Heterotandem bicyclic peptide conjugates

Abstract

The present invention relates to a heterotandem bicyclic peptide complex comprising a first peptide ligand that binds to a component present on a cancer cell conjugated via a linker to a second peptide ligand that binds to one or more components present on a natural killer (NK) cell. The present invention also relates to the use of the heterotandem bicyclic peptide complex in the prevention, inhibition, or treatment of cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a heterotandem bicyclic peptide complex comprising a first peptide ligand that binds to a component present on a cancer cell and one or more second peptide ligands that bind to one or more components present on a natural killer (NK) cell, the first peptide ligand being conjugated to the second peptide ligand via a linker. The present invention also relates to the use of the heterotandem bicyclic peptide complex in the prevention, inhibition, or treatment of cancer. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are able to bind protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutic drugs. Indeed, several cyclic peptides have already been successfully used in the clinic, such as, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al., (2008), Nat Rev Drug Discov 7(7), 608-24). The excellent binding properties are due not only to the relatively large interaction surface formed between the peptide and the target, but also to the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are used with the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 (Wu et al., (2007), Science 330, 1066-71), a cyclic peptide (355 Å) with an Arg-Gly-Asp motif that binds to integrin αVb3 2 ) (Xiong et al., (2002), Science 296(5565), 151-5), or the cyclic peptide inhibitor upain-1 (603 Å) that binds to urokinase-type plasminogen activator. 2 (Zhao et al., (2007), J Struct Biol 160(1), 1-10), which bind to surfaces measuring several hundred square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides and experience less entropy loss when binding to targets, resulting in higher binding affinity. The reduced flexibility also results in a target-specific conformational fixation, increasing binding specificity compared to linear peptides. This effect is exemplified by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity against other MMPs when its ring is opened (Cherney et al., (1998), J Med Chem 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously tethered polypeptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara, (1985), J. Org. Chem; Timmerman et al., (2005), ChemBioChem). Meloen and coworkers used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclization of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al., (2005), ChemBioChem). Methods for the generation of candidate drug compounds, where the compounds are generated by linking cysteine-containing polypeptides to molecular scaffolds such as tris(bromomethyl)benzene, are disclosed in WO2004 / 077062 and WO2006 / 078161.

[0005] A phage display-based combinatorial approach has been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al., (2009), Nat Chem Biol 5(7), 502-7 and WO2009 / 098450). Briefly, a linear peptide containing three cysteine ​​residues and two random six amino acid regions (Cys-(Xaa) 6 -Cys-(Xaa) 6 A combinatorial library of cysteine ​​(Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the Invention

[0006] (Summary of the invention) In a first aspect of the present invention, (a) a first peptide ligand that binds to a component present on a cancer cell; (b) one or more second peptide ligands that bind to one or more components present on natural killer (NK) cells; The heterotandem bicyclic peptide complex is provided, wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, and at least two polypeptide loops are formed on the molecular scaffold.

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

[0008] In a further aspect of the present invention there is provided a heterotandem bicyclic peptide complex as defined herein for use in the prevention, suppression or treatment of cancer. [Brief description of the drawings]

[0009] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows the activation of NK cells by BCY15664 and BCY15911, as measured by upregulation of CD107a. [Diagram 2] FIG. 2 shows the NK cytotoxicity assay results for BCY17226. [Diagram 3] FIG. 3 shows the NK cytotoxicity assay results for BCY15664 and BCY15923. [Figure 4] FIG. 4 shows the NK cytotoxicity assay results for BCY15664. [Diagram 5] FIG. 5 shows the IFN-γ secretion assay results for BCY17226. [Figure 6] FIG. 6 shows the NK cytotoxicity assay results for BCY17225, BCY21686, and BCY21687. [Figure 7] FIG. 7 shows the NK cytotoxicity assay results for BCY17231, BCY17235, and BCY18731. [Figure 8] FIG. 8 shows the NK cytotoxicity assay results for BCY20793. [Figure 9] FIG. 9 shows the NK cytotoxicity assay results for BCY15924, BCY18042, and BCY18049. [Figure 10] FIG. 10 shows the NK cytotoxicity assay results for BCY18603 and BCY18604. [Figure 11] FIG. 11 shows the cytokine secretion assay results for BCY17225, BCY21686, and BCY21687. [Figure 12] FIG. 12 shows the cytokine secretion assay results for BCY18048. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention In a first aspect of the present invention, (a) a first peptide ligand that binds to a component present on a cancer cell; (b) one or more second peptide ligands that bind to one or more components present on natural killer (NK) cells; the heterotandem bicyclic peptide complex, each of said peptide ligands comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with said reactive groups of said polypeptide, and at least two polypeptide loops are formed on said molecular scaffold. is provided.

[0011] (First peptide ligand) The term "cancer cell" as referred to herein includes any cell known to be involved in cancer. Cancer cells arise when genes involved in the control of cell division are damaged. Carcinogenesis occurs through mutations and epimutations of the genetic material of normal cells that disrupt the normal balance between proliferation and cell death. This results in uncontrolled cell division and the evolution of these cells by natural selection in the body. Uncontrolled, and often rapid, cell proliferation can result in 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 sites (metastasis), and become life threatening.

[0012] 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.

[0013] In one embodiment, the component present on cancer cells is Nectin-4.

[0014] Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which includes four members. Nectins are cell adhesion molecules that play important roles in various biological processes, such as polarization, 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 the main receptors for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding nectin-1 (PVRL1) or nectin-4 (PVRL4) cause ectodermal dysplasia syndromes, which are associated with other abnormalities. Nectin-4 is expressed during fetal development. Its expression is more restricted in adult tissues than that of the other members of the family. Nectin-4 is a tumor-associated antigen in 50%, 49%, and 86% of breast, ovarian, and lung cancers, respectively, and is mainly associated with tumors with poor prognosis. Its expression is not detectable in corresponding normal tissues. In breast tumors, Nectin-4 is mainly expressed in triple-negative and ERBB2+ carcinomas. Detection of soluble forms of Nectin-4 in the serum of these cancer patients is associated with poor prognosis. Serum levels of Nectin-4 increase during metastatic progression and decrease after treatment. These results suggest that Nectin-4 may be a reliable target for the treatment of cancer. Accordingly, 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 that is currently in clinical investigation for the treatment of patients suffering from solid tumors.

[0015] In one embodiment, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand.

[0016] Suitable examples of Nectin-4 binding bicyclic peptide ligands are disclosed in WO2019 / 243832, the peptides of which are incorporated herein by reference.

[0017] In one embodiment, the Nectin-4 binding bicyclic peptide comprises: [ka] (In the formula, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; HArg represents homoarginine; and HyP represents trans-4-hydroxy-L-proline, or a pharma- ceutically acceptable salt thereof.

[0018] In another embodiment, the component present on cancer cells is EphA2.

[0019] Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosine residues. Ephs and their membrane-bound ephrin ligands (ephrins) control cell location and tissue organization (Poliakov et al., (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur in a state of high ligand oligomerization (Stein et al., (1998) Genes Dev 12, 667-678).

[0020] Among other pattern recognition functions, various Ephs and ephrins have been shown to play roles in vascular development. Knockout of EphB4 and ephrin-B2 results in a loss of ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrins has been observed in newly formed adult microvessels as well (Brantley-Sieders et al., (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0021] The deregulated re-emergence of some ephrins and their receptors in adults has also been observed to contribute to tumor invasion, metastasis, and neovascularization (Nakamoto et al., (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). Furthermore, several Eph family members have been found to be overexpressed in tumor cells derived from a variety of human tumors (Brantley-Sieders et al., supra; Marme, (2002) Ann Hematol 81 Suppl 2, S66; Booth et al., (2002) Nat Med 8, 1360-1).

[0022] EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHA2 gene.

[0023] EphA2 is expressed in a number of human cancers that are often correlated with disease progression, metastasis, and poor prognosis, including breast cancer (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 cancer (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), gastric cancer (Nakamura et al., (2005), Cancer Sci. 96, 301-308), and leukemia (Hu et al., (2013), J Thorac Oncol. 97, 301-308). 42-47; Yuan et al. (2009), Dig Dis Sci 54, 2410-2417), pancreatic cancer (Mudali et al. (2006), Clin Exp Metastasis 23, 357-365), prostate cancer (Walker-Daniels et al. (1999), Prostate 41, 275-280), liver cancer (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).

[0024] Although the full role of EphA2 in cancer progression is yet to be defined, there is evidence of its interaction at many stages of cancer progression, including tumor cell proliferation, survival, invasion, and angiogenesis. Downregulation of EphA2 expression suppresses tumor cancer cell proliferation (Binda et al. (2012), Cancer Cell 22, 765-780), while blocking EphA2 suppresses 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).

[0025] Antibody-drug conjugates with 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 a similar approach has 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).

[0026] In one embodiment, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

[0027] Suitable examples of EphA2-binding bicyclic peptide ligands are disclosed in WO2019 / 122860, WO2019 / 122861, and WO2019 / 122863, the peptides of which are incorporated herein by reference.

[0028] In one embodiment, the EphA2-binding bicyclic peptide ligand is: [ka] (In the formula, C i , C ii , and C iii represents a first (i) reactive group, a second (ii) reactive group, and a third (iii) reactive group; HyP represents trans-4-hydroxy-L-proline; and HArg represents homoarginine, or a pharma- ceutically acceptable salt thereof.

[0029] In a further embodiment, the EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal and / or a C-terminal modification and comprises: A-[HArg]-D-(SEQ ID NO:2) (referred to herein as BCY9594); (wherein HArg represents homoarginine), or a pharma- ceutically acceptable salt thereof.

[0030] In another embodiment, the component present on cancer cells is PD-L1.

[0031] 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. Expression of PD-L1 is involved in evading immune responses involved in chronic infections, such as chronic viral infections (including, for example, HIV, HBV, HCV, and HTLV, among others), chronic bacterial infections (including, for example, Helicobacter pylori, among others), and chronic parasitic infections (including, for example, Schistosoma mansoni). PD-L1 expression has been detected in numerous tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, myocytes, and placenta.

[0032] PD-L1 expression has also been implicated in suppressing antitumor immune activity. Although tumors express antigens that can be recognized by host T cells, immune elimination of tumors is rare. This failure is due in part to immune suppression by the tumor microenvironment. PD-L1 expression in many tumors is a component of this suppressive environment and acts in concert with other immunosuppressive signals. PD-L1 expression has been demonstrated in situ in a wide variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymic epithelium, and head and neck (Brown JA et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003 Cancer Res. 63:6501-5; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi 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, expression of programmed cell death protein 1 (also known as PD-1 and CD279), a receptor for PD-L1, is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al., 2003 Immunol. 171:4574-81).Most importantly, studies on the relationship between PD-L1 expression in tumors and disease outcome have shown that PD-L1 expression is strongly correlated with poor prognosis in renal, ovarian, bladder, breast, gastric, and pancreatic cancers (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). Furthermore, these studies suggest that higher levels of PD-L1 expression in tumors may promote tumor stage progression and deeper invasion into tissue structures.

[0033] The PD-1 pathway may also play a role in hematological 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 on some primary T-cell lymphomas, especially aplastic large cell T-lymphoma (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is highly expressed on T cells in angioimmunoblastic lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al., 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin's lymphoma, T cells associated with lymphocytes or histiocytic (L&H) cells express PD-1. Microarray analysis using gene readouts induced by PD-1 ligation suggests that tumor-associated T cells respond to PD-1 signals in situ in non-Hodgkin's lymphoma (Chemnitz JM et al., 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-associated adult T-cell leukemia and lymphoma (Shimauchi T et al., 2007 Int. J. Cancer 121:2585-90). These tumor cells are hyporesponsive to TCR signals.

[0034] Studies in animal models have shown that tumor PD-L1 inhibits T cell activation, suppresses 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 antitumor T cell responses. PD-L1 expression on tumor-associated bone marrow DC populations is upregulated by tumor environmental factors (Curiel TJ et al. 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in tumor-draining lymph nodes of B16 melanoma express IDO, which potently activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cellular contact with IDO-expressing DCs (Sharma MD et al. 2007 Clin. Invest. 117:2570-82).

[0035] In one embodiment, the first peptide ligand comprises a PD-L1 binding bicyclic peptide ligand.

[0036] In one embodiment, the PD-L1 binding bicyclic peptide ligand is: [ka] (In the formula, C i , C ii , and C iii represent a first (i) cysteine ​​group, a second (ii) cysteine ​​group, and a third (iii) cysteine ​​group, respectively, or a pharma- ceutically acceptable salt thereof.

[0037] In a further embodiment, the molecular scaffold is TATA and the PD-L1 binding bicyclic peptide ligand optionally comprises an N-terminal modification and / or a C-terminal modification, and Ac-D-[Harg]-(SEQ ID NO:3)-PSH (referred to herein as BCY11865) (wherein Harg represents homoarginine), or a pharma- ceutical acceptable salt thereof.

[0038] Suitable examples of PD-L1 binding bicyclic peptide ligands are disclosed in WO2020 / 128526 and WO2020 / 128527, the peptides of which are incorporated herein by reference.

[0039] In another embodiment, the component present on cancer cells is membrane type 1 matrix metallopeptidase 14 (MT1, also known as MMP14). MT1-MMP is a transmembrane metalloprotease that plays a major role in extracellular matrix remodeling, directly by degrading some of its components and indirectly by activating pro-MMP2. MT1-MMP is crucial for tumor angiogenesis (Sounni et al., (2002) FASEB J. 16(6), 555-564) and is overexpressed in various solid tumors. Thus, the MT1-MMP-binding bicyclic peptides of the invention have particular utility for targeted treatment of cancer, particularly solid tumors such as non-small cell lung cancer. In one embodiment, the bicyclic peptides of the invention are specific for human MT1-MMP. In a further embodiment, the bicyclic peptides of the invention are specific for mouse MT1-MMP. In yet further embodiments, the bicyclic peptides of the invention are specific for human MT1-MMP and mouse MT1-MMP. In yet further embodiments, the bicyclic peptides of the invention are specific for human MT1-MMP, mouse MT1-MMP and canine MT1-MMP.

[0040] In one embodiment, the MT1-binding bicyclic peptide ligand is: [ka] (In the formula, C i , C ii , and C iii represent a first (i) cysteine ​​group, a second (ii) cysteine ​​group, and a third (iii) cysteine ​​group, respectively, or a pharma- ceutically acceptable salt thereof.

[0041] In a further embodiment, the molecular scaffold is TATA and the MT1-binding bicyclic peptide ligand optionally comprises an N-terminal and / or a C-terminal modification and: LPP-(SEQ ID NO:4) (referred to herein as BCY14320) or a pharma- ceutical acceptable salt thereof.

[0042] Suitable examples of MT1-binding bicyclic peptide ligands are disclosed in WO2016 / 067035, the peptides of which are incorporated herein by reference.

[0043] In another embodiment, the component present on cancer cells is prostate-specific membrane antigen (PSMA).

[0044] Prostate-specific membrane antigen (PSMA), also known as glutamic acid carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamic peptidase I (NAALADase I), and NAAG peptidase, is an enzyme that, in humans, is encoded by the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750 amino acids and weighs approximately 84 kDa.

[0045] Human PSMA is highly expressed in the prostate, approximately 100-fold higher than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, increasing 8-12-fold over levels in noncancerous prostate cells. Because of this high expression, PSMA is being developed as a potential biomarker for the treatment and imaging of some cancers. In human prostate cancer, high tumor expression is associated with shorter progression-free times and a greater proportion of patients who relapse.

[0046] In one embodiment, the first peptide ligand comprises a PSMA-binding bicyclic peptide ligand.

[0047] Suitable examples of PSMA-binding bicyclic peptide ligands are WO2019 / 243455 and WO2020 / 120980, the peptides of which are incorporated herein by reference.

[0048] (Second Peptide Ligand) It will be appreciated that one or more second peptide ligands are required to bind to one or more components present on natural killer (NK) cells. It will also be appreciated that when more than one second peptide ligand is present, the multiple second peptide ligands may bind to the same or different targets within the NK cell. Thus, in one embodiment, the second bicyclic peptide ligand is specific for the same target within the NK cell. In a further embodiment, the heterotandem bicyclic peptide complex comprises at least two identical second bicyclic peptide ligands. This "identical" means that the second bicyclic peptide has the same amino acid sequence, and, of particular importance, the identical amino acid sequence refers to the binding portion of the second bicyclic peptide (e.g., the binding position of that sequence may vary). In this embodiment, each second bicyclic peptide in the heterotandem bicyclic peptide complex will bind to the exact same epitope on the same target on an NK cell, such that the target-bound complex will form a homodimer (if the heterotandem bicyclic peptide complex contains two identical second bicyclic peptides), a homotrimer (if the heterotandem bicyclic peptide complex contains three identical second bicyclic peptides), or a homotetramer (if the heterotandem bicyclic peptide complex contains four identical second bicyclic peptides), and so on.

[0049] In another embodiment, the heterotandem bicyclic peptide complex comprises at least two different second bicyclic peptide ligands, where "different" means that the second bicyclic peptides have different amino acid sequences. In this embodiment, the different second bicyclic peptides in the heterotandem bicyclic peptide complex bind to different epitopes on NK cells, such that target-bound complexes will be biparatopic (if the heterotandem bicyclic peptide complex comprises two different second bicyclic peptides), triparatopic (if the heterotandem bicyclic peptide complex comprises three different second bicyclic peptides), or tetraparatopic (if the heterotandem bicyclic peptide complex comprises four different second bicyclic peptides), etc.

[0050] Without being bound by theory, it is believed that the resulting heterotandem bicyclic peptide complex can activate different targets on NK cells, such as different target receptors, by heterocrosslinking the receptors. Thus, in one embodiment, the second bicyclic peptide ligands are specific for different targets on NK cells. In this embodiment, it will be appreciated that the heterotandem bicyclic peptide complex includes at least two different second bicyclic peptide ligands (i.e., second bicyclic peptide ligands having different amino acid sequences). In this embodiment, each of the second bicyclic peptides in the heterotandem bicyclic peptide complex will bind to a different epitope on a NK cell, such that the target-bound complex will be a bispecific heterotandem bicyclic peptide complex (if the heterotandem bicyclic peptide complex comprises two different second bicyclic peptides), a trispecific multimeric binding complex (if the heterotandem bicyclic peptide complex comprises three different second bicyclic peptides), or a tetraspecific heterotandem bicyclic peptide complex (if the heterotandem bicyclic peptide complex comprises four different second bicyclic peptides), etc.

[0051] (NKp46-binding bicyclic peptide) Natural killer (NK) cells are members of the innate immune system that occupy a small fraction of peripheral blood mononuclear cells. As frontline responders, these immune cells detect and eliminate unhealthy cells, bridging the gap between innate and adaptive immune responses. Their unique properties make NK cells excellent candidates to enhance therapeutic approaches in tumor immunity and autoimmunity.

[0052] NK cells play a role in immune surveillance through various inhibitory and activating receptors. These activating and inhibitory receptors on the surface of NK cells are a complex means through which NK cell activity is balanced in healthy individuals. NK cells recognize MHC class I molecules on the surface of healthy cells and inhibit the elimination of these healthy cells through inhibitory receptors. During times of stress, infection, or transformation, NK cells recognize unhealthy cells through the loss of MHC class I on their cell surface and the induction of NK cell receptor ligands that bind to activating receptors. Recognition of non-self by NK cells initiates a cytotoxic response, with the release of cytokines and cytotoxic molecules leading to the elimination of unhealthy cells.

[0053] NK cell activity is due to a complex mechanism involving both activating and inhibitory signals. Several studies have provided evidence for the central role of NK cell receptors in natural cytotoxicity and their utility in cancer therapy. An open challenge is to further understand and enhance NK cell-mediated tumor cell recognition and killing. Reports suggest that tumor cells utilize many mechanisms to reduce NK activity and that the presence and availability of NK cells correlates with favorable patient prognosis (Pasero et al. (2015) Oncotarget 6(16), 14360-14373; Stringaris et al. (2014) Haematologica 99(5), 836-847). Through therapeutic intervention, the potential of NK cells to mediate immune responses to combat cancer and autoimmune diseases could be harnessed.

[0054] In one embodiment, the one or more components present on a natural killer (NK) cell are natural cytotoxic receptors present on the surface of the NK cell. In a further embodiment, the one or more components present on a natural killer (NK) cell are natural cytotoxic receptors selected from NKp30, NKp44, and NKp46. In an even further embodiment, the one or more components present on a natural killer (NK) cell is NKp46.

[0055] Natural cytotoxicity receptors (NCRs) are a family of stimulatory receptors expressed on the surface of NK cells that initiate NK activation and NK cell-mediated cytotoxicity. The NCR family consists of three members: NKp30, NKp44, and NKp46. Although the cellular ligand for NKp46 is unknown, a role for NKp46 in antitumor immunity has been demonstrated. NK cell activation of NKp46 via viral antigens leads to tumor rejection (Chinnery et al., 2012). The NKp46 receptor, through interaction with its ligand, induces NK cells to engage and direct cytotoxicity, as shown by the use of anti-NKp46 blocking antibodies that inhibit the ability of NK cells to lyse targets (Arnon et al., 2004). The amount of NCR expression on the surface of NK cells also increases NK cytotoxicity. A strong correlation has been identified between the density of NCR expression and the ability of NK cells to kill target cells, including a wide variety of tumor cells (Moretta et al., 2006). In AML and cervical cancer and its precursor lesions, tumor cells are resistant to NK cytotoxicity due to insufficient amounts of NCR or NCR ligands (Costello et al., 2002; Garcia-Iglesias et al., 2009). In many solid tumors, NK cells are downregulated by the tumor microenvironment, including tumors that shed NCR ligands and immunoedit, impeding the ability of NK cells to recognize, infiltrate, and kill tumor cells (Nayyar et al., 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 poor cytotoxicity of NK cells from AML patients.Furthermore, in solid tumors such as prostate cancer, a decrease in the expression of several activating receptors (CD16, NKp30, NKp46, NKG2D, and DNAM-1) and an increase in the inhibitory receptor CD85j have been reported (Pesaro et al., 2016). In contrast, Gautheir et al. (2019) identified NKp46 as a potential candidate for targeting activating receptors on NK cells in cancer and demonstrated no statistically significant downregulation of NKp46 in the periphery in patients with SCCHN, breast cancer, liver cancer, lung cancer, kidney cancer, and metastatic melanoma. Furthermore, persistent expression of NKp46 in multiple solid tumors associated with downregulation of other activating receptors, e.g., NKG2D, NKp30, and NKp44, as well as low expression of CD16 on tumor-infiltrating lymphocytes, has been reported in cancers, e.g., acute myeloid leukemia, breast cancer, and lung cancer (Fauriat et al., 2007; Mamessier et al., 2011; Platonova et al., 2011; Levi et al., 2015; Kim et al., 2010; MacFarlane et al., 2017). Thus, NKp46 was shown to be a specific NK surface marker suitable for therapeutic applications, allowing NK cells to identify and target tumors.

[0056] In one embodiment, the one or more NKp46-binding bicyclic peptide ligands are: [ka] (In the formula, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively; Cba represents β-cyclobutylalanine; dA represents D-alanine; and PYA represents pentynic acid), or a pharma- ceutically acceptable salt thereof.

[0057] In a further embodiment, the molecular scaffold is TATA and the one or more NKp46-binding bicyclic peptide ligands optionally comprise an N-terminal and / or C-terminal modification and: Ac-(SEQ ID NO:5)-[K(PYA)] (referred to herein as BCY17224); A-(SEQ ID NO:6)-A-[dK(PYA)] (referred to herein as BCY15452); A-(SEQ ID NO:7)-A-[K(PYA)] (referred to herein as BCY15686); A-(SEQ ID NO:8)-A-[K(PYA)] (referred to herein as BCY15687); A-(SEQ ID NO:9)-A-[K(PYA)] (referred to herein as BCY18004); A-(SEQ ID NO:10)-A (referred to herein as BCY17662); and A-(SEQ ID NO:11)-A-[K(PYA)] (referred to herein as BCY18005) wherein PYA represents pentynic acid, or a pharma- ceutically acceptable salt thereof.

[0058] (CD16a-binding bicyclic peptide) In one embodiment, the one or more components present on a natural killer (NK) cell are Fc receptors present on the surface of the NK cell. In a further embodiment, the one or more components present on a natural killer (NK) cell are low affinity Fcγ receptors (FcγR) selected from FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB. In an even further embodiment, the one or more components present on a natural killer (NK) cell are FcγRIIIA (also known as CD16a).

[0059] Fc receptors are expressed on the surface of many leukocytes. In humans, five classical low affinity Fcγ receptors (FcγR) (FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB) bind to the Fc portion of immunoglobulin G (IgG) and are mediators of inflammation through immune cell activation and suppression (Muta et al., 1994; Ravetch et al., 2001). FcγRIIIA (CD16a) is activated by engagement with the Fc portion of IgG molecules and is crucial for the antibody-dependent cellular cytotoxicity (ADCC) process. ADCC is a mechanism by which antigen-specific antibodies direct NK cells to kill cancer cells expressing the antigen (Arnould et al., 2006). Given its important role in the antitumor effects of IgG1 mAbs, several studies have shown that the antitumor part of the effect of trastuzumab, a human IgG1 anti-human epidermal growth factor receptor 2 (HER-2) antibody, is mediated by ADCC, similar to the EGFR antibody cetuximab in metastatic colorectal patients (Zhang et al., 2007, 2020; Wu et al., 2003). Similar results have been reported for rituximab, a chimeric IgG1 mAb against the B cell differentiation antigen CD20 (Manches et al., 2003; Clynes et al., 2000). The utility of CD16 expression in inducing immune cells to promote tumor cell death was shown in overexpression studies. Upon retroviral transduction of Ig-Fc, expression of IgFc on the surface of B16 melanoma cells results in tumor killing in vivo (Riddle et al., 2005). The role of FcγR engagement in tumor targeting of NK cells was also demonstrated in studies whereby chimeric antigen receptor T cells expressing CD16 scFv targeted antibody-coated tumor cells. Transfer of CD16-CarT to EGFR or CD20 tumor-bearing mice treated with cetuximab or rituximab enhanced immune cell targeting and ADCC killing in an in vivo model, thereby eradicating tumor immune evasion (Rataj et al., 2019, Caratelli et al., 2017).

[0060] The contribution of FcγR genes to autoimmune diseases has attracted much attention, and functional FcγR polymorphisms have been reported to play an important role in the pathogenesis of autoimmune diseases (Salmon et al., 2001; Morgan et al., 2003; Wu et al., 1997). FcγR knockout mouse models indicate that both activating and inhibitory FcγRs affect the development of autoimmune diseases (Nabbe et al., 2003; Kleinau et al., 2000; Bolland et al., 2000). Variations in FcγR expression greatly affect the signal threshold mediated by IgG immune complexes. In particular, proinflammatory and anti-inflammatory cytokines can regulate the expression levels of activating and inhibitory FcγRs and affect the threshold of immune cell responses to IgG immune complexes (Pricop et al., 2001; Boruchov et al., 2005). FCGR3A and FCGR3B copy number variations were associated with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in Taiwanese patients (Chen et al., 2014). Elevated copy number of FCGR3A was demonstrated to be a risk factor for SLE. High frequencies of cytokine-producing FcγRIIIA-positive dendritic cells (DCs) were observed in SLE patients, especially in those with active disease, suggesting that FcγRIIIA-mediated proinflammatory cytokine production in DCs may contribute to disease pathogenesis (Henriques et al., 2012). The high density of activating FcγRIIIA on the surface of immune cells (NK cells, monocytes, DCs, macrophages, and a subset of T cells) may tip the delicate balance of the immune response toward intense inflammation, resulting in the development of SLE (Chen et al., 2014). A correlation between low copy number of FCGR3A and low expression of CD16A in NK cells has been demonstrated, suggesting that copy number of FCGR3A has physiological significance in NK cell function. Most importantly, FCGR3A deficiency is associated with two different autoimmune diseases (SLE and RA), suggesting that functional defects of FcγRIIIA may be a common risk factor for various autoimmune diseases. This disease association suggests that modulation of FcγRIIIA function may be an important therapeutic target for lupus nephritis (Chen et al., 2014).

[0061] Therapeutic innovation as a means to block inflammation in autoimmune diseases through FcγR interactions offers some hope. Previous studies support the potential of FcγR inhibition as a therapeutic strategy to suppress immune complex-mediated events in autoimmune diseases (Clarkson et al., 1986; Flaherty et al., 2012). IgG Fc fragments by themselves have demonstrated good efficacy in animal models of RA and in humans with thrombocytopenia (ITP) or Kawasaki disease (Anthony et al., 2008; Debre M et al., 1993; Hsu et al., 1993). Multivalent Fc constructs have been shown to suppress immune complex processes in murine models of thrombocytopenia and arthritis (Ortiz et al., 2016). Furthermore, infusion of soluble FcγR3a and 2a can suppress immune complex-driven inflammation in a murine lupus model (Li et al., 2014). Blocking immune complex formation on NK cells is an avenue to be explored for reducing inflammatory activation and therefore autoimmune disease.

[0062] In one embodiment, the one or more CD16a binding bicyclic peptide ligands are: [ka] (In the formula, C i , C ii , and C iii represent a first, second, and third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0063] In a further embodiment, the molecular scaffold is TBMT and the one or more CD16a binding bicyclic peptide ligands optionally comprise an N-terminal and / or a C-terminal modification and: A-(SEQ ID NO:12)-A-[K(PYA)] (referred to herein as BCY13886); A-(SEQ ID NO:13)-A-[K(PYA)] (referred to herein as BCY20361); and A-(SEQ ID NO:14)-A-[K(PYA)] (referred to herein as BCY13883) wherein PYA represents pentynic acid, or a pharma- ceutically acceptable salt thereof.

[0064] In one embodiment, the heterotandem bicyclic peptide complex comprises one (i.e., a single) second peptide ligand that binds to a component present on a natural killer (NK) cell. In a further embodiment, the single second peptide ligand is an NKp46-binding bicyclic peptide ligand as defined herein, or a CD16a-binding bicyclic peptide as defined herein.

[0065] In another embodiment, the heterotandem bicyclic peptide complex comprises two second peptide ligands that bind to components present on natural killer (NK) cells. In a further embodiment, the two second peptide ligands are both of which are NKp46-binding bicyclic peptide ligands as defined herein; or both being CD16a-binding bicyclic peptides as defined herein; or one NKp46-binding bicyclic peptide ligand as defined herein and one CD16a-binding bicyclic peptide ligand as defined herein.

[0066] In yet a further embodiment, when the heterotandem bicyclic peptide complex comprises two NKp46-binding bicyclic peptide ligands as defined herein, the peptide ligands are identical (i.e., share the same peptide sequence).

[0067] In yet a further embodiment, when the heterotandem bicyclic peptide complex comprises two CD16a-binding bicyclic peptide ligands as defined herein, the peptide ligands are identical (i.e., share the same peptide sequence).

[0068] In another embodiment, the heterotandem bicyclic peptide complex comprises three second peptide ligands that bind to components present on natural killer (NK) cells. In a further embodiment, the three second peptide ligands are each an NKp46-binding bicyclic peptide ligand as defined herein. In an even further embodiment, the three NKp46-binding bicyclic peptide ligands as defined herein are identical (i.e., share the same peptide sequence).

[0069] (Linker) It will be understood that the first peptide ligand may be conjugated to one or more second peptide ligands via any suitable linker. Typically, the linker design will be presented in such a way that two or more full bicyclic peptides can bind unhindered to their respective targets, either alone or simultaneously to both target receptors. Furthermore, the linker should allow simultaneous binding to both targets while maintaining an appropriate distance between the target cells that results in the desired functional outcome. The properties of the linker can be adjusted to increase the length, rigidity, or solubility to optimize the desired functional outcome. The linker may also be designed to allow more than one bicyclic binding to the same target. Increasing the valency of either binding peptide may serve to increase the affinity of the heterotandem to the target cell or may help induce oligomerization of one or both of the target receptors.

[0070] In one embodiment, the linker is a linear linker. Without being bound by theory, it is believed that a linear linker has the advantage that it allows for one first peptide at one end and one second peptide at the other end.

[0071] In a further embodiment, the linear linker is [ka] , is selected from.

[0072] In one embodiment, the linker is a branched linker. Without being bound by theory, it is believed that branched linkers have the advantage of allowing for a first peptide at one end and two or more second peptides at the remaining end.

[0073] In a further embodiment, the branched linker is [ka] TIFF2024503632000010.tif152170TIFF2024503632000011.tif214170TIFF2024503632000012.tif136170.

[0074] In one particular embodiment, the branched linker is [ka] , is.

[0075] (heterotandem complex) In one particular embodiment, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand bound to a TATA scaffold, the one or more second peptide ligands comprise two NKp46-binding bicyclic peptide ligands bound to a TATA scaffold, and the heterotandem complex is a complex listed in Table A1, Table A2, and Table A3. (Table A1(EphA2:NKp46;1:1)) [Table 1]

[0076] The heterotandem bicyclic peptide conjugate BCY17225 was synthesized by the synthesis of azide-PEG- 5 It consists of the EphA2-specific peptide BCY9594 linked to one NKp46-specific peptide (BCY17224) via a -acid linker. [ka]

[0077] The heterotandem bicyclic peptide conjugate BCY18731 was synthesized by the synthesis of azide-PEG- 24 It consists of the EphA2-specific peptide BCY9594 linked to one NKp46-specific peptide (BCY17224) via a -acid linker. [ka]

[0078] (Table A2(EphA2:NKp46;1:2)) [Table 2]

[0079] The heterotandem bicyclic peptide conjugate BCY15664 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY15452. [ka]

[0080] The heterotandem bicyclic peptide conjugate BCY15923 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY15686. [ka]

[0081] The heterotandem bicyclic peptide conjugate BCY17226 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY17224. [ka]

[0082] The heterotandem bicyclic peptide conjugate BCY15924 is a tandem bicyclic peptide conjugate comprising N-(acid-PEG) 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY15687. [ka]

[0083] The heterotandem bicyclic peptide conjugate BCY18042 is prepared by reacting N-(acid-PEG) 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY18004. [ka]

[0084] The heterotandem bicyclic peptide conjugate BCY18048 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY17662. [ka]

[0085] The heterotandem bicyclic peptide conjugate BCY18049 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY18005. [ka]

[0086] (Table A3(EphA2:NKp46;1:3)) [Table 3]

[0087] The heterotandem bicyclic peptide conjugate BCY21686 is a peptide having a structure similar to that of methane-N-(PEG 5 -acid)-tri(MeOPr-amide-PEG 4 -azido) linkers to three NKp46-specific peptides (all of which are BCY17224). [ka]

[0088] The heterotandem bicyclic peptide conjugate BCY21687 is a peptide having a structure similar to that of methane-N-(PEG 10 -acid)-tri(MeOPr-amide-PEG 10 -azido) linkers to three NKp46-specific peptides (all of which are BCY17224). [ka]

[0089] In one particular embodiment, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand bound to a TATA scaffold, and the one or more second peptide ligands comprise two CD16a-binding bicyclic peptide ligands bound to a TBMT scaffold, and the heterotandem complex is selected from the complexes listed in Table B1. (Table B1(EphA2:CD16a;1:2)) [Table 4]

[0090] The heterotandem bicyclic peptide conjugate BCY15911 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two CD16a-specific peptides, both of which are BCY13886. [ka]

[0091] The heterotandem bicyclic peptide conjugate BCY20810 was synthesized by the synthesis of N-(acid-PEG)- 10 )-N-Bis(PEG 10 -azido) linkers to two CD16a-specific peptides, both of which are BCY20361. [ka]

[0092] In one particular embodiment, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand bound to a TATA scaffold and the two second peptide ligands comprise one NKp46-binding bicyclic peptide ligand bound to a TATA scaffold and one CD16a-binding bicyclic peptide ligand bound to a TBMT scaffold, and the heterotandem complex is selected from the complexes listed in Table C. (Table C(EphA2:NKp46:CD16a;1:1:1)) [Table 5]

[0093] The heterotandem bicyclic peptide conjugate BCY17231 is a tandem bicyclic peptide conjugate having a structure similar to that of the N-(PEG 2-aminophenyl)-N-phenyl-N-phenyl-2-propanediol (N-phenylenediamine) as shown in the figure below. 3 -Acid)-N-(PEG 3 -Azide)-N-(PEG 3 It consists of the EphA2-specific peptide BCY9594 linked to an NKp46-specific peptide (BCY17224) and a CD16a-specific peptide (BCY13883) via a (-NH-AcAz) linker. [ka]

[0094] The heterotandem bicyclic peptide complex BCY17235 is a tandem bicyclic peptide complex that is synthesized by 23 ] 3 It consists of the EphA2-specific peptide BCY9594 linked via linkers to an NKp46-specific peptide (BCY17224) and a CD16a-specific peptide (BCY13883). [ka]

[0095] The heterotandem bicyclic peptide conjugate BCY20793 is a tandem bicyclic peptide conjugate having a structure similar to that of the N-(PEG 2-aminophenyl)-N-phenyl-N-phenyl-2-propanediol (N-phenylenediamine) as shown in the figure below. 3 -Acid)-N-(PEG 3-Azide)-N-(PEG 3 It consists of the EphA2-specific peptide BCY9594 linked to an NKp46-specific peptide (BCY17224) and a CD16a-specific peptide (BCY20361) via a (-NH-AcAz) linker. [ka]

[0096] In one particular embodiment, the first peptide ligand comprises an MT1-binding bicyclic peptide ligand bound to a TATA scaffold, and the one or more second peptide ligands comprise two NKp46-binding bicyclic peptide ligands bound to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table D. (Table D(MT1:NKp46;1:2)) [Table 6]

[0097] The heterotandem bicyclic peptide conjugate BCY18604 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linkers to two NKp46-specific peptides, both of which are BCY17224. [ka]

[0098] In one particular embodiment, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand bound to a TATA scaffold and the one or more second peptide ligands comprise two NKp46-binding bicyclic peptide ligands bound to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table E. (Table E(PD-L1:NKp46;1:2)) [Table 7]

[0099] The heterotandem bicyclic peptide conjugate BCY18603 was synthesized by the synthesis of N-(acid-PEG)- 3 )-N-Bis(PEG 3 -azido) linker to two NKp46-specific peptides (both of which are BCY17224). [ka]

[0100] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, for example, peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetics, and biochemistry methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th ed., John Wiley & Sons, Inc., incorporated herein by reference).

[0101] (Nomenclature) (Numbering) When referring to amino acid residue positions within the peptides of the invention, a cysteine ​​residue (C i , C ii , and C iii ) are omitted from the numbering since they are invariant. The numbering of amino acid residues within the peptides of the invention is therefore represented as follows: -C i-N 1 -L 2 -Q 3 -A 4 -P 5 -C ii -M 6 -Q 7 -T 8 -G 9 -K 10 -V 11 -C iii -(Sequence number: 1).

[0102] For the purposes of this description, all bicyclic peptides are considered to be cyclized with TATA or TBMT to give trisubstituted structures. Cyclization with TATA or TBMT removes the first, second, and third reactive groups (i.e., C i , C ii , and C iii ) occurs on

[0103] (Molecular format) N- or C-terminal extensions to the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO:X) It is symbolized as follows.

[0104] (Reverse peptide sequence) In view of the disclosure in Nair et al., (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein will also find utility in their retro-inverso forms. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus and vice versa) and the stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids and vice versa). For the avoidance of doubt, reference to an amino acid, either as its full name or as the one-letter or three-letter designation of that amino acid, is intended herein to be represented as an L-amino acid, unless otherwise indicated. When such an amino acid is intended to be represented as a D-amino acid, the amino acid is preceded by a lower case d in square brackets, e.g., [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0105] (Advantages of peptide ligands) Multifunctional NK cell engager (NKCE) approaches combine and enhance the immune surveillance capabilities of NK cells with their cytotoxic functions. Bispecific killer engagers (BiKE), trispecific killer engagers (TRiKE), or quadrispecific killer engagers (TetraKE) are small artificial antibody molecules designed to create bonds between effector NK cells and targeted tumor cells (Felices et al., 2016; Moore et al., 2011). Importantly, mAb-mediated activation of NCRs activates NK cytotoxicity against many types of target cells. Cross-linking induced by specific mAbs leads to potent NK cell activation, resulting in cytotoxicity and increased cytokine production. These engagers include anti-CD16 antibodies that bind CD16 and activate NK cell cytotoxicity, and antibodies or antigens against tumor cells. An example of BiKE is CD16×CD33, which enhances NK activity against the CD33+ HL60 AML cell line in vitro (Gleason et al., 2014). TRiKE and TetraKE use the cytokine interleukin-15 (IL-15) molecule as a link between antibodies and show higher cytotoxicity and proinflammatory cytokine production than BiKE (Davis et al., 2017; Felices et al., 2019). To maximize NK antitumor responses, Vivier et al.'s group recently demonstrated the efficacy of a multifunctional NKCE consisting of an Fc fragment for CD16 binding, activation of the NK cell receptor NKp46, and two antibody domains targeting specific tumor antigens such as CD19, CD20, and EGFR (Gauthier et al., 2019). In in vivo models, NKCE has been shown to enhance NK cell infiltration into tumors and promote tumor clearance, and has demonstrated enhanced efficacy over current clinically used antibodies, such as rituximab and cetuximab. Furthermore, by harnessing the activation capacity of multiple stimulatory receptors on NK cells (CD16 and NKp46), inhibition was overcome and full NK cell activity was achieved (BenShumel, 2020; Tarzona, 2020; Davis et al., 2017).

[0106] The particular bicyclic peptides of the present invention have many advantageous properties that allow them to be considered suitable drug-like molecules for injection, inhalation, nasal, ophthalmic, oral or topical administration. Such advantageous properties include: - species cross-reactivity (which is usually required for preclinical pharmacodynamic and pharmacokinetic evaluation); - Protease stability (bicyclic peptide ligands should in most circumstances demonstrate stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic peptide lead candidates can not only be developed in animal models but also reliably administered to humans); - a desirable solubility profile (which is a function of the ratio of "charged and hydrophilic residues" to "hydrophobic residues" and intra- / inter-molecular hydrogen bonds, which is important for formulation and absorption purposes); and - Optimal plasma half-life within the circulation (depending on the clinical indication and treatment regimen, there will be a desire to develop bicyclic peptides for short or long in vivo exposure times in the management setting of either chronic or acute disease stages. The optimal exposure time will be governed by the need for sustained exposure (for maximum therapeutic efficacy) versus short exposure times to minimize toxic effects resulting from sustained exposure of the drug).

[0107] (Peptide Ligand) A peptide ligand as referred to herein refers to a peptide covalently attached to a molecular scaffold. Typically, such a peptide contains two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond with the scaffold, and an arc-shaped sequence between the reactive groups, called a loop sequence, because the peptide forms a loop when it binds to the scaffold. In this case, the peptide contains at least three reactive groups selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine, and forms at least two loops on the scaffold.

[0108] (Reactive group) The molecular scaffold of the present invention may be attached to a polypeptide via a functional or reactive group on the polypeptide. These are typically formed from the side chains of certain amino acids found in the polypeptide polymer. Such reactive groups may be cysteine ​​side chains, lysine side chains, or N-terminal amine groups, or any other suitable reactive group, such as penicillamine. Details of suitable reactive groups can be found in WO2009 / 098450.

[0109] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartic acid or glutamic acid, the guanidinium group of arginine, the phenolic group of tyrosine, or the hydroxyl group of serine. Unnatural amino acids can provide a wide range of reactive groups, including azide, keto-carbonyl, alkyne, vinyl, or aryl halide groups. The amino and carboxyl groups at the termini of polypeptides can also serve as reactive groups to form covalent bonds with molecular scaffolds / molecular cores.

[0110] The polypeptides of the invention contain at least three reactive groups. They can also contain four or more reactive groups. The more reactive groups used, the more loops can be formed in the molecular scaffold.

[0111] In one preferred embodiment, a polypeptide having three reactive groups is generated. The reaction of the polypeptide with a molecular scaffold / molecular core having a three-fold symmetry generates a single product isomer. This generation of a single product isomer is preferred for several reasons. The nucleic acid of the compound library only codes for the primary sequence of the polypeptide, but does not code for the isomeric state of the molecule formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer isolated in the screening or selection process. The generation of a single product isomer is also advantageous when a specific member of the library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold generates a single product isomer, not a mixture of isomers.

[0112] In another embodiment, a polypeptide having four reactive groups is generated. The reaction of said polypeptide with a molecular scaffold / molecular core having tetrahedral symmetry generates two product isomers. Even though two different product isomers are encoded by one and the same nucleic acid, the isomeric properties of the isolated isomers can be determined by chemically synthesizing both isomers, separating the two isomers, and testing both isomers for binding with target ligands.

[0113] In one embodiment, at least one of the reactive groups of the polypeptide of the present invention is orthogonal to the remaining reactive groups. The use of an orthogonal reactive group allows the orthogonal reactive group to be directed to a specific site on the molecular core. The linking strategy involving the orthogonal reactive group can be used to limit the number of product isomers formed. In other words, by selecting a reactive group for one or more of the at least three bonds that is separate or different from the reactive group selected for the remaining of the at least three bonds, it is possible to effectively achieve the attachment or orientation of specific reactive groups of the polypeptide to specific positions on the molecular scaffold in a specific order.

[0114] In another embodiment, the reactive group of the polypeptide of the invention is reacted with a molecular linker, where the linker can react with the molecular scaffold such that the linker is interposed between the molecular scaffold and the polypeptide in the final bound state.

[0115] In some embodiments, the amino acids that are members of a library or set of polypeptides can be substituted by any natural or unnatural amino acid. Excluded from these substitutable amino acids are those that have a functional group for linking the polypeptide to the molecular core, such that only the loop sequence can be substituted. Substitutable polypeptide sequences include either random sequences, constant sequences, or sequences with random and constant amino acids. Any amino acid that has a reactive group is placed at a defined position in the polypeptide, since the position of these amino acids determines the loop size.

[0116] In one embodiment, the polypeptide having three reactive groups has the sequence (X) l Y(X) m Y(X) n Y(X) o where Y represents an amino acid having a reactive group, X represents a random amino acid, m and n are numbers from 3 to 6 that define the length of the intervening polypeptide segments (which may be the same or different), and l and o are numbers from 0 to 20 that define the length of the adjacent polypeptide segments.

[0117] Alternatives to thiol-mediated conjugation can be used to attach molecular scaffolds to peptides via covalent interactions. Alternatively, these techniques can be used to modify or attach additional moieties (e.g., small molecules of interest different from the molecular scaffold) to polypeptides after they have been selected or isolated according to the invention - in this embodiment, obviously, the attachment does not have to be covalent, but can include non-covalent attachment. These methods can be used instead of (or in combination with) thiol-mediated methods by producing phage displaying proteins and peptides with unnatural amino acids with the required chemical reactive groups in combination with small molecules with complementary reactive groups, or by incorporating unnatural amino acids into chemically or recombinantly synthesized polypeptides, if the molecules are generated after the selection / isolation step. Further details can be found in WO2009 / 098450 or Heinis et al., Nat Chem Biol 2009, 5(7), 502-7.

[0118] In one embodiment, the reactive groups are selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine residues.

[0119] (Pharmaceutical acceptable salts) It will be understood that salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of that ligand.

[0120] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods, such as those described in "Pharmaceutical Salts: Properties, Selection, and Use", P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. Typically, such salts can be prepared by reacting the free acid or free base form of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0121] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclohexane ... laminic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid, etc.), glutamic acid (e.g., L-glutamic acid, etc.), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, salts acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, The salts include mono- or di-salts formed with acids selected from the group consisting of acetic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0122] One particular group of salts consists of the salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0123] If the compound is anionic or has a functional group which may be anionic (e.g., -COOH becomes -COO - Salts can be formed with organic or inorganic bases that provide suitable cations. Examples of suitable inorganic cations include, but are not limited to, Li + , Na + , and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , N.H. 2 R 2 + , N.H.R. 3 + , N.R. 4 + ) are included. Some suitable examples of substituted ammonium ions are those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4 + It is.

[0124] When the compounds of the present invention contain amine groups, they may form quaternary ammonium salts, for example, by reaction with alkylating agents by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0125] (Modified derivative) It will be appreciated that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of suitable such modified derivatives include N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more sterically equivalent or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural sterically equivalent or isoelectronic amino acids); addition of a spacer group; substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; substitution of one or more amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; addition of one or more amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues, ... replacement of one or more peptide bonds with an alternative bond; alteration of the peptide backbone length; replacement of the hydrogen on the alpha carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate / aspartate and tyrosine with amine-, thiol-, carboxylic acid- and phenol-reactive reagents suitable for functionalizing these amino acids, and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, e.g., introduction or substitution of amino acids bearing an azide or alkyne group, which allows functionalization of an alkyne or azide-containing moiety, respectively.

[0126] In one embodiment, the modified derivatives include N-terminal and / or C-terminal modifications. In a further embodiment, the modified derivatives include N-terminal modifications using suitable amino-reactive chemistry and / or C-terminal modifications using suitable carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modifications include the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator, or a chromophore.

[0127] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, an N-terminal cysteine ​​group (referred to herein as C iThe N-terminal end of the acetyl group (referred to as "acetyl group") is capped with acetic anhydride or other suitable reagent during peptide synthesis to yield an N-terminally acetylated molecule. This embodiment offers the advantage of eliminating an aminopeptidase recognition point, avoiding potential degradation of the bicyclic peptide.

[0128] In another embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of an effector group and promotes retention of efficacy of the bicyclic peptide against its target.

[0129] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine ​​group (herein referred to as C iii The C-terminal amidated group (referred to as "C-terminal amidated group") is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment offers the advantage of eliminating a recognition point for carboxypeptidases, reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0130] In one embodiment, the modified derivatives include the substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids can be selected that have sterically isomeric / isoelectronic side chains that are not recognized by degradative proteases and do not adversely affect the targeting efficacy in any way.

[0131] Alternatively, unnatural amino acids with constrained amino acid side chains may be used, such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. These in particular relate to proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids (a simple derivative is amino-cyclopropyl carboxylic acid).

[0132] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of an N-terminal cysteine ​​(C i) and / or C-terminal cysteine ​​(C iii ) including the addition of a spacer group to

[0133] In one embodiment, the modified derivative comprises the substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises the substitution of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage that the pharmaceutical stability profile of the resulting bicyclic peptide ligand is improved.

[0134] In one embodiment, the modified derivative comprises the substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In another embodiment, the modified derivative comprises the substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of "charged amino acid residues" versus "hydrophobic amino acid residues" is an important property of bicyclic peptide ligands. For example, hydrophobic amino acid residues will affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) will affect the interaction of the peptide with the phospholipid membrane of the cell surface. The combination of the two will affect the half-life, volume of distribution, and exposure of the peptide drug and can be tailored to the clinical evaluation endpoint. Furthermore, the correct combination and number of "charged amino acid residues" versus "hydrophobic amino acid residues" will reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

[0135] In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues, which is believed to enhance proteolytic stability by steric hindrance and by the properties of D-amino acids to stabilize the β-turn conformation (Tugyi et al., (2005) PNAS, 102(2), 413-418).

[0136] In one embodiment, the modified derivative comprises the removal of any amino acid residue and substitution with alanine, this embodiment offers the advantage of removing a potential site(s) of proteolytic attack.

[0137] It should be noted that each of the above modifications serves to deliberately improve the potency or stability of the peptide. Further potency improvements based on the modifications could be achieved by the following mechanisms: - Higher affinity is achieved by incorporating hydrophobic moieties that exploit the hydrophobic effect to reduce the dissociation rate; - the incorporation of charged groups that take advantage of long-range ionic interactions, increasing the rate of association and resulting in higher affinity (see, e.g., Schreiber et al., "Rapid, electrostatically assisted association of proteins," (1996), Nature Struct. Biol. 3, 427-31); and - incorporating additional constraints into the peptide, for example by precisely constraining the amino acid side chains so as to minimize the entropic loss upon target binding, by restricting the torsion angles of the backbone so as to minimize the entropic loss upon target binding, and by introducing additional cyclizations into the molecule for the same reasons (for reviews see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0138] (isotopic variants) The present invention includes all pharma- ceutically acceptable (radio)isotopically labeled peptide ligands of the invention in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, as well as peptide ligands of the invention in which a metal chelating group (called an "effector") capable of bearing a relevant (radio)isotope is conjugated, and peptide ligands of the invention in which a particular functional group is covalently substituted with a relevant (radio)isotope or an isotopically labeled functional group.

[0139] Examples of isotopes suitable for inclusion within the peptide ligands of the present invention include hydrogen isotopes, e.g. 2 H(D) and 3 H(T), carbon isotopes, e.g. 11 C. 13 C and 14 C, chlorine isotopes, e.g. 36 Cl, fluorine isotopes, e.g. 18 F, iodine isotopes, e.g. 123 I, 125 I and 131 I, nitrogen isotopes, e.g. 13 N and 15 N, oxygen isotopes, e.g. 15 O. 17 O and 18 O, phosphorus isotopes, e.g. 32 P, sulfur isotopes, e.g. 35 S, copper isotopes, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y, as well as lutetium isotopes, e.g. 177 Lu, as well as bismuth isotopes, e.g. 213 Contains Bi.

[0140] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies and for clinically assessing the presence and / or absence of Nectin-4 targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. This detection or identification method may employ compounds that are labeled with labeling agents, such as, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). The radioisotope tritium, i.e. 3 H(T), and carbon-14, i.e. 14 C is particularly useful for this purpose given its ease of incorporation and the means for detection available.

[0141] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), may be preferable in some circumstances because it may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0142] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, can be utilized in Positron Emission Topography (PET) studies for examining target occupancy.

[0143] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the Examples herein, substituting appropriate isotopically labeled reagents in place of the non-labeled reagents already utilized.

[0144] (Molecular scaffolds) Molecular scaffolds are described, for example, in WO2009 / 098450 and the documents cited therein, in particular WO2004 / 077062 and WO2006 / 078161.

[0145] As mentioned in said document, the molecular scaffold may be a small molecule, for example a small organic molecule.

[0146] In one embodiment, the molecular scaffold may be a polymer. In one embodiment, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates.

[0147] In one embodiment, the molecular scaffold comprises a reactive group that can react with a functional group(s) of a polypeptide to form a covalent bond.

[0148] The molecular scaffold can contain chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0149] The molecular scaffolds of the invention comprise chemical groups that allow a functional group of a polypeptide of an encoded library of the invention to form a covalent bond with the molecular scaffold, the chemical group being selected from a wide range of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

[0150] Scaffold reactive groups that can be used to react with thiol groups of cysteines on molecular scaffolds are alkyl halides (also called halogenoalkanes or haloalkanes).

[0151] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups used to selectively attach compounds to cysteines in proteins are maleimides, alpha-unsaturated carbonyl-containing compounds, and alpha-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an alpha-beta unsaturated carbonyl-containing compound is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of an α-halomethylcarbonyl-containing compound is N,N',N''-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a naturally occurring amino acid with similar reactivity to cysteine ​​and can be used in the same reactions. Therefore, whenever cysteine ​​is mentioned, it is generally permissible to use selenocysteine ​​instead, unless the context suggests otherwise.

[0152] In one embodiment, the molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one, also known as triacryloylhexahydro-s-triazine (TATA): [ka] .

[0153] From the above, the C i , C ii , and C iii After cyclization on the cysteine ​​residue, the molecular scaffold forms a trisubstituted 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropan-1-one derivative of TATA having the following structure: [ka] (where * indicates the attachment points of the three cysteine ​​residues).

[0154] In another embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)-s-triazine (TBMT): [ka] .

[0155] From the above, the C i , C ii , and C iii After cyclization on the cysteine ​​residues, the molecular scaffold forms a trisubstituted TBMT derivative having the following structure: [ka] (where * indicates the attachment points of the three cysteine ​​residues).

[0156] (synthesis) The peptides of the invention may be synthetically produced by standard techniques and then reacted with the molecular scaffold in vitro. Standard chemical methods can be used to carry this out. This allows for rapid large-scale production of soluble material for further downstream experimentation or validation. Such methods can be accomplished using conventional chemical methods such as those disclosed in Timmerman et al., supra.

[0157] The present invention therefore also relates to the production of a polypeptide or conjugate selected as described herein, which production comprises any further steps as explained below. In one embodiment, these steps are carried out on the final product polypeptide / conjugate produced by chemical synthesis.

[0158] Optionally, amino acid residues in the polypeptide of interest may be substituted during preparation of the conjugate or complex.

[0159] The peptides can also be extended to incorporate, for example, additional loops, thereby introducing multiple specificities.

[0160] To elongate a peptide, it may simply be chemically extended at its N- or C-terminus or within a loop with orthogonally protected lysines (and analogues) using standard solid- or solution-phase chemistries. Activated or activatable N- or C-termini may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be by fragment condensation or by native chemical ligation as described, for example, in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation, Science 266:776-779) or enzymatically using subtiligase as described, for example, in (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20;91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, 15 Nov. 2008, pp. 6000-6003).

[0161] Alternatively, peptides can be extended or modified by further conjugation via disulfide bonds. This has the added advantage that the first and second peptides can dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) is added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; then, an additional cysteine ​​or thiol is added to the N-terminus or C-terminus of the first peptide, so that this cysteine ​​or thiol can only react with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0162] Similar techniques can be applied to the synthesis / coupling of two bicyclic bispecific macrocycles as well to generate tetraspecific molecules.

[0163] Furthermore, the addition of other functional or effector groups may be achieved similarly using appropriate chemical methods, coupling at the N-terminus or C-terminus or via a side chain, In one embodiment, the coupling is carried out in such a manner as not to block the activity of either entity.

[0164] Pharmaceutical Composition In a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0165] Typically, the peptide ligands may be used in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous liquids, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride solution, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants, if necessary to keep the polypeptide complex in suspension, may be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0166] Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives can also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack, (1982), Remington's Pharmaceutical Sciences, 16th Edition).

[0167] The peptide ligands of the invention can be used as compositions administered separately or in combination with other drugs. These can include antibodies, antibody fragments, and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin, or cisplatin, and immunotoxins. Pharmaceutical compositions can also include "cocktails" of various cytotoxic or other drugs in conjunction with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities, e.g., polypeptides selected with different targeting ligands, whether pooled or not prior to administration.

[0168] The route of administration of the pharmaceutical composition of the present invention may be any of those generally known to those skilled in the art. For therapy, the peptide ligands of the present invention may be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or, also appropriately, by direct injection using a catheter. Preferably, the pharmaceutical composition according to the present invention is administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, the concomitant administration of other drugs, contraindications, and other parameters taken into account by the clinician.

[0169] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be utilized. Those skilled in the art will recognize that lyophilization and reconstitution can result in varying degrees of activity loss, and that concentration levels may need to be adjusted upward to compensate for this loss.

[0170] Compositions containing the peptide ligands or cocktails thereof can be administered for prophylactic and / or therapeutic treatments. In a particular therapeutic application, an amount adequate to achieve at least partial inhibition, pacification, modulation, killing, or any other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dosage will depend on the severity of the disease and the general state of the patient's own immune system, but will generally range from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses in the range of 0.05 to 2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof will also be administered at similar or slightly lower doses.

[0171] Compositions comprising peptide ligands according to the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in a mammal. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove target cell populations from heterogeneous populations of cells. Blood from a mammal can be combined ex vivo with the selected peptide ligand, thereby killing or otherwise removing undesirable cells from the blood according to standard techniques, and then returned to the mammal.

[0172] (therapeutic use) In a further aspect of the present invention there is provided a heterotandem bicyclic peptide complex as defined herein for use in the prevention, suppression or treatment of cancer.

[0173] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and various types of cancers, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other cancers), such as bladder and urinary tract cancer, breast cancer, carcinomas of the digestive tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, carcinomas of the lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancers (e.g., tongue, oral cavity, larynx, pharynx, and bladder cancer), and tumors of the esophagus, stomach, small intestine, colon, rectum, and anus). head, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses); ovarian, fallopian tube, peritoneal, vaginal, vulvar, penile, cervical, myometrial, endometrial, thyroid (e.g., follicular thyroid carcinoma), adrenal, prostate, skin, and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and premalignant hematological disorders and borderline malignant disorders, e.g., hematological malignancies and related disorders of the lymphatic system (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], diffuse large intestine ... B-cell lymphomas such as de novo large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and related disorders of the myeloid system (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, polycythemia vera, myeloproliferative disorders such as essential thrombocythemia and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia; tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma);These include endocrine tumors (e.g., pituitary, adrenal, islet cell, parathyroid, carcinoid, and medullary carcinoma of the thyroid); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas), as well as pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors) or syndromes, congenital or not, that predispose a patient to malignancies (e.g., xeroderma pigmentosum);

[0174] In further embodiments, the cancer is selected from hematopoietic malignancies, e.g., selected from non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

[0175] References herein to the term "prevention" include administration of the protective composition prior to the induction of disease. "Suppression" refers to administration of the composition after an inductive event but prior to the clinical appearance of disease. "Treatment" includes administration of the protective composition after disease symptoms are manifest.

[0176] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease. The use of animal model systems is facilitated by the present invention, which allows for the development of polypeptide ligands that can cross-react with human and animal targets, thereby permitting the use of animal models.

[0177] The invention will now be further described with reference to the following examples. EXAMPLES

[0178] (Example) In general, some of the heterotandem bicyclic peptide conjugates of the present invention can be prepared according to the following general method. [ka]

[0179] Degas all solvents with N 2 The mixture is stirred at 40° C. for 16 h. The reaction mixture is then concentrated under reduced pressure to remove the solvent and purified by preparative HPLC to give intermediate 2.

[0180] A mixture of intermediate 2 (1.0 equiv.) and bicyclic 2 (2.0 equiv.) was dissolved in t-BuOH / H 2 2O (1:1), then CuSO 4 (1.0 equiv.), VcNa (4.0 equiv.), and THPTA (2.0 equiv.). Finally, 0.2 M NH 4 HCO 3 The pH is adjusted to 8 by dropwise addition of N. The reaction mixture is incubated at 40 °C for 16 h with N 2 Stir under atmosphere. The reaction mixture was directly purified by preparative HPLC.

[0181] Heterotandem bicyclic peptide conjugates prepared using this method are listed below. [Table 8]

[0182] More detailed experimental details of selected heterotandem bicyclic peptide complexes of the present invention are provided herein below.

[0183] Example 1: Synthesis of intermediate BP23825-BCY9594 [ka] To a mixture of compound 1 (BP23825, 60.0 mg, 96.2 μmol, 1.0 equiv.) and DMF (3 mL), DIEA (12.4 mg, 96.2 μmol, 16.8 μL, 1.0 equiv.) and HATU (38.4 mg, 101 μmol, 1.05 equiv.) were added and the mixture was stirred for 5 min. Then, BCY9594 (243 mg, 101 μmol, 1.05 equiv.) was added to the mixture and the mixture was subjected to N 2 After purging with N, the mixture was cooled to 40 °C for 16 h. 2 The mixture was stirred under atmosphere. LC-MS showed that compound 1 was completely consumed and one main peak with the desired m / z was detected. The reaction mixture was purified by preparative HPLC to give BP23825-BCY9594 (154 mg, 48.1 μmol, 50.0% yield, 94.0% purity) as a white solid. Calculated MW: 3006.48, observed m / z: 1002.8 ([M+3H] 3+ ), 1504.4([M+2H] 2+ ).

[0184] Example 2: Synthesis of BCY15664 [ka] A mixture of compound 1 (20.0 mg, 6.65 μmol, 1.0 eq.), compound 2 (30.0 mg, 13.97 μmol, 2.1 eq.), and THPTA (2.9 mg, 6.65 μmol, 1.0 eq.) was degassed with N 2 Purged with t-BuOH / H 2 Dissolved in 200 ml of CuSO (1:1, 1 mL). 4 (0.4 M, 16.6 μL, 1.0 equiv.) and Vc (4.7 mg, 26.61 μmol, 4.0 equiv.) were added to a water bath containing 100 mL of 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H O), and the solution turned bright yellow. The reaction mixture was incubated at 25 °C for 1 h with N 2Stirred under atmosphere. LC-MS showed that BP23825-BCY9594 was completely consumed and one main peak with the desired m / z was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC to give BCY15664 (15.0 mg, 1.97 μmol, 29.6% yield, 97.7% purity) as a white solid. Calculated MW: 7317.3, observed m / z: 1220.3 ([M+6H] 6+ ), 915.5([M+8H] 8+ ).

[0185] Example 3: Synthesis of BCY15911 [ka] A mixture of compound 1 (20.0 mg, 6.65 μmol, 1.0 eq.), compound 2 (31.6 mg, 16.63 μmol, 2.5 eq.), and THPTA (2.9 mg, 6.65 μmol, 1.0 eq.) was degassed with N 2 Purged with t-BuOH / H 2 Dissolved in 200 ml of CuSO (1:1, 1 mL). 4 An aqueous solution of (0.4 M, 16.6 μL, 1.0 eq.) and Vc (4.7 mg, 26.61 μmol, 4.0 eq.) was prepared with N 2 The solution was adjusted to pH 8, turning it bright yellow. The reaction mixture was incubated at 25 °C for 1 h under N 2 Stirred under atmosphere. LC-MS showed that BP23825-BCY9594 was completely consumed and one main peak with the desired m / z was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC to give BCY15911 (10.4 mg, 1.50 μmol, 22.61% yield, 94% purity) as a white solid. Calculated MW: 6814.8, observed m / z: 1364.0 ([M+5H] 5+ ), 1137.0([M+6H] 6+ ).

[0186] Example 4: Synthesis of BCY15923 [ka] A mixture of compound 1 (20.0 mg, 6.65 μmol, 1.0 eq.), compound 2 (35.0 mg, 16.79 μmol, 2.5 eq.), and THPTA (2.9 mg, 6.65 μmol, 1.0 eq.) was added to t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 1 mL) and pre-degassed with N 2 Purge three times with CuSO 4 (0.4 M, 16.6 μL, 1.0 equiv.) and ascorbic acid (4.69 mg, 26.61 μmol, 4 equiv.) in water. 2 The solution was diluted with 0.2 M NH 4 HCO 3 The pH was adjusted to 8 by dropwise addition of 100 ml of ethyl acetate, and the solution turned bright yellow. The reaction mixture was incubated at 25 °C for 2 h with N 2 Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 7191.31, observed m / z: 1439.3 ([M / 5+H] + ), 1199.6([M / 6+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY15923 (4 mg, 5.28e-1 umol, 7.94% yield, 95% purity) as a white solid.

[0187] Example 5: Synthesis of BCY17226 [ka] A mixture of compound 1 (20.0 mg, 6.65 μmol, 1.0 eq.), compound 2 (30.5 mg, 14.64 μmol, 2.2 eq.), and THPTA (5.8 mg, 13.30 μmol, 2.0 eq.) was added to t-BuOH / H 2 HO (1:1, 1.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4(0.4 M, 24.9 μL, 1.5 eq.) and VcNa (4.0 mg, 19.96 μmol, 3.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25–30 °C for 0.5 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 7169.18, observed m / z: 1434.9 ([M / 5+H] + ), 1195.9([M / 6+H] + It was shown that one main peak containing 4,4-dichlorophenyl 1 ...

[0188] Example 6: Synthesis of BCY15924 [ka] A mixture of compound 1 (5 mg, 1.66 μmol, 1.0 eq.), compound 2 (6.97 mg, 3.33 μmol, 2.0 eq.), and THPTA (1.45 mg, 3.33 μmol, 2.0 eq.) was diluted with t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 6.24 μL, 1.5 eq.) and VcNa (988.40 μg, 4.99 μmol, 3.0 eq.) was 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 7195.55, observed m / z: 1440.1 ([M / 5+H] + ), 1200.2([M / 6+H] + ), 1029.1([M / 7+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY15924 (3.4 mg, 4.52e-1 μmol, 27.16% yield, 95.6% purity) as a white solid.

[0189] Example 7: Synthesis of BCY18042 [ka] A mixture of compound 1 (15 mg, 4.99 μmol, 1.0 eq.), compound 2 (28.08 mg, 10.48 μmol, 2.1 eq.), and THPTA (4.34 mg, 9.98 μmol, 2.0 eq.) was mixed with t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 18.71 μL, 1.5 eq.) and VcNa (2.97 mg, 14.97 μmol, 3.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 6999.35, observed m / z: 1400.7 ([M / 5+H] + ), 1167.6([M / 6+H] + ), 1000.9([M / 7+H] +)) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY18042 (3.9 mg, 5.15e-1 μmol, 10.32% yield, 96.0% purity) as a white solid.

[0190] Example 8: Synthesis of BCY18048 [ka] A mixture of compound 1 (15 mg, 4.99 μmol, 1.0 eq.), compound 2 (21.99 mg, 10.48 μmol, 2.1 eq.), and THPTA (4.34 mg, 9.98 μmol, 2.0 eq.) was added to t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 25.0 μL, 2.0 eq.) and VcNa (2.97 mg, 14.97 μmol, 3.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 7203.30, observed m / z: 1441.3 ([M / 5+H] + ), 1201.3([M / 6+H] + ), 901.7([M / 8+H] + It was shown that one main peak with 1,2-dichlorophenyl 1,4-dichlorophenyl 1,5-dichlorophenyl 1,6 ...

[0191] Example 9: Synthesis of BCY18049 [ka] A mixture of compound 1 (15 mg, 4.99 μmol, 1.0 eq.), compound 2 (24.41 mg, 10.48 μmol, 2.1 eq.), and THPTA (4.34 mg, 9.98 μmol, 2.0 eq.) was added to t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 18.71 μL, 1.5 eq.) and VcNa (2.97 mg, 14.97 μmol, 3.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 7666.02, observed m / z: 959.3 ([M / 8+H] + ), 1096.2([M / 7+H] + ), 1278.6([M / 6+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY18049 (10.6 mg, 1.37 μmol, 27.41% yield, 98.9% purity) as a white solid.

[0192] Example 10: Synthesis of BCY18603 (Preparation of intermediate BP23825-BCY11865) [ka] A mixture of compound 1 (15.0 mg, 24.0 μmol, 1.0 equiv), HATU (10.1 mg, 26.5 μmol, 1.1 equiv), and DIEA (9.3 mg, 72.1 μmol, 12.6 μL, 3.0 equiv) was dissolved in DMF (1.0 mL). The reaction mixture was stirred at 25-30 °C for 6 min, and then compound 2 (68.5 mg, 26.5 μmol, 1.1 equiv) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed the desired m / z (MW: 3196.7, observed m / z: 1066.6 [M / 3+H]). + , 800.2 [M / 4+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The soluble crude product was then purified by preparative HPLC (TFA conditions). Compound 3 (BP23825-BCY11865, 43.0 mg, 13.4 μmol, 55.8% yield, 97.4% purity) was obtained as a white solid.

[0193] (Preparation of BCY18603) [ka] A mixture of compound 3 (15.0 mg, 4.69 μmol, 1.0 eq.), compound 4 (20.5 mg, 9.85 μmol, 2.1 eq.), and THPTA (2.0 mg, 4.69 μmol, 1.0 eq.) was mixed with t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 17.6 μL, 1.5 equiv.) and VcNa (2.8 mg, 14.1 umol, 3.0 equiv.) in water. 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 7359.3, observed m / z: 1472.9 [M / 5+H] + , 1227.5 [M / 6+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY18603 (13.4 mg, 1.82 umol, 38.8% yield, 97.8% purity) as a white solid.

[0194] Example 11: Synthesis of BCY18604 (Preparation of intermediate BP23825-BCY14320) [ka] A mixture of compound 1 (20.0 mg, 32.1 μmol, 1.0 equiv), HATU (13.4 mg, 35.3 μmol, 1.1 equiv) and DIEA (12.4 mg, 96.2 μmol, 16.8 μL, 3.0 equiv) was dissolved in DMF (1.0 mL). The reaction mixture was stirred at 25-30 °C for 6 min, then compound 2 (74.4 mg, 35.3 μmol, 1.1 equiv) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed the desired m / z (MW: 2714.27, observed m / z: 1358.1 [M / 2+H]). + , 905.9 [M / 3+H] + , 679.5 [M / 4+H] + ) was detected. The reaction mixture was filtered to remove the insoluble residue. The residue was then purified by preparative HPLC (TFA condition). Compound 3 (BP23825-BCY14320, 42.3 mg, 15.6 μmol, 48.5% yield, 97.7% purity) was obtained as a white solid.

[0195] (Preparation of BCY18604) [ka] A mixture of compound 3 (15.0 mg, 5.53 μmol, 1.0 eq.), compound 4 (24.2 mg, 11.6 umol, 2.1 eq.), and THPTA (2.4 mg, 5.53 μmol, 1.0 eq.) was added to t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 20.7 uL, 1.5 equiv.) and VcNa (3.3 mg, 16.6 μmol, 3.0 equiv.) in water. 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 6876.9, observed m / z: 1376.4 [M / 5+H] + , 1147.3 [M / 6+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY18604 (19.1 mg, 2.77 μmol, 50.0% yield, 97.1% purity) as a white solid.

[0196] Example 12: Synthesis of BCY20810 (Preparation of BP25635-BCY9594) [ka] A mixture of compound 1 (50.0 mg, 32.3 μmol, 1.0 equiv), HATU (14.7 mg, 38.7 μmol, 1.2 equiv) and DIEA (8.3 mg, 64.5 μmol, 11.2 μL, 2.0 equiv) was dissolved in NMP (0.5 mL). The reaction mixture was stirred at 25-30 °C for 5 min, then compound 2 (85.2 mg, 35.5 μmol, 1.1 equiv) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed the desired m / z (MW: 3931.5, observed m / z: 1310.5 [M / 3+H]). + , 983.8 [M / 4+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The soluble crude product was then purified by preparative HPLC (TFA conditions). Compound 3 (BP25635-BCY9594, 34.6 mg, 8.80 μmol, 27.2% yield, 96.8% purity) was obtained as a white solid.

[0197] (Preparation of BCY20810) [ka] A mixture of compound 3 (15.0 mg, 3.82 μmol, 1.0 eq.), compound 4 (15.6 mg, 7.63 μmol, 2.0 eq.) and THPTA (3.3 mg, 7.63 μmol, 2.0 eq.) was diluted with t-BuOH / H 2 HO (1:1, 0.3 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 14.3 μL, 1.5 eq.) and VcNa (2.3 mg, 11.4 μmol, 3.0 eq.) was prepared with N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25–30 °C for 0.5 h with N2O. 2Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 8028.3, observed m / z: 1338.7 [M / 6+H] + , 1147.7 [M / 7+H] + , 1004.4 [M / 8+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The soluble crude product was purified by preparative HPLC (TFA condition) to give BCY20810 (3.0 mg, 3.73e-1 μmol, 9.76% yield, 95.2% purity) as a white solid.

[0198] Example 13: Synthesis of BCY21686 (Preparation of intermediate BP25517-BCY9594) [ka] A mixture of compound 1 (10.0 mg, 7.19 μmol, 1.0 equiv), HATU (3.3 mg, 8.6 μmol, 1.2 equiv), and DIEA (2.8 mg, 21.6 μmol, 3.8 uL, 3.0 equiv) was dissolved in NMP (0.5 mL). The reaction mixture was stirred at 25-30 °C for 5 min, then compound 2 (19.0 mg, 7.91 μmol, 1.1 equiv) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed the desired m / z (calculated MW: 3773.3, observed m / z: 1258.4 [M / 3+H]). + , 944.1 [M / 4+H] + , 755.5 [M / 5+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was then purified by preparative HPLC (TFA conditions). Compound 3 (BP25517-BCY9594, 9.5 mg, 2.52 μmol, 35.0% yield, 95.3% purity) was obtained as a white solid.

[0199] (Preparation of BCY21686) [ka] A mixture of compound 3 (9.5 mg, 2.52 μmol, 1.0 eq.), compound 4 (15.7 mg, 7.55 μmol, 3.0 eq.), and THPTA (2.2 mg, 5.04 μmol, 2.0 eq.) was diluted with t-BuOH / H 2 HO (1:1, 0.3 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 12.6 μL, 2.0 eq.) and VcNa (2.0 mg, 10.0 μmol, 4.0 eq.) was prepared with N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25–30 °C for 0.5 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 10017.3, observed m / z: 1431.7 [M / 7+H] + , 1252.9 [M / 8+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY21686 (12.8 mg, 1.23 μmol, 48.8% yield, 96.4% purity) as a white solid.

[0200] Example 14: Synthesis of BCY21687 (Preparation of intermediate BP25668-BCY9594) [ka] A mixture of compound 1 (15.0 mg, 6.24 μmol, 1.0 equiv), HATU (2.8 mg, 7.49 μmol, 1.2 equiv), and DIEA (2.4 mg, 18.7 μmol, 3.3 μL, 3.0 equiv) was dissolved in NMP (0.5 mL). The reaction mixture was stirred at 25-30 °C for 5 min, and then compound 2 (16.5 mg, 6.86 μmol, 1.1 equiv) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed the desired m / z (MW: 4786.5, observed m / z: 1197.4 [M / 4+H]). + ) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was then purified by preparative HPLC (TFA conditions). Compound 3 (BP25668-BCY9594, 13.6 mg, 2.84 μmol, 45.51% yield, 95.5% purity) was obtained as a white solid.

[0201] (Preparation of BCY21687) [ka] A mixture of compound 3 (13.6 mg, 2.84 μmol, 1.0 eq.), compound 4 (17.3 mg, 8.32 μmol, 3.0 eq.), and THPTA (2.5 mg, 5.68 μmol, 2.0 eq.) was mixed with t-BuOH / H 2 HO (1:1, 0.3 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.9 mg, 5.68 μmol, 2.0 equiv.) and VcNa (2.2 mg, 11.37 μmol, 4.0 equiv.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25–30 °C for 0.5 h with N2O. 2Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 11030.5, observed m / z: 1379.6 [M / 8+H] + , 1226.5 [M / 9+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The soluble crude product was purified by preparative HPLC (TFA condition) to give BCY21687 (14.5 mg, 1.31 μmol, 46.1% yield, 94.5% purity) as a white solid.

[0202] Example 15: Synthesis of BCY17231 (Preparation of intermediate BP24527-BCY9594) [ka] A mixture of compound 1 (100.0 mg, 143 μmol, 1.0 equiv), HATU (59.9 mg, 157.6 μmol, 1.1 equiv), and DIEA (55.56 mg, 429.9 μmol, 74.88 uL, 3.0 equiv) was dissolved in DMF (5.0 mL). The reaction mixture was stirred at 25-30 °C for 6 min, then compound 2 (378 mg, 157 μmol, 1.1 equiv) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed the desired m / z (MW: 3080.59, observed m / z: 1027.9 [(M / 3+H] + , 771.2 [(M / 4+H] + ) was detected. The reaction mixture was filtered to remove the insoluble residue. The residue was then purified by preparative HPLC (TFA condition). Compound 3 (BP24527-BCY9594, 342 mg, 111.02 μmol, 77.4% yield, 91.5% purity) was obtained as a white solid.

[0203] (Preparation of intermediate BCY17230) [ka] A mixture of compound 3 (100.0 mg, 32.5 μmol, 1.0 eq.), compound 4 (74.3 mg, 35.7 μmol, 1.1 eq.), and THPTA (14.1 mg, 32.5 μmol, 1.0 eq.) was added to t-BuOH / H 2 HO (1:1, 4.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 81.1 μL, 2.0 equiv.) and VcNa (12.86 mg, 64.92 μmol, 2.0 equiv.) were added to a water bath containing 100 mL of N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 5161.94, observed m / z: 1291.6 ([M / 4+H] + ), 1033.4([M / 5+H] + ), 861.4([M / 6+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to obtain compound 5 (BCY17230, 65.0 mg, 12.1 μmol, 37.28% yield, 96.1% purity) as a white solid.

[0204] (Preparation of intermediate BCY17233) [ka] To a solution of compound 5 (65.0 mg, 12.1 μmol, 1.0 equiv) in DCM (0.80 mL) was added TEA (0.2 mL). The mixture was stirred at 25° C. for 0.5 h. LC-MS showed that compound 5 was completely consumed and had the desired m / z (MW: 5061.9, observed m / z: 1266.1 [M / 4+H]). + , 1013.2[M / 5+H] +, 844.5 [M / 6+H] + ) was detected. The reaction mixture was filtered, and then the soluble fraction was purified by preparative HPLC (TFA condition). Compound 6 (36.8 mg, 7.13 μmol, 56.64% yield, 98.1% purity) was obtained as a white solid.

[0205] (Preparation of intermediate BCY17234) [ka] To a solution of compound 6 (BCY17233, 36.8 mg, 7.13 μmol, 1.0 equiv.) and AcAz (1.73 mg, 8.72 μmol, 1.2 equiv.) in DMF (1.0 mL) was added DIEA (1.9 mg, 14.4 μmol, 2.5 μL, 2.0 equiv.). The mixture was stirred at 25° C. for 0.5 h. LC-MS showed that compound 6 was completely consumed and had the desired m / z (MW: 5141.35, observed m / z: 1029.8 [M / 5+H]). + , 1286.9[M / 4+H] + ) was detected. The reaction mixture was filtered, and then the soluble fraction was purified by preparative HPLC (TFA condition). Compound 7 (BCY17231, 25.0 mg, 4.65 μmol, 63.96% yield, 95.7% purity) was obtained as a white solid.

[0206] (Preparation of BCY17231) [ka] A mixture of compound 7 (22 mg, 4.28 μmol, 1.0 eq.), BCY13883 (9.31 mg, 4.70 μmol, 1.1 eq.), and THPTA (1.86 mg, 4.28 μmol, 1.0 eq.) was mixed with t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4(0.4 M, 10.69 μL, 1.0 eq.) and VcNa (1.69 mg, 8.55 μmol, 2.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 7 was completely consumed and had the desired m / z (calculated MW: 7123.1, observed m / z: 1425.3 ([M / 5+H] + ), 1188.0([M / 6+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY17231 (4.8 mg, 6.29e-1 μmol, 14.70% yield, 93.2% purity) as a white solid.

[0207] Example 16: Synthesis of BCY20793 [ka] A mixture of compound 1 (17.4 mg, 3.38 μmol, 1.0 eq.), BCY20361 (7.61 mg, 3.72 μmol, 1.1 eq.), and THPTA (1.47 mg, 3.38 μmol, 1.0 eq.) was mixed with t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 8.46 μL, 1.0 eq.) and VcNa (1.34 mg, 6.76 μmol, 2.0 eq.) was prepared with N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 7193.2, observed m / z: 1439.3 ([M / 5+H] + ), 1199.7([M / 6+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY20793 (8.0 mg, 1.05 μmol, 30.96% yield, 94.1% purity) as a white solid.

[0208] Example 17: Synthesis of BCY17235 (Preparation of intermediate COM00000116-BCY17224) [ka] A mixture of COM00000116 (50.0 mg, 13.8 μmol, 1.0 equiv.), BCY17224 (23.0 mg, 11.1 μmol, 0.8 equiv.), and THPTA (30.1 mg, 69.3 μmol, 5.0 equiv.) was dissolved in t-BuOH / H 2 HO (1:1, 10 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 13.8 μL, 0.4 eq.) and VcNa (54.8 mg, 277.1 μmol, 20.0 eq.) was 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25–30 °C for 3 h with N2O. 2 Stirred under atmosphere. LC-MS showed the desired mass (calculated MW: 5690.6, observed m / z: 1416.3 [(M-28) / 4+H] +) was shown. The crude product was purified by preparative HPLC (TFA condition), but the purity was low. Compound 3 (COM00000116-BCY17224, 43.9 mg, 53.1% purity) was obtained as a white solid.

[0209] (Preparation of intermediates COM00000116-BCY17224-BCY15017) [ka] A mixture of compound 3 (COM00000116-BCY17224, 29.8 mg, 5.24 μmol, 1.0 equiv.), compound 4 (BCY15017, 13.0 mg, 5.24 μmol, 1.0 equiv.), and THPTA (4.5 mg, 10.5 μmol, 2.0 equiv.) was mixed with t-BuOH / H 2 HO (1:1, 1 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 13.1 μL, 1.0 eq.) and VcNa (3.1 mg, 15.7 μmol, 3.0 eq.) was 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirred under atmosphere. LC-MS showed desired m / z (calculated MW: 8171.45, observed m / z: 1362.6 ([M / 6+H] + ), 1168.3([M / 7+H] + ) was detected. The reaction mixture was filtered to remove insoluble residues. The soluble crude product was purified by preparative HPLC (TFA conditions) to obtain compound 5 (COM00000116-BCY17224-BCY15017, 8.7 mg, 1.06 μmol, 20.2% yield, 78.7% purity) as a white solid.

[0210] (Preparation of BCY17235) [ka] Compound 5 (COM00000116-BCY17224-BCY15017, 4.2 mg, 5.14e-1 μmol, 1.0 equivalent), compound 6 (BCY13883, 1.0 mg, 5.14e-1 μmol, 1.0 equivalent), and THPTA (0.2 mg, 5.14e-1 t-BuOH / H 2 HO (1:1, 0.2 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 1.3 μL, 1.0 eq.) and VcNa (0.2 mg, 1.03 μmol, 2.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25–30 °C for 0.5 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 5 was completely consumed and had the desired m / z (calculated MW: 10149.8, observed m / z: 1269.3 ([M / 8+H] + ), 1128.4([M / 9+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The soluble crude product was purified by preparative HPLC (TFA condition) to give BCY17235 (1.8 mg, 1.77e-1 μmol, 34.4% yield, 91.6% purity) as a white solid.

[0211] Example 18: Synthesis of BCY17225 [ka] A mixture of compound 1 (20 mg, 7.36 μmol, 1.0 eq.), compound 2 (BCY17224, 16.85 mg, 8.09 μmol, 1.1 eq.), and THPTA (3.20 mg, 7.36 μmol, 1.0 eq.) was mixed with t-BuOH / H 2HO (1:1, 1.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 An aqueous solution of (0.4 M, 18.4 μL, 1.0 eq.) and VcNa (2.92 mg, 14.72 μmol, 2.0 eq.) was prepared with N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 1 was completely consumed and had the desired m / z (calculated MW: 4799.47, observed m / z: 961.0 ([M / 5+H] + ), 1200.9([M / 4+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY17225 (19.5 mg, 3.88 μmol, 52.73% yield, 95.5% purity) as a white solid.

[0212] Example 19: Synthesis of BCY18731 (Preparation of intermediate N3-PEG24-BCY9594) [ka] A mixture of compound 1 (17.45 mg, 13.75 μmol, 1.1 equiv.), DIEA (3.23 mg, 24.99 μmol, 4.35 uL, 2.0 equiv.) was dissolved in DMF (1.0 mL). The reaction mixture was stirred at 25-30 °C for 6 min, then compound 2 (30.0 mg, 12.50 μmol, 1.0 equiv.) was added to the reaction mixture. The reaction mixture was stirred at 25-30 °C for 0.5 h. LC-MS showed that compound 2 was completely consumed and the desired m / z (MW: 3555.1, observed m / z: 1186.1 [(M / 3+H] +) was detected. The reaction mixture was filtered to remove the insoluble residue. The residue was then purified by preparative HPLC (TFA condition). Compound 3 (N3-PEG24-BCY9594, 23.8 mg, 6.69 μmol, 52.40% yield, 97.8% purity) was obtained as a white solid.

[0213] (Preparation of BCY18731) [ka] A mixture of compound 3 (N3-PEG24-BCY9594, 23.8 mg, 6.69 μmol, 1.0 eq.), compound 4 (BCY17224, 15.33 mg, 7.36 μmol, 1.1 eq.), and THPTA (2.91 mg, 6.69 μmol, 1.0 eq.) was mixed with t-BuOH / H 2 HO (1:1, 2.0 mL, pre-degassed with N 2 (purged three times with 1000 mL of ethyl acetate) and then dissolved in CuSO 4 (0.4 M, 16.74 μL, 1.0 eq.) and VcNa (2.65 mg, 13.39 μmol, 2.0 eq.) in N 2 The solution was diluted with 0.2 M NH 4 HCO 3 (1:1 t-BuOH / H 2 The pH was adjusted to 8 by dropwise addition of 2,4-dichloromethane (in H2O), and the solution turned bright yellow. The reaction mixture was incubated at 25-30 °C for 1 h with N2O. 2 Stirring was performed under atmosphere. LC-MS showed that compound 3 was completely consumed and had the desired m / z (calculated MW: 5636.47, observed m / z: 1410.0 ([M / 5+H] + ),1128.3([M / 5+H] + ), 940.4([M / 6+H] + )) was detected. The reaction mixture was filtered to remove insoluble residues. The crude product was purified by preparative HPLC (TFA condition) to give BCY18731 (14.7 mg, 2.50 μmol, 37.4% yield, 96.0% purity) as a white solid.

[0214] (Biological Data) 1. Degranulation Assay Tumor cells are recognized and killed by CD8+ T-cytotoxic cells and NK cells through the secretion of immune lytic granules that kill target cells. This process involves the fusion of the granule membrane with the cytoplasmic membrane of immune effector cells, resulting in surface exposure of CD107a (LAMP1). Membrane expression of CD107a constitutes a marker of immune cell activation and cytotoxic degranulation. Heterotandem bicyclic peptide complexes of EphA2 / NKp46 or EphA2 / CD16a were assessed for NK cell activation using a degranulation assay.

[0215] On the day of the experiment, medium was prepared by supplementing RPMI-1640 (Gibco™ 11875-093; with L-glutamine) with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI). This is referred to herein as working medium. Human peripheral blood mononuclear cells (PBMCs) previously isolated from whole blood were rapidly thawed in a water bath and washed once with 10 mL of pre-warmed working medium at 500 rpm for 5 min. The PBMC pellet was then diluted with 5 × 10 6 The cells were resuspended in working medium to a concentration of 1 × 10 cells / mL and placed horizontally in a coated tissue culture flask (T-183; CELLTREAT Scientific 229351) overnight (12-18 hours). Furthermore, human lung cancer cell line A549 (ATCC® CCL-185; cells were grown and maintained according to the manufacturer's recommendations) expressing ephrin type A receptor (EphA2) was detached from the culture vessel using trypsin / EDTA and washed once with 15 mL of pre-warmed working medium at 500 rpm for 5 minutes. The cell pellet was then diluted to 1 × 10 5The cells were resuspended in working medium at a concentration of 100 cells / mL. Then, 100 μL of the cell suspension was plated onto a flat-bottom tissue culture coated 96-well plate (Greiner CellStar® 655180) and left to stand overnight (12 to 18 hours).

[0216] After overnight incubation, PBMCs were removed from the flasks and washed once with 10 mL of pre-warmed working medium at 500 rpm for 5 min. NK cells were then isolated from the total PBMC population using a negative isolation kit (STEMCELL Technologies 17955) according to the manufacturer's recommendations. The NK cell pellet was then diluted to 1 × 10 5 The cells were resuspended in working medium at a concentration of 1000 cells / mL. 100 μL of the cell suspension was then plated onto a 96-well plate containing overnight quiescent A549 cells.

[0217] Heterotandem bicyclic peptide complexes were diluted in working medium and titrated in a 1:4 dilution series onto the corresponding cell plates at a recommended starting concentration of 300 nM or 5 μM to perform a 12-point serial dilution. In addition, the protein transport inhibitor GolgiStop™ (BD Biosciences 554715) was added as recommended by the manufacturer. Plates were then incubated at 37 °C, 5 % CO 2 The cells were incubated at 500 rpm for 4 hours. Samples were then washed once with 200 μL 1× phosphate buffered saline (PBS; Gibco™ 10-010-023) for 5 minutes at 500 rpm. Cells were resuspended in 200 μL PBS and transferred to a 96-well V-bottom polypropylene plate (Greiner Bio-One 651201). Samples were then centrifuged at 500 rpm for 5 minutes and the supernatant was removed.

[0218] For flow cytometry Zombie Aqua™ Fixable Viability Dye (BioLegend® 423102) samples were prepared by diluting 1:1000 in PBS and adding 100 μL of Viability Dye to each well and incubating for 30 min at 4 °C in the dark. The wells were then washed with 100 μL of PBS at 500 rpm for 5 min and the supernatant was removed. Next, human TruStain FcX™ block (BioLegend® 422302) was prepared by diluting 1.5 μL of FcX in 25 μL of staining buffer (1×PBS supplemented with 2% FBS). Fc block solution (25 μL / well) was incubated for 10 min at room temperature (RT) in the dark. An antibody master mix cocktail was prepared by diluting 1.5 μL of the following antibodies per 100 μL of staining buffer: FITC anti-human CD45 (BioLegend® 304038; clone HI30), Brilliant Violet 605™ anti-human CD3 (BioLegend® 344836; clone SK7), PE / Cyanine 7 anti-human CD56 (BioLegend® 362510; clone 5.1H11), PE anti-human NKp46 (BioLegend® 331908; clone 9E2), and Brilliant Violet 421™ anti-human CD107a (BioLegend® 328626; clone H4A3). Cells were resuspended in the master mix cocktail (100 μL) and incubated at 4° C. for 30 min in the dark. Cells were then washed three times with 100 μL staining buffer at 500 rpm for 5 min, and the supernatant was removed. Cells were resuspended in 200 μL staining buffer and kept in the dark at 4° C. until they were read on a BD FACSCelesta™ flow cytometer and FCS files were analyzed with FlowJo™.

[0219] The data presented in Table 1 and Figure 1 demonstrate a dose-dependent upregulation of CD107a on the surface of NK cells following treatment with BCY15664 and BCY15911. The non-binding controls BCY15667 and BCY15666 do not elicit an upregulation above baseline.

[0220] The data presented in Table 1 and Figure 1 show dose- and tumor antigen-dependent upregulation of CD107a on the surface of NK cells following treatment with BCY15664 and BCY15911. A non-binding control (BCY15667) in which both the EphA2 bicyclic peptide and the NKp46 bicyclic peptide are composed of all D-amino acids does not elicit upregulation above baseline. Furthermore, a control in which only the EphA2 bicyclic peptide is composed of all D-amino acids (BCY15666), which is therefore capable of binding to NKp46 but not EphA2, does not elicit upregulation above baseline.

[0221] (Table 1: Concentration causing half-maximal effect (EC 50 )) [Table 9]

[0222] 2. NK Cytotoxicity Assay Heterotandem bicyclic peptide conjugates were evaluated for NK functional readouts (cytotoxicity and cytokine secretion) in NK cell line-tumor cell line cocultures.

[0223] NK cells were isolated from the total PBMC population purified from whole blood using a negative isolation kit (STEMCELL Technologies 17955). The NK cell pellet was then cultured at 4 × 10 ng / mL in DMEM medium (Corning™ 10-013-CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI), and 50 IU / mL human IL-2 (Miltenyi Biotec® 130-097-748). 5 For the NK cytotoxicity assay, 50 μL (2 × 10 4 ) cell suspension, 50 μl (4 × 10 3 ) HT1080-luc cells (ATCC® CCL-121-luc2) (in DMEM (Gibco™ 11875-093; with L-glutamine) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI)) were plated in a 96-well plate (Grenier® Bio One™ 655090). Test substances or antibodies (InvivoGen®, hegfr-mab1) were diluted in DMEM medium (Corning® 10-013-CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI) and titrated in a 1 / 5 dilution series into the corresponding cell plates (50 μl) to perform 8-point serial dilutions at a recommended starting concentration of 10 nM. Plates were then incubated at 37 °C, 5% CO 2Plates were incubated at 250×g for 5 min and 100 μl of supernatant was removed. Samples were then incubated with 50 μl of Bright-Glo™ Luciferase Assay System (Promega™ E2620) for 10 min. Emission excited at 570 nm was read using a CLARIOstar® plate reader with MARS Data Analysis Software™. Data were fitted to a four-parameter nonlinear regression using GraphPad Prism™ 8.0.2 to determine EC 50 The value was calculated.

[0224] Selected heterotandem bicyclic peptide complexes of the invention were tested in this assay and the results are shown in Figures 2-4 and 6-10.

[0225] Figure 2 shows that BCY17226 elicits a dose-dependent NK cell response and kills EphA2+ve HT1080-luc tumor cell line. No enhanced dose-dependent effect on tumor cell death is observed with the non-conjugated heterotandem bicyclic peptide complex (BCY15667) compared to NK:HT1080-luc co-cultures without the addition of heterotandem bicyclic peptide complex. An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1) was used in the assay as a positive reference control for NK-induced cytotoxicity. The mean luminescence without NK-TICA (referring to "without heterotandem bicyclic peptide complex") is shown for convenience at 0.00001 pM as a reference. EC 50 = 6.1 pM was calculated using 4-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0226] FIG. 3 shows that BCY15664 and BCY15923 elicit a dose-dependent NK cell response and kill the EphA2+ve HT1080-luc tumor cell line. No enhanced dose-dependent effect on tumor cell death is observed with the non-conjugated heterotandem bicyclic peptide complex (BCY15667) compared to NK:HT1080-luc cocultures without the addition of heterotandem bicyclic peptide complex. An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1) was used in the assay as a positive reference control for NK-induced cytotoxicity. The mean luminescence without NK-TICA (referring to "without heterotandem bicyclic peptide complex") is shown at 0.00001 pM for convenience as a reference. BCY15664 (NKp46 epitope 1, EC 50 =21 pM) or BCY15923 (NKp46 epitope 2, EC 50 = 44 pM) was calculated using four-parameter logistic regression using GraphPad Prism™ 8.0.2 software.

[0227] Figure 4 shows that tumor antigen-bound bicyclic peptides are required in the heterotandem bicyclic peptide complex construct to elicit enhanced NK cytotoxic activity. No enhanced dose-dependent effect on tumor cell death is observed with the non-binding EphA2 / non-binding NKp46 heterotandem bicyclic peptide complex (BCY15667) compared to NK:HT1080-luc coculture without the addition of the heterotandem bicyclic peptide complex. No enhanced dose-dependent effect on tumor cell death is observed with the non-binding EphA2 / binding NKp46 heterotandem bicyclic peptide complex (BCY15666) compared to NK:HT1080-luc coculture without the addition of the heterotandem bicyclic peptide complex. An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1) was used in the assay as a positive reference control for NK-induced cytotoxicity. The average luminescence without NK-TICA (referring to "without heterotandem bicyclic peptide complex") is shown for convenience as 0.001 nM for reference. EC 50= 16 pM was calculated using 4-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0228] FIG. 6 shows that varying the valency of bicyclic NKp46 in NK-TICA constructs enhances NK cytotoxic activity. 50 =3.6pM), BCY21686_01_01(EC 50 =1.3 pM) an enhanced dose-dependent effect on tumor cell death was observed compared to NK:HT1080-luc cocultures without the addition of NK-TICA. No enhanced dose-dependent effect on tumor cell death was observed with non-binding NK-TICA (BCY15667_01_01). An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1) was used as a positive reference control (EC 50 = 0.053 pM) was used in the assay. The average luminescence without NK-TICA is shown at 0.005 pM for convenience as a reference. 50 Values ​​were calculated using four-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0229] FIG. 7 shows that varying the spacer length of the NK-TICA construct enhances dose-dependent NK cytotoxicity. 50 =18pM), BCY17231_01_01(EC 50 =21 pM) and BCY17235_04_01 (EC 50=14 pM) an enhanced dose-dependent effect on tumor cell death was observed compared to NK:HT1080-luc cocultures without the addition of NK-TICA. No enhanced effect on NK cytotoxicity was observed with unconjugated NK-TICA (BCY15667_01_01). An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1, 6.7 nM) was used in the assay as a positive reference control for NK-induced cytotoxicity. The mean luminescence without NK-TICA is shown at 0.05 pM for convenience as a reference. The EC 50 Values ​​were calculated using four-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0230] FIG. 8 shows enhanced dose-dependent tumor killing by NK cells treated with CD16-binding bicyclic and NKp46-binding bicyclic NK-TICA constructs (BCY20793_01_01). NK cell tumoricidal activity was observed with BCY20793_01_01 (EC 50 = 6.5 pM). An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1) was used as a positive reference control for NK-induced cytotoxicity (EC 50 = 0.45 pM) was used in the assay. No enhanced dose-dependent effect on tumor cell death is observed with unbound NK-TICA (BCY15667_01_01) compared to NK:HT1080-luc cocultures without NK-TICA. The mean luminescence without NK-TICA is shown at 0.02 pM for convenience as a reference. The EC 50 Values ​​were calculated using four-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0231] FIG. 9 shows that alternative NKp46-binding bicyclic forms of NK-TICA induced enhanced killing of HT1080-luc cells. NKp46-binding NK-TICA construct BCY18049_01_01 (EC 50 = unstable), BCY18042_01_01(EC50 = unstable), and BCY15924_01_01 (EC 50 =0.5nM) enhanced NK cytotoxicity of HT1080-luc tumor cell line compared to NK:HT1080-luc coculture without NK-TICA addition. BCY15667_01_01 (non-binding NK-TICA) has no activity compared to NK:HT1080-luc coculture without NK-TICA addition. Mean luminescence without NK-TICA is shown at 0.001 pM for convenience as a reference. An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1, 6.7 nM) was used in the assay as a positive reference control for NK-induced cytotoxicity. EC 50 Values ​​were calculated using four-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0232] FIG. 10 shows that NK-TICA containing an additional tumor-binding bicyclic arm within the NKp46-binding NK-TICA construct induced enhanced killing of HT1080-luc cells. 50 = 3.0 pM) or the MT1-linked bicyclic NK-TICA construct (BCY18604_01_01; EC 50 =7.9 pM) enhanced NK cytotoxicity of the HT1080-luc tumor cell line compared to NK:HT1080-luc cocultures without the addition of NK-TICA. A non-binding control NK-TICA construct (BCY15667_01_02) has no activity compared to NK:HT1080-luc cocultures without the addition of NK-TICA. The mean luminescence without NK-TICA is shown at 0.005 pM for convenience as a reference. An ADCC-capable anti-EGFR antibody (InvivoGen®, hegfr-mab1, 6.7 nM) was used as a positive reference control (EC 200) for NK-induced cytotoxicity. 50 = 0.70 pM) was used in the assay. 50 Values ​​were calculated using four-parameter logistic regression using GraphPad Prism™ 8.0.2.

[0233] 3. Cytokine Secretion Assay Heterotandem bicyclic peptide conjugates were evaluated for NK functional readouts (cytotoxicity and cytokine secretion) in NK cell line-tumor cell line cocultures.

[0234] NK cells were isolated from the total PBMC population purified from whole blood using a negative isolation kit (STEMCELL Technologies® 17955). The NK cell pellet was then cultured at 4 × 10 ng / mL in DMEM medium (Corning™ 10-013-CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI), and 50 IU / mL human IL-2 (Miltenyi Biotec® 130-097-748). 5 For NK cell cytokine secretion assays, the cells were resuspended at a concentration of 2 × 10 4 NK cells (in 50 μL of cell suspension) were added to 50 μl of (4 × 10 4) HT1080-luc cells (ATCC® CCL-121-luc2) (in DMEM (Gibco™ 11875-093; with L-glutamine) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI)) were plated in 96-well U-bottom plates (Grenier Bio One™ 650180). Test substances or antibodies (InvivoGen®, hegfr-mab1) were diluted in DMEM medium (Corning® 10-013-CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Corning® 35-011-CV), 10 mM HEPES (Gibco™ 15-630-080), and 1% penicillin-streptomycin (Corning™ 30-002-CI) and titrated in a 1 / 5 dilution series into the corresponding cell plates (50 μl) to perform 8-point serial dilutions at a recommended starting concentration of 10 nM. Plates were then incubated at 37 °C, 5% CO 2The plates were incubated at 4°C for 4 hours. After incubation, the plates were centrifuged at 250×g for 5 minutes and 100 μl of the supernatant was collected. Samples were either immediately stored at −20° C. or cytokine levels were assessed. Interferon-γ or TNF-α levels were measured in 50 μl of the collected supernatant using a Human IFN-γ Quantikine™ ELISA Kit (R&D Systems, DIF50C) or a Human TNF-α Quantikine™ ELISA Kit (R&D Systems, DTA00D). Data at 450 nm wavelength was acquired on a CLARIOstar® plate reader and fitted to a 4-parameter nonlinear regression using the CLARIOstar® plate reader and MARS Data Analysis™ software, and analyzed with GraphPad Prism™ 8.0.2 software to quantify cytokine levels. Alternatively, TNF-α and IFN-γ cytokines were measured in 25 ul of harvested supernatant using a Luminex assay: Human XL Cytokine Discovery Premixed Kit (R&D Systems, FCSTM18-05) Luminex 200™ flow device and xPONENT™ analysis software.

[0235] Selected heterotandem bicyclic peptide complexes of the invention were tested in this assay and the results are shown in FIGS.

[0236] Figure 5 demonstrates that NK cells were co-cultured with the HT1080-luc tumor cell line in the presence of BCY17226 or the non-conjugated heterotandem bicyclic peptide complex BCY15667. As a positive control, an ADCC-capable anti-EGFR antibody (InvivoGen, hegfr-mab1) was used. Released cytokines (IFN-γ) were measured by ELISA (R&D systems, DIF50C) using a CLARIOstar™ plate reader and MARS Data Analysis™ software applying 4-parameter nonlinear regression.

[0237] Figure 11 shows that NK cells produce TNF-α and IFN-γ when co-cultured with HT1080-luc tumor cell line in the presence of 2 nM NKp46-linked bicyclic NK-TICA constructs of various NKp46 bicyclic valencies. Cytokine production from NK cells was observed with the addition of BCY00017225_01_02, BCY21686_01_01, BCY21687_01_01 compared to the non-linked NK-TICA construct (BCY15667_01_01). An ADCC-capable anti-EGFR antibody (InvivoGen, hegfr-mab1) was used as a positive reference control for NK-induced cytokines (1.34 nM). Released cytokines (IFN-γ and TNF-α) were measured by Luminex assay: Human XL Cytokine Discovery Premixed Kit (R&D Systems, FCSTM18-05) using a Luminex 200 flow device and xPONENT analysis software.

[0238] Figure 12 shows that NK cells secrete cytokines when co-cultured with HT1080-luc tumor cell line in the presence of 10 nM of NKp46-binding bicyclic NK-TICA constructs with structural modifications. NKp46-binding bicyclic NK-TICA (BCY18048_01_01) induced IFN-γ and TNF-α secretion compared to no cytokine production with non-binding NK-TICA (BCY15667_01_01 or BCY15666_01_01) treatment. As a positive control, an ADCC-capable anti-EGFR antibody (InvivoGen, hegfr-mab1) was used at 6.7 nM. Released cytokines (IFN-γ and TNF-α) were measured by ELISA (R&D systems, DIF50C, DTA00D) using a CLARIOstar plate reader and MARS Data Analysis software applying 4-parameter nonlinear regression.

[0239] (List of References) [Table 10] TIFF2024503632000078.tif244170TIFF2024503632000079.tif217170

Claims

1. (a) a first peptide ligand that binds to a component present on a cancer cell; (b) one or more second peptide ligands that bind to one or more components present on natural killer (NK) cells; The heterotandem bicyclic peptide complex, wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, and at least two polypeptide loops are formed on the molecular scaffold.

2. The heterotandem bicyclic peptide conjugate of claim 1, wherein the reactive group is a cysteine ​​residue.

3. The heterotandem bicyclic peptide complex of claim 1 or 2, wherein the one or more components present on the natural killer (NK) cell are natural cytotoxic receptors present on the surface of the NK cell, such as NKp30, NKp44, and NKp46, in particular NKp46, and wherein the one or more second peptide ligands comprise one or more NKp46-binding bicyclic peptide ligands.

4. The one or more NKp46-binding bicyclic peptide ligands include: 【Chemistry 1】 (In the formula, C i , C ii , and C iii represents the first, second, and third cysteine ​​residues, respectively; Cba represents β-cyclobutylalanine; dA represents D-alanine; and PYA represents pentynic acid), or a pharma- ceutically acceptable salt thereof; In particular, the molecular scaffold is TATA, the one or more NKp46-binding bicyclic peptide ligands optionally comprise an N-terminal and / or a C-terminal modification, and Ac-(SEQ ID NO:5)-[K(PYA)] (referred to herein as BCY17224); A-(SEQ ID NO:6)-A-[dK(PYA)] (referred to herein as BCY15452); A-(SEQ ID NO:7)-A-[K(PYA)] (referred to herein as BCY15686); A-(SEQ ID NO:8)-A-[K(PYA)] (referred to herein as BCY15687); A-(SEQ ID NO:9)-A-[K(PYA)] (referred to herein as BCY18004); A-(SEQ ID NO:10)-A (referred to herein as BCY17662); and A-(SEQ ID NO:11)-A-[K(PYA)] (referred to herein as BCY18005) wherein PYA represents pentynic acid, or a pharma- ceutically acceptable salt thereof.

5. The heterotandem bicyclic peptide complex of claim 1 or 2, wherein the one or more components present on the natural killer (NK) cell are Fc receptors present on the surface of the NK cell, such as low affinity Fcγ receptors (FcγR) selected from FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB, in particular FcγRIIIA (also known as CD16a), e.g., the one or more second peptide ligands comprise one or more CD16a-binding bicyclic peptide ligands.

6. The one or more CD16a-binding bicyclic peptide ligands include: 【Chemistry 2】 (In the formula, C i , C ii , and C iii represents a first, second, and third cysteine ​​residue, respectively), or a pharma- ceutically acceptable salt thereof; In particular, the molecular scaffold is TBMT, the one or more CD16a binding bicyclic peptide ligands optionally comprise an N-terminal and / or a C-terminal modification, and A-(SEQ ID NO:12)-A-[K(PYA)] (referred to herein as BCY13886); A-(SEQ ID NO:13)-A-[K(PYA)] (referred to herein as BCY20361); and A-(SEQ ID NO:14)-A-[K(PYA)] (referred to herein as BCY13883) wherein PYA represents pentynic acid, or a pharma- ceutically acceptable salt thereof.

7. 7. The heterotandem bicyclic peptide complex of any one of claims 1 to 6, wherein 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.

8. The heterotandem bicyclic peptide complex of any one of claims 1 to 7, wherein the component present on a cancer cell is EphA2, for example, the first peptide comprises an EphA2-binding bicyclic peptide ligand.

9. The EphA2-binding bicyclic peptide ligand is: 【Chemistry 3】 (In the formula, C i , C ii , and C iii represents a first (i) reactive group, a second (ii) reactive group, and a third (iii) reactive group; HyP represents trans-4-hydroxy-L-proline; and HArg represents homoarginine; or a pharma- ceutically acceptable salt thereof; In particular, the molecular scaffold is TATA, the EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal modification, and A-[HArg]-D-(SEQ ID NO:2) (referred to herein as BCY9594) (wherein HArg represents homoarginine), or a pharma- ceutically acceptable salt thereof.

10. 8. The heterotandem bicyclic peptide complex of any one of claims 1 to 7, wherein the component present on a cancer cell is PD-L1, e.g., the first peptide comprises a PD-L1-binding bicyclic peptide ligand.

11. The PD-L1 binding bicyclic peptide ligand is: 【Chemistry 4】 (In the formula, C i , C ii , and C iii represent a first (i) cysteine ​​group, a second (ii) cysteine ​​group, and a third (iii) cysteine ​​group, respectively, or a pharma- ceutically acceptable salt thereof; In particular, the molecular scaffold is TATA, the PD-L1 binding bicyclic peptide ligand optionally comprises an N-terminal modification and / or a C-terminal modification, and Ac-D-[Harg]-(SEQ ID NO:3)-PSH (referred to herein as BCY11865) wherein Harg represents homoarginine, or a pharma- ceutically acceptable salt thereof.

12. The heterotandem bicyclic peptide complex of any one of claims 1 to 7, wherein the component present on a cancer cell is MT1, e.g., the first peptide comprises an MT1-binding bicyclic peptide ligand.

13. The MT1-binding bicyclic peptide ligand is: 【Chemistry 5】 (In the formula, C i , C ii , and C iii represents a first (i) cysteine ​​group, a second (ii) cysteine ​​group, and a third (iii) cysteine ​​group, respectively, or a pharma- ceutically acceptable salt thereof; In particular, the molecular scaffold is TATA, the MT1-binding bicyclic peptide ligand optionally comprises an N-terminal and / or a C-terminal modification, and LPP-(SEQ ID NO:4) (referred to herein as BCY14320) or a pharma- ceutical acceptable salt thereof.

14. 14. The heterotandem bicyclic peptide complex of any one of claims 1 to 13, which is any of the complexes listed in Tables A1, A2, A3, B1, C, D and E.

15. The heterotandem bicyclic peptide complex according to any one of claims 1 to 14, wherein the pharma- ceutically acceptable salt is selected from the free acid or a sodium, potassium, calcium, or ammonium salt.

16. A pharmaceutical composition comprising the heterotandem bicyclic peptide complex of any one of claims 1 to 15 in combination with one or more pharma- ceutically acceptable excipients.

17. A heterotandem bicyclic peptide complex according to any one of claims 1 to 15, or a pharmaceutical composition according to claim 16, for use in the prevention, suppression or treatment of cancer.

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