q], the groups can be the same or different.

[0219] In another embodiment, the branch connector is selected from:

[0220]

[0221] N-(acid-PEG3)-N-bis(PEG3-azide);

[0222]

[0223] N-(acid-PEG) 10 )-N-Bis(PEG) 10 -azide);

[0224]

[0225] N-(PEG3-acid)-N-(PEG3-azide)-N-(PEG3-NH-AcAz);

[0226]

[0227] Benzotriic acid-[Peg] 10 ]3;

[0228]

[0229] TCA-[Peg 10 ]3;

[0230]

[0231] TCA-[Peg 23 ]3;

[0232]

[0233] Tet-[Peg 10 ]4;

[0234]

[0235] Methane-N-(PEG5-acid)-tris(MeOPr-amide-PEG4-azide);

[0236]

[0237] Methane-N-(PEG) 10-acid)-tris(MeOPr-amide-PEG 10 -azide); and

[0238]

[0239] Bis-N-aminopropyl-glycine-(PEG5)2.

[0240] In one specific implementation, the branch connector is:

[0241]

[0242] N-(acid-PEG3)-N-bis(PEG3-azide).

[0243] As explained above, those skilled in the art will understand that, when conjugated with a bicyclic peptide ligand as described herein, the azide group, or each azide group, will typically react with an alkyne group on one of the bicyclic peptide ligands (e.g., with an alkyne group contained in the K(PYA) moiety as described herein), and the carboxylic acid or NHS-ester, or each carboxylic acid or NHS-ester, will typically react with an amine group on another bicyclic peptide ligand (e.g., with an N-terminal amino group of the polypeptide as described herein). For example, when the branched linker is:

[0244]

[0245] Linkers in heterotandem bicyclic peptide complexes can have the following forms:

[0246]

[0247] Wherein NH-Bic1 represents the amino group (e.g., N-terminal amino group) of the first bicyclic peptide ligand and each Bic2 The alkyne group (e.g., K(PYA) group) indicates the reaction of the second bicyclic peptide ligand; other linkers behave similarly.

[0248] Multimer binding complexes

[0249] As described above, in some embodiments, the multimeric binding complex comprises at least one bicyclic peptide ligand as described herein. In some embodiments, the multimeric binding complex comprises at least two bicyclic peptide ligands as described herein. In some embodiments, the bicyclic peptide ligands are linked together by a linker, such as a linker described herein.

[0250] In some embodiments, such multimeric binding complexes further comprise one or more effector groups and / or functional groups, such as one or more cytotoxic agents, chelating agents (e.g., radiochelating agents), chromophores, and / or fluorophores. In some embodiments, the one or more effector groups and / or functional groups, such as one or more cytotoxic agents, chelating agents (e.g., radiochelating agents), chromophores, and / or fluorophores, are directly linked to one of the two or more bicyclic peptide ligands. In some embodiments, the one or more effector groups and / or functional groups, such as one or more cytotoxic agents, chelating agents (e.g., radiochelating agents), chromophores, and / or fluorophores, are linked to a linker between the two or more bicyclic peptide ligands.

[0251] In some embodiments, the multimeric binding complex comprises at least one bicyclic peptide ligand selected from BCY24188 and BCY26631. In some embodiments, the multimeric binding complex comprises at least two bicyclic peptide ligands, each selected from BCY24188 and BCY26631. In some embodiments, the multimeric binding complex comprises two BCY24188 bicyclic peptide ligands linked together by a linker. In some embodiments, the multimeric binding complex comprises two BCY26631 bicyclic peptide ligands linked together by a linker.

[0252] In some embodiments, the two bicyclic peptide ligand moieties are linked together via a reaction with an N-(amino-PEG2)-N-bis(PEG3-azide) linker. In some embodiments, the linker may be:

[0253]

[0254] Therefore, linkers in polymer-bound complexes can have the following forms:

[0255]

[0256] Wherein EF is an effector group and / or functional group as described herein, such as a cytotoxic agent, chelating agent (e.g., a radiochelating agent), chromophore, or fluorophore (e.g., a fluorophore as described herein), for example, wherein the effector group comprises a carboxylic acid or an activated derivative thereof prior to reaction with the linker, which reacts with the NH2 group of the linker to form the -NH-C(O)-EF moiety; and each Bic2 The term refers to an alkyne group (e.g., a K(PYA) group) representing the reaction of the bicyclic peptide ligand as described herein. In other words, when included in a multimeric binding complex, the linker comprises an N-(amide-PEG2)-N-bis(PEG3-azide) linker.

[0257] In some embodiments, the multimeric binding complex comprises two BCY24188 bicyclic peptide ligands linked together by an N-(amido-PEG2)-N-bis(PEG3-azide) linker. In some embodiments, the multimeric binding complex comprises two BCY24188 bicyclic peptide ligands, each linked via a C-terminal K(PYA) linker to an N-(amido-PEG2)-N-bis(PEG3-azide) linker, which is connected to a fluorophore as described herein. In some embodiments, the fluorophore is AF647. An example of such a multimeric binding complex is BCY28671.

[0258] In some embodiments, the multimeric binding complex comprises two BCY26631 bicyclic peptide ligands linked together by an N-(amido-PEG2)-N-bis(PEG3-azide) linker. In some embodiments, the multimeric binding complex comprises two BCY26631 bicyclic peptide ligands, each linked via a C-terminal K(PYA) linker to an N-(amido-PEG2)-N-bis(PEG3-azide) linker, which is connected to a fluorophore as described herein. In some embodiments, the fluorophore is AF647. An example of such a multimeric binding complex is BCY28672.

[0259] Therefore, this document provides fluorescent bicyclic peptide dimer complexes comprising a fluorophore as described herein, the fluorophore being linked to two NKp46-specific peptides (both BCY24188) via an N-(amide-PEG2)-N-bis(PEG3-azide) linker. In some embodiments, the fluorophore is AF647, and the complex is BCY28671. This document also provides fluorescent bicyclic peptide dimer complexes comprising a fluorophore as described herein, the fluorophore being linked to two NKp46-specific peptides (both BCY26631) via an N-(amide-PEG2)-N-bis(PEG3-azide) linker. In some embodiments, the fluorophore is AF647, and the complex is BCY28672. Such complexes are therapeutically useful, for example, as clinical reagents for PD readout.

[0260]

[0261] Therefore, this paper provides a multimer-binding complex.

[0262]

[0263] Or its salt, wherein the fluorophore is as described herein; for example, when the fluorophore is AF647, the complex may be BCY28671:

[0264]

[0265] Or its salt.

[0266] It also provides multimeric binding complexes.

[0267]

[0268] Or its salt, wherein the fluorophore is as described herein; for example, when the fluorophore is AF647, the complex may be BCY28672:

[0269]

[0270] Or its salt.

[0271] Hetero-serial complexes

[0272] In one specific embodiment, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand linked to the TATA backbone, and one or more second peptide ligands comprise two NKp46-binding bicyclic peptide ligands linked to the TATA backbone, and the heterotandem complex is a complex listed in Table A or a pharmaceutically acceptable salt thereof.

[0273] Table A (EphA2 : NKp46; 1 :2)

[0274]

[0275] The heterotandem bicyclic peptide complex BCY27047 consists of an EphA2-specific peptide BCY13118 linked to two NKp46-specific peptides (both BCY26631) via an N-(acid-PEG3)-N-bis(PEG3-azide) linker, as illustrated in the figure.

[0276]

[0277] BCY27047

[0278] To avoid any confusion, the structure of BCY27047 can be represented as follows:

[0279]

[0280] in

[0281] L 1 for

[0282]

[0283] L 2 for

[0284]

[0285] L 3 for

[0286]

[0287] BCY27047 can be provided in the form of a pharmaceutically acceptable salt, which is also provided herein.

[0288] The heterotandem bicyclic peptide complex BCY26129 consists of an EphA2-specific peptide BCY13118 linked to two NKp46-specific peptides (both BCY24188) via an N-(acid-PEG3)-N-bis(PEG3-azide) linker, as illustrated in the figure.

[0289]

[0290] BCY26129

[0291] To avoid any confusion, the structure of BCY26129 can be represented as follows:

[0292]

[0293] in

[0294] L 1 for

[0295]

[0296] L 2 for

[0297]

[0298] L 3 for

[0299]

[0300] BCY26129 may be provided in the form of a pharmaceutically acceptable salt, which is also provided herein.

[0301] Number

[0302] When referring to the positions of amino acid residues within the peptides of the present invention, reactive groups, namely cysteine ​​residues, are omitted from the numbering, as they are constant. Therefore, the numbering of amino acid residues within the peptides of the present invention is represented as follows:

[0303] -[dC]-Y1-[Cba]2-P3-D4-Y5-L6-C-X7-D8-E9-Y 10 -C- (SEQ ID NO: 1)

[0304] For the purposes of this specification, unless otherwise stated, it is assumed that all bicyclic peptides are cyclized with TATA and produce a trisubstituted structure. Cyclization with TATA occurs on the first, second, and third cysteine ​​groups. As used herein, the term "cysteine ​​group" in relation to cyclization of the molecular backbone as described herein may refer to D-cysteine ​​([dC]) and L-cysteine ​​(C) residues that may be present in the polypeptide contained in the peptide ligand.

[0305] Molecular form

[0306] The N- or C-terminus of the double-loop core sequence is extended to the left or right side of the sequence, separated by a hyphen. For example, the N-terminal biotin-G-Sar5 tail would be represented as:

[0307] [Biot]-G-[Sar5]-A-(SEQ ID NO: X).

[0308] [Biot] represents biotin, and [Sar] represents sarcosine.

[0309] Reverse peptide sequence

[0310] Based on the disclosure in Nair et al. (2003) J. Immunol. 170(3), 1362-1373, it is assumed that the peptide sequences disclosed herein will also be used in their retro-inverso form. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa), and their stereochemistry is also reversed (i.e., D-amino acids become L-amino acids, and vice versa).

[0311] Peptide ligand definition

[0312] As mentioned in this article, peptide ligands refer to peptides, peptides, or peptide mimics that are covalently bound to the molecular backbone. Typically, such peptides, peptides, or peptide mimics contain a peptide with natural or non-natural amino acids, two or more reactive groups capable of forming covalent bonds with the backbone (e.g., cysteine, homocysteine ​​(hCys, (S)-2-amino-4-thioalkylbutyric acid), βCys ((R)-3-amino-3-mercaptopropionic acid), penicillamine (Pen, (R)-2-amino-3-mercapto-3-methylbutyric acid), Dap ((S)-2,3-diaminopropionic acid), or N-alkyl-Dap). (e.g., N-methyl-Dap, (S)-2-amino-3-(methylamino)propionic acid) and a sequence present between the reactive groups, the sequence being referred to as a cyclic sequence because it forms a ring when the peptide, peptide class, or peptide mimic binds to the backbone. In the case of the present invention, the peptide, peptide class, or peptide mimic typically contains three cysteine ​​residues and forms two rings on the backbone. Thus, in some embodiments, this disclosure provides peptide ligands, bicyclic peptide ligands, or complexes containing peptide ligands or bicyclic peptide ligands as provided herein, wherein one or more cysteine ​​residues of one or more polypeptides contained in the peptide ligand, bicyclic peptide ligand, or complex are replaced by homocysteine ​​(hCys), βCys, penicillamine (Pen), Dap, or N-methyl-Dap. In some embodiments, SEQ ID NO: One or more (e.g., 1, 2, or 3) cysteine ​​residues (e.g., L-cysteine ​​and / or D-cysteine) in any one of SEQ ID NO: 6 or 7 are replaced with homocysteine ​​(hCys), βCys, penicillamine (Pen), Dap, or N-methyl-Dap. In some embodiments, one or more (e.g., 1, 2, or 3) cysteine ​​residues (e.g., L-cysteine ​​and / or D-cysteine) in any one of SEQ ID NO: 6 or 7 are replaced with homocysteine ​​(hCys), βCys, penicillamine (Pen), Dap, or N-methyl-Dap. For the avoidance of doubt, in the complexes comprising two or more peptide ligands (e.g., two or more bicyclic peptide ligands) provided herein, each ligand is independently and optionally modified in such a manner that different ligands may be modified or not modified, and different modified ligands may contain different modifications.

[0313] Peptide specificity

[0314] As explained above, in some embodiments, the provided peptide and ligand containing such peptide (described in more detail herein) are specific to NK cells. In some embodiments, the provided complex (e.g., a heterotandem binding complex) contains a peptide and ligand containing such peptide (described in more detail herein), said peptide and ligand being specific to EphA2, Nectin-4, PD-L1, MT1, or PSAM. In some embodiments, the provided complex (e.g., a heterotandem binding complex) contains one or more first peptides or ligands containing such first peptides (described in more detail herein) specific to EphA2 and one or more second peptides or ligands containing such second peptides (described in more detail herein) specific to NK cells.

[0315] As used herein, the term "specificity" ("specific binding," etc.) in its broadest sense refers to a peptide or peptide ligand that binds to its biological target. In some embodiments, the peptide or peptide ligand binds to its biological target in a specific manner; that is, the binding to the biological target is not nonspecific. In some embodiments, peptides exhibiting nonspecific binding are heterogeneous; that is, the peptide is capable of binding to a variety of different biological substances, typically including both the target target and off-target binding sites, such as binding sites on cell types other than the target cell type. Therefore, in some embodiments, peptides or peptide ligands selected or designed to bind specifically to their intended target do not exhibit heterogeneous binding to off-target binding sites.

[0316] In some embodiments, binding to the target is binding to a specific epitope on the target. The peptide or peptide ligand can be designed to be specific to a specific epitope, or can be determined by suitable screening methods, such as display techniques (e.g., phage display), which can be used to develop high-affinity binders to a given target (e.g., epitope). Alternatively, the peptide or peptide ligand can be identified as having specific binding to a biological target (such as a cellular target) without knowing the specific epitope it binds to. In some embodiments, peptides or peptide ligands that specifically bind to a target or epitope have high affinity for the target or epitope. In some embodiments, the binding affinity of a peptide or peptide ligand to its epitope can be determined by its dissociation constant (…). K D Also written Kd This indicates that peptides or peptide ligands that specifically bind to biological targets typically have a binding affinity for that target of less than 10 µM, for example, less than 1 µM. K D Typically, peptides or peptide ligands that specifically bind to a biological target will have nanomolar amounts of [something related to that target] K DSuch values ​​are less than 100 nm, less than 20 nm, less than 10 nm, or even less than 1 nm. Binding affinity can be determined by methods known in the art, such as SPR and competition assays. Some suitable assays are described in the examples.

[0317] In some implementations, peptides or peptide ligands that specifically bind to a biological target will have a higher affinity (lower affinity) for that particular biological target than for other biologically binding epitopes. K D For example, peptides or peptide ligands that bind specifically to NK cells will typically bind to NK cells with a higher affinity than to other cell types (e.g., binding to one or more epitopes expressed by NK cells, such as NKp30, NKp44, NKp46, e.g., NKp46). Similarly, peptides or peptide ligands that bind specifically to EphA2 will typically bind to EphA2 with a higher affinity than to other epitopes. In some embodiments, peptides or peptide ligands that bind specifically to a biological target will bind to the target with an affinity at least twice, for example at least five times, for example at least ten times, for example at least twenty times, for example at least fifty times, for example at least one hundred times, for example at least one thousand times, or even more than that the peptide binds to any other off-target binding object.

[0318] Advantages of the peptide ligand

[0319] Certain bicyclic peptides of the present invention possess numerous advantageous properties that make them suitable pharmaceutically analogous molecules for injection, inhalation, nasal administration, ocular administration, oral administration, or topical application. These advantageous properties include:

[0320] - Species cross-reactivity. This is a common requirement for preclinical pharmacodynamic and pharmacokinetic evaluations;

[0321] - Protease stability. The bicyclic peptide ligand should exhibit stability against plasma proteases, epithelial (“membrane-anchored”) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc., in most cases. Protease stability should be maintained across different species so that the bicyclic peptide lead candidate can be developed in animal models and confidently administered to humans;

[0322] - The desired solubility profile. This depends on the ratio of charged and hydrophilic to hydrophobic residues and the intramolecular / intermolecular H bonds, which are important for formulation and absorption purposes; and

[0323] - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides with short or prolonged in vivo exposure times for managing chronic or acute disease states. Optimal exposure time will be controlled by the requirement for continuous exposure (to obtain maximum therapeutic efficiency) and the requirement for short exposure time to minimize the toxicological effects resulting from continuous exposure to the agent.

[0324] Pharmaceutically acceptable salts

[0325] It should be understood that salt form is within the scope of this invention, and references to peptide ligands and complexes containing such peptide ligands include salt forms of said ligands and complexes.

[0326] The salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety using conventional chemical methods, such as in... Pharmaceutical Salts: Properties, Selection, and Use P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, hardcover, 388 pages, August 2002, describes the method. Generally, such salts can be prepared by reacting the free acidic or basic form of these compounds with a suitable base or acid in water, in an organic solvent, or in a mixture of both.

[0327] Acid addition salts (monosal or diosal) can be formed from a variety of inorganic and organic acids. Examples of acid addition salts include monosal or diosal forms with acids selected from the group consisting of: 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, butyric acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, octanoic acid, cinnamic acid, citric acid, cyclohexanesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactopyric acid, gentian acid, glucoheponic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutarate. Glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), hydroxyethanesulfonic 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, dihydroxynaphthoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanate, p-toluenesulfonic acid, undecenoic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

[0328] A specific group of salts consists of salts formed from the following: acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, ethanesulfonic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid, ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid, and lactobionic acid. One specific salt is a hydrochloride salt. Another specific salt is an acetate salt.

[0329] If the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be -COO), - This allows it to form salts with organic or inorganic bases that produce suitable cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li. + Na + and K + Alkaline earth metal cations such as Ca 2+ and Mg 2+ ; and other cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+). +) and substituted ammonium ions (e.g., NH3R) + NH2R2 + NHR3 + NR4 + Examples of suitable substituted ammonium ions are 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. A common example of a quaternary ammonium ion is N(CH3)4. + .

[0330] In the case where the peptides of the present invention contain amine functional groups, these peptides can form quaternary ammonium salts, for example, by reacting with an alkylating agent according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the present invention.

[0331] The peptides (including peptide ligands and their complexes) of the present invention can exist as zwitterions. Such compounds can also be provided in the form of pharmaceutically acceptable salts. Suitable salts include those that form with pharmaceutically acceptable acids, said acids being negatively charged groups such as COO. - The zwitterion provides a proton and a counterion to balance the positive charge on the positively charged group, such as the quaternary nitrogen atom. Suitable pharmaceutically acceptable acids include hydrochloric acid, sulfonic acids (including methanesulfonic acid and toluenesulfonic acid), ascorbic acid, and citric acid. Hydrochloric acid and sulfonic acids are preferred, especially hydrochloric acid. Alternatively, the zwitterion can be combined with a pharmaceutically acceptable base, such as alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides.

[0332] Modified derivatives

[0333] It should be understood that derivatives modified with peptide ligands as defined herein are within the scope of this invention.

[0334] In some implementations, the modified derivatives include functional fragments, derivatives, and variants of the sequences provided herein.

[0335] As those skilled in the art will understand, the amino acid sequence fragments include deletion variants of such sequences, wherein one or more, such as at least 1, 2, 3, 4, or 5 amino acids, are deleted. The deletion may occur at the C-terminus or N-terminus of the reference sequence or within the reference sequence.

[0336] Derivatives of amino acid sequences include modified sequences, including in vivo or in vitro modified sequences. Many different protein modifications are known to those skilled in the art, and include modifications that introduce new functions into amino acid residues, modifications that protect reactive amino acid residues, or modifications that couple amino acid residues to chemical motifs such as reactive functional groups for linking to such amino acid residues. Exemplary modifications that can be made to the provided peptides, ligands, and related complexes are described in more detail herein.

[0337] Derivatives of the amino acid sequence include addition variants of such sequences, wherein one or more amino acids, such as at least 1, 2, 3, 4, or 5 amino acids, are added to or introduced into the reference sequence. Additions can occur at the C-terminus or N-terminus of the reference sequence or within the reference sequence. This document provides some examples of peptides of SEQ ID NO 1 with additional amino acids added thereto.

[0338] Variations of the amino acid sequence include sequences in which one or more amino acids in the reference sequence, such as at least 1, 2, 3, 4, or 5 amino acid residues, are replaced by one or more substitute residues. Variations of the amino acid sequence also include sequences carrying naturally occurring amino acids and / or non-natural amino acids.

[0339] The variants, derivatives, and fragments of the aforementioned amino acid sequence generally retain at least some of the activities / functions of the reference sequence. In a preferred embodiment, the variants, derivatives, and fragments substantially retain their (multiple) biological functions as described herein. Thus, in one embodiment, the variants, derivatives, and fragments retain the binding specificity of the reference sequence, i.e., the ability to specifically bind to biological targets such as NKp30, NKp44, NKp46, for example, NKp46. In one such embodiment, the variants, derivatives, and fragments bind to the same epitopes as the reference sequence. In another embodiment, the variants, derivatives, and fragments retain the binding affinity of the reference sequence. Preferably, the variants, derivatives, and fragments of the reference sequence have increased / improved activity / function compared to the reference sequence.

[0340] In some implementations, variants, derivatives, or fragments of an amino acid sequence are expressed as a percentage of their identity with a reference sequence. Methods for determining the percentage of identity are conventional procedures within the scope of those skilled in the art. Suitable methods include CLUSTAL W (Thompson et al., Nucleic Acids Research, 22(22) 4673-4680 (1994)) and iterative refinement (Gotoh, J. Mol. Biol. 264(4) 823-838 (1996)); and the methods described in Altschul et al., Bull. Math. Bio. 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-19, 1992. In the exemplary method, two amino acid sequences are aligned using a vacancy opening penalty of 10, a vacancy extension penalty of 1, and the Henikoff and Henikoff “blosum 62” scoring matrix (as above) to optimize the alignment score. The identity percentage is then calculated as: [100 x (T / L)]; where T = the total number of identical matches, and L = the length of the longer sequence plus the number of vacancy introduced into the longer sequence to align the two sequences.

[0341] In some implementations, variants, derivatives, or fragments of the reference sequence have at least 60% identity with the reference sequence, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more.

[0342] Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and / or C-terminal modification; 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 isomeric or isoelectronic amino acids; replacement of one or more nonpolar amino acid residues with other non-natural isomeric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; replacement of one or more amino acid residues with one or more substitute amino acids such as alanine, or replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacement of one or more peptide bonds with substitute bonds; peptide backbone length modification; substitution of the hydrogen on the α-carbon of one or more amino acid residues with another chemical group; modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid, and tyrosine with suitable amine, thiol, carboxylic acid, and phenolic reagents to functionalize the amino acids; and introduction or replacement of orthogonally reactive amino acids suitable for functionalization, such as amino acids carrying azido or alkyne groups, which respectively allow for partial functionalization with alkyne or azido groups.

[0343] In one embodiment, the modified derivative comprises N-terminal and / or C-terminal modifications. In another embodiment, the modified derivative comprises an N-terminal modification using a suitable amino-reaction chemistry and / or a C-terminal modification using a suitable carboxyl-reaction chemistry. In yet another embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to cytotoxic agents, radiochelates, or chromophores.

[0344] In another embodiment, the modified derivative comprises an N-terminal modification. In yet another 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 to produce an N-terminal acetylated molecule. This embodiment offers the advantage of removing a potential recognition site for aminopeptidases and avoids the possibility of bicyclic peptide degradation.

[0345] In an alternative embodiment, N-terminal modification includes the addition of a spacer group that promotes the conjugation of effector groups and the retention of the bicyclic peptide's potency toward its target.

[0346] In another embodiment, the modified derivative comprises a C-terminal modification. In yet another 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-terminal amidated molecule. This embodiment offers the advantage of removing a potential recognition site for carboxypeptidases and reduces the likelihood of hydrolytic degradation of the bicyclic peptide protein.

[0347] In one embodiment, the modified derivative comprises replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids with isotropic / isoelectronic side chains can be selected, which are neither recognized by degrading proteases nor have any adverse effect on target efficacy.

[0348] Alternatively, non-natural amino acids with restricted amino acid side chains can be used, thereby hindering the conformational and spatial lysis of nearby peptide bonds. In particular, these involve proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cyclic amino acids, with simple derivatives being amino-cyclopropylcarboxylic acid.

[0349] In one embodiment, the modified derivative includes the addition of a spacer group. In another embodiment, the modified derivative includes the addition of a spacer group to an N-terminal cysteine ​​and / or a C-terminal cysteine.

[0350] In one embodiment, the modified derivative comprises replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues. In another embodiment, the modified derivative comprises replacing tryptophan residues with naphthylalanine or alanine residues. These embodiments provide the advantage of improving the pharmaceutical stability properties of the resulting bicyclic peptide ligand.

[0351] In one embodiment, the modified derivative comprises replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The proper balance between charged and hydrophobic amino acid residues is an important characteristic of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (particularly arginine) can affect the interaction of the peptide with phospholipid membranes on cell surfaces. The combination of both can affect the half-life, volume of distribution, and exposure of the peptide drug and can be adjusted according to clinical endpoints. Furthermore, the proper combination and amount of charged and hydrophobic amino acid residues can reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

[0352] In one embodiment, the modified derivative comprises replacing one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase protein hydrolytic stability through steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0353] In one embodiment, the modified derivative includes the removal of any amino acid residues and substitution with alanine, such as D-alanine. This embodiment offers the advantage of identifying key binding residues and removing potential proteolytic attack sites(s).

[0354] It should be noted that each of the above modifications is intended to intentionally improve the potency or stability of the peptide. Further potency improvements based on modifications can be achieved through the following mechanisms:

[0355] - Introducing a hydrophobic portion that utilizes hydrophobic effects and results in a lower dissociation rate enables higher affinity;

[0356] - Introducing charged groups that utilize long-range ion interactions leads to faster binding rates and higher affinities (see, for example, Schreiber et al.). Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0357] - Additional restrictions are introduced into peptides by, for example, properly restricting the side chains of amino acids to minimize entropy loss during target binding, restricting the torsion angle of the main chain to minimize entropy loss during target binding, and introducing additional cyclization into the molecule for the same reason.

[0358] (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0359] Isotopic variants

[0360] This invention includes all pharmaceutically acceptable (radioactive) isotope-labeled peptide ligands of this invention, wherein one or more atoms are replaced by atoms having the same atomic number but different atomic mass or mass number from those commonly found in nature, and peptide ligands of this invention wherein a metal chelating group (referred to as an "effectant") capable of retaining the relevant (radioactive) isotope is attached, and peptide ligands of this invention wherein certain functional groups are covalently replaced by relevant (radioactive) isotope or isotope-labeled functional groups.

[0361] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, such as... 2 H(D) and 3 H(T); isotopes of carbon, such as 11 C 13 C and14 C; isotopes of chlorine, such as 36 Cl; isotopes of fluorine, such as 18 F; isotopes of iodine, such as 123 I, 125 I and 131 I; isotopes of nitrogen, such as 13 N and 15 N; isotopes of oxygen, such as 15 O、 17 O and 18 O; isotopes of phosphorus, such as 32 P; isotopes of sulfur, such as 35 S; isotopes of copper, such as 64 Cu; isotopes of gallium, such as 67 Ga or 68 Ga; isotopes of yttrium, such as 90 Y; and isotopes of lutetium, such as 177 Lu; and isotopes of bismuth, such as 213 Bi.

[0362] Certain isotope-labeled peptide ligands of the present invention, such as peptide ligands incorporating radioactive isotopes, can be used for drug and / or substrate tissue distribution studies and for clinical assessment of the presence and / or absence of targets on diseased tissues. The peptide ligands of the present invention can also possess valuable diagnostic properties because they can be used to detect or determine the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or determination methods can use compounds labeled with labeling agents such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, jellyfish luminescent proteins, and luciferase), etc. Radioactive isotope tritium (i.e.... 3 H (T)) and carbon-14 (i.e. 14 C) Given its ease of introduction and the ease of detection, it is particularly suitable for this purpose.

[0363] Using heavier isotopes such as deuterium (i.e. 2 H(D) substitution can provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements, and is therefore preferred in some cases.

[0364] Using positron emission isotopes such as 11 C 18 F, 15 O and 13 N substitution can be used in positron emission tomography (PET) studies to examine target occupancy.

[0365] The isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples, using appropriate isotopically labeled reagents instead of previously used unlabeled reagents.

[0366] Molecular scaffold

[0367] In some embodiments, the peptides disclosed herein are linked to a molecular backbone. In one embodiment, the molecular backbone comprises a non-aromatic molecular backbone. "Non-aromatic molecular backbone" as used herein refers to any molecular backbone as defined herein that does not contain an aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring system. Therefore, in some embodiments, the peptide is linked to a non-aromatic molecular backbone. In other embodiments, the peptide is linked to an aromatic molecular backbone.

[0368] Suitable examples of non-aromatic molecular skeletons are described in Heinis et al. (2014). Angewandte Chemie, International Edition 53(6) 1602-1606.

[0369] As noted in the aforementioned literature, the molecular skeleton can be a small molecule, such as a small organic molecule.

[0370] In one embodiment, the molecular backbone can be a macromolecule. In one embodiment, the molecular backbone is a macromolecule composed of amino acids, nucleotides, or carbohydrates.

[0371] In one embodiment, the molecular backbone includes reactive groups capable of reacting with multiple functional groups of the polypeptide to form covalent bonds.

[0372] The molecular skeleton can contain chemical groups that form bonds with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, acid anhydrides, succinimides, maleimides, haloalkyl groups, and haloacyl groups.

[0373] In one embodiment, the molecular skeleton is 1,1',1''-(1,3,5-triazine-1,3,5-triyl)triprop-2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA)).

[0374]

[0375] TATA.

[0376] Therefore, after cyclization with the bicyclic peptide of the present invention (e.g., on a cysteine ​​residue), the molecular skeleton forms a trisubstituted 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropyl-1-one derivative of TATA having the following structure:

[0377] ,

[0378] It can also be described as

[0379] ,

[0380] in This indicates the junction of three cysteine ​​residues.

[0381] In an alternative embodiment, the molecular skeleton is 2,4,6-tris(bromomethyl)-s-triazine (TBMT):

[0382]

[0383] TBMT.

[0384] Therefore, after cyclization of the bicyclic peptide of the present invention onto the cysteine ​​residue, the molecular backbone forms a trisubstituted derivative of TBMT having the following structure:

[0385]

[0386] It can also be described as

[0387]

[0388] in This indicates the junction of three cysteine ​​residues.

[0389] In an alternative embodiment, the molecular skeleton is 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine (TATB):

[0390]

[0391] TATB.

[0392] Therefore, after cyclization of the bicyclic peptide of the present invention onto the cysteine ​​residue, the molecular backbone forms a trisubstituted 1,3,5-tris(bromoacetyl)hexahydro-1,3,5-triazine derivative of TATB having the following structure:

[0393]

[0394] It can also be described as

[0395]

[0396] in This indicates the junction of three cysteine ​​residues.

[0397] Synthesis

[0398] The peptides of this invention can be synthesized using standard techniques and then reacted in vitro with a molecular backbone. Standard chemical methods can be used when performing this operation. This enables the rapid, large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be implemented using conventional chemical methods, such as those disclosed in Timmerman et al. (ibid.).

[0399] Therefore, the present invention also relates to the preparation of selected peptides or conjugates as described herein, wherein the preparation includes optional additional steps as explained below. In one embodiment, these steps are performed on a final product peptide / conjugate prepared by chemical synthesis.

[0400] Optionally, when preparing conjugates or complexes, amino acid residues in the target polypeptide can be substituted.

[0401] Peptides can also be extended to introduce, for example, another ring, and thus introduce a variety of specificities.

[0402] To extend peptides, standard solid-phase or solution-phase chemistry methods can be used to simply chemically extend them by using orthogonally protected lysine (and analogues) at their N-terminus or C-terminus or within the ring. Standard (biological) conjugation techniques can be used to introduce an activated or activatable N- or C-terminus. Alternatively, addition can be made by fragment condensation or native chemical linking, as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymatic addition, such as using subtiligase, as described in (Chang et al. ProcNatl Acad Sci US A. Dec 20, 1994; 91(26):12544-8 or Hikari et al. Bioorganic & Medicinal Chemistry Letters Vol. 18, No. 22, Nov 15, 2008, pp. 6000-6003).

[0403] Alternatively, the peptide can be extended or modified via further conjugation via disulfide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other once in the reducing environment of the cell. In this case, the molecular backbone (e.g., TATA, TATB, or TBMT) can be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; then, additional cysteine ​​or thiol can be attached to the N or C terminus of the first peptide such that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide, forming a disulfide-linked bicyclic peptide-peptide conjugate.

[0404] Furthermore, the addition of other functional groups or effector groups can be achieved in the same manner, using appropriate chemical methods, by coupling at the N or C terminus or via side chain coupling. In one embodiment, coupling is performed in a way that does not inhibit the activity of either entity.

[0405] In some embodiments, the synthesis of peptide ligands as provided herein may include solid-phase synthesis of peptides as described herein. In some embodiments, solid-phase synthesis includes Fmoc solid-phase peptide synthesis (e.g., as described in more detail herein). In some embodiments, the synthesized peptide is cyclized with a molecular backbone as described herein. In some embodiments, the cyclized backbone is purified, for example, by lyophilization. In some embodiments, the synthesis of complexes as described herein includes reacting the cyclized peptide as described herein with a linker. In some embodiments, the reaction of the cyclized peptide with the linker involves the reaction of an azide group (e.g., on the linker) with an alkyne group (e.g., on the peptide). In some embodiments, the reaction of the azide group with the alkyne group is carried out in the presence of a suitable azide-alkyne cycloaddition catalyst. In some embodiments, a suitable catalyst comprises CuSO4. In some embodiments, the reaction of the azide group with the alkyne group is carried out under an inert atmosphere (e.g., N2). In some embodiments, the reaction of the cyclized peptide with the linker involves the reaction of an amine group (e.g., on the peptide) with a carboxylic acid group or an activated derivative thereof (e.g., an NHS-ester group) (e.g., on the linker). In some embodiments, the reaction between the amine group and the carboxylic acid group or their activated derivatives is carried out in the presence of a suitable coupling agent. In some embodiments, the suitable coupling agent comprises a base.

[0406] In some embodiments, the synthesis of BCY27047 comprises reacting BCY13118, or a salt thereof as defined herein, with an N-(acid-PEG3)-N-bis(PEG3-azide) linker to produce the product. In some embodiments, the product is BCY14964 as defined herein. In some embodiments, the synthesis comprises reacting the product of the reaction of BCY13118, or a salt thereof, with an N-(acid-PEG3)-N-bis(PEG3-azide) linker (e.g., BCY14964) with BCY 26631 to form BCY27047.

[0407] In some embodiments, the synthesis of BCY26129 comprises reacting BCY13118 or a salt thereof, as defined herein, with an N-(acid-PEG3)-N-bis(PEG3-azide) linker to produce the product. In some embodiments, the product is BCY14964, as defined herein. In some embodiments, the synthesis comprises reacting the product of the reaction of BCY13118 or a salt thereof with an N-(acid-PEG3)-N-bis(PEG3-azide) linker (e.g., BCY14964) with BCY 24188 to form BCY26129.

[0408] Pharmaceutical compositions

[0409] According to another aspect of the invention, pharmaceutical compositions are provided comprising a combination of a peptide ligand, a bicyclic peptide ligand, a multimeric binding complex, or a heterotandem binding complex as defined herein, and one or more pharmaceutically acceptable excipients. In some embodiments, pharmaceutical compositions are provided comprising BCY27047 and one or more pharmaceutically acceptable excipients. In some embodiments, pharmaceutical compositions are provided comprising BCY26129 and one or more pharmaceutically acceptable excipients. In some embodiments, pharmaceutical compositions are provided comprising BCY28671 or BCY28672 and one or more pharmaceutically acceptable excipients.

[0410] Typically, the peptide ligands of the present invention are used in purified form with pharmacologically suitable excipients or carriers. These excipients or carriers typically include aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including saline and / or buffer media. Parenteral media include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, and lactated Ringer's solution. If it is necessary to maintain the polypeptide complex in suspension, suitable physiologically acceptable adjuvants may be selected from thickeners such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin, and alginate.

[0411] Intravenous carriers include fluids and nutritional supplements, as well as electrolyte supplements, such as those based on Ringer's dextran. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th edition).

[0412] The peptide ligands of the present invention can be used as a composition administered alone or in combination with other pharmaceutical agents. These agents may include antibodies, antibody fragments, and various immunotherapeutic agents such as cyclosporine, methotrexate, doxorubicin, or cisplatin and immunotoxins. Further examples of other pharmaceutical agents that can be administered alone or in combination with the peptide ligands of the present invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic agents, and antithrombotic factors. Pharmaceutical compositions may include a “cocktail” of various cytotoxic agents or other agents combined with the protein ligands of the present invention, or even combinations of peptides selected according to the present invention with different specificities, such as peptides using different target ligand selections, whether or not they are pooled prior to administration.

[0413] The administration route of the pharmaceutical composition according to the invention can be any route commonly known to those skilled in the art. For treatment, the peptide ligand of the invention can be administered to any patient according to standard techniques. Administration can be performed by any suitable means, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary routes, or, suitably, direct infusion via catheter. Preferably, the pharmaceutical composition according to the invention is administered intravenously. The dosage and frequency of administration will depend on the patient's age, sex and condition, concurrent administration of other drugs, contraindications, and other parameters to be considered by the clinician.

[0414] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective and can be employed using lyophilization and reconstitution techniques known in the art. Those skilled in the art will understand that lyophilization and reconstitution can result in varying degrees of activity loss, and that levels may need to be adjusted upwards to compensate for this.

[0415] Compositions containing the peptide ligands of the present invention or mixtures thereof can be administered for prophylactic and / or therapeutic treatment. In some therapeutic applications, a sufficient amount to achieve at least partial inhibition, suppression, regulation, killing, or some other measurable parameter of a selected cell population is defined as a “therapeuticly effective dose.” The amount required to achieve this dose will depend on the severity of the disease, but typically ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, more commonly from 0.05 to 2.0 mg / kg / dose. For prophylactic applications, compositions containing the peptide ligands of the present invention or mixtures thereof may also be administered at similar or slightly lower doses.

[0416] Compositions containing peptide ligands according to the invention can be used in preventative and therapeutic settings to help alter, inactivate, kill, or remove selected target cell populations in mammals. Furthermore, the peptide ligands described herein can be used in vitro or selectively in vitro to kill, deplete, or otherwise effectively remove target cell populations from heterogeneous cell collections. Blood from mammals can be combined in vitro with selected peptide ligands, thereby killing unwanted cells or otherwise removing them from the blood according to standard techniques for return to the mammal.

[0417] Therapeutic uses

[0418] The polypeptide ligands selected according to the method of the present invention and complexes containing such polypeptide ligands can be used for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, etc. Ligands with selected levels of specificity can be used in applications involving testing in non-human animals where cross-reactivity is desired, or in diagnostic applications where careful control of cross-reactivity with homologs or parahomologs is required. In some applications, such as vaccine applications, the ability to induce an immune response to a predetermined range of antigens can be utilized to tailor vaccines to specific diseases and pathogens.

[0419] Peptide ligands that are substantially pure with at least 90 to 95% homogeneity are preferably used for administration to mammals, and peptide ligands with 98 to 99% or higher homogeneity are most preferably used for pharmaceutical purposes, especially when the mammal is human. Once partially purified or purified to the desired homogeneity, the selected peptide can be used for diagnostic or therapeutic purposes (including in vitro) or for the development and administration of assay procedures, immunofluorescence staining, etc. (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

[0420] According to another aspect of the invention, peptide ligands, bicyclic peptide ligands, multimeric binding complexes or heterotandem binding complexes as defined herein are provided for the prevention, inhibition or treatment of diseases or disorders mediated by NK cells.

[0421] According to another aspect of the invention, a method for preventing, inhibiting, or treating diseases or disorders mediated by NK cells is provided, the method comprising administering to a patient in need a peptide ligand, a bicyclic peptide ligand, a multimeric binding complex, or a heterotandem binding complex as defined herein.

[0422] According to another aspect of the invention, the use of peptide ligands, bicyclic peptide ligands, multimeric binding complexes or heterotandem binding complexes as defined herein in the preparation of medicaments for the prevention, inhibition or treatment of NK cell-mediated diseases or disorders is provided.

[0423] In one implementation, the NK cell-mediated diseases or disorders are selected from inflammatory disorders, autoimmune diseases, and cancer. In another implementation, the NK cell-mediated diseases or disorders are selected from: rheumatoid arthritis (RA), bone erosion, intraperitoneal abscess, inflammatory bowel disease, allogeneic graft rejection, psoriasis, angiogenesis, atherosclerosis, asthma, multiple sclerosis, systemic lupus erythematosus (SLE), ocular surface disorders (such as dry eye), ankylosing spondylitis, psoriatic arthritis, and cancers (such as multiple myeloma and breast cancer).

[0424] In another embodiment, the NK cell-mediated disease or disorder is selected from cancer. According to another aspect, peptide ligands, bicyclic peptide ligands, multimeric binding complexes, or heterotandem binding complexes as defined herein are provided for the prevention, inhibition, or treatment of diseases or disorders selected from inflammatory disorders, autoimmune diseases, and cancer. According to another aspect, methods for the prevention, inhibition, or treatment of diseases or disorders selected from inflammatory disorders, autoimmune diseases, and cancer are provided, the methods comprising administering a peptide ligand, bicyclic peptide ligand, multimeric binding complex, or heterotandem binding complex as defined herein to a patient in need. According to another aspect, the use of peptide ligands, bicyclic peptide ligands, multimeric binding complexes, or heterotandem binding complexes as defined herein in the preparation of a medicament for the prevention, inhibition, or treatment of diseases or disorders selected from inflammatory disorders, autoimmune diseases, and cancer is provided.

[0425] Examples of cancers (and their benign counterparts) that can be treated (or suppressed) include, but are not limited to, tumors of epithelial origin (adenomas and various types of carcinoma, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other cancers), such as bladder and urinary tract cancers, breast cancers, and gastrointestinal cancers (including esophageal cancer, stomach cancer, etc.). Cancers that can cause various types of cancer, including: gastric cancer, small bowel cancer, colon cancer, rectal cancer, and anal cancer; liver cancer (hepatocellular carcinoma); gallbladder cancer and biliary tract cancer; exocrine pancreatic cancer; kidney cancer; lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma); head and neck cancer (e.g., tongue cancer, buccal carcinoma, laryngeal cancer, pharyngeal cancer, nasopharyngeal carcinoma, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer); ovarian cancer; fallopian tube cancer; peritoneal cancer; vaginal cancer; vulvar cancer; penile cancer; cervical cancer; myometrial cancer; endometrial cancer; thyroid cancer (e.g., follicular thyroid carcinoma); adrenal cancer; prostate cancer; skin cancer; and adnexal cancer (e.g., melanoma). Basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysdifferentiated nevus; hematologic malignancies (i.e., leukemia, lymphoma) and pre-malignant hematologic disorders as well as borderline malignancies, including hematologic malignancies and lymphoid-related conditions (e.g., acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of unknown cause, plasmacytoma, etc. Multiple myeloma and post-transplant lymphoproliferative disorders, as well as hematologic malignancies and myeloid-related conditions (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as erythrocytosis, essential thrombocythemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome and promyelocytic leukemia); mesenchymal tumors, such as sarcomas of soft tissue, bone or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fat Sarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); tumors of endocrine glands (e.g., pituitary gland tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary thyroid carcinoma); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas);And pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, nephroblastoma, and primitive neuroectodermal tumors); or congenital or other forms of syndromes that predispose patients to malignant tumors (e.g., xeroderma pigmentosum).

[0426] The term "prevention" as used herein includes the application of a protective composition prior to the onset of disease. "Inhibition" refers to the application of the composition after the triggering event, but before the clinical onset of disease. "Treatment" includes the application of a protective composition after the symptoms of disease have become apparent.

[0427] Animal model systems are available that can be used to screen the effectiveness of peptide ligands in protecting against or treating diseases. This invention facilitates the use of animal model systems that allow for the development of peptide ligands that can cross-react with human and animal targets, thus enabling the use of animal models.

[0428] The present invention will be further described below with reference to the following embodiments.

[0429] Example

[0430] Materials and methods

[0431] Preparation of bicyclic peptide ligands (general method)

[0432] Bicyclic peptides were synthesized on Rink amide resin using standard Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis, either by manual coupling (for large-scale) or using a Biotage Syroll automated peptide synthesizer (for small-scale). After TFA-based cleavage from the resin, the peptide was precipitated with diethyl ether and dissolved in a 50:50 acetonitrile / water solution. The crude peptide (~1 mM concentration) was then cyclized with 1.3 equivalents of the backbone using ammonium bicarbonate (100 mM) as the base. Completion of cyclization was determined by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) or LC-MS. Once complete, the cyclization reaction was quenched with N-acetylcysteine ​​(10 equivalents relative to the peptide), and the solution was lyophilized. The residues were dissolved in a suitable solvent and purified by RP-HPLC. Peptide fractions of sufficient purity and correct molecular weight (verified by MALDI-TOF and HPLC or LC-MS) were collected and lyophilized. The concentration was determined by UV absorption using the extinction coefficient at 280 nm based on the Trp / Tyr content.

[0433] Unless otherwise stated, all amino acids are referred to in the L-configuration. Amino acids with the prefix "d" (i.e., dC or dA) mentioned herein refer to amino acids in the D-configuration.

[0434] Examples

[0435] Example 1 : Preparation of hetero-serial bicyclic peptide complex BCY27047

[0436] Procedure for preparing intermediate BCY14964

[0437]

[0438] A mixture of BP-23825 (155.5 mg, 249.40 μmol, 1.2 equivalents) and HATU (95.0 mg, 249.92 μmol, 1.2 equivalents) was dissolved in NMP (1.0 mL), and the pH of the solution was adjusted to 8 by dropwise addition of DIEA (64.6 mg, 499.83 μmol, 87.0 μL, 2.4 equivalents), and then the solution was allowed to stir at 25 °C for 5 min. BCY13118 (500.0 mg, 207.83 μmol, 1.0 equivalents) was dissolved in NMP (5.0 mL), and then added to the reaction solution, with the pH of the resulting solution adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25 °C for 45 min. LC-MS showed complete consumption of BCY13118 and a main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC to give BCY14964 as a white solid (1.35 g, 403.46 μmol, 64.7% yield, 90% purity). Calculated MW: 3011.53, observed m / z: 1506.8 ([M+2H]). 2+ ), 1005.0([M+3H] 3+ ).

[0439] Procedure for preparing BCY27047

[0440]

[0441] THPTA (11.4 mg, 26.4 μmol, 2.00 equivalent) was added to a solution of compound 1 (BCY14964, 41.2 mg, 13.2 μmol, 1.00 equivalent, TFA) in DMF (0.50 mL) (pre-degassed and purged three times with N2). CuSO4 was then added under N2. 5H₂O (4.00 M, 6.60 μL, 2.00 equivalents) and VcNa (4.00 M, 19.8 μL, 6.00 equivalents). The reaction mixture was stirred at 20 °C for 0.5 h under a N₂ atmosphere. LC-MS showed that compound 1 (BCY14964) was completely consumed and a result with the expected m / z (calculated MW: 7230.3, observed MW: 7230.3). m / z : 1447.1([M / 5+H]) + The main peak of the product was observed. The crude product was purified by preparative HPLC to obtain BCY27047 as a white solid (31.2 mg, 3.97 μmol, 30.1% yield, 93.48% purity, TFA).

[0442] Example 2: Preparation of hetero-serial bicyclic peptide complex BCY26129

[0443] Procedure for preparing BCY26129

[0444]

[0445] THPTA (8.09 mg, 18.6 μmol, 2.00 equivalent) was added to a solution of compound 1 (BCY14964, 97.9% purity, 29.7 mg, 9.31 μmol, 1.00 equivalent) in DMF (0.30 mL) (pre-degassed and purged three times with N2). CuSO4 was then added under N2. 5H₂O (4.00 M, 4.66 μL, 2.00 equivalents) and VcNa (4.00 M, 13.9 μL, 6.00 equivalents). The reaction mixture was stirred at 20 °C for 0.5 h under a N₂ atmosphere. LC-MS showed complete consumption of compound 1 and detected a result with the expected m / z (calculated MW: 7174.2, observed MW: 7174.2). m / z : 1435.8([M / 5+H]) + The main peak of the product was observed. The crude product was purified by preparative HPLC to obtain BCY26129 as a white solid (31.6 mg, 4.17 μmol, 44.8% yield, 96.21% purity, TFA).

[0446] Biological data

[0447] 1. NKp46 SPR assay

[0448] Human NKp46 (ACROBiosystems, catalog number NC1-H5257) assay peptide targeting the Fc tag.

[0449] For peptide binding analysis, the Biacore 8k+ instrument was used with the CM5 chip (Cytiva). All experiments were performed at 25°C. Anti-human IgG (Fc) antibody (Cytiva) was immobilized on all flow cells using a standard amine coupling chemistry method. The run buffer was HBS-N (Cytiva, 10 mM HEPES, 0.15 M NaCl, pH 7.4) at a flow rate of 10 μL / min. The carboxymethyl dextran surface was activated for 420 s with a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) (Cytiva). The anti-human IgG (Fc) antibody was diluted to 12.5 μg / mL in 10 mM sodium acetate at pH 5.0 and injected onto the chip surface for 360 s. Residual activating groups were blocked by injection with 1 M ethanolamine (pH 8.5) for 420 s. The 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 in flow cell 2 of the sensor chip at a flow rate of 10 μL / min. The buffer was then replaced with PBS-P+ (Cytiva) supplemented with 2% DMSO. Peptide dilutions were prepared in this buffer to obtain a final concentration of 2% DMSO. The flow rate was 50 μL / min, with association for 60 s and dissociation for 400 s. Data were corrected for DMSO volume exclusion effect. All data were double-referenced against blank injection and reference surface responses using standard processing procedures. Data processing and kinetic fitting were performed using Biacore Insight evaluation software (Cytiva) with extended screening and characterization extensions. Data were fitted using a steady-state affinity 1:1 binding model to determine K. D Where appropriate, use a 1:1 kinetic model fit to determine the kinetic constants.

[0450] The selected peptides of the present invention were tested in this assay, and the results are shown in Table 1.

[0451] Table 1: SPR assay data of the selected bicyclic peptides in this invention

[0452]

[0453] 2. Binding to purified NK

[0454] The affinity of NKp46 dicyclic peptide conjugated with AF 647 (available from Lumiprobe) for negative NK cells selected from healthy peripheral blood and cryopreserved or selected from fresh peripheral blood was evaluated using flow cytometry.

[0455] On the day of the experiment, a culture medium, referred to herein as working medium, was prepared by supplementing RPMI-1640 (Gibco™ 11875-093; containing 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). Previously isolated human NK cells (AllCells, LP-CR-CD56+-NS-10M) were rapidly thawed in a water bath and washed once at 500 rpm for 5 minutes in 10 mL of pre-warmed working medium. The cell pellet was then resuspended in working medium to a final volume of 5 x 10⁻⁶. 6 A concentration of cells / mL was established. Subsequently, 100 μL of cell suspension was plated in 96-well V-bottom polypropylene plates (Greiner Bio-One 651201). Sample preparation for flow cytometry: A fixable viable dye (eBioscience™ catalog number 65-0865-18) was prepared as a 1:1000 dilution in PBS, and 100 μL of the viable dye was added to each well and incubated in the dark at 4°C for 30 min. The wells were then washed with 100 μL of PBS at 500 rpm for 5 min, and the supernatant was discarded. Next, the bicyclic peptide was diluted in working medium and added to the corresponding cell plates at a starting concentration of 50 nM, followed by 8-point serial dilutions at a 1:5 ratio. The plates were then incubated at 37°C and 5% CO2 for 1 h. After incubation, the plates were centrifuged at 500 rpm for 5 min, and the supernatant was discarded. The sample was then washed once at 500 rpm for 5 minutes in 200 μL of 1X phosphate-buffered saline (PBS; Gibco™ 10-010-023), and the supernatant was discarded.

[0456] Resuspend the cells in 200 μL of staining buffer and keep them in the dark at 4°C until read by a BDFACSSymphony™ flow cytometer.

[0457] Flow cytometry results were analyzed using a gating strategy in FlowJo™ software to evaluate lymphocyte populations, then debris was excluded, gating was performed only on single and live cells, and the geometric mean of AF 647 in the live NK population was determined. Binding affinity was calculated using four-parameter logistic regression in Prism GraphPad™ 8.0.2 software.

[0458] Figure 1 The data shown illustrate the dose-dependent binding of BCY28671 to the surface of NK cells. The data were obtained from a single representative NK donor (n=3).

[0459] Figure 2 The data shown illustrate the dose-dependent binding of BCY28672 to the surface of NK cells. The data were obtained from a single representative NK donor (n=3).

[0460] The data shown in Table 2 demonstrate that AF 647-tagged NKp46 BCY28671 and BCY28672 induce dose-dependent binding to NK cells.

[0461] Table 2: Binding affinity (Kb) of AF 647 NKp46 dimer to NK cells d,app )

[0462]

[0463] 3. Binding to PBMC

[0464] The affinity of AF 647-conjugated NKp46 dicyclic peptide for primary healthy peripheral blood mononuclear cells (PBMCs) was evaluated using flow cytometry.

[0465] On the day of the experiment, a culture medium, referred to herein as working medium, was prepared using 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) supplemented with RPMI-1640 (Gibco™ 11875-093; containing L-glutamine). Human PBMCs previously isolated from whole blood were rapidly thawed in a water bath and washed once at 500 rpm for 5 minutes in 10 mL of pre-warmed working medium. The cell pellet was then resuspended in working medium to a final volume of 5 x 10⁻⁶ cells / mL. 6 A concentration of cells / mL was then established. Subsequently, 100 μL of the cell suspension was plated in 96-well V-bottom polypropylene plates (Greiner Bio-One 651201).

[0466] Sample preparation for flow cytometry: A 1:1000 dilution of the immobilizable viable dye (eBioscience™ catalog number 65-0865-18) in PBS was prepared, and 100 μL of the viable dye was added to each well and incubated in the dark at 4°C for 30 min. Subsequently, the wells were washed with 100 μL PBS at 500 rpm for 5 min, and the supernatant was discarded. Next, human TruStain FcX™ blocking solution (BioLegend® 422302) was prepared by diluting 1.5 μL of FcX in 25 μL staining buffer (1X PBS supplemented with 2% FBS). The Fc blocking solution (25 μL / well) was incubated in the dark at room temperature (RT) for 10 min. Prepare the antibody master mix by diluting less than 1.5 μL of antibody per 100 μL of staining buffer: BUV737™ anti-human CD3 (BD ​​biosciences® 612750; clone UCHT1) and BUV395 anti-human CD56 (BD biosciences® 612750; clone NCAM16.2). Resuspend the cells in the master mix (100 μL) and incubate in the dark at 4°C for 30 min. Subsequently, wash the cells three times for 5 min at 500 rpm in 100 μL of staining buffer and discard the supernatant.

[0467] The bicyclic peptide was diluted in working medium and added to the appropriate cell culture plates at an initial concentration of 50 nM, followed by 8-point serial dilutions at a 1:5 ratio. The plates were then incubated at 37°C and 5% CO2 for 1 hour. After incubation, the plates were centrifuged at 500 rpm for 5 minutes, and the supernatant was discarded. The samples were then washed once at 500 rpm for 5 minutes in 200 μL of 1X phosphate-buffered saline (PBS; Gibco™ 10-010-023), and the supernatant was discarded.

[0468] Resuspend the cells in 200 μL of staining buffer and keep them in the dark at 4°C until read by a BDFACSFortessa™ flow cytometer.

[0469] Flow cytometry results were analyzed in FlowJo™ software using a gating strategy to evaluate lymphocyte populations, then debris was excluded, gating was performed only on single and live cells, and the geometric mean of AF 647 in the CD3+ and CD3- / CD56+ populations was determined. Binding affinity was calculated using four-parameter logistic regression in Prism Graph Pad™ 8.0.2 software.

[0470] Figure 3The data shown illustrate the dose-dependent binding of the AF-647-labeled NKp46 bicyclic peptide dimer BCY28671 to CD56+ (NK cells) derived from human PBMCs. Data were obtained from a single representative PBMC donor (n=4). BCY18807 is a non-bound bicyclic peptide dimer composed of all D-amino acids.

[0471] Figure 4 The data shown illustrate the dose-dependent binding of the AF-647-labeled NKp46 bicyclic peptide dimer BCY28672 to CD56+ (NK cells) derived from human PBMCs. Data were obtained from a single representative PBMC donor (n=4). BCY18807 is a non-bound bicyclic peptide dimer composed of all D-amino acids.

[0472] The data shown in Table 3 demonstrate that AF 647-tagged NKp46 BCY28671 and BCY28672 only elicit dose-dependent binding with NK cells (CD56+ dissociated). In contrast, the non-AF 647-tagged NKp46 BCY18807 does not elicit binding in any PBMC population.

[0473] Table 3: Binding affinity (Kb) of AF 647 NKp46 dimer to subsets in PBMC cells d,app )

[0474]

[0475] 4. NK cytotoxicity assay

[0476] Evaluation of NK function readout (i.e. cytotoxicity) of heterotandem bicyclic peptide complexes in NK-tumor cell line cocultures.

[0477] NK cells were isolated from the total PBMC population purified from whole blood using a negative isolation kit (STEMCELL™ Technologies 17955). Alternatively, CD56+ NK cells were also harvested from peripheral blood, negatively selected, and cryopreserved (AllCells, LP, CR, CD56+, NS, 10M, Custom). The NK cell pellet was then precipitated at 4 x 10⁻⁶ cells / mL. 5The concentration of cells / mL was resuspended in RPMI-1640 (Gibco™ 11875-093; containing L-glutamine) containing 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 50 IU / mL human IL-2 (Miltenyi Biotec® 130-097-748). For NK cell cytotoxicity assays, 50 μL (2 x 10⁻⁶ cells / mL) was resuspended in RPMI-1640 (Gibco™ 11875-093; containing L-glutamine). 4 The cell suspension was plated in a 96-well plate (Grenier® Bio One™ 655090) containing 50 μl (1 x 10⁻⁶) of DMEM (Gibco™ 11875-093; containing L-glutamine). 3 The DMEM contains 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). The test material was diluted in DMEM medium (Corning™ 10-013-CV) containing 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) and added to the appropriate cell plate (50 μl) at the recommended starting concentration of 30 or 10 nM, and serially titrated 10 times at a 1:4 ratio. The plates were then incubated at 37°C and 5% CO2 for 24 h. After incubation, the plates were centrifuged at 250 xg for 5 min and 100 μl of supernatant was discarded. The sample was then incubated with 50 μl of a Bright-Glo™ luciferase assay system (Promega™ E2620) for 10 min. The emission at 570 nm excitation was read using a CLARIOstar® plate reader with MARS Data Analysis Software™. The data was fitted to a four-parameter nonlinear regression in GraphPad Prism™ 8.0.2 to generate EC. 50 value.

[0478] The selected heterotandem bicyclic peptide complex of the present invention was tested in this assay, and the results are shown in Figures 5-8middle.

[0479] Figure 5 This demonstrates that BCY27047 elicits a dose-dependent NK cell response to kill the EphA2+ve A431-luc tumor cell line. No enhanced dose-dependent effect on tumor cell killing was observed in the absence of the heterotandem bicyclic peptide complex (BCY15666) compared to the NK:A431-luc coculture without the heterotandem bicyclic peptide complex. Mean luminescence without NK-TICA (referring to "BCY-free") is shown arbitrarily at 2.9 pM for reference. EC50 of BCY27047 was calculated using four-parameter logistic regression with GraphPad Prism™ 8.0.2. 50 =5.3 nM.

[0480] Figure 6 This demonstrates that BCY26129 elicits a dose-dependent NK cell response to kill the EphA2+ve A431-luc tumor cell line. No enhanced dose-dependent effect on tumor cell killing was observed in the absence of the heterotandem bicyclic peptide complex (BCY15666) compared to the NK:A431-luc coculture without the heterotandem bicyclic peptide complex. Mean luminescence without NK-TICA (referring to "BCY-free") is shown arbitrarily at 2.9 pM for reference. EC50 of BCY26129 was calculated using four-parameter logistic regression with GraphPad Prism™ 8.0.2. 50 = 7.6 nM.

[0481] Figure 7 This demonstrates that BCY27047 elicits a dose-dependent NK cell response to kill the EphA2+ve HT1080-luc tumor cell line. No enhanced dose-dependent effect in tumor cell killing was observed in the absence of the heterotandem bicyclic peptide complex (BCY15667) compared to the NK:HT1080-luc coculture without the heterotandem bicyclic peptide complex. Mean luminescence without NK-TICA (referring to "BCY-free") is arbitrarily displayed at 5e-16 M for reference. EC50 of BCY27047 was calculated using four-parameter logistic regression with GraphPad Prism™ 8.0.2. 50 =2.1 pM.

[0482] Figure 8This demonstrates that BCY26129 elicits a dose-dependent NK cell response to kill the EphA2+ve HT1080-luc tumor cell line. No enhanced dose-dependent effect in tumor cell killing was observed in the absence of the heterotandem bicyclic peptide complex (BCY15667) compared to the NK:HT1080-luc coculture without the heterotandem bicyclic peptide complex. Mean luminescence without NK-TICA (referring to "0 BCY") is arbitrarily displayed at 5e-16 M for reference. EC50 of BCY26129 was calculated using four-parameter logistic regression with GraphPad Prism™ 8.0.2. 50 =22.9 pM.

[0483] 5. Cytokine secretion assay

[0484] Evaluation of NK function readout (i.e. cytokine secretion) of heterotandem bicyclic peptide complexes in NK-tumor cell line co-cultures.

[0485] NK cells were isolated from the total PBMC population purified from whole blood using a negative isolation kit (STEMCELL™ Technologies 17955). Alternatively, CD56+ NK cells were also obtained from peripheral blood, negatively selected, and cryopreserved (Allcells, LP, CR, CD56+, NS, 10M, Custom). The NK cell pellet was then precipitated at 4 x 10⁻⁶ cells / mL. 5 The cells / mL concentration was resuspended in RPMI-1640 (Gibco™ 11875-093; containing L-glutamine) containing 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 50 IU / mL human IL-2 (Miltenyi Biotec® 130-097-748). For NK cell cytokine secretion assays, 2 x 10 cells / mL of the cell suspension were resuspended in RPMI-1640 (Gibco™ 11875-093; containing L-glutamine) containing 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 50 IU / mL human IL-2 (Miltenyi Biotec® 130-097-748). 5 NK cells were plated in a 96-well U-shaped substrate (GrenierBio One™ 650180), the substrate containing 50 μl (4 x 10⁻⁶) of DMEM (Gibco™ 11875-093; containing L-glutamine). 4HT1080-luc cells (ATCC® CCL-121-Luc2) were used in DMEM containing 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). The test material was diluted in DMEM medium (Corning™ 10-013-CV) containing 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) and added to the appropriate cell plate (50 μl) at the recommended starting concentration of 10 nM, followed by 8-point serial titrations at a 1:5 ratio. The plates were then incubated at 37°C and 5% CO2 for 4–6 hours. After incubation, the plates were centrifuged at 250 xg for 5 minutes, and 100 μl of supernatant was collected. The samples were immediately stored at -70°C or used to evaluate cytokine levels. Interferon-γ (IFNg) or TNF-α (TNFa) levels were measured in 25 μl of the collected supernatant using the V-PLex kit (2pLex with IFNg and TNFa) pro-inflammatory panels (Mesoscale Diagnostics K151QOD, K151QWD). Data were collected on a MESO® QuickPLex SQ 120MM reader with TeamLink™, ProductLink™, and InstrumentLink™ connectivity 1.0.38, and fitted and analyzed to a four-parameter nonlinear regression in GraphPad Prism™ 8.0.2 software to quantify cytokine levels.

[0486] The selected heterotandem bicyclic peptide complex of the present invention was tested in this assay, and the results are shown in Figures 9-12 middle.

[0487] Figure 9 This image shows NK cells co-cultured with the HT1080-luc tumor cell line in the presence of either BCY27047 or the unbound isotandem bicyclic peptide complex BCY15667. The amount of released cytokines (IFNg) was measured using the V-PLex kit (IFNg) pro-inflammatory panel (Mesoscale Diagnostics K151QOD). Data were collected on a MESO® QuickPLex SQ 120MM reader with TeamLink™, ProductLink™, and InstrumentLink™ connectivity 1.0.38, and fitted and analyzed to a four-parameter nonlinear regression in GraphPad Prism™ 8.0.2 software to quantify cytokine levels. EC BCY27047 50 =8.4 pM.

[0488] Figure 10 The image shows NK cells co-cultured with the HT1080-luc tumor cell line in the presence of BCY26129 or the non-conjugated heterotandem bicyclic peptide complex BCY15667. The amount of released cytokines (IFNg) was measured using the V-PLex kit (IFNg) pro-inflammatory panel (Mesoscale Diagnostics K151QOD). Data were collected on a MESO® QuickPLex SQ 120MM reader with TeamLink™, ProductLink™, and InstrumentLink™ connectivity 1.0.38, and fitted and analyzed to a four-parameter nonlinear regression in GraphPad Prism™ 8.0.2 software to quantify cytokine levels. EC BCY26129 50 =7.6 pM.

[0489] Figure 11 This study demonstrates TNF-α production by NK cells when co-cultured with the HT1080-luc tumor cell line in the presence of BCY27047 or the unbound isotandem bicyclic peptide complex BCY15667. The released cytokine (TNF-α) was measured by MSD assay: V-PLex Kit (TNFa) pro-inflammatory panel (Mesoscale Diagnostics K151QWD). Data were collected on a MESO® QuickPLex SQ 120MM reader with TeamLink™, ProductLink™, and InstrumentLink™ connectivity 1.0.38, and fitted and analyzed to a four-parameter nonlinear regression in GraphPad Prism™ 8.0.2 software to quantify cytokine levels. EC50 of BCY27047... 50 =8.8 pM.

[0490] Figure 12This study demonstrates TNF-α production by NK cells when co-cultured with the HT1080-luc tumor cell line in the presence of BCY26129 or the unbound isotandem bicyclic peptide complex BCY15667. The released cytokine (TNF-α) was measured by MSD assay: V-PLex Kit (TNFa) pro-inflammatory panel (Mesoscale Diagnostics K151QWD). Data were collected on a MESO® QuickPLex SQ 120MM reader with TeamLink™, ProductLink™, and InstrumentLink™ connectivity 1.0.38, and fitted and analyzed to a four-parameter nonlinear regression in GraphPad Prism™ 8.0.2 software to quantify cytokine levels. EC50 of BCY26129... 50 =9.5 pM.

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[0548] The following are the numbering aspects of this invention:

[0549] 1. A peptide ligand specific to natural killer (NK) cells, comprising an amino acid sequence, said amino acid sequence being:

[0550] [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1);

[0551] Where X represents dNva or dA, and Cba represents cyclobutylalanine and Nva represents valine.

[0552] Or its pharmaceutically acceptable salt.

[0553] 2. The peptide ligand according to aspect 1, wherein X represents dNva, and the peptide ligand comprises the following sequence:

[0554] [dC]Y[Cba]PDYLC[dNva]DEYC (SEQ ID NO: 2);

[0555] Nva represents valine.

[0556] Or its pharmaceutically acceptable salt.

[0557] 3. The peptide ligand according to aspect 1, wherein X represents [dA], and the peptide ligand comprises the following sequence:

[0558] [dC]Y[Cba]PDYLC[dA]DEYC (SEQ ID NO: 3);

[0559] Cba represents cyclobutylalanine.

[0560] Or its pharmaceutically acceptable salt.

[0561] 4. The peptide ligand according to any one of aspects 1 to 3, wherein the peptide ligand further comprises an N-terminal and / or C-terminal addition and comprises an amino acid sequence selected from:

[0562] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4), and

[0563] Ac-([dC]Y[Cba]PDYLC[dA]DEYC)-[K(PYA)] (SEQ ID NO: 5),

[0564] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0565] Or its pharmaceutically acceptable salt.

[0566] 5. The peptide ligand according to any one of aspects 1 to 3, wherein the peptide ligand further comprises an N-terminal and / or C-terminal addition and comprises an amino acid sequence, said amino acid sequence being:

[0567] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4),

[0568] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0569] Or its pharmaceutically acceptable salt.

[0570] 6. A bicyclic peptide ligand comprising a peptide ligand according to any one of aspects 1 to 5 and a molecular backbone, wherein three cysteine ​​residues of the peptide ligand are covalently bonded to the molecular backbone to form two cyclic sequences.

[0571] 7. The bicyclic peptide ligand according to aspect 6, wherein the molecular backbone is a derivative of TATA having the following structure:

[0572]

[0573] in This indicates the connection point of the three cysteine ​​residues.

[0574] 8. The bicyclic peptide ligand according to aspect 6 or 7, wherein the bicyclic peptide comprises a molecular backbone, the molecular backbone being a derivative of TATA having the following structure:

[0575]

[0576] in The linker represents the three cysteine ​​residues, and the peptide ligand comprises an amino acid sequence selected from the following:

[0577] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (its bicyclic peptide is referred to herein as BCY26631), and

[0578] Ac-([dC]Y[Cba]PDYLC[dA]DEYC)-[K(PYA)] (SEQ ID NO: 5) (its bicyclic peptide is referred to in this article as BCY24188),

[0579] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0580] Or its pharmaceutically acceptable salt.

[0581] 9. The bicyclic peptide ligand according to aspect 6 or 7, wherein the bicyclic peptide comprises a molecular backbone, the molecular backbone being a derivative of TATA having the following structure:

[0582]

[0583] in The linker represents the three cysteine ​​residues, and the peptide ligand comprises an amino acid sequence as follows:

[0584] Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (its bicyclic peptide is referred to herein as BCY26631), and

[0585] Where Cba represents cyclobutylalanine, Nva represents valine, and PYA represents pentynoic acid.

[0586] Or its pharmaceutically acceptable salt.

[0587] 10. The peptide ligand according to any one of aspects 1 to 5 or the bicyclic peptide ligand according to any one of aspects 6 to 9, wherein the pharmaceutically acceptable salt is selected from free acids or sodium, potassium, calcium or ammonium salts.

[0588] 11. A multimeric binding complex comprising at least two bicyclic peptide ligands according to any one of aspects 6 to 9, wherein the peptide ligands may be the same or different.

[0589] 12. A heterotandem bicyclic peptide complex comprising:

[0590] (a) A first bicyclic peptide ligand that binds to a component present on cancer cells; the first bicyclic peptide ligand is conjugated to via a linker.

[0591] (b) One or more second bicyclic peptide ligands that are specific to natural killer (NK) cells according to any one of aspects 6 to 9.

[0592] 13. The heterotandem bicyclic peptide complex according to aspect 12, wherein the first bicyclic peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

[0593] 14. The heterotandem bicyclic peptide complex according to aspect 13, wherein the EphA2-binding bicyclic peptide ligand comprises an amino acid sequence, said amino acid sequence being:

[0594] C[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 6);

[0595] HyP represents trans-4-hydroxy-L-proline and 1Nal represents 1-naphthylalanine.

[0596] Or its pharmaceutically acceptable salt.

[0597] 15. The heterotandem bicyclic peptide complex according to aspect 13 or 14, wherein the molecular backbone is a derivative of TATA having the following structure:

[0598]

[0599] in The junction of the three cysteine ​​residues is indicated, and the EphA2-binding bicyclic peptide ligand optionally includes N-terminal and / or C-terminal modifications and comprises:

[0600] A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 7) (its bicyclic peptide is referred to as BCY13118 in this article),

[0601] Where HArg represents high arginine, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine.

[0602] Or its pharmaceutically acceptable salt.

[0603] 16. The heterotandem bicyclic peptide complex according to any one of aspects 12 to 15, wherein the linker is:

[0604] (a) Straight-chain connectors, selected from:

[0605]

[0606] Azide-PEG5-acid; and

[0607]

[0608] Azide-PEG24-acid; or

[0609] (b) A branch connector, selected from:

[0610]

[0611] N-(acid-PEG3)-N-bis(PEG3-azide);

[0612]

[0613] N-(acid-PEG) 10 )-N-Bis(PEG) 10 -azide);

[0614]

[0615] N-(PEG3-acid)-N-(PEG3-azide)-N-(PEG3-NH-AcAz);

[0616]

[0617] Benzotriic acid-[Peg] 10 ]3;

[0618]

[0619] TCA-[Peg 10 ]3;

[0620]

[0621] TCA-[Peg 23 ]3;

[0622]

[0623] Tet-[Peg 10 ]4;

[0624]

[0625] Methane-N-(PEG5-acid)-tris(MeOPr-amide-PEG4-azide);

[0626]

[0627] Methane-N-(PEG) 10 -acid)-tris(MeOPr-amide-PEG 10 -azide); and

[0628]

[0629] Bis-N-aminopropyl-glycine-(PEG5)2.

[0630] 17. The heterotandem bicyclic peptide complex according to any one of aspects 12 to 16, wherein the linker is:

[0631]

[0632] N-(acid-PEG3)-N-bis(PEG3-azide).

[0633] 18. The heterotandem bicyclic peptide complex according to any one of aspects 12 to 17, wherein the complex is selected from BCY27047:

[0634]

[0635] Or BCY26129:

[0636] .

[0637] 19. A pharmaceutical composition comprising a peptide ligand according to any one of aspects 1 to 5, or a bicyclic peptide ligand according to any one of aspects 6 to 10, or a multimeric binding complex according to aspect 11, or an heterotandem bicyclic peptide complex according to any one of aspects 12 to 18, and a combination of one or more pharmaceutically acceptable excipients.

[0638] 20. A peptide ligand according to any one of aspects 1 to 5, or a bicyclic peptide ligand according to any one of aspects 6 to 10, or a multimeric binding complex according to aspect 11, or an heterotandem bicyclic peptide complex according to any one of aspects 12 to 18, or a pharmaceutical composition according to aspect 19, for the prevention, inhibition, or treatment of diseases or disorders mediated by natural killer (NK) cells.

[0639] 21. A peptide ligand according to any one of aspects 1 to 5, or a bicyclic peptide ligand according to any one of aspects 6 to 10, or a multimeric binding complex according to aspect 11, or an heterotandem bicyclic peptide complex according to any one of aspects 12 to 18, or a pharmaceutical composition according to aspect 19, for the prevention, inhibition, or treatment of diseases or disorders selected from inflammatory disorders, autoimmune diseases, and cancer.

Claims

1. A peptide ligand comprising a polypeptide having an amino acid sequence of: [dC]Y[Cba]PDYLCXDEYC (SEQ ID NO: 1); wherein X represents dNva or dA, wherein dNva represents D-norvaline, dA represents D-alanine, dC represents D-cysteine, and Cba represents cyclobutylalanine; or a pharmaceutically acceptable salt thereof.

2. The peptide ligand of claim 1, wherein X represents dNva and the peptide ligand comprises a polypeptide of the sequence: [dC]Y[Cba]PDYLC[dNva]DEYC (SEQ ID NO: 2); wherein dNva represents D-norvaline, dC represents D-cysteine, and Cba represents cyclobutylalanine, or a pharmaceutically acceptable salt thereof.

3. The peptide ligand of claim 1, wherein X represents [dA] and the peptide ligand comprises a polypeptide of the sequence: [dC]Y[Cba]PDYLC[dA]DEYC (SEQ ID NO: 3); wherein dA represents D-alanine, dC represents D-cysteine, and Cba represents cyclobutylalanine, or a pharmaceutically acceptable salt thereof.

4. The peptide ligand of any one of claims 1 to 3, wherein the polypeptide comprises one or more N-terminal and / or C-terminal additions.

5. The peptide ligand of claim 4, comprising a polypeptide having an amino acid sequence selected from the group consisting of: Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4), and Ac-([dC]Y[Cba]PDYLC[dA]DEYC)-[K(PYA)] (SEQ ID NO: 5), wherein dNva represents D-norvaline, dA represents D-alanine, dC represents D-cysteine, Cba represents cyclobutylalanine, and PYA represents pentynoic acid, or a pharmaceutically acceptable salt thereof.

6. The peptide ligand of any one of claims 1 to 3, comprising a polypeptide of the sequence: Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4), wherein dC represents D-cysteine, Cba represents cyclobutylalanine, dNva represents D-norvaline and PYA represents pentynoic acid, or a pharmaceutically acceptable salt thereof.

7. The peptide ligand of any one of claims 4 to 6, wherein the C-terminus is amidated.

8. The peptide ligand of any one of claims 1 to 7, wherein the polypeptide is linked to a molecular scaffold.

9. A bicyclic peptide ligand comprising the peptide ligand of any one of claims 1 to 8, wherein the polypeptide is linked to a molecular scaffold, wherein three cysteine residues of the peptide ligand form covalent bonds with the molecular scaffold to form two loop sequences. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 10. The peptide ligand of claim 8 or the bicyclic peptide ligand of claim 9, wherein the molecular scaffold is a derivative of TATA having the structure: ， wherein represents the point of attachment to the polypeptide; optionally, represents the point of attachment of three cysteine residues of the polypeptide.

11. The bicyclic peptide ligand of claim 9 or claim 10, comprising a molecular scaffold which is a derivative of TATA having the structure: ， wherein denotes the point of attachment of the three cysteine residues, and the peptide ligand comprises a polypeptide having an amino acid sequence selected from the group consisting of: Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (wherein the bicyclic peptide is referred to herein as BCY26631), and Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (wherein the bicyclic peptide is referred to herein as BCY26631), and wherein Cba represents cyclobutylalanine, dNva represents D-norvaline, dC represents D-cysteine and PYA represents pentyne acid, or a pharmaceutically acceptable salt thereof.

12. The bicyclic peptide ligand of any one of claims 9 to 11, wherein the bicyclic peptide comprises a molecular scaffold which is a derivative of TATA having the structure: , wherein denotes the point of attachment of the three cysteine residues, and the peptide ligand comprises a polypeptide having an amino acid sequence of: Ac-([dC]Y[Cba]PDYLC[dNva]DEYC)-[K(PYA)] (SEQ ID NO: 4) (wherein the bicyclic peptide is referred to herein as BCY26631), and wherein Cba represents cyclobutylalanine, dNva represents D-norvaline, dC represents D-cysteine and PYA represents pentyne acid, or a pharmaceutically acceptable salt thereof.

13. The peptide ligand of any one of claims 1 to 8 or 10 or the bicyclic peptide ligand of any one of claims 9 to 12, wherein the pharmaceutically acceptable salt is selected from the free acid or a sodium, potassium, calcium or ammonium salt.

14. The peptide ligand of any one of claims 1 to 8, 10 or 13 or the bicyclic peptide ligand of any one of claims 9 to 13, which is specific for natural killer (NK) cells.

15. A multimeric binding complex comprising at least two bicyclic peptide ligands, which can be the same or different, wherein at least one bicyclic peptide ligand is as defined in any one of claims 9 to 14; optionally, wherein the multimeric binding complex comprises one or more cytotoxic agents, chelating agents, chromophores and / or fluorophores.

16. The multimeric binding complex of claim 15, comprising at least two bicyclic peptide ligands of any one of claims 9 to 14, wherein the peptide ligands can be the same or different; optionally, wherein the multimeric binding complex comprises one or more cytotoxic agents, chelating agents, chromophores and / or fluorophores.

17. A hetero-serial bicyclic peptide complex comprising: (a) a first bicyclic peptide ligand capable of binding to a component present on a cancer cell; the first bicyclic peptide ligand being linked to (b) one or more second bicyclic peptide ligands according to any one of claims 9 to 14, wherein the one or more second bicyclic peptide ligands can be the same or different.

18. The hetero-serial bicyclic peptide complex of claim 17, comprising: (a) a first bicyclic peptide ligand that binds to a component present on a cancer cell; the first bicyclic peptide ligand is conjugated to (b) one or more second bicyclic peptide ligands according to any one of claims 9 to 14.

19. The hetero-serial bicyclic peptide complex of claim 17 or 18, wherein the first bicyclic peptide ligand binds to EphA2, Nectin-4, PD-L1, MT1, or PSMA.

20. The hetero-serial bicyclic peptide complex of any one of claims 17 to 19, wherein the first bicyclic peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

21. The hetero-serial bicyclic peptide complex of claim 20, wherein the EphA2-binding bicyclic peptide ligand comprises a polypeptide having the amino acid sequence: C[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 6); wherein HyP represents trans-4-hydroxy-L-proline and d1Nal represents D-1- naphthylalanine, or a pharmaceutically acceptable salt thereof.

22. The hetero-serial bicyclic peptide complex of claim 21, wherein the EphA2- binding bicyclic peptide ligand comprises one or more N-terminal and / or C-terminal modifications.

23. The hetero-serial bicyclic peptide complex of any one of claims 18 to 22, wherein the polypeptide of the first bicyclic peptide ligand is linked to a molecular scaffold.

24. The hetero-serial bicyclic peptide complex of claim 23, wherein the molecular scaffold is a derivative of TATA having the structure: ， wherein represents the point of attachment to the polypeptide; optionally, represents the point of attachment of three cysteine residues of the polypeptide.

25. The hetero-serial bicyclic peptide complex of any one of claims 17 to 24, wherein the first bicyclic peptide ligand comprises an EphA2-binding bicyclic peptide ligand comprising a molecular scaffold that is a derivative of TATA having the structure: ， wherein represents the point of attachment of the three cysteine residues, and the EphA2 binding bicyclic peptide ligand optionally comprises an N-terminal and / or C-terminal modification and comprises: A[HArg]DC[HyP]LVNPLCLEP[d1Nal]WTC (SEQ ID NO: 7) (wherein the bicyclic peptide is referred to herein as BCY13118), wherein HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, and d1Nal represents D-1-naphthylalanine, or a pharmaceutically acceptable salt thereof.

26. The hetero-serial bicyclic peptide complex of any one of claims 18 to 25, wherein the linker is a linear linker or a branched linker, optionally wherein the branched linker comprises three or four branches and is capable of binding to three or four bicyclic peptide ligands.

27. The hetero-interstrand bicyclic peptide complex of any one of claims 18-26, wherein the linker comprises one or more PEG groups, optionally wherein the linker comprises one or more PEG n groups, wherein each n is independently an integer from about 2 to about 25. Optionally, wherein the linker comprises one or more azido groups capable of reacting with one or more alkyne groups of the one or more peptide ligands; and / or one or more carboxylic acid groups or activated derivatives thereof capable of reacting with one or more amine groups of the one or more peptide ligands.

28. The hetero-serial bis-cycle peptide complex of any one of claims 18 to 27, wherein the first bis-cycle peptide ligand is conjugated to the one or more second bis-cycle peptide ligands by a linker selected from the group consisting of: (a) a linear linker selected from the group consisting of: azide-PEG5-acid; and azido-PEG24-acid; or (b) a branched linker selected from the group consisting of: N-(acid-PEG3)-N-bis(PEG3-azido); N-(acid-PEG 10 )-N-bis(PEG 10 -azide); N-(acid-PEG3)-N-bis(PEG3-azido); benzene tricarboxylic acid - [Peg 10 ]3; TCA-[Peg 10 ]3; TCA-[Peg 23 ]3; Tet-[Peg 10 ]4; methane-N-(PEG5-acid)-tris(MeOPr-amide-PEG4-azido); Methane-N-(PEG 10 -acid)-tris(MeOPr-amide-PEG 10 -azide); and bis-N-aminopropyl-glycine-(PEG5)2.

29. The hetero-serial bis-cycle peptide complex of any one of claims 18 to 28, wherein the linker is: N-(acid-PEG3)-N-bis(PEG3-azido).

30. A hetero-serial bis-cycle peptide complex selected from the group consisting of BCY27047: wherein L1is L2is L3is or a pharmaceutically acceptable salt thereof; or BCY26129: wherein L 1 To L 2 To L 3 To or a pharmaceutically acceptable salt thereof.

31. A pharmaceutical composition comprising the peptide ligand of any one of claims 1 to 8, or the bis-cycle peptide ligand of any one of claims 9 to 14, or the multimeric binding complex of claim 15, or the hetero-serial bis-cycle peptide complex of any one of claims 16 to 30, in combination with one or more pharmaceutically acceptable excipients.

32. The peptide ligand of any one of claims 1 to 8, or the bis-cycle peptide ligand of any one of claims 9 to 14, or the multimeric binding complex of claim 15, or the hetero-serial bis-cycle peptide complex of any one of claims 16 to 30, or the pharmaceutical composition of claim 31, for use in the prevention, inhibition or treatment of a disease or disorder mediated by natural killer (NK) cells.

33. The peptide ligand of any one of claims 1 to 8, or the bis-cycle peptide ligand of any one of claims 9 to 14, or the multimeric binding complex of claim 15, or the hetero-serial bis-cycle peptide complex of any one of claims 16 to 30, or the pharmaceutical composition of claim 31, for use in the prevention, inhibition or treatment of a disease or disorder selected from the group consisting of inflammatory disorders, autoimmune diseases and cancer.

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