Heterotandem bicyclic peptide complex

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 two or more second peptide ligands that bind to a component present on an immune cell, and the present invention also relates to the use of the heterotandem bicyclic peptide complex in the prevention, suppression, or treatment of cancer.

Description

Technical Field

[0001] (Field of the Invention) 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 two or more second peptide ligands that bind to a component present on an immune cell. The present invention also relates to the use of the heterotandem bicyclic peptide complex in the prevention, suppression, or treatment of cancer.

Background Art

[0002] (Background of the Invention) Cyclic peptides can bind to protein targets with high affinity and target specificity and are, therefore, an attractive molecular class for the development of therapeutic agents. Indeed, some cyclic peptides are already successful in being used in clinics, such as the antibacterial peptide vancomycin, the immunosuppressive drug 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. Usually, macrocyclic molecules bind to surfaces of several hundred square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ; Wu et al. (2007), Science 330, 1066 - 71), the cyclic peptide having an Arg - Gly - Asp motif that binds to integrin αVb3 (355 Å 2 )(Xiong et al. (2002), Science 296(5565), 151 - 5), or the cyclic peptide inhibitor upain - 1 that binds to urokinase - type plasminogen activator (603 Å 2 ; Zhao et al. (2007), J Struct Biol 160(1), 1 - 10).

[0003] Due to this cyclic three-dimensional arrangement, the peptide macrocyclic molecule is less flexible than the linear peptide, resulting in a smaller entropy loss when binding to the target, and consequently, a higher binding affinity. The reduced flexibility also leads to the fixation of the target-specific three-dimensional structure and increases the binding specificity compared to linear peptides. This effect is exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8) that loses its selectivity for 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 having multiple peptide rings, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously linked polypeptides having cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops on a synthetic scaffold for structural mimicking of the protein surface (Timmerman et al. (2005), ChemBioChem). Methods for making candidate drug compounds, where the compounds are made by linking a cysteine-containing polypeptide to a molecular scaffold such as tris(bromomethyl)benzene, are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

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

Summary of the Invention

[0006] (Summary of the Invention) According to a first aspect of the present invention, (a) a first peptide ligand that binds to a component present on a cancer cell; is linked via a linker to (b) two or more second peptide ligands that bind to components present on immune cells; conjugated to : comprising, 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 a covalent bond with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, a heterotandem bicyclic peptide complex is provided.

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

[0008] According to 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)

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[0010] (Detailed Description of the Invention) According to a first aspect of the present invention, (a) a first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) two or more second peptide ligands that bind to a component present on an immune cell; conjugated thereto comprising a polypeptide containing at least three reactive groups separated by at least two loop sequences, wherein each of the peptide ligands comprises at least three reactive groups separated by at least two loop sequences, and a molecular scaffold that forms a covalent bond with the reactive groups of the polypeptide, resulting in the formation of at least two polypeptide loops on the molecular scaffold, a hetero tandem bicyclic peptide complex is provided.

[0011] According to one aspect of the invention that may be mentioned, (a) a first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) two or more second peptide ligands that bind to components present on immune cells; conjugated to comprising a polypeptide containing at least three cysteine residues separated by at least two loop sequences, wherein each of the peptide ligands comprises at least three cysteine residues separated by at least two loop sequences, and a molecular scaffold that forms a covalent bond with the cysteine residues of the polypeptide, resulting in the formation of at least two polypeptide loops on the molecular scaffold, a hetero tandem bicyclic peptide complex is provided.

[0012] (the first peptide ligand) The reference herein to the term "cancer cell" includes any cell known to be involved in cancer. Cancer cells are produced when genes involved in the regulation of cell division are damaged. Carcinogenesis results from mutations and epigenetic mutations in the genetic material of normal cells that disrupt the normal balance between growth and cell death. As a result, uncontrolled cell division and the evolution of these cells by natural selection in the body occur. Uncontrolled and often rapid cell growth can give rise to benign or malignant tumors (cancers). 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 (metastasize), and be life-threatening.

[0013] 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.

[0014] In one embodiment, the component present on the cancer cells is nectin-4.

[0015] Nectin-4 is a surface molecule belonging to the nectin family of proteins that includes four members. Nectins are cell adhesion molecules that play important roles in various biological processes such as the polarity, proliferation, differentiation, and migration of epithelial, endothelial, immune, and nerve cells during development and in the adult stage. They are involved in some 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 associated with other abnormalities. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of the family. Nectin-4 is a tumor-associated antigen on tumors with mainly poor prognosis in 50%, 49%, and 86% of breast cancer, ovarian cancer, and lung cancer, respectively. Its expression is not detected in the corresponding normal tissues. In breast tumors, nectin-4 is mainly expressed in triple-negative and ERBB2+ cancers. Detection of soluble form of nectin-4 in the sera of patients with these cancers is associated with poor prognosis. The level of serum nectin-4 increases during the metastatic progression phase and decreases after treatment. These results suggest that nectin-4 can be a reliable target for cancer treatment. Therefore, several anti-nectin-4 antibodies have been described in the prior art. In particular, enfortumab vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting nectin-4 and is currently under clinical investigation for the treatment of patients with solid tumors.

[0016] In one embodiment, the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand.

[0017] Suitable examples of the nectin-4 binding bicyclic peptide ligand are disclosed in WO 2019 / 243832, the peptide of which is incorporated herein by reference.

[0018] In one embodiment, the nectin-4 binding bicyclic peptide ligand is

Chemical formula

[0019] In a further embodiment, the nectin-4 binding bicyclic peptide ligand is

Chemical formula

[0020] In a further embodiment, the nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification and SEQ ID NO: 1 (referred to herein as BCY8116); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 1) (referred to herein as BCY8846); [PYA]-(SEQ ID NO: 1) (referred to herein as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO: 1) (referred to herein as BCY11016); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 2) (referred to herein as BCY11942); Ac-(SEQ ID NO: 3) (referred to herein as BCY8831); SEQ ID NO: 4 (referred to herein as BCY11414); [PYA]-[B-Ala]-(SEQ ID NO: 14) (referred to herein as BCY11143); Palmitic acid - yGlu - yGlu-(SEQ ID NO: 14) (referred to herein as BCY12371); Ac-(SEQ ID NO: 14) (referred to herein as BCY12024); Ac-(SEQ ID NO: 16) (referred to herein as BCY12364); Ac-(SEQ ID NO: 18) (referred to herein as BCY12366); and Ac-(SEQ ID NO: 19) (referred to herein as BCY12367); (wherein PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units), or a pharmaceutically acceptable salt thereof : comprising.

[0021] In yet a further embodiment, the nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification and SEQ ID NO: 1 (referred to herein as BCY8116); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 1) (referred to herein as BCY8846); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 2) (referred to herein as BCY11942); Ac-(SEQ ID NO: 3) (referred to herein as BCY8831); and SEQ ID NO: 4 (referred to herein as BCY11414); (wherein PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units), or a pharmaceutically acceptable salt thereof :.

[0022] In yet a further embodiment, the nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO: 1 (referred to herein as BCY8116).

[0023] In an alternative embodiment, the component present on the cancer cell is EphA2.

[0024] Eph receptor tyrosine kinases (Ephs) belong to a large family of receptor tyrosine kinases (RTKs) that phosphorylate proteins on tyrosine residues. Ephs and their membrane-bound ephrin ligands control cell arrangement and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur at higher ligand oligomerization states (Stein et al. (1998) Genes Dev 12, 667-678).

[0025] Among other patterning functions, various Ephs and ephrins have been shown to play roles in angiogenesis. Knockout of EphB4 and ephrin-B2 results in the inability to remodel the capillary bed into vessels (Poliakov et al., supra) and embryonic lethality. Persistent expression of several Eph receptors and ephrins has also been observed in newly formed adult microvessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0026] Deregulated reappearance of several ephrins and their receptors in the adult 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 various human tumors (Brantley-Sieders et al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0027] EPH receptor A2 (ephrin type-A receptor 2) is a protein encoded by the EPHA2 gene in humans.

[0028] EphA2 is upregulated in a number of human cancers that are often correlated with disease progression, metastasis, and poor prognosis, such as 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, 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).

[0029] Although the exact role of EphA2 in cancer progression has not been fully elucidated, there is evidence of its involvement in many stages of cancer progression, including tumor cell proliferation, survival, invasion, and angiogenesis. Downregulation of EphA2 expression suppresses the growth of tumor cancer cells (Binda et al. (2012) Cancer Cell 22, 765-780), while blockade of EphA2 inhibits VEGF-induced cell migration (Hess et al. (2001) Cancer Res. 61, 3250-3255), sprouting 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).

[0030] Antibody-drug conjugates with EphA2 have been shown to significantly reduce tumor growth in xenograft models in rats and mice (Jackson et al. (2008) Cancer Research 68, 9367-9374), and similar approaches have been attempted in humans, but the treatment had to be discontinued due to treatment-related adverse events (Annunziata et al. (2013) Invest New drugs 31, 77-84).

[0031] In one embodiment, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

[0032] Suitable examples of EphA2-binding bicyclic peptide ligands are disclosed in WO 2019 / 122860, WO 2019 / 122861, and WO 2019 / 122863, the peptides of which are incorporated herein by reference.

[0033] In one embodiment, the EphA2-binding bicyclic peptide ligand is

Chemical formula

Chemical formula

Chemical formula

[0034] In one particular embodiment, the EphA2-binding bicyclic peptide ligand is

Chemical formula

[0035] In one alternative specific embodiment, the EphA2-binding bicyclic peptide ligand is [Chemical formula] (where C i , C ii , and C iii represent the first (i), second (ii), and third (iii) cysteine groups, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine) comprises an amino acid sequence that is, or a pharmaceutically acceptable salt thereof.

[0036] In a further embodiment, the EphA2-binding bicyclic peptide ligand optionally comprises N-terminal and / or C-terminal modifications and A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); [B-Ala]-[Sar 10 -A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY6099); [PYA]-A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY11813); Ac-A-[HArg]-D-(SEQ ID NO: 24)-[K(PYA)] (referred to herein as BCY11814); Ac-A-[HArg]-D-(SEQ ID NO: 24)-K (referred to herein as BCY12734); [NMeAla]-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY13121); [Ac]-(SEQ ID NO: 24)-L[dH]G[dK] (referred to herein as BCY13125); [PYA]-[B-Ala]-[Sar 10 -VGP-(SEQ ID NO: 25) (referred to herein as BCY8941); Ac-A-[HArg]-D-(SEQ ID NO: 26) (referred to herein as BCY11815); Ac-A-[HArg]-D-(SEQ ID NO: 27) (referred to herein as BCY11816); Ac-A-[HArg]-D-(SEQ ID NO: 28) (referred to herein as BCY11817); Ac-A-[HArg]-D-(SEQ ID NO: 29) (referred to herein as BCY12735); (Palmitoyl-Glu-LysN3)[PYA]A[HArg]D-(SEQ ID NO: 29) (hereinafter known as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO: 30) (referred to herein as BCY12736); Ac-A-[HArg]-D-(SEQ ID NO: 31) (referred to herein as BCY12737); A-[HArg]-D-(SEQ ID NO: 32) (referred to herein as BCY12738); A-[HArg]-E-(SEQ ID NO: 32) (referred to herein as BCY12739); A-[HArg]-D-(SEQ ID NO: 33) (referred to herein as BCY12854); A-[HArg]-D-(SEQ ID NO: 34) (referred to herein as BCY12855); A-[HArg]-D-(SEQ ID NO: 35) (referred to herein as BCY12856); A-[HArg]-D-(SEQ ID NO: 35)-[dA] (referred to herein as BCY12857); (SEQ ID NO: 35)-[dA] (referred to herein as BCY12861); [NMeAla]-[HArg]-D-(SEQ ID NO: 35) (referred to herein as BCY13122); [dA]-ED-(SEQ ID NO: 35) (referred to herein as BCY13126); [dA]-[dA]-D-(SEQ ID NO: 35) (referred to herein as BCY13127); AD-(SEQ ID NO: 35) (referred to herein as BCY13128); A-[HArg]-D-(SEQ ID NO: 36) (referred to herein as BCY12858); A-[HArg]-D-(SEQ ID NO: 37) (referred to herein as BCY12859); Ac-(SEQ ID NO: 37)-[dK] (referred to herein as BCY13120); A-[HArg]-D-(SEQ ID NO: 38) (referred to herein as BCY12862); A-[HArg]-D-(SEQ ID NO: 39) (referred to herein as BCY12863); [dA]-[HArg]-D-(SEQ ID NO: 39)-[dA] (referred to herein as BCY12864); (SEQ ID NO: 40)-[dA] (referred to herein as BCY12865); A-[HArg]-D-(SEQ ID NO: 41) (referred to herein as BCY12866); A-[HArg]-D-(SEQ ID NO: 42) (referred to herein as BCY13116); A-[HArg]-D-(SEQ ID NO: 43) (referred to herein as BCY13117); A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); [dA]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13123); [d1Nal]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13124); A-[HArg]-D-(SEQ ID NO: 47) (referred to herein as BCY13130); A-[HArg]-D-(SEQ ID NO: 48) (referred to herein as BCY13131); A-[HArg]-D-(SEQ ID NO: 49) (referred to herein as BCY13132); A-[HArg]-D-(SEQ ID NO: 50) (referred to herein as BCY13134); A-[HArg]-D-(SEQ ID NO: 51) (referred to herein as BCY13135); (SEQ ID NO: 154)-[dK] (referred to herein as BCY13129); A[HArg]D-(SEQ ID NO: 155) (referred to herein as BCY13133); A[HArg]D-(SEQ ID NO: 156) (referred to herein as BCY13917); A[HArg]D-(SEQ ID NO: 157) (referred to herein as BCY13918); A[HArg]D-(SEQ ID NO: 158) (referred to herein as BCY13919); A[HArg]D-(SEQ ID NO: 159) (referred to herein as BCY13920); A[HArg]D-(SEQ ID NO: 160) (referred to herein as BCY13922); A[HArg]D-(SEQ ID NO: 161) (referred to herein as BCY13923); A[HArg]D-(SEQ ID NO: 162) (referred to herein as BCY14047); A[HArg]D-(SEQ ID NO: 163) (referred to herein as BCY14048); and A[HArg]D-(SEQ ID NO: 164) (referred to herein as BCY14313); (where PYA represents 4-pentynoic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, HArg represents homoarginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, and palmitoyl-Glu-LysN3[PYA] is [Chem.] : (represented by), or a pharmaceutically acceptable salt thereof : contains.

[0037] In one particular embodiment, the EphA2-binding bicyclic peptide ligand optionally contains N-terminal and / or C-terminal modifications and A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); (where HArg represents homoarginine), or a pharmaceutically acceptable salt thereof : contains.

[0038] In one alternative particular embodiment, the EphA2-binding bicyclic peptide ligand optionally contains N-terminal and / or C-terminal modifications and A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); (where HArg represents homoarginine), or a pharmaceutically acceptable salt thereof : contains.

[0039] In an alternative embodiment, the component present on the cancer cell is PD-L1.

[0040] Programmed cell death 1 ligand 1 (PD-L1) is a 290-amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in the avoidance of immune responses associated with chronic infections, such as chronic viral infections (e.g., including in particular HIV, HBV, HCV, and HTLV), chronic bacterial infections (e.g., including in particular Helicobacter pylori), and chronic parasitic infections (e.g., including Schistosoma mansoni). PD-L1 expression has been detected in several tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta.

[0041] PD-L1 expression is also involved in the suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immunological clearance of tumors is rare. Some of these deficiencies are due to immunosuppression 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 shown in situ in a wide variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymic epithelium, head, and neck (Brown JA et al., 2003 Immunol. 170:1257-66; Dong H et al., 2002 Nat. Med. 8:793-800; Hamanishi J et al., 2007 Proc. Natl. Acad. Sci. 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, the expression of programmed cell death protein 1 (also known as PD-1 and CD279), the 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 correlating PD-L1 expression in tumors with disease outcome have shown that PD-L1 expression strongly correlates with an unfavorable prognosis in renal cancer, ovarian cancer, bladder cancer, breast cancer, gastric cancer, and pancreatic cancer (Hamanishi J et al., 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al., 2007 Cancer 109:1499-505; Konishi J et al., 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al., 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al., 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al., 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). Furthermore, these studies suggest that higher levels of PD-L1 expression in tumors may promote tumor stage progression and invasion into deeper tissue architecture.

[0042] The PD-1 pathway can also play a role in hematological malignancies. PD-L1 is expressed in a number of myeloma cells but not in normal plasma cells (Liu J et al., 2007 Blood 110:296-304). PD-L1 is expressed in some primary T-cell lymphomas, particularly anaplastic large cell T-cell lymphoma (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is highly expressed in the T cells of angioimmunoblastic lymphoma, and PD-L1 is expressed in the associated follicular dendritic cell network (Dorfman DM et al., 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, the T cells associated with lymphocyte or histiocyte (L&H) cells express PD-1. Microarray analysis using the readout of genes induced by PD-1 ligation has suggested that tumor-associated T cells respond to PD-1 signals in situ in Hodgkin lymphoma (Chemnitz JM et al., 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed in the CD4 T cells of HTLV-1-mediated adult T-cell leukemia and lymphoma (Shimauchi T et al., 2007 Int. J. Cancer 121: 2585-90). These tumor cells are poorly responsive to TCR signals.

[0043] Studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis, and in some cases, leads to an increase in 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 the anti-tumor T cell response. PD-L1 expression in the population of tumor-associated bone marrow DCs is upregulated by tumor environmental factors (Curiel TJ et al., 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in the tumor-draining regional lymph nodes of B16 melanoma express IDO, which strongly activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cell contact with IDO-expressing DCs (Sharma MD et al., 2007 Clin. Invest. 117:2570-82).

[0044] In one embodiment, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand.

[0045] Suitable examples of PD-L1-binding bicyclic peptide ligands are disclosed in WO 2020 / 128526 and WO 2020 / 128527, the peptides of which are incorporated herein by reference.

[0046] In one embodiment, the PD-L1-binding bicyclic peptide ligand is

Chemical formula

[0047] In a further embodiment, the PD-L1 binding bicyclic peptide ligand optionally comprises N-terminal and / or C-terminal modifications, and [PYA]-[B-Ala]-[Sar 10 -SDK-(SEQ ID NO: 52) (referred to herein as BCY10043); Ac-D-[HArg]-(SEQ ID NO: 52)-PSH (referred to herein as BCY11865); Ac-SDK-(SEQ ID NO: 53) (referred to herein as BCY11013); Ac-SDK-(SEQ ID NO: 53)-PSH (referred to herein as BCY10861); Ac-D-[HArg]-(SEQ ID NO: 54)-PSH (referred to herein as BCY11866); Ac-D-[HArg]-(SEQ ID NO: 55)-PSH (referred to herein as BCY11867); Ac-D-[HArg]-(SEQ ID NO: 56)-PSH (referred to herein as BCY11868); Ac-D-[HArg]-(SEQ ID NO: 57)-PSH (referred to herein as BCY11869); Ac-SD-[HArg]-(SEQ ID NO: 58)-PSHK (referred to herein as BCY12479); and Ac-SD-[HArg]-(SEQ ID NO: 59)-PSHK (referred to herein as BCY12477); (wherein PYA represents 4-pentynoic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, and HArg represents homoarginine), or a pharmaceutically acceptable salt thereof :.

[0048] In an alternative embodiment, the component present on the cancer cell is prostate specific membrane antigen (PSMA).

[0049] Prostate-specific membrane antigen (PSMA) (glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), and also known as NAAG peptidase) is an enzyme encoded by the FOLH1 (folate hydrolase 1) gene in humans. Human GCPII contains 750 amino acids and has a weight of approximately 84 kDa.

[0050] Human PSMA is expressed at a level approximately 100-fold higher in the prostate than in most other tissues. In some prostate cancers, PSMA is the second most highly upregulated gene product, with an 8- to 12-fold increase compared to levels in non-cancerous prostate cells. Due to this high expression, PSMA is being developed as a potential biomarker for the treatment and imaging of some cancers. In human prostate cancer, tumors with higher expression are associated with patients suffering from a faster progression time and a larger percentage of recurrences.

[0051] In one embodiment, the first peptide ligand comprises a PSMA-binding bicyclic peptide ligand.

[0052] Suitable examples of PSMA-binding bicyclic peptide ligands are disclosed in WO 2019 / 243455 and WO 2020 / 120980, the peptides of which are incorporated herein by reference.

[0053] (Second peptide ligand) References herein to the term "immune cell" include any cell within the immune system. Suitable examples include white blood cells such as lymphocytes (e.g., T lymphocytes or T cells, B cells, or natural killer cells). In one embodiment, the T cell is CD8 or CD4. In a further embodiment, the T cell is CD8. Other examples of immune cells include dendritic cells, follicular dendritic cells, and granulocytes.

[0054] In one embodiment, the component present on the immune cell is CD137.

[0055] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its aliases are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, and inducer of lymphocyte activation (ILA). CD137 can be expressed by activated T cells, and most can be expressed by CD8+ T cells rather than CD4+ T cells. Furthermore, CD137 expression is found in dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells of the vascular wall at the site of inflammation. One characterized activity of CD137 is its co-stimulatory activity on activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. Furthermore, it can enhance immune activity and cause tumors in mice to disappear.

[0056] CD137 is a T cell co-stimulatory receptor induced upon TCR activation (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu. Rev, Immunol., 23:23-68 (2005)). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described for antigen-presenting cells including B cells, monocytes / macrophages, and dendritic cells (Watts et al., Annu. Rev. Immunol, 23:23-68 (2005)). When interacting with its ligand, CD137 results in increased TCR-induced T cell proliferation, cytokine production, functional maturation, and extended survival of CD8+ T cells (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et al., Annu. Rev. Immunol, 23:23-68 (2005)).

[0057] Signaling via CD137 by either an agonistic monoclonal antibody (mAb) to CD137L or CD137 results in increased TCR-induced T cell proliferation, cytokine production, and functional maturation, as well as extended CD8+ T cell survival. These effects result from (1) activation of the NF-κB, c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and (2) regulation of anti-apoptotic and cell cycle-related gene expression.

[0058] Experiments conducted in both CD137-deficient and CD137L-deficient mice further demonstrated the importance of CD137 co-stimulation in the development of a fully competent T cell response.

[0059] IL-2 and IL-15-activated NK cells express CD137, and ligation of CD137 by an agonistic mAb stimulates NK cell proliferation and IFN-γ secretion but not their cytolytic activity.

[0060] Furthermore, CD137-stimulated NK cells promote the proliferation of activated T cells in vitro.

[0061] Based on its co-stimulatory function, agonistic mAbs to CD137 have been shown to promote rejection of cardiac and skin allografts, eradicate established tumors, expand primary anti-viral CD8+ T cell responses, and increase T cell cytolytic capacity. These studies support the view that CD137 signaling promotes T cell functions that can enhance immunity against tumors and infections.

[0062] In one embodiment, the two or more second peptide ligands include a CD137-binding bicyclic peptide ligand.

[0063] Suitable examples of CD137-binding bicyclic peptide ligands are disclosed in WO 2019 / 025811, the peptide of which is incorporated herein by reference.

[0064] In one embodiment, the CD137-binding bicyclic peptide ligand has the amino acid sequence:

Chemical formula

[0065] In a further embodiment, the CD137-binding bicyclic peptide ligand has the amino acid sequence:

Chemical formula

[0066] In one embodiment, the bicyclic peptide ligand is other than the amino acid sequence

Chemical formula

[0067] In one particular embodiment that may be mentioned, the CD137-binding bicyclic peptide ligand has the amino acid sequence: [Chem.] (wherein C i , C ii , and C iii represent first, second, and third cysteine residues, respectively, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, and Nle represents norleucine), or a pharmaceutically acceptable salt thereof.

[0068] In a further embodiment, the CD137-binding bicyclic peptide ligand comprises N- and / or C-terminal modifications and Ac-A-(SEQ ID NO: 5)-Dap (referred to herein as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (referred to herein as BCY7741); Ac-(SEQ ID NO: 6)-Dap (referred to herein as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (referred to herein as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (referred to herein as BCY8045); Ac-(SEQ ID NO: 8)-A (referred to herein as BCY8919); Ac-(SEQ ID NO: 9)-A (referred to herein as BCY8920); Ac-(SEQ ID NO: 10)-A (referred to herein as BCY8927); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); (SEQ ID NO: 11)-A (referred to herein as BCY14601); Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); Ac-[dA]-(SEQ ID NO: 13)-[dA]-NH2 (referred to herein as BCY11506); Ac-(SEQ ID NO: 60)-Dap(PYA) (referred to herein as BCY11144); Ac-A-(SEQ ID NO: 61)-K (referred to as BCY11613 in this specification); Ac-(SEQ ID NO: 62)-Dap(PYA) (referred to as BCY12023 in this specification); Ac-(SEQ ID NO: 63) (referred to as BCY12149 in this specification); Ac-(SEQ ID NO: 64) (referred to as BCY12143 in this specification); Ac-(SEQ ID NO: 65) (referred to as BCY12147 in this specification); Ac-(SEQ ID NO: 66) (referred to as BCY12145 in this specification); Ac-(SEQ ID NO: 67) (referred to as BCY12146 in this specification); Ac-(SEQ ID NO: 68) (referred to as BCY12150 in this specification); Ac-(SEQ ID NO: 69) (referred to as BCY12352 in this specification); Ac-(SEQ ID NO: 72)-[1,2-diaminoethane] (referred to as BCY12358 in this specification); [Palmitic acid]-[γGlu]-[γGlu]-(SEQ ID NO: 73) (referred to as BCY12360 in this specification); Ac-(SEQ ID NO: 75) (referred to as BCY12381 in this specification); Ac-(SEQ ID NO: 76) (referred to as BCY12382 in this specification); Ac-(SEQ ID NO: 77)-K (referred to as BCY12357 in this specification); Ac-(SEQ ID NO: 78)-[dA] (referred to as BCY13095 in this specification); [Ac]-(SEQ ID NO: 78)-K (referred to as BCY13389 in this specification); Ac-(SEQ ID NO: 79)-[dA] (referred to as BCY13096 in this specification); and Ac-(SEQ ID NO: 80) (referred to as BCY13097 in this specification); (Here, Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid), or a pharmaceutically acceptable salt thereof : comprises.

[0069] In yet a further embodiment, the CD137-binding bicyclic peptide ligand comprises N- and / or C-terminal modifications, and Ac-A-(SEQ ID NO: 5)-Dap (referred to herein as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (referred to herein as BCY7741); Ac-(SEQ ID NO: 6)-Dap (referred to herein as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (referred to herein as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (referred to herein as BCY8045); Ac-(SEQ ID NO: 8)-A (referred to herein as BCY8919); Ac-(SEQ ID NO: 9)-A (referred to herein as BCY8920); Ac-(SEQ ID NO: 10)-A (referred to herein as BCY8927); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); and Ac-[dA]-(SEQ ID NO: 13)-[dA]-NH2 (referred to herein as BCY11506); (Here, Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid), or a pharmaceutically acceptable salt thereof : comprises.

[0070] In one embodiment, the bicyclic peptide ligand is other than BCY11506 which has been shown not to bind to CD137.

[0071] In a further possible embodiment, the CD137-binding bicyclic peptide ligand comprises N- and / or C-terminal modifications and Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); (wherein Ac represents an acetyl group), or a pharmaceutically acceptable salt thereof :.

[0072] In an alternative embodiment, the component present on the immune cell is OX40.

[0073] The OX40 receptor (also known as tumor necrosis factor receptor superfamily, member 4 (TNFRSF4) and also known as CD134 receptor) is a member of the TNF receptor superfamily of receptors that is not constitutively expressed on resting naive T cells, unlike CD28. OX40 is a secondary costimulatory immune checkpoint molecule that is expressed 24 - 72 hours after activation; its ligand OX40L is also not expressed on resting antigen-presenting cells but is expressed after their activation. Expression of OX40 is dependent on complete activation of T cells; in the absence of CD28, expression of OX40 is delayed and at a four-fold lower level.

[0074] OX40 has no effect on the proliferative capacity of CD4+ cells for the first three days, but after this time, proliferation begins to decline and cells die at a high rate because they are unable to maintain high levels of PKB activity and expression of Bcl-2, Bcl-XL, and survivin. OX40L binds to the OX40 receptor on T-cells, preventing T-cells from dying and then increasing cytokine production. OX40, by virtue of its ability to enhance survival, plays a crucial role in maintaining the immune response towards a memory response after the first few days. OX40 plays a crucial role in both Th1-mediated and Th2-mediated responses in vivo.

[0075] OX40 binds to TRAF2, 3, and 5 and PI3K through unknown functions. TRAF2 is required for survival and memory cell generation via NF-κB, while TRAF5 appears to have a more negative or regulatory role as knockout animals have higher levels of cytokines and are more susceptible to Th2-mediated inflammation. TRAF3 may have a crucial role in OX40-mediated signaling. CTLA-4 is downregulated after OX40 engagement in vivo, and OX40-specific TRAF3 DN deficiency was partially overcome by CTLA-4 blockade in vivo. TRAF3 is associated with OX40-mediated memory T cell expansion and survival, suggesting that CTLA-4 downregulation may be a putative control element for enhancing initial T cell expansion via OX40 signaling.

[0076] In one embodiment, OX40 is mammalian OX40. In a further embodiment, the mammalian OX40 is human OX40 (hOX40).

[0077] OX40 peptides are used primarily (but not exclusively) to prevent, suppress, or treat early or advanced human malignancies, including solid tumors such as cancer, e.g., non-small cell lung cancer (NSCLC), breast cancer including triple-negative breast cancer (TNBC), ovarian cancer, prostate cancer, bladder cancer, urothelial cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), melanoma, pancreatic cancer, and other progressive solid tumors where immunosuppression blocks antitumor immunity. Other solid and non-solid malignancies for which OX40 peptides are used as therapeutic agents include, but are not limited to, B-cell lymphomas including low-grade / follicular non-Hodgkin lymphoma and acute myeloid leukemia (AML).

[0078] In one embodiment, the two or more second peptide ligands include OX40-binding bicyclic peptide ligands.

[0079] Preferred examples of the OX40-binding bicyclic peptide ligand are disclosed in International Patent Application No. PCT / GB2020 / 051144, the peptide of which is incorporated herein by reference.

[0080] In one embodiment, the OX40-binding bicyclic peptide ligand is

Chemical formula

[0081] In a further embodiment, the OX40-binding bicyclic peptide ligand further comprises N- and / or C-terminal modifications and A-(SEQ ID NO: 82)-A-[Sar6]-[KBiot] (referred to herein as BCY10551); A-(SEQ ID NO: 82)-A (referred to herein as BCY10371); A-(SEQ ID NO: 84)-A-[Sar6]-[KBiot] (referred to herein as BCY10552); [Biot]-G-[Sar5]-A-(SEQ ID NO: 84)-A (referred to herein as BCY10479); A-(SEQ ID NO: 84)-A (referred to herein as BCY10378); [Biot]-G-[Sar5]-A-(SEQ ID NO: 85)-A (referred to herein as BCY11371); A-(SEQ ID NO: 85)-A (referred to herein as BCY10743); [Biot]-G-[Sar5]-A-(SEQ ID NO: 87)-A (referred to herein as BCY10482); A-(SEQ ID NO: 87)-A-[Sar6]-[KBiot] (referred to herein as BCY10549); A-(SEQ ID NO: 87)-A-K(Pya) (referred to herein as BCY11607); Ac-A-(SEQ ID NO: 87)-A-K(Pya) (referred to herein as BCY12708); A-(SEQ ID NO: 87)-A (referred to herein as BCY10351); A-(SEQ ID NO: 88)-A-[Sar6]-[KBiot] (referred to herein as BCY11501); A-(SEQ ID NO: 88)-A (referred to herein as BCY10729); A-(SEQ ID NO: 89)-A-[Sar6]-[KBiot] (referred to herein as BCY10550); A-(SEQ ID NO: 89)-A (referred to herein as BCY10361); A-(SEQ ID NO: 90)-A-[Sar6]-[KBiot] (referred to herein as BCY10794); A-(SEQ ID NO: 90)-A (referred to herein as BCY10349); [Biot]-G-[Sar5]-A-(SEQ ID NO: 91)-A (referred to herein as BCY11369); A-(SEQ ID NO: 91)-A (referred to herein as BCY10331); A-(SEQ ID NO: 92)-A (referred to herein as BCY10375); A-(SEQ ID NO: 93)-A (referred to herein as BCY10364); A-(SEQ ID NO: 94)-A (referred to herein as BCY10365); A-(SEQ ID NO: 95)-A (referred to herein as BCY10366); A-(SEQ ID NO: 96)-A (referred to herein as BCY10367); A-(SEQ ID NO: 97)-A (referred to herein as BCY10368); A-(SEQ ID NO: 98)-A (referred to as BCY10369 in this specification); A-(SEQ ID NO: 99)-A (referred to as BCY10374 in this specification); A-(SEQ ID NO: 100)-A (referred to as BCY10376 in this specification); A-(SEQ ID NO: 101)-A (referred to as BCY10737 in this specification); A-(SEQ ID NO: 102)-A (referred to as BCY10738 in this specification); A-(SEQ ID NO: 103)-A (referred to as BCY10739 in this specification); A-(SEQ ID NO: 104)-A (referred to as BCY10740 in this specification); A-(SEQ ID NO: 105)-A (referred to as BCY10741 in this specification); A-(SEQ ID NO: 106)-A (referred to as BCY10742 in this specification); A-(SEQ ID NO: 107)-A (referred to as BCY10380 in this specification); A-(SEQ ID NO: 108)-A (referred to as BCY10370 in this specification); A-(SEQ ID NO: 109)-A (referred to as BCY10372 in this specification); A-(SEQ ID NO: 110)-A (referred to as BCY10373 in this specification); A-(SEQ ID NO: 111)-A (referred to as BCY10379 in this specification); A-(SEQ ID NO: 112)-A (referred to as BCY10377 in this specification); A-(SEQ ID NO: 113)-A (referred to as BCY10744 in this specification); A-(SEQ ID NO: 114)-A (referred to as BCY10343 in this specification); A-(SEQ ID NO: 115)-A (referred to as BCY10350 in this specification); A-(SEQ ID NO: 116)-A (referred to as BCY10352 in this specification); A-(SEQ ID NO: 117)-A (referred to as BCY10353 in this specification); A-(SEQ ID NO: 118)-A (referred to as BCY10354 in this specification); A-(SEQ ID NO: 119)-A (referred to as BCY10730 in this specification); A-(SEQ ID NO: 120)-A (referred to as BCY10731 in this specification); A-(SEQ ID NO: 121)-A (referred to as BCY10339 in this specification); A-(SEQ ID NO: 122)-A (referred to as BCY10340 in this specification); A-(SEQ ID NO: 123)-A (referred to as BCY10342 in this specification); A-(SEQ ID NO: 124)-A (referred to as BCY10345 in this specification); A-(SEQ ID NO: 125)-A (referred to as BCY10347 in this specification); A-(SEQ ID NO: 126)-A (referred to as BCY10348 in this specification); A-(SEQ ID NO: 127)-A (referred to as BCY10720 in this specification); A-(SEQ ID NO: 128)-A (referred to as BCY10721 in this specification); A-(SEQ ID NO: 129)-A (referred to as BCY10722 in this specification); A-(SEQ ID NO: 130)-A (referred to as BCY10723 in this specification); A-(SEQ ID NO: 131)-A (referred to as BCY10724 in this specification); A-(SEQ ID NO: 132)-A (referred to as BCY10725 in this specification); A-(SEQ ID NO: 133)-A (referred to as BCY10726 in this specification); A-(SEQ ID NO: 134)-A (referred to as BCY10727 in this specification); A-(SEQ ID NO: 135)-A (referred to as BCY10728 in this specification); A-(SEQ ID NO: 136)-A (referred to as BCY10360 in this specification); A-(SEQ ID NO: 137)-A (referred to as BCY10363 in this specification); A-(SEQ ID NO: 138)-A (referred to as BCY10732 in this specification); A-(SEQ ID NO: 139)-A (referred to as BCY10733 in this specification); A-(SEQ ID NO: 140)-A (referred to as BCY10734 in this specification); A-(SEQ ID NO: 141)-A (referred to as BCY10735 in this specification); A-(SEQ ID NO: 142)-A (referred to as BCY10736 in this specification); A-(SEQ ID NO: 143)-A (referred to as BCY10336 in this specification); A-(SEQ ID NO: 144)-A (referred to as BCY10337 in this specification); A-(SEQ ID NO: 145)-A (referred to as BCY10338 in this specification); A-(SEQ ID NO: 146)-A (referred to as BCY10346 in this specification); A-(SEQ ID NO: 147)-A (referred to as BCY10357 in this specification); A-(SEQ ID NO: 148)-A (referred to as BCY10362 in this specification); A-(SEQ ID NO: 149)-A (referred to as BCY10332 in this specification); A-(SEQ ID NO: 150)-A (referred to as BCY10717 in this specification); A-(SEQ ID NO: 151)-A (referred to as BCY10718 in this specification); A-(SEQ ID NO: 152)-A (referred to as BCY10334 in this specification); and A-(SEQ ID NO: 153)-A (referred to as BCY10719 in this specification); For example: A-(SEQ ID NO: 87)-A-K(Pya) (referred to as BCY11607 in this specification); (Here, Pya represents a 4-pentinoyl moiety) : comprises an amino acid sequence selected from

[0082] In one embodiment, two or more of the second peptides are specific for the same immune cell. In a further embodiment, each of the two or more second peptides is specific for the same binding site or target on the same immune cell. In an alternative embodiment, each of the two or more second peptides is specific for a different binding site or target on the same immune cell. In an alternative embodiment, two or more of the second peptides are specific for two different immune cells (i.e., CD137 and OX40). In a further embodiment, each of the two or more second peptides is specific for the same binding site or target on the two different immune cells. In an alternative embodiment, each of the two or more second peptides is specific for a different binding site or target on the two different immune cells.

[0083] In one embodiment, each of the two or more second peptides has the same peptide sequence.

[0084] In one embodiment, the heterotandem bicyclic peptide complex comprises two second peptide ligands. Thus, according to a further aspect of the invention, (a) a first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) two second peptide ligands that bind to components present on immune cells; conjugated to : is provided, wherein each of the peptide ligands comprises a polypeptide containing at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms a covalent bond with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, a heterotandem bicyclic peptide complex.

[0085] According to a further aspect of the invention that may be mentioned, (a) A first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) Two second peptide ligands that bind to a component present on an immune cell; : comprising, wherein the peptide ligand comprises a polypeptide comprising at least three cysteine residues separated by at least two loop sequences and a molecular scaffold that forms a covalent bond with the cysteine residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, a heterotandem bicyclic peptide complex is provided.

[0086] In an alternative embodiment, the heterotandem bicyclic peptide complex comprises three second peptide ligands. Thus, according to a further aspect of the invention, (a) A first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) Three second peptide ligands that bind to a component present on an immune cell; : comprising, 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 a covalent bond with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, a heterotandem bicyclic peptide complex is provided.

[0087] According to a further aspect of the invention that may be mentioned, (a) A first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) Three second peptide ligands that bind to a component present on an immune cell; : comprising a polypeptide containing at least three cysteine residues separated by at least two loop arrays for each of the peptide ligands, and a molecular scaffold that forms a covalent bond with the cysteine residues of the polypeptide, resulting in the formation of at least two polypeptide loops on the molecular scaffold, a heterotandem bicyclic peptide complex is provided.

[0088] In a further embodiment, each of the two or more second peptides comprises the same peptide sequence, and the peptide sequence is Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928) (wherein Ac represents an acetyl group), or a pharmaceutically acceptable salt thereof.

[0089] In yet a further embodiment, the heterotandem bicyclic peptide complex comprises two second peptide ligands, and both of the two second peptides Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928) (wherein Ac represents an acetyl group), or a pharmaceutically acceptable salt thereof.

[0090] (Linker) It will be understood that the first peptide ligand can be conjugated to two or more second peptide ligands via any suitable linker. Typically, the linker is designed such that the three bicyclic peptides are presented in a form that can bind to their respective targets while binding to both target receptors simultaneously without being hindered by each other. Further, the linker should maintain an appropriate distance between the target cells that results in the desired functional outcome while allowing binding to both targets simultaneously. The nature of the linker can be adjusted to increase length, rigidity, or solubility in order to optimize the desired functional outcome. The linker can also be designed to allow multiple bicyclic bindings to the same target. Increasing the valency of either binding peptide can serve to increase the affinity of the heterotandem for the target cell or can help to induce oligomerization of one or both of the target receptors.

[0091] In one embodiment, the linker is a branched linker that allows one first peptide at one end and two or more second peptides at the other end.

[0092] In a further embodiment, the branched linker is

Chemical formula

[0093] In one particular embodiment, the branched linker is

Chemical formula

[0094] (heterotandem complex) In one specific embodiment, the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand conjugated to a TATA scaffold, and the two or more second peptide ligands comprise two CD137 binding bicyclic peptide ligands conjugated to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table A: Table A (nectin-4:CD137; 1:2)

Table 1

[0095] In one embodiment, the heterotandem bicyclic peptide complex is selected from BCY11027, BCY11863, and BCY11864:. In a further embodiment, the heterotandem bicyclic peptide complex is selected from BCY11863 and BCY11864:

[0096] The heterotandem bicyclic peptide complex BCY11863 consists of a nectin-4 specific peptide BCY8116 linked via an N-(acid-PEG3)-N-bis(PEG3-azide) linker to two CD137 specific peptides (both BCY8928), as shown pictorially

Chemical formula

[0097] CD137 is a homotrimeric protein, and its natural ligand CD137L exists as a homotrimer that is either expressed on immune cells or secreted. The biology of CD137 is highly dependent on multimerization to induce CD137 activity in immune cells. One way to generate CD137 multimerization is through cell crosslinking of CD137-specific agonists via interaction with specific receptors present on another cell. The advantage of the heterotandem complex of the present invention is that the presence of two or more peptide ligands specific for immune cell components such as CD137 results in more effective clustering of CD137. For example, data showing that BCY11863 exhibited strong CD137 activation in a CD137 reporter assay are shown in FIGS. 1 and 1 in this specification. Further, data showing that BCY11863 induced strong IL-2 and IFN-γ cytokine secretion in a PBMC-4T1 co-culture assay are shown in FIGS. 2 and 5 in this specification. Further, data showing that BCY11863 exhibited an excellent PK profile with a terminal-phase half-life of 4.1 hours in Sprague-Dawley rats and 5.3 hours in cynomolgus monkeys are shown in FIGS. 3 and 7 in this specification.

[0098] The heterotandem bicyclic peptide complex BCY11027 consists of using a picture, [Chemical formula] : shown as such, a nectin-specific peptide BCY11015 linked to two CD137-specific peptides (both BCY8928) via a TCA-[Peg 10 3 linker.

[0099] The data shown in Figure 13 demonstrate that the nectin-4 / CD137 heterotandem BCY11027 induces target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors. Treatment with BCY11027 induced nectin-4-dependent changes in several immune markers (normalized to vehicle) and nectin-4-dependent changes in %CD8+ ki67+ T cells in patient-derived samples, which correlated with the level of nectin-4 expression.

[0100] In an alternative specific embodiment, the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand conjugated to a TATA scaffold, and the two or more second peptide ligands comprise two CD137 binding bicyclic peptide ligands conjugated to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table B: Table B (nectin-4:CD137; 1:3) [Table 2]

[0101] In one specific embodiment, the first peptide ligand comprises an EphA2 binding bicyclic peptide ligand conjugated to a TATA scaffold, and the two or more second peptide ligands comprise two CD137 binding bicyclic peptide ligands conjugated to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table C: Table C (EphA2:CD137; 1:2) [Table 3] TIFF0007704732000030.tif245170TIFF0007704732000031.tif232170TIFF0007704732000032.tif245170TIFF0007704732000033.tif246170TIFF0007704732000034.tif238170TIFF0007704732000035.tif245170

[0102] In one embodiment, the heterotandem bicyclic peptide complex is selected from BCY12491, BCY12730, BCY13048, BCY13050, BCY13053, and BCY13272.

[0103] In one embodiment, the heterotandem bicyclic peptide complex is selected from BCY12491, BCY12730, BCY13048, BCY13050, and BCY13053.

[0104] In a further embodiment, the heterotandem bicyclic peptide complex is BCY12491.

[0105] The heterotandem bicyclic peptide complex BCY12491 consists of an EphA2-specific peptide BCY9594 linked via an N-(acid-PEG3)-N-bis(PEG3-azide) linker to two CD137-specific peptides (both BCY8928), as shown using a drawing

Chemical formula

[0106]

[0107] In an alternative embodiment, the heterotandem bicyclic peptide complex is BCY13272.

[0108] The heterotandem bicyclic peptide complex BCY13272 is shown using a drawing

Chemical formula

[0109] Data showing that BCY13272 produces a significant anti-tumor effect in a mouse MC38 tumor model are shown in Figure 18 herein.

[0110] In one specific embodiment, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand bound to a TATA scaffold, and the two or more second peptide ligands comprise two CD137-binding bicyclic peptide ligands bound to the TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table D: Table D (PD-L1:CD137; 1:2) [Table 4]

[0111] In one specific embodiment, the first peptide ligand comprises a nectin-4-binding bicyclic peptide ligand bound to a TATA scaffold, and the two or more second peptide ligands comprise two OX40-binding bicyclic peptide ligands bound to the TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table E: Table E (nectin-4:OX40; 1:2) [Table 5]

[0112] In one specific embodiment, the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand conjugated to a TATA scaffold, one of the two or more second peptide ligands comprises an OX40 binding bicyclic peptide ligand conjugated to a TATA scaffold, and the other of the two or more second peptide ligands comprises a CD137 binding bicyclic peptide ligand conjugated to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table F: Table F (nectin-4:OX40:CD137; 1:1:1) [Table 6]

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, e.g., in the fields of peptide chemistry, cell culture, and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for methods of molecular biology, genetics, and biochemistry (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Edition, John Wiley & Sons, which are incorporated herein by reference).

[0114] (Nomenclature) (Numbering) When referring to the positions of amino acid residues within the compounds of the present invention, cysteine residues (C i , C ii , and C iii ) are invariant and are therefore omitted from the numbering, and thus the numbering of the amino acid residues in SEQ ID NO: 1 is referred to as follows: C i -P1-1Nal2-dD3-C ii -M4-HArg5-D6-W7-S8-T9-P 10 -HyP 11 -W 12 -C iii (SEQ ID NO: 1).

[0115] For the purposes of this description, all bicyclic peptides are considered to be cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) or 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) to yield a trisubstituted structure. Cyclization with TBMB and TATA occurs on C i 、C ii 、and C iii above.

[0116] (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, the N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO: X) is represented as.

[0117] (Reverse peptide sequence) In view of the disclosure in the literature of Nair et al. (2003) J Immunol 170(3), 1362-1373, the peptide sequences disclosed herein are expected to find utility even in their retro-inverso forms. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus and the C-terminus becomes the N-terminus), and its stereochemistry is similarly reversed (i.e., D-amino acids become L-amino acids and L-amino acids become D-amino acids). To avoid misunderstanding, any reference to an amino acid, either by its formal name or by its one-letter or three-letter notation of the amino acid, is intended to be represented as an L-amino acid herein unless otherwise specified. When such an amino acid is intended to be represented as a D-amino acid, a small letter d is prefixed to the amino acid within square brackets, e.g., [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0118] (Advantages of Peptide Ligands) Certain heterotandem bicyclic peptide complexes of the present invention have several advantageous properties that can be regarded as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral, or topical administration. Such advantageous properties include the following: -Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations; -Protease stability. The heterotandem bicyclic peptide complex should ideally exhibit stability against plasma proteases, epithelial ("membrane-bound") proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained among different species so that not only can heterotandem bicyclic peptide lead candidates be developed in animal models, but they can also be administered to humans with confidence; -Desirable solubility profile. This is a function of the ratio of charged and hydrophilic residues to hydrophobic residues and intramolecular / intermolecular H-bonds, which is important for formulation and absorption purposes; - Selectivity. Certain heterotandem bicyclic peptide complexes of the invention exhibit better selectivity than other targets; - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop heterotandem bicyclic peptide complexes for short-term exposure in acute disease management settings or heterotandem bicyclic peptide complexes with enhanced retention in circulation, so it is optimal for the management of more chronic disease states. Other factors that drive the desired plasma half-life are the requirement for sustained exposure for maximum therapeutic efficiency and the associated toxicity due to the sustained exposure of the drug.

[0119] Importantly, data are presented herein showing antitumor resistance when the selected heterotandem bicyclic peptide complex is administered at a frequency that does not maintain plasma concentrations above the in vitro EC 50 of the compound. This is in contrast to a greater recombinant biologics-based (i.e., antibody-based) approach to CD137 agonism or bispecific CD137 agonism (Segal et al., Clin Cancer Res., 23(8):1929-1936 (2017), Claus et al., Sci Trans Med., 11(496): eaav5989, 1-12 (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889 (2019)). Without being bound by theory, the reason for this observation is thought to be due to the fact that the heterotandem bicyclic complex has a relatively low molecular weight (usually, <15 kDa), that it is fully synthetic, and that it is a tumor-targeting agonist of CD137. Thus, it has a relatively short plasma half-life but good tumor permeability and retention. Data fully supporting these advantages are presented herein. For example, antitumor resistance is shown in a syngeneic rodent model of mice with humanized CD137 either daily or every three days. Furthermore, intraperitoneal pharmacokinetic data show that the plasma half-life is less than 3 hours, whereby the circulating concentration of the complex is below the in vitro EC 50is predicted to always be below. Furthermore, the tumor pharmacokinetic data indicate that the level of the heterotandem bicyclic complex in tumor tissue can be higher and more sustained compared to the plasma level.

[0120] This observation will be understood to form an important further aspect of the present invention. Thus, according to a further aspect of the present invention, there is provided a method of treating cancer, comprising administering the heterotandem bicyclic peptide complex as defined herein at a dosing frequency that does not maintain a plasma concentration of the complex above the in vitro EC 50 of the complex.

[0121] - Immunological memory. Coupling a bicyclic peptide ligand that binds to cancer cells with a bicyclic peptide ligand that binds to immune cells provides a synergistic advantage of immunological memory. Data are presented herein showing that the selected heterotandem bicyclic peptide complexes of the present invention not only eradicated tumors, but also that none of the vaccinated complete responder mice developed tumors when the tumorigenic agent was re-administered (see Figure 5). This indicates that treatment with the selected heterotandem bicyclic peptide complexes of the present invention induced immunogenic memory in complete responder mice. This has important clinical advantages for preventing recurrence once the tumor is initially controlled and eradicated.

[0122] (peptide ligand) The peptide ligands referred to herein refer to peptides covalently bound to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine residues) capable of forming a covalent bond with the scaffold, and a sequence that lies between the reactive groups, called a loop sequence, which forms a loop when the peptide binds to the scaffold. In this case, the peptide comprises at least three reactive groups selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine, and forms at least two loops on the scaffold.

[0123] (Reactive group) The molecular scaffold of the present invention may be attached to a polypeptide via a functional group or a reactive group on the polypeptide. These are typically formed from the side chains of specific amino acids found in polypeptide polymers. Such reactive groups may be cysteine side chains, lysine side chains, or N-terminal amino groups, or any other suitable reactive group, for example, penicillamine. Details of suitable reactive groups can be found in WO 2009 / 098450.

[0124] 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 guanidium group of arginine, the phenol 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 terminal amino and carboxyl groups of the polypeptide can also serve as reactive groups to form covalent bonds with the molecular scaffold / molecular core.

[0125] The polypeptide of the present invention contains at least three reactive groups. The polypeptide can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.

[0126] In a preferred embodiment, a polypeptide having three reactive groups is produced. By reacting the polypeptide with a molecular scaffold / molecular core having three-fold rotational symmetry, a single product isomer is produced. The production of a single product isomer is preferred for several reasons. The nucleic acids of the compound library encode only the primary sequence of the polypeptide and do not encode the molecular species of the different states formed upon reaction of the polypeptide with the molecular core. When only one product isomer can be formed, the attribution of the nucleic acid to the product isomer is clearly defined. When multiple product isomers are formed, the nucleic acid cannot provide information regarding the nature of the product isomer isolated in a screening or selection process. Information on a single product isomer is also advantageous when a particular member of the library of the present invention is synthesized. In this case, a chemical reaction of the polypeptide with the molecular scaffold produces a single product isomer rather than a mixture of isomers.

[0127] In another embodiment, a polypeptide having four reactive groups is produced. By reacting the polypeptide with a molecular scaffold / molecular core having tetrahedral symmetry, two product isomers are produced. Even if two different product isomers are encoded by one and the same nucleic acid, the nature of the isolated isomers can be determined by chemically synthesizing both isomers, separating the two isomers, and testing both isomers for binding to a target ligand.

[0128] In one embodiment of the present invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the orthogonal reactive group to be directed to a specific site on the molecular core. Using a ligation strategy involving orthogonal reactive groups, the number of product isomers formed can be limited. In other words, by selecting a distinct or different reactive group for one or more of at least three bonds relative to the remaining ones of at least three bonds, a specific order of binding or orientation of a specific reactive group of the polypeptide to a specific position on the molecular scaffold can be effectively achieved.

[0129] In another embodiment, the reactive group of the polypeptide of the invention reacts with a molecular linker, in which case the linker can react with the molecular scaffold such that the linker enters between the molecular scaffold in the final bound state and the polypeptide.

[0130] In some embodiments, the amino acids of a library or set of members of the polypeptide can be exchanged for any natural or non-natural amino acid. Those having a functional group for cross-linking the polypeptide to the molecular core are excluded from these exchangeable amino acids so that only the loop sequences are exchangeable. The exchangeable polypeptide sequences have either a random sequence, a defined sequence, or a sequence having both random and defined amino acids. Since the positions of these amino acids determine the loop size, any amino acids having a reactive group are in defined positions within the polypeptide.

[0131] In one embodiment, a 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 represent numbers from 3 to 6 that define the length of intervening polypeptide segments (which may be the same or different), and l and o represent numbers from 0 to 20 that define the length of adjacent polypeptide segments.

[0132] Instead of using thiol-mediated conjugation, a molecular scaffold can be attached to a peptide via a covalent interaction. Alternatively, these techniques can be used in the modification or attachment of such additional moieties (e.g., small molecules other than the molecular scaffold) to polypeptides after they have been selected or isolated according to the present invention - in this embodiment, clearly, the attachment need not be covalent and can include non-covalent bonds. These methods can be by producing phage presenting proteins and peptides having unnatural amino acids with the necessary chemical reactive groups in combination with small molecules having complementary reactive groups, or by incorporating unnatural amino acids chemically or recombinantly synthesized into polypeptides when the molecules are made after the selection / isolation step, to be used instead of (or in combination with) the thiol-mediated method. Further details can be found in WO 2009 / 098450 or in the literature of Heinis et al., Nat Chem Biol 2009, 5(7), 502-7.

[0133] In one embodiment, the reactive group is selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine residues.

[0134] (pharmaceutically acceptable salts) Salt forms are within the scope of the present invention, and it will be understood that reference to a peptide ligand includes salt forms of the ligand.

[0135] The salts of the present invention can be synthesized from the parent compounds containing basic or acidic moieties by the methods described in conventional chemical methods, for example, Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Usually, such salts can be prepared by reacting the free acid or base form of these compounds with an appropriate base or acid in water or an organic solvent, or in a mixture of the two.

[0136] Acid addition salts (mono- or di-salts) can be formed with a wide variety of both inorganic and organic acids. 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, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic 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, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, 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, and mono- or di-salts formed with an acid selected from the group consisting of acylated amino acids and cation exchange resins.

[0137] One specific group of salts consists of salts formed with 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 specific salt is the hydrochloride salt. Another specific salt is the acetate salt.

[0138] If the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO - ), a salt can be formed with an organic or inorganic base to generate a suitable cation. Examples of suitable inorganic cations include alkali metal ions such as Li + , Na + , and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + , but are not limited to these. Examples of suitable organic cations include ammonium ions (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R + , NH2R2 + , NHR3 + , NR4 + ), but are not limited to these. Examples of some suitable substituted ammonium ions include those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + .

[0139] If the compounds of the present invention contain an amine function, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0140] (Modified derivative) It will be understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within a bicyclic peptide ligand; substitution of one or more peptide bonds with alternative bonds; modification of the peptide backbone length; substitution of hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid, and tyrosine with suitable amine, thiol, carboxylic acid, and phenol-reactive reagents to functionalize the amino acid, and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, for example, introduction of amino acids having azide or alkyne groups that enable functionalization with moieties having alkyne or azide, respectively: one or more modifications selected from the foregoing are included.

[0141] In one embodiment, the modified derivative includes N-terminal and / or C-terminal modifications. In a further embodiment, here, the modified derivative includes N-terminal modification using suitable amine-reactive chemistry and / or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification includes addition of an effector group including, but not limited to, a cytotoxic agent, a radiochelating agent, or a chromophore.

[0142] In a further embodiment, the modified derivative includes N-terminal modification. In a further embodiment, the N-terminal modification includes an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (designated C in this specification) iThe group (referred to as such) is capped with acetic anhydride or other suitable reagents during peptide synthesis, resulting in a molecule with an acetylated N-terminus. This embodiment provides the advantage of removing potential recognition points for aminopeptidases and avoiding the possibility of degradation of the bicyclic peptide.

[0143] In an alternative embodiment, the N-terminal modification includes the addition of a molecular spacer group that facilitates the conjugation of an effector group and the retention of the potency of the bicyclic peptide against its target.

[0144] In a further embodiment, the modified derivative includes a C-terminal modification. In a further embodiment, the C-terminal modification includes an amide group. In this embodiment, the C-terminal cysteine group (herein, C iii The group (referred to as such) is synthesized as an amide during peptide synthesis, resulting in a molecule with an amidated C-terminus. This embodiment provides the advantage of removing potential recognition points for carboxypeptidases and reducing the possibility of proteolysis of the bicyclic peptide.

[0145] In one embodiment, the modified derivative includes substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids having isosteric / isoelectronic side chains that are neither recognized by degradative proteases nor have any detrimental effects on target potency may be selected.

[0146] Alternatively, non-natural amino acids with constrained amino acid side chains may be used such that proteolytic hydrolysis of nearby peptide bonds is sterically and conformationally hindered. In particular, these relate to proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., amino isobutyric acid, Aib), and cyclic amino acids that are simple derivatives of aminocyclopropylcarboxylic acid.

[0147] In one embodiment, the modified derivative includes the addition of a spacer group. In a further embodiment, the modified derivative includes an N-terminal cysteine (C i) and / or the addition of a spacer group to the C-terminal cysteine (C iii ) including the addition of a spacer group.

[0148] In one embodiment, the modified derivative comprises 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 substitution of tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0149] In one embodiment, the modified derivative comprises substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. These two combinations can affect the half-life, volume of distribution, and exposure of the peptide drug and can be adjusted according to the clinical endpoint. Furthermore, the correct combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0150] In one embodiment, the modified derivative comprises substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is thought to enhance proteolytic stability due to steric hindrance and the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0151] In one embodiment, the modified derivative includes removal of any amino acid residue and substitution with alanine. This embodiment has the advantage of removing potential proteolytic attack sites.

[0152] It should be noted that each of the above modifications serves to intentionally improve the potency or stability of the peptide. Further improvement in potency based on modification can be achieved by the following mechanisms: - Incorporating hydrophobic moieties that utilize the hydrophobic effect to achieve higher affinity and result in a lower dissociation rate; - Incorporating charged groups that utilize long-range ionic interactions to result in a faster association rate and higher affinity (see, for example, Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - Incorporating additional constraints into the peptide, for example, by correctly constraining the side chains of amino acids, constraining the backbone torsion angles, and introducing additional cyclization within the molecule so that the loss of entropy is minimized upon target binding. (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0153] Examples of the modified heterotandem bicyclic peptide complexes of the present invention include those listed in Tables G and H below: Table G: (EphA2:CD137; 1:2) [Table 7] Table H: (Nectin-4:CD137; 1:2)

Table 8

[0154] (Isotope variation) The present invention includes all (radioactive) isotope-labeled peptide ligands acceptable as medicaments of the present invention, in which one or more atoms are replaced by atoms having the same atomic number but different atomic mass or mass number from the atomic mass or mass number usually found in nature, and peptide ligands of the present invention (referred to as "effectors") to which a metal chelate group capable of retaining related (radioactive) isotopes is attached, and peptide ligands of the present invention in which specific functional groups are covalently replaced by related (radioactive) isotopes or isotope-labeled functional groups.

[0155] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention are isotopes of hydrogen, such as 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C, 13 C and 14 C, isotopes of chlorine, such as 36 Cl, isotopes of fluorine, such as 18 F, isotopes of iodine, such as 123 I, 125 I, and 131 I, isotopes of nitrogen, such as 13 N and 15 N, isotopes of oxygen, such as 15 O, 17 O, and 18 O, isotopes of phosphorus, such as 32 P, isotopes of sulfur, such as 35 S, isotopes of copper, such as 64 Cu, isotopes of gallium, such as 67 Ga or 68 Ga, isotopes of yttrium, such as 90 Y, and isotopes of lutetium, such as 177 Lu, and isotopes of bismuth, such as 213 Bi.

[0156] Certain isotope-labeled peptide ligands of the present invention, for example, those incorporating a radioisotope, are useful in studies of the tissue distribution of drugs and / or substrates and in clinically assessing the presence and / or absence of nectin-4 targets on diseased tissue. The peptide ligands of the present invention can further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods can use compounds labeled with labeling agents such as, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). Tritium, a radioisotope, i.e., 3 H(T) and carbon-14, i.e., 14 C are particularly useful for this purpose in view of the ease of their incorporation and the availability of means for detection.

[0157] Substitution with heavier isotopes such as deuterium, i.e., 2 H(D) can result in certain therapeutic advantages obtained as a result of greater metabolic stability, e.g., increased in vivo half-life or reduced required dosage, and is therefore, in some situations, preferably.

[0158] 11 C, 18 F, 15 O, and 13 N and substitution with positron-emitting isotopes such as can be useful in positron emission to Mo graphy (PET) tests for examining target occupancy.

[0159] Isotope-labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by a process similar to those described in the appended examples using appropriate isotope-labeled reagents in place of the unlabeled reagents previously utilized.

[0160] (Molecular scaffold) Molecular scaffolds are described, for example, in WO 2009 / 098450 and the references cited therein, in particular WO 2004 / 077062 and WO 2006 / 078161.

[0161] As described in the foregoing documents, the molecular scaffold may be a low molecule such as a low organic molecule.

[0162] 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.

[0163] In one embodiment, the molecular scaffold includes a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0164] The molecular scaffold may include 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.

[0165] In one embodiment, the molecular scaffold may include, or consist of, hexahydro-1,3,5-triazine, particularly 1,3,5-triacryloylhexahydro-1,3,5-triazine ("TATA"), or a derivative thereof.

[0166] The molecular scaffold of the present invention contains a chemical group that enables the functional group of the polypeptide of the encoded library of the present invention to form a covalent bond with the molecular scaffold. The chemical group is 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.

[0167] The scaffold reaction groups that can be used on a molecular scaffold for reacting with the thiol group of cysteine are alkyl halides (also named halogenoalkanes or haloalkanes).

[0168] Examples include bromomethylbenzene (scaffold reaction group exemplified by TBMB) or iodoacetamide. Other scaffold reaction groups used for selectively coupling a compound to cysteine in a protein are maleimide, α,β-unsaturated carbonyl-containing compounds, and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an α,β-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 natural amino acid having reactivity similar to that of cysteine and can be used in the same reaction. Therefore, whenever cysteine is mentioned, generally, selenocysteine can be used instead, unless otherwise suggested by the context.

[0169] (Synthesis) The peptides of the present invention can be synthetically produced by standard techniques and then reacted with a molecular scaffold in vitro. When doing this, standard chemistry can be used. This enables the rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be achieved using conventional chemistries such as those disclosed in the literature of Timmerman et al. (supra).

[0170] Accordingly, the present invention also relates to the manufacture of a polypeptide or conjugate selected as described herein, where the manufacture includes any further steps as described below. In one embodiment, these steps are carried out on the polypeptide conjugate of the final product made by chemical synthesis.

[0171] Optionally, the amino acid residues in the polypeptide of interest may be substituted when producing the conjugate or complex.

[0172] The peptide can also be extended, for example, to incorporate another loop and thus introduce multiple specificities.

[0173] To extend the peptide, it may simply be chemically extended at its N - or C - terminus or within a loop using orthogonally protected lysine (and analogs) using standard solid - phase or liquid - phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N - or C - terminus. Alternatively, the addition may be effected enzymatically, for example, by fragment condensation or native chemical ligation as described in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation. Science 266:776 - 779) or using subtiligase as described, for example, in (Chang et al., Proc Natl Acad Sci U S A. 1994 Dec 20; 91(26):12544 - 8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, November 15, 2008, pp. 6000 - 6003).

[0174] Alternatively, the peptide may be extended or modified by further conjugation via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other within the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with three cysteine groups; subsequently, additional cysteine or thiol can be added to the N - or C - terminus of the first peptide such that this cysteine or thiol reacts only with the free cysteine or thiol of the second peptide to form a disulfide - linked bicyclic peptide - peptide conjugate.

[0175] Similar techniques are equally applicable to the synthesis / coupling of two bicyclic bispecific macrocyclic molecules that potentially give rise to a quad - specific molecule.

[0176] Furthermore, the addition of other functional or effector groups may be achieved in the same manner by coupling at the N- or C-terminus, or via the side chain, using appropriate chemistry. In one embodiment, the coupling is performed in such a way as not to block the activity of either entity.

[0177] (Pharmaceutical composition) According to a further aspect of the invention, there is provided a pharmaceutical composition comprising the peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0178] Generally, the peptide ligand is utilized in a purified form together with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, and lactated Ringer's. Suitable physiologically acceptable adjuvants may be selected from thickening agents such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin, and alginate, if necessary to keep the polypeptide complex in suspension.

[0179] Parenteral vehicles include fluids and nutrient replenishers and electrolyte replenishers, for example, those based on Ringer's dextrose. Also, preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may be present (Mack (1982), Remington's Pharmaceutical Sciences, 16th edition).

[0180] The peptide ligand of the invention may be used as a separately administered composition or in combination with other agents. These include antibodies, antibody fragments, and various immunotherapeutic agents, such as CloExamples include sporin, methotrexate, adriamycin, or cisplatin, and immunotoxins. The pharmaceutical composition can be a "cocktail" of various cytotoxic agents or other drugs combined with the protein ligand of the present invention, or a combination of selected polypeptides according to the present invention with different specificities, such as polypeptides selected using different target ligands, whether pooled or not before administration.

[0181] The route of administration of the pharmaceutical composition according to the present invention may be any generally known to those skilled in the art. For therapy, the peptide ligand of the present invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, via the pulmonary route, or, equally 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 are determined by the patient's age, sex, and condition, the simultaneous administration of other drugs, contraindications, and other parameters considered by the clinician.

[0182] The peptide ligand of the present invention can be lyophilized before storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective and can utilize lyophilization and reconstitution techniques known in the art. It will be understood by those skilled in the art that lyophilization and reconstitution can result in varying degrees of loss of activity and that it may be necessary to adjust the level upwards to compensate.

[0183] The composition containing the peptide ligand of the present invention or a cocktail thereof can be administered for prophylactic and / or therapeutic treatment. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, regulation, killing, or some other measurable parameter of a selected population of cells is defined as a "therapeutically effective dose". The amount required to achieve this dosage depends on the severity of the disease and the general state of the patient's own immune system, but generally ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, and a dosage of 0.05 to 2.0 mg / kg is more commonly used. For prophylactic use, the composition containing the present peptide ligand or a cocktail thereof may also be administered at a similar or slightly lower dosage.

[0184] The composition containing the peptide ligand according to the present invention can be utilized in prophylactic and therapeutic settings to assist in the alteration, inactivation, killing, or removal of a selected target cell population in a mammal. Furthermore, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove the target cell population from a heterogeneous cell aggregate. Blood derived from a mammal can be combined ex vivo with a selected peptide ligand, thereby killing unwanted cells or otherwise removing them from the blood for return to the mammal according to standard techniques.

[0185] (Therapeutic use) According to 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.

[0186] Examples of cancers (and their benign counterparts) that can be treated (or suppressed) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other carcinomas), for example, of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary tract, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma), head and neck (e.g., cancers of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and appendage cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (i.e., leukemias, lymphomas) and borderline malignancies including pre-malignant blood disorders and hematologic malignancies and related diseases of the lymphoid lineage (e.g., acute lymphoblastic leukemia [ALL], chronic lymphocytic leukemia [CLL], B cell lymphoma, e.g., diffuse 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 hematologic malignancies and related diseases of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], eosinophilic syndrome, myeloproliferative disorders, e.g., polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, for example, sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi sarcoma, Ewing sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumor, benign and malignant histiocytoma, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and schwannoma);Endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid); tumors of the eye and its adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, undifferentiated embryonal cell tumor, cystic teratoma, and choriocarcinoma); and pediatric and fetal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and undifferentiated neuroectodermal tumor); or congenital or other syndromes that predispose a patient to malignancy (e.g., xeroderma pigmentosum), but are not limited thereto.;

[0187] In a further embodiment, the cancer is selected from hematopoietic malignancies selected from, for example, non-Hodgkin lymphoma (NHL), Burkitt 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 lymphoma (HL), and chronic myeloid leukemia (CML).;

[0188] References herein to the term "prevention" include the administration of a prophylactic composition prior to the induction of the disease. "Suppression" refers to the administration of a composition after an inductive event but prior to the clinical appearance of the disease. "Treatment" includes the administration of a prophylactic composition after the manifestation of disease symptoms.;

[0189] Animal model systems are available that can be used to screen for the efficacy of peptide ligands in the prevention of disease or the treatment of disease. The use of animal model systems is facilitated by the present invention, which enables the development of polypeptide ligands that can cross-react with human and animal targets.;

[0190] The present invention will be further described below with reference to the following examples.;

Examples

[0191] (Example) Generally, some of the heterotandem bicyclic peptide complexes of the present invention can be prepared according to the following general method: [Chemical formula]

[0192] All solvents were degassed and purged three times with N2. A DMF solution of BP-23825 (1.0 equivalent), HATU (1.2 equivalents), and DIEA (2.0 equivalents) was mixed for 5 minutes, and then bicyclic 1 (1.2 equivalents) was added. The reaction mixture was stirred at 40 °C for 16 hours. Thereafter, the reaction mixture was concentrated under reduced pressure to remove the solvent and purified by preparative HPLC to obtain intermediate 2.

[0193] A mixture of intermediate 2 (1.0 equivalent) and bicyclic 2 (2.0 equivalents) was dissolved in t-BuOH / H2O (1:1), and then CuSO4 (1.0 equivalent), VcNa (4.0 equivalents), and THPTA (2.0 equivalents) were added. Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. The reaction mixture was stirred at 40 °C for 16 hours under a N2 atmosphere. The reaction mixture was directly purified by preparative HPLC.

[0194] The heterotandem bicyclic peptide complexes prepared using this method are listed below: [Table 9] TIFF0007704732000045.tif243170TIFF0007704732000046.tif153170

[0195] More detailed experiments on the selected heterotandem bicyclic peptide complexes of the present invention are provided herein below:

[0196] (Example 1: Synthesis of BCY11863) [Chemical formula] (Preparation of Compound 2) [Chemical formula] A mixture of N-(acid-PEG3)-N-bis(PEG3-azide) (70.0 mg, 112.2 μmol, 1.0 equivalent), HATU (51.2 mg, 134.7 μmol, 1.2 equivalents), and DIEA (29.0 mg, 224.4 μmol, 40 μL, 2.0 equivalents) was dissolved in DMF (2 mL) and mixed for 5 minutes. Then, BCY8116 (294.0 mg, 135.3 μmol, 1.2 equivalents) was added. The reaction mixture was stirred at 40 °C for 16 hours. By LC-MS, a very small amount of compound 2 remained (MW: 2172.49, observed m / z: 1087.1), and one major peak with the desired m / z (MW: 2778.17, observed m / z: 1389.3 ([(M / 2 + H + ), 926.7 ([(M / 3 + H + )) was shown to be detected. The reaction mixture was concentrated under reduced pressure to remove the solvent, resulting in a residue. Then, the residue was purified by preparative HPLC (neutral conditions). Compound 2 (194.5 mg, 66.02 μmol, 29.41% yield, 94.3% purity) was obtained as a white solid.

[0197] (Preparation of BCY11863) [Chemical formula] A mixture of compound 2 (100.0 mg, 36.0 μmol, 1.0 equivalent) and BCY8928 (160.0 mg, 72.0 μmol, 2.0 equivalents) was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 180 μL, 1.0 equivalent), VcNa (28.5 mg, 143.8 μmol, 4.0 equivalents), and THPTA (31.2 mg, 71.8 μmol, 2.0 equivalents) were added. Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. All solvents here were degassed and purged three times with N2. The reaction mixture was stirred at 40 °C for 16 hours under a N2 atmosphere. By LC-MS, BCY8928 remained, and the desired m / z (calculated MW: 7213.32, observed m / z: 1444.0 ([M / 5 + H]+ )) was also shown to be detected. The reaction mixture was purified directly by preparative HPLC. By the first purification, BCY11863 (117.7 mg, 15.22 μmol, 42.29% yield, 93.29% purity) was obtained as the TFA salt, while the less pure fractions were purified again by preparative HPLC (TFA conditions) to give BCY11863 (33.2 mg, 4.3 μmol, 11.92% yield, 95.55% purity) as the TFA salt.

[0198] (Example 2: Synthesis of BCY12491) [Chemical Structure] (General Procedure for the Preparation of BP-23825-BCY9594) [Chemical Structure] To a mixture of Compound 1 (BP-23825, 60.0 mg, 96.2 μmol, 1.0 equiv) in DMF (3 mL) was added DIEA (12.4 mg, 96.2 μmol, 16.8 μL, 1.0 equiv) and HATU (38.4 mg, 101 μmol, 1.05 equiv), and the mixture was stirred for 5 minutes. Then, BCY9594 (243 mg, 101 μmol, 1.05 equiv) was added to the mixture, which was purged three times with N2 and then stirred at 40 °C for 16 hours under a N2 atmosphere. LC-MS indicated that Compound 1 was completely consumed and one major peak with the desired m / z was detected. Purification of the reaction mixture by preparative HPLC gave (BP-23825)-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 / 3+H] + , 1504.4 [M / 2+H] +

[0199] (General Procedure for the Preparation of Compound BCY12491) [Chemical Structure] A mixture of Compound 1 (56.0 mg, 18.6 μmol, 1.0 equiv), BCY8928 (83.0 mg, 37.2 μmol, 2.0 equiv), and THPTA (17.0 mg, 39.1 μmol, 2.1 equiv) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 three times), and then CuSO4 (0.4 M, 94.0 μL, 2.0 equiv) and VcNa (15.0 mg, 74.5 μmol, 4.0 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 3 h under a N2 atmosphere. LC-MS showed that Compound 3 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA conditions) to give BCY12491 (59.2 mg, 7.79 μmol, 41.81% yield, 97.9% purity) as a white solid. Calculated MW: 7441.63, observed m / z: 1861.1 ([M / 4+H] + ), 1489.0 ([M / 5+H] + ).

[0200] (Example 3: Synthesis of BCY12730)

Chemical Structure

Chemical Structure

[0201] (Example 4: Synthesis of BCY13048) [Chemical formula] (Procedure for the preparation of BP-23825-BCY12860) [Chemical formula] A mixture of BP-23825 (12.0 mg, 19.24 μmol, 1.2 equiv) and HATU (7.32 mg, 19.24 μmol, 1.2 equiv) was dissolved in NMP (0.3 mL), and then the pH of this solution was adjusted to 8 by dropwise addition of DIEA (5.12 mg, 40.26 μmol, 7 μL, 2.4 equiv). Then the solution was activated at 40 °C for 5 minutes. Compound 2 (33.0 mg, 16.03 μmol, 1.0 equiv) was dissolved in NMP (0.5 mL), and then added dropwise to the activated solution, and the pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 40 °C for 0.5 h. By LC-MS, it was shown that BCY12860 was completely consumed and one major peak with the desired m / z (MW: 2667.12, observed m / z: 1334.2 ([(M / 2+H + )、889.8([(M / 3+H + )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent, giving a residue. Then the residue was purified by preparative HPLC (neutral conditions). BP-23825-BCY12860 (26.5 mg, 7.88 μmol, 49.12% yield, 79.26% purity) was obtained as a white solid.

[0202] (Procedure for the preparation of BCY13048)

Chemical Structure

[0203] (Example 5: Synthesis of BCY13050)

Chemical formula

Chemical formula

[0204] (Procedure for the preparation of BCY13050) [Chemical formula] A mixture of Compound 3 (20.9 mg, 6.92 μmol, 1.0 equiv), Compound 4 (32.2 mg, 14.54 μmol, 2.1 equiv), and THPTA (7.0 mg, 15.93 μmol, 2.3 equiv) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 three times). Then, CuSO4 (0.4 M, 39 μL, 2.3 equiv) and VcNa (6.3 mg, 31.85 μmol, 4.6 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 1 h under a N2 atmosphere. LC-MS indicated that Compound 4 remained and one major peak with the desired m / z (calculated MW: 7453.66, observed m / z: 1864.2([M / 4+H]+ It was shown that )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. Purification of the crude product by preparative HPLC (TFA conditions) gave BCY13050 (6.0 mg, 0.77 μmol, 11.24% yield, 96.7% purity) as a white solid.

[0205] (Example 6: Synthesis of BCY13053) [Chemical formula] (Procedure for the preparation of BCY12865 - BP23825) [Chemical formula] BP - 23825 (14.0 mg, 22.45 μmol, 1.2 equiv) and HATU (8.5 mg, 22.35 μmol, 1.2 equiv) were first dissolved in 0.5 mL of NMP, and then DIEA (7.8 μL, 44.77 μmol, 2.4 equiv) was added. The mixture was stirred at 25 °C for 6 minutes, and then BCY12865 (40.0 mg, 18.65 μmol, 1.0 equiv) was added. The reaction mixture was stirred at 25 °C for 0.5 h. By LC - MS, one peak with the desired m / z was shown (calculated MW: 2750.21, observed m / z: 1375.5 ([M / 2 + H] + )) was shown. Purification of the reaction mixture by preparative HPLC (TFA conditions) gave Compound 1 (15.9 mg, 5.78 μmol, 31.0% yield, 96.69% purity) as a white solid.

[0206] (Procedure for the preparation of BCY13053) [Chemical formula] Compound 1 (15.9 mg, 5.78 μmol, 1.0 equiv) and BCY8928 (26.0 mg, 11.72 μmol, 2.1 equiv) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 29.0 μL, 2.0 equiv), VcNa (4.6 mg, 23.2 μmol, 4.0 equiv), and THPTA (5.1 mg, 11.7 μmol, 2.0 equiv) were added. Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged three times with N2. The reaction mixture was stirred at 40 °C for 16 h under a N2 atmosphere. By LC-MS, it was shown that Compound 1 was completely consumed and one major peak with the desired m / z (calculated MW: 7185.38, observed m / z: 1796.7 ([M / 4+H] + )) was detected. Purification of the reaction mixture by preparative HPLC (TFA conditions) gave BCY13053 (21.8 mg, 3.03 μmol, 52.84% yield, 98.01% purity) as a white solid.

[0207] (Example 7: Synthesis of BCY13341)

Chemical formula

Chemical formula

[0208] (Procedure for the preparation of BCY13341) [Chemical formula] A mixture of Compound 2 (20.6 mg, 7.49 μmol, 1.00 equiv), BCY12353 (31.5 mg, 15.08 μmol, 2.01 equiv), and THPTA (7.0 mg, 16.11 μmol, 2.15 equiv) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2 three times), and then CuSO4 (0.4 M, 37.5 μL, 2.00 equiv) and VcNa (6.0 mg, 30.29 μmol, 4.04 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 1 h under a N2 atmosphere. By LC-MS, one major peak with the desired m / z was shown (calculated MW: 6929.13, observed m / z: 1386.5 ([M / 5+H] + ) and 1155.8 ([M / 6+H] + ). The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (performed under TFA conditions for the first time and AcOH conditions for the second time) to give BCY13341 (10.3 mg, 1.49 μmol, 19.85% yield, 93.48% purity) as a white solid.

[0209] (Example 8: Synthesis of BCY13343)

Chemical Structure

Chemical Structure

[0210] (Procedure for the preparation of BCY13343) [Chemical formula] A mixture of Compound 3 (25.2 mg, 9.45 μmol, 1.0 equiv), Compound 4 (40.4 mg, 19.37 μmol, 2.05 equiv), and THPTA (9.5 mg, 21.73 μmol, 2.3 equiv) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged 3 times with N2), and then CuSO4 (0.4 M, 54.3 μL, 2.3 equiv) and VcNa (8.7 mg, 43.51 μmol, 2.5 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 25 °C for 1 h under an N2 atmosphere. By LC-MS, Compound 3 was also completely consumed and one major peak with the desired m / z (calculated MW: 6846.04, observed m / z: 1370.3([M / 5+H+ It was shown that ))) was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. When the crude product was purified by preparative HPLC (TFA conditions), BCY13343 (28.2 mg, 3.61 μmol, 38.23% yield, 87.7% purity) was obtained as a white solid.

[0211] (Example 9: Synthesis of BCY11027)

Chemical formula

Chemical formula

[0212] (Procedure for the preparation of BCY11027)

Chemical formula

[0213] (Example 10: Synthesis of BCY12967) [Chemical formula] [Chemical formula] Compound 1 (20.0 mg, 7.20 μmol, 1.0 equiv) and BCY11607 (32.0 mg, 14.9 μmol, 2.1 equiv) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 36.0 μL, 2.0 equiv), VcNa (6.0 mg, 30.3 μmol, 4.2 equiv), and THPTA (6.4 mg, 14.7 μmol, 2.0 equiv) were added. Finally, 1 M NH4HCO3 was added to adjust the pH to 8. All solvents here were degassed and purged three times with N2. The reaction mixture was stirred at 40 °C for 16 h under a N2 atmosphere. By LC-MS, it was shown that Compound 2 was completely consumed and one major peak with the desired m / z (calculated MW: 7077.7, observed m / z: 1416.3 ([M / 5+H] +)、1180.4([M / 6 + H] + )、1011.9([M / 7 + H] + )) was shown. When the reaction mixture was purified by preparative HPLC (TFA conditions), BCY12967 (20.6 mg, 2.82 μmol, 39.17% yield, 96.82% purity) was obtained as a white solid.

[0214] (Example 11: Synthesis of BCY13272)

Chemical formula

Chemical formula

[0215] (General procedure for the preparation of compound BCY13272)

Chemical formula

[0216] (Example 12: Synthesis of BCY12733)

Chemical formula

Chemical formula

[0217] (Procedure for the preparation of BCY12733) [Chemical formula] [Chemical formula] A mixture of Compound 1 (5.0 mg, 9.77 μmol, 1.0 equiv), Compound 2 (2.4 mg, 1.08 μmol, 1.1 equiv), and THPTA (0.4 M, 3 μL, 1.0 equiv) was dissolved in t-BuOH / H2O (1:1, 0.5 mL, degassed and purged with N2), and then an aqueous solution of CuSO4 (0.4 M, 3 μL, 1.0 equiv) and VcNa (0.4 M, 3 μL, 1.0 equiv) was added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 1 h under a N2 atmosphere. LC-MS indicated that Compounds 3 and 4 also remained and the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY12733 (3.3 mg, 0.41 μmol, 42.42% yield, 94.60% purity) as a white solid. Calculated MW: 7307.33, observed m / z: 1827.1([M+4H] 4+ ), 1462.1([M+5H] 5+ ).

[0218] (Example 13: Synthesis of BCY14413)

Chemical formula

Chemical formula

[0219] (Procedure for the preparation of BCY14413)

Chem.

[0220] (Example 14: Synthesis of BCY14415)

Chemical formula

Chemical formula

[0221] (Example 15: Synthesis of BCY14416)

Chemical Structure

Chemical Structure

[0222] (Example 16: Synthesis of BCY14414)

Chemical formula

Chemical formula

[0223] (Procedure for the preparation of BCY14414) [Chemical formula] A mixture of BCY14798 (21.0 mg, 4.02 μmol, 1.0 equiv), BCY13389 (10.0 mg, 4.42 μmol, 1.1 equiv), and THPTA (1.8 mg, 4.02 μmol, 1.0 equiv) was dissolved in t-BuOH / 0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 5.0 μL, 0.5 equiv) and sodium ascorbate (2.8 mg, 16.06 μmol, 4.0 equiv) were added under N2. When the pH of this solution was adjusted to 7.5 by the dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / 0.2 M NH4HCO3), the solution turned pale yellow. The reaction mixture was stirred at 25 °C for 2 h under a N2 atmosphere. LC-MS indicated that BCY14798 was completely consumed, some BCY13389 remained, and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification of the crude product by preparative separation afforded BCY14414 (20 mg, 2.40 μmol, 59.73% yield, 90.9% purity) as a white solid. Calculated MW: 7503.74, observed m / z: 1251.5 ([M+5H] 5+ ), 1072.9 ([M+7H] 7+ ).

[0224] (Example 17: Synthesis of BCY14417)

Chemical Structure

Chemical Structure

[0225] (Example 18: Synthesis of BCY14418) [Chemical formula] (Procedure for the preparation of BCY14418) [Chemical formula] A mixture of BCY14414 (5.6 mg, 0.75 μmol, 1.0 equiv) and Alexa fluor® 488 (0.9 mg, 1.49 μmol, 2.0 equiv) was dissolved in DMF (0.3 mL). Then, the pH of this solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25 °C for 1.0 h. LC-MS showed that BCY14414 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification of the crude product by preparative HPLC gave BCY14418 (2.3 mg, 0.25 μmol, 32.89% yield, 85.6% purity) as a red solid. Calculated MW: 8020.19, observed m / z: 1337.2 ([M+6H] 6+ )。

[0226] (Example 19: Synthesis of BCY15217)

Chem.

Chem.

[0227] (Example 20: Synthesis of BCY15218)

Chem.

Chem.

[0228] (Example 21: Synthesis of BCY12979)

Chemical Structure

Chemical Structure

[0229] (Procedure for the preparation of BAPG-BCY9594) [Chemical formula] Compound 2 (47.6 mg, 15.78 μmol, 1.0 equiv) was first dissolved in 1 mL of DMF, and then piperidine (0.2 mL, 2.03 mmol, 128.0 equiv) was added. The mixture was stirred at 30 °C for 30 minutes. By LC-MS, one major peak with the desired m / z was shown (calculated MW: 2572.04, observed m / z: 1286.8 ([M+2H] 2+ ), 858.1 ([M+3H] 3+ ). The reaction mixture was purified by preparative HPLC to give Compound 3 (24.4 mg, 9.06 μmol, 57% yield, 95% purity) as a white solid.

[0230] (Procedure for the preparation of BCY9594-BAPG-PEG5-N3) [Chemical formula] Compound 3 (24.4 mg, 9.06 μmol, 1.0 equiv) and compound 4 (10.0 mg, 23.13 μmol, 2.4 equiv) were dissolved in 2 mL of MeCN / H2O (1:1), and 1 M NaHCO3 was added to adjust the pH to 8. The mixture was stirred at 25 °C for 2 h. By LC-MS, it was shown that compound 3 was completely consumed and one major peak with the desired m / z (calculated MW: 3206.71, observed m / z: 1069.7 ([M+3H] 3+ ) was detected. The reaction mixture was purified by preparative HPLC to give compound 5 (12.8 mg, 3.99 μmol, 42.08% yield, 88.62% purity) as a white solid.

[0231] (Procedure for the preparation of BCY12979) [Chemical formula] Compound 5 (12.8 mg, 3.99 μmol, 1.0 equiv) and BCY8928 (18.0 mg, 8.12 μmol, 2.0 equiv) were first dissolved in 2 mL of t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 20.0 μL, 2.0 equiv), VcNa (3.2 mg, 16.1 μmol, 4.0 equiv), and THPTA (3.5 mg, 8.0 μmol, 2.0 equiv) were added. Finally, 1 M NH4HCO3 was added to adjust the pH to 8. All solvents here were degassed and purged with N2. The reaction mixture was stirred at 40 °C for 16 h under a N2 atmosphere. By LC-MS, it was shown that compound 5 was completely consumed and one major peak with the desired m / z was shown. The reaction mixture was purified by preparative HPLC to give BCY12979 (16.0 mg, 2.02 μmol, 51% yield, 96.6% purity) as a white solid. Calculated MW: 7641.87, observed m / z: 1911.2 ([M+4H] 4+ ), 1528.3 ([M+5H] 5+ ), 1247.5 ([M+6H] 6+ ), 1092.2 ([M+7H] 7+ ).

[0232] (Example 22: Synthesis of BCY10918) [Chemical Formula] (Procedure for the preparation of Compound 1) [Chemical Formula] A mixture of COM00000329 (102 mg, 58.76 μmol, 1.0 equiv), BCY11015 (92.6 mg, 41.13 μmol, 0.7 equiv), and THPTA (0.4 M, 146.9 μL, 1.0 equiv) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged with N2). Then, CuSO4 (0.4 M, 146.9 μL, 1.0 equiv) and VcNa (0.4 M, 293.8 μL, 2.0 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 25 - 30 °C for 12 h under a N2 atmosphere. LC-MS showed that COM00000329 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was purified directly by preparative HPLC. Compound 1 (60 mg, 13.61 μmol, 23.16% yield, 90.45% purity) was obtained as a white solid. Calculated MW: 3988.52, observed m / z: 1329.97 ([M + 3H] 3+ ), 990.56 ([M + 4H] 4+ ).

[0233] (Procedure for the preparation of BCY10918) [Chemical Formula] A mixture of Compound 1 (60 mg, 15.04 μmol, 1.0 equiv), BCY8928 (72.0 mg, 32.47 μmol, 2.2 equiv), and THPTA (0.4 M, 37.6 μL, 1.0 equiv) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 37.6 μL, 1.0 equiv) and VcNa (0.4 M, 75.2 μL, 2.0 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 25 - 30 °C for 12 h under a N2 atmosphere. LC-MS indicated that Compound 1 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was purified directly by preparative HPLC. BCY10918 (48 mg, 5.47 μmol, 36% yield, 96% purity) was obtained as a white solid. Calculated MW: 8423.67, observed m / z: 1404.27 ([M+6H] 6+ ), 1203.73 ([M+7H] 7+ ).

[0234] (Example 23: Synthesis of BCY10919)

Chemical formula

Chemical formula

[0235] (Example 24: Synthesis of BCY11021)

Chem.

Chem.

[0236] (Procedure for the preparation of BCY11021)

Chem.

[0237] (Example 25: Synthesis of BCY11022)

Chemical Structure

Chemical Structure

[0238] (Example 26: Synthesis of BCY11864) [Chemical formula] (Procedure for the preparation of BCY11864) [Chemical formula] A mixture of Compound 2 (5 mg, 1.80 μmol, 1.0 equiv), BCY7744 (9 mg, 3.85 μmol, 2.1 equiv), and THPTA (0.4 M, 9 μL, 1.0 equiv) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 9 μL, 2.0 equiv) and VcNa (0.4 M, 18 μL, 4.0 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 16 h under a N2 atmosphere. LC-MS indicated that BCY7744 remained and the desired m / z was detected. The reaction mixture was purified directly by preparative HPLC. BCY11864 (5.2 mg, 0.62 μmol, 34% yield, 89% purity) was obtained as a white solid. Calculated MW: 7453.44, observed m / z: 1490.70 ([M+5H] 5+ ).

[0239] (Example 27: Synthesis of BCY11780) [Chemical formula] (Procedure for the preparation of Compound 3) [Chemical formula] A mixture of Compound 1 (40.0 mg, 21.15 μmol, 1.0 equiv), Compound 2 (43.0 mg, 15.86 μmol, 0.75 equiv), and THPTA (10.0 mg, 21.20 μmol, 1.0 equiv) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (53.0 μL, 0.4 M, 1.0 equiv) and VcNa (0.4 M, 53.0 μL, 1.0 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 4 h, and LC-MS indicated that Compound 2 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was then concentrated under reduced pressure to remove the solvent, giving a residue. This was purified by preparative HPLC. Compound 3 (11.7 mg, 2.44 μmol, 11% yield, 96.2% purity) was obtained as a white solid. Calculated MW: 4607.33, observed m / z: 1152.36 ([M+4H] 4+ )。

[0240] (Procedure for the preparation of BCY11780) [Chemical formula] A mixture of compound 3 (11.7 mg, 2.54 μmol, 1.0 equiv), BCY8928 (11.8 mg, 5.33 μmol, 2.1 equiv), and THPTA (2.3 mg, 5.3 μmol, 2.0 equiv) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 12.7 μL, 2.0 equiv) and VcNa (0.4 M, 25.4 μL, 4.0 equiv) were added under N2. When the pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), the solution turned pale yellow. The reaction mixture was stirred at 40 °C for 4 h under a N2 atmosphere. LC-MS showed that compound 3 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification of the crude product by preparative HPLC gave BCY11780 (5.0 mg, 0.509 μmol, 20.03% yield, 92.0% purity) as a white solid. Calculated MW: 9042.48, observed m / z: 1292.8 ([M+7H] 7+ ), 1130.96 ([M+8H] 8+ ).

[0241] (Example 28: Synthesis of BCY13390) (Procedure for the preparation of BCY12476) [Chemical formula] A mixture of N-(Acid-PEG3)-N-bis(PEG3-azide) (70.0 mg, 112.2 μmol, 1.0 equiv), HATU (51.2 mg, 134.7 μmol, 1.2 equiv), and DIEA (29.0 mg, 224.4 μmol, 40 μL, 2.0 equiv) was dissolved in DMF (2 mL) and mixed for 5 min. Then, BCY8116 (294.0 mg, 135.3 μmol, 1.2 equiv) was added. The reaction mixture was stirred at 40 °C for 16 h. LC-MS showed one major peak with the desired m / z. The reaction mixture was concentrated under reduced pressure to remove the solvent, giving a residue. The residue was then purified by preparative HPLC. BCY12476 (194.5 mg, 66.02 μmol, 29% yield, 94% purity) was obtained as a white solid. Calculated MW: 2778.17, observed m / z: 1389.3 ([M+2H] 2+ ), 926.7 ([M+3H] 3+ ).

[0242]

Chemical Structure

Chemical Structure

[0243] (Procedure for the preparation of BCY13390)

Chem.

[0244] (Example 29: Synthesis of BCY13582)

Chemical formula

Chemical formula

[0245] (Example 30: Synthesis of BCY13583) [Chemical formula] (Procedure for the preparation of BCY13583) [Chemical formula] A mixture of BCY13390 (15.0 mg, 2.06 μmol, 1.0 equiv) and Alexa fluor® 488 NHS ester (2.5 mg, 4.12 μmol, 2.0 equiv) was dissolved in DMF (0.5 mL). Then, DIEA (2.6 mg, 20.63 μmol, 3.6 μL, 10 equiv) was added dropwise. The reaction mixture was stirred at 25 °C for 1 h. LC-MS indicated that BCY13390 remained and one major peak with the desired m / z was detected. Additional Alexa fluor® 488 NHS ester (2.0 mg, 3.09 μmol, 1.5 equiv) was added to the reaction mixture and the reaction mixture was stirred at 25 °C for an additional 1 h. HPLC indicated that BCY13390 was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification of the crude product by preparative HPLC gave BCY13583 (5 mg, 0.61 μmol, 29% yield, 95% purity) as a red solid. Calculated MW: 7787.9, observed m / z: 1948.8 ([M + 4H + H2O] 4+ ), 1558.6 ([M + 5H + H2O] 5+ ), 1299.1 ([M + 7H + H2O] 7+ ).

[0246] (Example 31: Synthesis of BCY13628)

Chemical Structure

Chemical Structure

[0247] (Example 32: Synthesis of BCY15155)

Chemical formula

Chemical formula

[0248] (Analytical data) The following heterotandem bicyclic peptide complexes of the present invention were analyzed using mass spectrometry and HPLC. The HPLC settings for the following analytical methods A - C were as follows: Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow rate: 1.0 ml / min Column: Gemini-NX C18 5um 110A 150*4.6mm Equipment: Agilent 1200 HPLC-BE(1-614)

[0249] The HPLC settings for the following analytical method D were as follows: Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow rate: 1.0 ml / min Column: Kintex 1.7um C18 100A 2.1mm*150mm Equipment: Agilent UPLC 1290

[0250] The gradient used is shown in the table below:

Table 10

Table 11

[0251] Additional analysis data was generated as follows:

Table 12

[0252] (Biological data) (1. CD137 reporter assay co - culture with tumor cells) A culture medium called R1 medium is prepared by adding 1% FBS to RPMI - 1640 (a component of Promega kit CS196005). Serial dilutions of the test article in R1 are prepared in a sterile 96 - well plate. 25 μL of the test article or R1 (as a background control) per well is added to the designated wells in a white cell culture plate. Tumor cells *Harvest and resuspend in Medium R1 at a concentration of 400,000 cells / mL. Add 25 (25) μL / well of tumor cells to a white cell culture plate. Thaw Jurkat cells (Promega kit CS196005, 0.5 mL) in a water bath and then add to 5 mL of pre-warmed Medium R1. Then add 25 (25) μL / well of Jurkat cells to a white cell culture plate. Incubate the cells and test article at 37 °C, 5% CO2 for 6 hours. At the end of 6 hours, add 75 μL / well of Bio-Glo™ reagent (Promega), incubate for 10 minutes, and then read luminescence with a plate reader (Clariostar, BMG). Calculate the fold change compared to cells only (Jurkat cells + cell line used in co-culture), plot as log(agonist) vs. response in GraphPad Prism, and determine the EC 50 (nM) and fold induction over background (Max).

[0253] The tumor cell types used in co-culture are NCI-H292 and HT1376, which have been shown to express nectin-4. The tumor cell types used in co-culture for EphA2 are A549, PC3, and HT29. The tumor cell type used in co-culture for PD-L1 is RKO.

[0254] The data shown in Figure 1A indicate that the nectin-4 / CD137 heterodimer (BCY11863) induces strong CD137 activation in the CD137 reporter assay and that this activation is dependent on the binding of the heterodimer to CD137. BCY11617, a molecule composed of all D-amino acids that abrogates binding of the CD137 bicyclic peptide, does not induce CD137 agonism.

[0255] EC 50 (nM) and fold induction derived by the heterodimeric bicyclic peptide complex in the CD137 reporter assay in co-culture with nectin-4 expressing tumor cell lines are reported in Table 1A below: Table 1A: Fold induction mediated by nectin-4 / CD137 heterodimeric cyclic peptide complex in CD137 reporter assay [Table 13]

[0256] EC induced by heterodimeric cyclic peptide complexes BCY11863 and clone analogs in CD137 reporter assay under co-culture with nectin-4 expressing tumor cell lines 50 (nM) are summarized in Table 1B below and visualized in Figure 1B. This data shows the ability of BCY11863 to induce CD137 agonism under co-culture with cell lines having various nectin-4 expressions. Table 1B: EC50 (nM) of fold induction relative to background mediated by nectin-4 / CD137 heterodimeric cyclic peptide complex in CD137 reporter assay [Table 14]

[0257] A summary of fold induction mediated by nectin-4 / CD137 heterodimeric peptide in CD137 reporter co-culture assay using NCI-H292 cells is shown in Table 2 below. All compounds were compared to plate control BCY10000 having an average EC 50 of 1.1 ± 0.5 nM and an Emax of 28 ± 11-fold relative to background. Table 2: Fold induction mediated by nectin-4 / CD137 heterodimeric cyclic peptide complex in CD137 reporter assay [Table 15]

[0258] The summary of induction fold mediated by nectin-4 / CD137 heterotandem peptide in the CD137 reporter co-culture assay using HT1376 tumor cells is shown in Table 2A below, and the EC50 (nM) and Emax (induction fold relative to background) are reported. Most of the nectin-4 / CD137 heterotandems have an EC50 of less than 1 nM. Table 2A: EC50 and Emax of the nectin-4 reporter assay [Table 16]

[0259] The data shown in Figure 16 indicate that the EphA2 / CD137 heterotandem BCY13272 induces strong CD137 activation in the CD137 reporter assay in the presence of EphA2-expressing cell lines (PC3, A549, and HT29), while the unbound control molecule (BCY13626) does not show CD137 activation.

[0260] The summary of induction fold mediated by EphA2 / CD137 heterotandem peptide in the CD137 reporter co-culture assay using PC3 cells is shown in Table 3A below. All compounds are compared to the plate control BCY9173, which has an average EC 50 of 0.54 nM and an Emax of 42-fold relative to background. Table 3A: Induction fold mediated by the EphA2 / CD137 heterotandem bicyclic peptide complex in the CD137 reporter assay [Table 17]

[0261] The EC 50 (nM) and induction fold mediated by BCY13272 in the CD137 reporter assay under co-culture with EphA2-expressing tumor cell lines are reported in Table 3B below: Table 3B: Activity of EphA2 / CD137 Heterodimeric Cyclic Peptide Complexes in CD137 Reporter Assays [Table 18]

[0262] The fold inductions mediated by EphA2 / CD137 heterodimeric peptides in CD137 reporter co - culture assays using PC3 tumor cells are summarized in Table 3C below, and the EC50 (nM) and Emax (fold induction relative to background) are reported. Most EphA2 / CD137 heterodimers have an EC50 of less than 1 nM. Table 3C: EC50 and Emax of EphA2 Reporter Assays [Table 19] TIFF0007704732000145.tif105170

[0263] The fold inductions mediated by PD - L1 / CD137 heterodimeric peptides in CD137 reporter co - culture assays using RKO cells are summarized in Table 4 below. Table 4: Fold Inductions Mediated by PD - L1 / CD137 Heterodimeric Cyclic Peptide Complexes in CD137 Reporter Assays [Table 20]

[0264] (2. Human PBMC - Tumor Cell Co - culture (Cytokine - Stimulated Assay) Assay) The tumor cell line was cultured according to the protocol recommended by the supplier. Cryopreserved PBMCs from healthy human donors were thawed, washed once in PBS at room temperature, and then resuspended in R10 medium. 100 μl of PBMCs (1,000,000 PBMC / ml) and 100 μl of tumor cells (100,000 tumor cells / ml) (effector:target cell ratio (E:T) 10:1) were plated into each well of a 96-well flat-bottom plate for the co-culture assay. 100 ng / ml of soluble anti-CD3 mAb (clone OKT3) was added to the cultures on day 0 to stimulate human PBMCs. The test article, control compound, or vehicle control was diluted in R10 medium and 50 μL was added to each well to bring the final volume per well to 250 μL. The plates were covered with breathable film and incubated in a humidified chamber at 37 °C containing 5% CO2 for 3 days. Supernatants were harvested 48 hours after stimulation and human IL-2 and IFN-γ were detected by Luminex. Briefly, standards and samples were added to a black 96-well plate. A microparticle cocktail (provided in the Luminex kit, R&D Systems) was added and the plate was shaken for 2 hours at room temperature. The plate was washed 3 times using a magnetic holder. Thereafter, a biotin cocktail was added to the plate and shaken for 1 hour at RT. The plate was washed 3 times using a magnetic holder. A streptavidin cocktail was added to the plate and shaken for 30 minutes at RT. The plate was washed 3 times using a magnetic holder, resuspended in 100 μL of wash buffer, shaken for 2 minutes at RT, and read using a Luminex 2000. Raw data were analyzed using the built-in Luminex software to generate a standard curve, interpolate protein concentrations, and all other data analysis and graphing were performed using Excel and Prism software. The data represent one test using three independent donor PBMCs examined in experimental duplicates.

[0265] The data shown in FIGS. 2A and 2B indicate that nectin-4 / CD137 heterotandem (BCY11863) induces strong IL-2 and IFN-γ cytokine secretion in the PBMC-4T1 co-culture assay. BCY11617 is a negative control that binds to nectin-4 but not to CD137.

[0266] EC induced by the selected nectin-4 / CD137 heterotandem bicyclic peptide complex in a human PBMC co-culture (cytokine release) assay 50 (nM) and maximal IFN-γ cytokine secretion (pg / ml) are summarized in Table 4A below and visualized in FIG. 2C. This demonstrates the ability of BCY11863 to induce cytokine secretion in the presence of several different tumor cell lines expressing nectin-4. Table 4A: IFN-γ cytokine secretion EC induced by the selected nectin-4 / CD137 heterotandem bicyclic peptide complex in a human PBMC-4T1 co-culture (cytokine release) assay 50 [Table 21]

[0267] (Pharmacokinetics of the CD137 heterotandem bicyclic peptide complex in 3. SD rats) Each heterotandem bicyclic peptide complex formulated in 25 mM histidine HCl, 10% sucrose at pH 7 was administered to male Sprague-Dawley rats by IV bolus or IV infusion (over 15 minutes). Serial blood sampling (≈80 μL blood per time point) was performed at each time point from the submandibular or jugular vein. All blood samples were immediately transferred to pre-chilled microcentrifuge tubes containing 2 μL of K2-EDTA (0.5 M) as anticoagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at ≈4 °C and 3000 g. A precipitating agent containing internal standard was immediately added to the plasma, mixed well, and centrifuged at 12,000 rpm and 4 °C for 10 minutes. The supernatant was transferred to pre-labeled polypropylene microcentrifuge tubes and then snap frozen on dry ice. Samples were stored at ≤70 °C until analysis as needed. 7.5 μL of the supernatant sample was directly injected for LC-MS / MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte. Plasma concentration vs. time data were analyzed by a non-compartmental approach using the Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf), and graphs of the plasma concentration vs. time profiles were reported. The pharmacokinetic parameters of the experiment are as shown in Table 6A: Table 6A: Pharmacokinetic Parameters in SD Rats

Table 22

[0268] In particular, the pharmacokinetic parameters of BCY11863 are as shown in Table 6B: Table 6B: Pharmacokinetic Parameters in SD Rats

Table 23

[0269] The data in Table 6B above and in Figure 5 indicate that BCY11863 is a low-clearance molecule with a volume of distribution greater than plasma volume. Furthermore, the bioavailability obtained from SC administration of BCY11863 is high in rats. Table 6C: Pharmacokinetic Parameters of BCY11863 and Potential Metabolites in an SD Rat PK Study after Administration of a 100 mg / kg Dose by IV Administration

Table 24

[0270] The data in Table 6C and Figure 25 indicate that when BCY11863 is administered IV to SD rats, less than 1% of BCY11863 is metabolized to BCY15155. No significant conversion to BCY14602 is observed in the first 24 hours of the study.

[0271] (4. Pharmacokinetics of CD137 Heterodimeric Cyclic Peptide Conjugates in Cynomolgus Monkeys) Each heterodimeric cyclic peptide conjugate at 1 mg / kg formulated in 25 mM histidine HCl, 10% sucrose pH 7 was administered to the radial cutaneous vein of treated cynomolgus monkeys via intravenous infusion (15 or 30 minutes). Serial blood sampling (approximately 1.2 ml blood / time point) was performed from the peripheral vessels of restrained, non-sedated animals at each time point into potassium (K2) EDTA on wet ice *Put it into a commercially available tube containing 2H2O (0.85 - 1.15 mg) and treat it for plasma. Immediately after collecting the sample, centrifuge it (3,000×g, for 10 minutes at 2 - 8 °C). Transfer 0.1 mL of plasma to a labeled polypropylene microcentrifuge tube. Add 5 times the precipitant containing 100 ng / mL labelolol, 100 ng / mL dexamethasone, 100 ng / mL tolbutamide, 100 ng / mL verapamil, 100 ng / mL glyburide, and 100 ng / mL celecoxib in MeOH to the plasma immediately, mix well, and centrifuge at 12,000 rpm for 10 minutes at 2 - 8 °C. Transfer the supernatant sample to a pre-labeled polypropylene microcentrifuge tube and freeze it on dry ice. Store the sample at -60 °C or below until LC-MS / MS analysis. Aliquots of 40 μL of calibration standard samples, quality control samples, single blank samples, and double blank samples were added to 1.5 mL tubes. Each sample (except double blanks) was quenched with 200 μL IS1 respectively (double blank samples were quenched with 200 μL MeOH containing 0.5% tritonX - 100), then the mixture was vortex mixed well (for at least 15 seconds) with a vortexer and centrifuged at 12000 g for 15 minutes at 4 °C. Inject 10 μL of the supernatant for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte. Plasma concentration vs. time data were analyzed by a non-compartmental approach using the Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2, AUC(0 - last), AUC(0 - inf), MRT(0 - last), MRT(0 - inf), and graphs of plasma concentration vs. time profiles were reported. The pharmacokinetic parameters of the three bispecific compounds are as shown in Table 7. Table 7: Pharmacokinetic Parameters in Cynomolgus Monkeys

Table 25

[0272] Figure 3 shows the plasma concentration-time curves of BCY11863 obtained from 2 mg / kg IV administration in Sprague-Dawley rats (n = 3) and 1 mg / kg IV infusion in cynomolgus monkeys (n = 2). BCY11863 has a steady-state volume of distribution (Vdss) of 1.6 L / kg and a clearance of 7.7 mL / min / kg resulting in a terminal-phase half-life of 4.1 hours in rats. BCY11863 has a steady-state volume of distribution (Vdss) of 0.62 L / kg and a clearance of 3.3 mL / min / kg resulting in a terminal-phase half-life of 5.3 hours in cynomolgus monkeys.

[0273] Figure 12 shows the plasma concentration-time curves of BCY12491 obtained from a 15-minute 1 mg / kg IV infusion in cynomolgus monkeys (n = 2).

[0274] Figure 17 shows the plasma concentration-time curves of BCY13272 obtained from a 3.6 mg / kg IV infusion (15 minutes) in Sprague-Dawley rats (n = 3) and a 9.2 mg / kg IV infusion (15 minutes) in cynomolgus monkeys (n = 3). BCY13272 has a steady-state volume of distribution (Vdss) of 1.0 L / kg and a clearance of 7.5 mL / min / kg resulting in a terminal-phase half-life of 2.9 hours in rats. BCY13272 has a steady-state volume of distribution (Vdss) of 0.82 L / kg and a clearance of 4.1 mL / min / kg resulting in a terminal-phase half-life of 8.9 hours in cynomolgus monkeys.

[0275] (5. Pharmacokinetics of CD137 Heterodimeric Cyclic Peptide Conjugates in CD1 Mice) Six male CD-1 mice were administered each heterodimeric bicyclic peptide complex at 15 mg / kg formulated in 25 mM histidine HCl, 10% sucrose pH 7 by intraperitoneal or intravenous administration. Serial blood sampling (approx. 80 μL blood / time point) was performed from the submandibular or saphenous vein at each time point. All blood samples were immediately transferred to pre-chilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5 M) as anticoagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at approximately 4°C, 3000 g. A precipitating agent containing internal standard was immediately added to the plasma, mixed well, and centrifuged at 12,000 rpm, 4°C for 10 minutes. The supernatant was transferred to pre-labeled polypropylene microcentrifuge tubes and then rapidly frozen on dry ice. Samples were stored at 70°C or below until analysis as needed. 7.5 μL of the supernatant sample was injected directly for LC-MS / MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte. Plasma concentration vs. time data were analyzed by a non-compartmental approach using the Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf), and graphs of plasma concentration vs. time profiles were reported.

[0276] Figure 11 shows the plasma concentration vs. time curves of BCY11863 and BCY12491 obtained from 15 mg / kg IP administration in CD1 mice (n = 3) and the terminal phase plasma half-lives of BCY11863 and BCY12491. Table 7A: Pharmacokinetic Parameters in CD-1 Mice

Table 26

[0277] The data in Figure 11 above and Table 7A show that BCY11863 can be administered as an IV bolus and IP in mice. The bioavailability obtained from the IP administration of BCY11863 is high in mice. The PK parameters obtained from the IV test indicate that this is a low clearance molecule with a volume of distribution larger than the plasma volume.

[0278] Figure 17 shows the plasma concentration-time curve of BCY13272 obtained from a 5.5 mg / kg IV administration in CD1 mice (n = 3); the volume of distribution (Vdss) of BCY13272 is 1.1 L / kg and the clearance is 7.5 mL / min / kg, resulting in a terminal phase half-life of 2.9 hours.

[0279] (6. Antitumor Activity of BCY11863 in a Syngeneic Necl-4 Overexpressing MC38 Tumor Model (MC38#13)) 1 x 10 6 syngeneic necl-4 overexpressing MC38 cells (MC38#13) were inoculated into the flanks of 6-8 week old C57BL / 6J-hCD137 female mice. When the tumors reached an average size of 72 mm 3 , the mice were randomly assigned to receive vehicle or BCY11863 (intraperitoneal administration). BCY11863 was administered daily (QD) or every 3 days (Q3D) at either 1 mg / kg or 10 mg / kg (n = 6 mice / treatment cohort). Mice receiving QD administration received 16 doses of BCY11863 and mice receiving Q3D administration received 10 doses of BCY11863. Tumor growth was monitored by caliper measurement until day 69 after treatment initiation. The results of this experiment can be seen in Figure 4. In this experiment, a significant decrease in tumor growth (p < 0.05, two-way ANOVA with Dunnett's multiple comparison test) was observed in two treatment cohorts by day 7, and by day 14, all treatment groups were significantly different from the vehicle group. By day 48, 22 out of 24 BCY11863-treated animals had a complete response to treatment and no palpable tumors remained.

[0280] Based on the circulating plasma half-life of BCY11863 in mice after IP injection (2.5 hours), the plasma trough levels were near zero after both BCY11863 doses (1 and 10 mg / kg) and dosing intervals (QD and Q3D), and thus intermittent dosing provides sufficient but not continuous plasma exposure of BCY11863 to result in significant antitumor activity that leads to a durable complete response.

[0281] (7. BCY11863 treatment induces immunogenic memory against the nectin-4 overexpressing MC38 tumor model) On day 69, five animals that had completely responded to BCY11863 treatment were re-challenged with 1 × 10 6 MC38#13 cells. A cohort of five untreated C57BL / 6J-hCD137 female mice was inoculated with 1 × 10 6 MC38#13 cells as a control. The results of this experiment can be seen in Figure 5. In this experiment, all five untreated C57BL / 6J-hCD137 female mice that were inoculated developed tumors by day 13 after inoculation, whereas none of the inoculated complete responder mice developed tumors. This indicates that animals that achieved a complete antitumor response as a result of BCY11863 treatment developed immunogenic memory.

[0282] (8. BCY11863 shows antitumor activity in a syngeneic nectin-4 overexpressing CT26 tumor model (CT26#7)) The flanks of 6-8-week-old BALB / c-hCD137 female mice were inoculated with 3 × 10 5 syngeneic nectin-4 overexpressing CT26 cells (CT26#7). When the tumors reached an average size of 70 mm 3 , mice were randomly assigned to receive vehicle or 5 mg / kg BCY11863 intraperitoneally every 3 days (total of 6 doses). Tumor growth was monitored by caliper measurement until day 14 after treatment initiation. The results of this experiment can be seen in Figure 6. In this experiment, BCY11863 treatment significantly (p < 0.0001, Student's t-test) reduced tumor growth after day 7.

[0283] Based on the circulating plasma half-life of BCY11863 in mice at the time of IP injection (2.5 hours), plasma exposure is not continuous throughout the dosing period, indicating that the by no means continuous plasma exposure of BCY11863 is sufficient to result in significant antitumor activity.

[0284] (9. Total T cells and CD8+ T cells increase CT26#7 tumor tissue 1 hour after the last (6th) Q3D administration of BCY11863) One hour after the last vehicle or BCY11863 administration, CD26#7-bearing mice were sacrificed, tumors were excised, processed for single cell suspension, and stained for flow cytometry analysis of total T cells (CD45+CD3+), CD8+ T cells (CD45+CD3+CD8+), CD4+ T cells (CD45+CD3+CD4+), and regulatory T cells (Treg; CD45+CD3+CD4+Foxp3+). The results of this experiment can be seen in Figure 7. In this experiment, it was found that treatment with BCY11863 resulted in a significant increase in total T cells (p < 0.0001, Student's t-test) and CD8+ T cells (p < 0.0001, Student's t-test), as well as a significant increase in the CD8+ T cell / Treg ratio (p < 0.05, Student's t-test).

[0285] This indicates that treatment with BCY11863 can result in a local increase in the level of T cells in tumor tissue after intermittent dosing.

[0286] (10. Pharmacokinetic profile of BCY11863 in plasma and tumor tissue of CT26#7 syngeneic tumor-bearing animals after a single intravenous (iv) administration of 5 mg / kg of BCY11863) To the flanks of 6-8 week-old female BALB / c mice, 3 × 10 5 syngeneic nectin-4 overexpressing CT26 cells (CT26#7) were inoculated. When the tumors reached an average of approximately 400 mm 3When the size reached, mice were randomly assigned to receive a single intravenous administration of vehicle or 5 mg / kg BCY11863. Mouse cohorts (n = 3 / time point) were sacrificed at 0.25, 0.5, 1, 2, 4, 8, and 24 hours time points, plasma was collected, and tumor tissues were analyzed for BCY11863. For tumor BCY11863 content analysis, tumor homogenates were prepared by homogenizing tumor tissues with 10 volumes (w:v) of homogenization solution (MeOH / 15 mM PBS (1:2, v:v)). 40 μL of the sample was quenched with 200 μL IS1, the mixture was mixed by vortexing at 800 rpm for 10 minutes, and centrifuged at 3220 g at 4 °C for 15 minutes. The supernatant was transferred to another clean 96-well plate, centrifuged at 3220 g at 4 °C for 5 minutes, and then 10.0 μL of the supernatant was injected for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte. For plasma BCY11863 content analysis, blood samples were collected into K2-EDTA tubes and immediately processed to plasma by centrifugation at approximately 4 °C at 3000 g. 40 μL of plasma sample was quenched with 200 μL IS1, the mixture was mixed by vortexing at 800 rpm for 10 minutes, and centrifuged at 3220 g at 4 °C for 15 minutes. The supernatant was transferred to another clean 96-well plate, centrifuged at 3220 g at 4 °C for 5 minutes, and then 10.0 μL of the supernatant was injected for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte.

[0287] The results of this experiment can be seen in Figure 8. In this experiment, the plasma T of BCY11863 1 / 2 (1.65 hours) and tumor T 1 / 2 (13.4 hours), it can be seen that BCY11863 was retained in the tumor tissue after the plasma BCY11863 was eliminated from the circulation.

[0288] (11. Antitumor Activity of BCY12491 in the Syngeneic MC38 Tumor Model) 1×10 6 syngeneic MC38 cells were inoculated into the flanks of 6- to 8-week-old C57BL / 6J-hCD137 female mice. When the tumors reached an average size of 76 mm 3 , the mice were randomly assigned to receive vehicle or BCY12491 (intraperitoneal administration). BCY12491 was administered daily (QD) or every 3 days (Q3D) at either 5 mg / kg or 15 mg / kg (n = 6 mice / treatment cohort). Mice receiving QD dosing received 22 doses of BCY12491, and mice receiving Q3D dosing received 8 doses of BCY12491. Tumor growth was monitored by caliper measurement until day 73 after treatment initiation. The results of this experiment can be seen in Figure 9. In this experiment, the effect of BCY12491 on tumor growth became apparent within the first 2 weeks of the dosing period, with tumor growth decreasing and a reduction in the volume of many of the treated tumors being observed. By day 41, 15 out of 24 BCY12491-treated animals had a complete response to treatment and no palpable tumors remained.

[0289] Based on the circulating plasma half-life of BCY12491 in mice after IP injection (2.5 hours), the plasma trough levels were near zero after both BCY12491 doses (5 and 15 mg / kg) and dosing intervals (QD and Q3D), and thus intermittent dosing provides plasma exposure that is never continuous but is sufficient to result in significant antitumor activity that leads to a durable complete response.

[0290] (12. EphA2 / CD137 heterotandem bicyclic peptide conjugates BCY12491, BCY13272, BCY12723, BCY13050, BCY13048, and BCY13047 induce IFN-γ cytokine secretion in the MC38 co-culture assay) The mouse mammary tumor cell line MC38 was cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× penicillin / streptomycin, 10 mM HEPES, and 2 mM L-glutamine (referred to as R10 medium herein). Cryopreserved PBMCs from healthy human donors were thawed, washed once in room temperature PBS containing benzonase, and then resuspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× penicillin / streptomycin, 10 mM HEPES, and 2 mM L-glutamine (referred to as R10 medium herein). 100 μl of PBMCs (1,000,000 PBMC / ml) and 100 μl of tumor cells (100,000 tumor cells / ml) (effector:target cell ratio (E:T) 10:1) were plated into each well of a 96-well flat-bottom plate for the co-culture assay. 100 ng / ml of soluble anti-CD3 mAb (clone OKT3) was added to the cultures on day 0 to stimulate human PBMCs. Test articles, control compounds, or vehicle controls were diluted in R10 medium and 50 μL was added to each well to bring the final volume per well to 250 μL. The plates were covered with breathable film and incubated in a humidified chamber at 37 °C containing 5% CO2 for 2 days. Supernatants were harvested 24 and 48 hours after stimulation and human IFN-γ was detected by Luminex. Briefly, standards and samples were added to a black 96-well plate. A microparticle cocktail (provided in the Luminex kit, R&D Systems) was added and the plate was shaken for 2 hours at room temperature. The plate was washed 3 times using a magnetic holder. Thereafter, a biotin cocktail was added to the plate and shaken for 1 hour at RT. The plate was washed 3 times using a magnetic holder. A streptavidin cocktail was added to the plate and shaken for 30 minutes at RT. The plate was washed 3 times using a magnetic holder, resuspended in 100 μL of wash buffer, shaken for 2 minutes at RT, and read using a Luminex 2000. Raw data were analyzed using the built-in Luminex software to generate a standard curve, interpolate protein concentrations, and all other data analysis and graphing were performed using Excel and Prism software.The data represent one experiment using three independent donor PBMCs examined in experimental duplicates.

[0291] The data shown in Figure 10 indicate that the EphA2 / CD137 heterotandem bicyclic peptide complex BCY12491 induces IFN-γ cytokine secretion in an MC38 co-culture assay at an EC of 34 pM (Figure 10A = donor 228769) or 85 pM (Figure 10B = donor 228711) using PBMCs from two different human donors. 50 BCY12762 is a heterotandem bicyclic peptide complex that binds to EphA2 with the same affinity but does not bind to CD137.

[0292] Similarly, PBMCs from healthy donors were co-cultured with EphA2-expressing cancer cells (MC38 and HT-1080) at a 5:1 ratio in the presence of anti-CD3 and BCY13272. The supernatants were analyzed for cytokines (IL-2 and IFNγ) by Luminex after 48 hours. The data are shown in Table 8 and represent PBMCs from one donor (from a total of n = 4 or 5 individual experiments). Table 8: EC50 of IL-2 cytokine secretion induced by the EphA2 / CD137 heterotandem bicyclic complex in a human PBMC-MC38 / HT-1080 co-culture assay [Table 27]

[0293] The data shown in Figure 26 and tabulated in Table 8A indicate that the EphA2 / CD137 heterotandem bicyclic peptide complex induces IFN-γ cytokine secretion in an MC38 co-culture assay with sub-nanomolar potency. Table 8A: EC50 and Emax of IFNγ secretion induced by the EphA2 / CD137 heterotandem bicyclic complex in a human PBMC-MC38 co-culture assay [Table 28]

[0294] (13. Target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors) Primary patient-derived tumor cells from Discovery Life Sciences (DLS) were slowly thawed in pre-warmed wash medium freshly supplemented with 10 mL of benzonase. Cells were maintained in culture for 2 days using a 3D spheroid kit (cat# 655840) from Greiner. Briefly, tumor cells were counted with trypan blue using a hemocytometer. Cells were centrifuged at 1500 rpm for 5 minutes, washed, and the pellet was resuspended in 1×10 6Resuspend them in 100 μL of N3D nanoshuffle per cell. To magnetize them, spin down the cells at 1500 rpm for 5 minutes and resuspend; repeat this process a total of 4 times. After the last spin, resuspend the cells in an appropriate amount of fresh lung DTC medium (DLS) to obtain 50,000 - 100,000 cells per well at 100 μL / well. A Greiner cell-repellent 96-well plate (cat #655976) was used for this experiment. If there were visible cell clumps or cell debris, filter the sample through a 70 - 100 μm filter before plating. Take at least 50,000 cells per sample for the day 0 flow cytometry panel, stain, fix, and store these cells at 4°C for subsequent flow analysis. Prepare control / test compound dilutions in Lung DTC medium at 2× in separate plates and add 100 μL / well of these 2× drug solutions to the wells as described by the plate map. Then, place the assay plate on a 96-well magnetic spheroid drive in a humidified chamber at 37°C, 5% CO₂. At 24 hours, remove the magnetic spheroid drive. At 48 hours, collect the medium for cytokine analysis and collect the cells for the day 2 flow cytometry panel. Quantify the cytokines using a custom cytokine / chemokine panel (IP-10, granzyme B, IFNγ, IL-2, IL-6, TNFα, IL-8, MIP-1a, MIP-1b, MCP-1, IL-10, MIG) from R&D systems on a Luminex reader. Flow panel: day 0 = live / dead, CD45, EpCAM, nectin 4, CD3, CD4, CD8, CD137; day 2 = live / dead, CD45, EpCAM, nectin 4, CD3, CD8, Ki67, and beads for counting. Analyze the flow data with Flowjo software.

[0295] The data shown in Figure 13 demonstrate that nectin-4 / CD137 heterotandem BCY11027 induces target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors. Treatment with BCY11027 induced nectin-4-dependent changes in several immune markers (normalized to vehicle) and nectin-4-dependent changes in %CD8+ ki67+ T cells in patient-derived samples, which correlated with the level of nectin-4 expression.

[0296] (14. Promega OX40 Cell Activity Assay under Co-Culture with Tumor Cells) Promega developed an OX40 cell activity assay (Promega CS197704) that uses NF-κB luciferase luminescence as a readout of OX40 activation in Jurkat cells. On the day of the experiment, thaw FBS and prepare the medium by adding 5% FBS to RPMI-1640. Thaw OX40 Jurkat cells in a water bath and then add 500 μl of the cells to 11.5 ml of pre-warmed 5% FBS RPMI-1640 medium. Add 55 μl of the cells per well to a white cell culture plate. Harvest the tumor cells from the culture. 4T1 is a nectin-4-negative mouse mammary epithelial carcinoma cell line. It was genetically modified to express mouse nectin-4 on the cell surface (4T1 nectin-4 positive; clone 4T1-D02). Culture the tumor cells in RPMI1640 medium (RPMI working medium) supplemented with 10% heat-inactivated FBS, 1× penicillin / streptomycin, 1× L-glutamine, 20 mM HEPES, and 1× NEAA in vitro until 80% confluence. Treat the tumor cells with trypsin and wash twice at 1500 rpm for 5 minutes in pre-warmed RPMI1640 working medium at 37°C. Count the cells and resuspend them in R5 medium at 2,000,000 cells / mL (for 10,000 cells / well). Add 5 μL of the tumor cells per well.

[0297] Next, dilute the agonist at the concentration that gives the maximum induction fold, and then gradually reduce the amount in a sterile 96-well plate. Prepare sufficient reagents for duplicate samples, and then perform a 1 / 3 dilution series or a 1 / 10 dilution series. Include the positive control OX40L trimer (AcroBiosystems, R&D systems) and the negative control monomer or unbound peptide. Add 20 μl of the agonist as duplicate samples or 5% FBS RPMI-1640 only as a background control.

[0298] Incubate the cells together with the agonist at 37 °C and 5% CO2 for 6 hours. After 6 hours, thaw Bio-Glo™ and develop the assay at room temperature. Add 80 μl of Bio-Glo™ per well and incubate for 5 - 10 minutes. Read the luciferase signal using the MARS program on a CLAIROStar plate reader and normalize the induction fold to the background (media only). Analyze the data by transforming the data to x = log(X), and then plot log(agonist) vs. response variable slope (4-parameter) to calculate the EC 50 value.

[0299] The results of this assay are shown in Table 9 and Figure 14. In this assay, it can be seen that the BCY12967 nectin-4:OX40 compound showed strong OX40 agonism compared to OX40L and the unbound control peptide BCY12968 when in co-culture with nectin-4 positive 4T1-D02 cells. Table 9: EC obtained from the Promega OX40 cell activity assay under co-culture with tumor cells 50 value

Table 29

[0300] (15. Administration of the 1:2 heterotandem complex of EphA2:CD137 induces a dramatic immune response in a mouse tumor model) Six- to eight-week-old female C57BL / 6J-hCD137 mice [B-hTNFRSF9 (CD137) mice; Biocytogen] were subcutaneously implanted with 1 × 10 6 MC38 cells. When the mean tumor volume reached approximately 240 mm 3 , the mice were randomly assigned to treatment groups and treated intravenously (IV) with vehicle (25 mM histidine, 10% sucrose, pH 7), IV with 15 mg / kg of BCY12491 (a 1:2 heterotandem complex of EphA2:CD137), IV with 15 mg / kg of BCY13626 (a non-binding control of EphA2), or intraperitoneally with 2 mg / kg of anti-CD137 (urelumab analog) (n = 6 / treatment cohort). All treatments were administered Q3D for three doses, and 1 hour after the last dose, tumor tissue was harvested. A portion of the tumor tissue was used for RNA isolation for transcriptional analysis, and a portion of the tumor tissue was used for preparation of formalin-fixed paraffin-embedded (FFPE) samples for immunohistochemical (IHC) analysis. RNA was isolated from tumor tissue using the RNAeasy kit [Qiagen], and transcriptional analysis was performed from 100 ng RNA / tumor using the nCounter Mouse PanCancer IO 360 panel (Nanostring). Data were analyzed using nSolver analysis software (Nanostring). CD8+ tumor-infiltrating cells were stained in FFPE tissue sections using an anti-mouse CD8 antibody (Abcam, # ab217344) and the Ventana Discovery OmniMap anti-rabbit HRP kit (Ventana #760 4310).

[0301] The results of this test are shown in FIGS. 15A - D. In this test, by transcriptional analysis, compared to tumors from vehicle - treated mice, significant increases in immune cell scores such as the cytotoxic cell score (FIG. 15A), macrophage cell (FIG. 15B), and T - cell score (FIG. 15C) in tumor tissues were observed when treated with EphA2 BCY12491. Treatment with anti - CD137 antibody also significantly increased the cytotoxic cell score and T - cell score in tumor tissues, but to a lesser extent than BCY12491. In tumor tissues from non - binding control (BCY13626) - treated animals, no change in immune cell scores was seen. By IHC analysis of CD8+ cells in tumor tissues, strong infiltration of CD8+ cells in tumors from BCY12491 - treated mice was shown compared to tumors from vehicle - or non - binder BCY13626 - treated mice (FIG. 15D). A certain degree of increase in CD8+ cell infiltration was also observed in tumors from anti - CD137 antibody - treated mice. These changes in immune cell scores and CD8+ cells in tumor tissues indicate that agonism of CD137 in tumor tissues by the EphA2:CD137 1:2 heterotandem complex BCY12491 results in significant modulation (increase) of tumor - infiltrating immune cells and immune responses.

[0302] (16. Anti - tumor activity of BCY13272 in the syngeneic MC38 tumor model) Six - to eight - week - old female C57BL / 6J - hCD137 mice [B - hTNFRSF9(CD137) mice; Biocytogen] were subcutaneously implanted with 1×10 6 individual MC38 cells. When the average tumor volume reached approximately 80 mm 3 ³, the mice were randomly assigned to treatment groups (n = 6 / cohort) and treated intravenously (IV) with vehicle (25 mM histidine, 10% sucrose, pH 7), IV with 8 mg / kg of BCY13272, 0.9 mg / kg of BCY13272, and 0.1 mg / kg of BCY13272. All treatments were administered twice a week (BIW) for a total of six doses. Tumor growth was monitored from the start of treatment until day 28. Complete responder animals (n = 7) were followed up until day 62 after the start of treatment, and 2×10 6Re-antigen administration was performed by transplanting individual MC38 tumor cells, and tumor growth was monitored for 28 days. At the same time, age-matched untreated control huCD137 C57Bl / 6 mice (n = 5) were transplanted with 2 × 10 6 individual MC38 tumor cells and monitored for 28 days.

[0303] The results of this experiment can be seen in Figure 18. In this experiment, BCY13272 brought about significant anti-tumor activity, and complete responses were observed at dose levels of 0.9 mg / kg (2 out of 6 complete responders) and 8 mg / kg (5 out of 6 complete responders) (Figure 18A). Different from age-matched untreated control huCD137 C57Bl / 6 mice (tumor engraftment rate 100%), tumor regrowth was not observed in BCY13272 complete responder animals (Figure 18B). These data indicate that BCY13272 has significant anti-tumor activity and that BCY13272 treatment can bring about immunogenic memory in complete responder animals.

[0304] (17. Binding of BCY13272 to EphA2 and CD137 measured by SPR) (a) CD137 Biacore experiments were performed to determine the k a (M -1 s -1 )、k d (s -1 )、K D (nM) values for the heterotandem peptide binding to human CD137 protein. Recombinant human CD137 (R&D systems) was resuspended in PBS and biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the protocol proposed by the manufacturer. The protein was desalted using a spin column to remove uncoupled biotin and taken in PBS.

[0305] For the analysis of peptide binding, a Biacore T200 or Biacore 3000 instrument was used together with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine coupling chemistry at 25 °C using HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, carboxymethyl dextran sulfate was activated by injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) for 7 minutes at a flow rate of 10 μl / min. For capture of streptavidin, the protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl onto the activated chip surface. The remaining activated groups were blocked by injection of 1 M ethanolamine (pH 8.5) for 7 minutes, and biotinylated CD137 was captured to a level of 270 - 1500 RU. The buffer was changed to PBS / 0.05% Tween 20 and a dilution series of the peptide was prepared in this buffer containing a final DMSO concentration of 0.5%. The maximum peptide concentration was 500 nM and six additional two- or three-fold dilutions were made. SPR analysis was performed at 25 °C at a flow rate of 90 μl / min with 60 seconds of association and 900 seconds of dissociation. After each cycle, a regeneration step (10 μl of 10 mM glycine pH 2) was utilized. The data were corrected for volume effects excluding DMSO as necessary. All data were double-referenced against blank injections and a reference surface using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). The data were fitted using a simple 1:1 binding model, taking into account mass transport effects as appropriate.

[0306] (b) EphA2 Biacore experiments were performed to determine the k of BCY13272 binding to human EphA2 protein a (M -1 s -1 )、kd (s -1 )、K D (nM) value was determined.

[0307] EphA2 was biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 hour in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 containing 3× molar excess of biotin with respect to the protein. After dialysis of the reaction mixture into PBS, the degree of labeling was determined using a Fluorescent Biotin Quantitation Kit (Thermo). For analysis of peptide binding, a Biacore T200 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized onto the chip using standard amine coupling chemistry at 25 °C using HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, carboxymethyldextran sulfate was activated by injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min for 7 minutes. For capture of streptavidin, the protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injection of 120 μl onto the activated chip surface. The remaining activated groups were blocked by injection of 1 M ethanolamine (pH 8.5):HBS-N (1:1) for 7 minutes. The buffer was changed to PBS / 0.05% Tween 20 and biotinylated EphA2 was captured to a level of 500 - 1500 RU using a dilution of the protein to 0.2 μM in the buffer. In this buffer containing a final DMSO concentration of 0.5%, a dilution series of the peptide was prepared with a maximum peptide concentration of 50 or 100 nM and six additional two-fold dilutions were made. SPR analysis was performed at 25 °C at a flow rate of 90 μl / min with 60 seconds of association and 900 - 1200 seconds of dissociation. The data were corrected for volume effects excluding DMSO. All data were double referenced against blank injections and a reference surface using standard procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). The data were fitted using a simple 1:1 binding model, taking into account mass transport effects as appropriate.

[0308] Figure 20A shows a sensorgram demonstrating that BCY13272 binds to EphA2 (human) with an affinity of 2.0 nM. Figure 20B shows a sensorgram demonstrating that BCY13272 binds to CD137 (human) with high affinity. Due to the presence of two CD137-binding bicyclics in BCY13272, the dissociation rate of the immobilized CD137 protein is very slow, and the reported K D may be an overestimate (Figure 19B).

[0309] (18. Binding of BCY11863 to nectin-4 and CD137 across four preclinical species) The binding of BCY11863 to its primary targets, nectin-4 and CD137, was characterized using surface plasmon resonance (SPR).

[0310] (a) Nectin-4 BCY11863 binds to cynomolgus monkey, rat, mouse, and human nectin-4 with a K D of 5 - 27 nM as measured by direct binding to the extracellular domain that was biotinylated and captured on the streptavidin sensor chip surface. Table 10: Binding affinity of BCY11863 for biotinylated nectin-4 extracellular domain: SPR data

Table 30

[0311] To understand whether the binding of BCY11863 to nectin-4 changed in the context of a ternary complex, i.e., when also bound to CD137, a multi-component SPR binding assay was developed. First, BCY11863 was captured on human CD137 immobilized on the SPR chip surface, and then nectin-4 from different species was passed over the chip to determine its affinity for the captured BCY11863 (see Figure 21C). The affinity for nectin-4 was generally maintained in the presence of CD137 binding as shown below: Table 11: Binding affinity of BCY11863 for nectin-4 extracellular domain using biotinylated human CD137 as a capture reagent [Table 31]

[0312] (b) CD137 The direct binding of BCY11863 to surface-bound CD137 results in a very slow k off due to the binding force caused by two CD137-binding bicyclic rings in BCY11863 and cannot be accurately measured by SPR (see Figure 21B). Furthermore, biotinylation of cynomolgus CD137 abrogates the binding of BCY11863, probably due to the modification of lysine on the cynomolgus protein that is important for BCY11863 binding. Therefore, a BCY11863 analog (BCY13582) containing a C-terminal biotinylated lysine was tested by SPR to determine the cross-species specificity of BCY11863. Using a reversible biotin capture kit, BCY13582 was captured on the sensor chip and the affinity for nectin-4 from different species was determined. By both strategies, it was shown that these BCY11863 analogs bind to human and cynomolgus CD137 with a K D < 10 nM and bind only negligibly to both mouse CD137 and rat CD137. Table 12: Binding affinity of biotinylated BCY11863 analogs for CD137 extracellular domain: SPR data [Table 32]

[0313] To understand whether the binding of BCY11863 to CD137 changes in the context of a ternary complex, i.e., when also bound to nectin-4, a dual-binding SPR binding assay was developed. First, BCY11863 was captured on human nectin-4 immobilized on the SPR chip surface, and then soluble CD137 from different species was passed over the chip to determine its affinity for the captured BCY11863 (see Figure 21D). The affinity for CD137 was generally maintained in the presence of nectin-4 binding as shown below: Table 13: Binding affinity of BCY11863 for CD137 ECD using biotinylated human nectin-4 as a capture reagent [Table 33]

[0314] Figure 21A shows one example of a sensorgram demonstrating that BCY11863 binds to nectin-4 (human) with an affinity of 4.1 nM. Figure 21B shows a sensorgram demonstrating that BCY11863 binds to CD137 (human) with high affinity. Due to the presence of two CD137-binding bicyclics in BCY11863, the dissociation rate of the immobilized CD137 protein is very slow, and the reported K D may be overestimated (Figure 21B). Figure 21C shows that BCY11863 binds to nectin-4 while the CD137 arm binds to the CD137 protein immobilized on the chip to form a ternary complex. Figure 21D shows that BCY11863 binds to CD137 while the nectin-4-binding arm binds to the nectin-4 protein immobilized on the chip to form a ternary complex. Figure 21E shows the ability of BCY13582 immobilized on an SPR chip to bind to human CD137.

[0315] (19. Selectivity of BCY11863 for nectin-4 and CD137) Necl-4 paralog screening: Biotinylated Necl-1 (2880-N1, R&D Systems), Necl-2 (2229-N2, R&D Systems), Necl-3 (3064-N3, R&D Systems), Necl-like-1 (3678-S4-050, R&D Systems), Necl-like-2 (3519-S4-050, R&D Systems), Necl-like-3 (4290-S4-050, R&D Systems), Necl-like-4 (4164-S4, R&D Systems), and Necl-like-5 (2530-CD-050, R&D Systems) were immobilized on streptavidin surfaces, and SPR was used to evaluate the binding of BCY11863 to these. BCY11863 did not show binding to these targets up to a concentration of 5000 nM.

[0316] CD137 paralog screening: SPR for soluble TNF family receptors OX40 and CD40 was used to evaluate the binding of streptavidin-captured BCY13582 (biotinylated BCY11863). BCY13582 did not bind to these targets up to a concentration of 100 nM.

[0317] Retrogenix microarray screening: Retrogenix cell microarray technology was used to screen for specific off-target binding interactions of biotinylated BCY11863 known as BCY13582.

[0318] Examination of the binding levels of the test peptide to immobilized untransfected HEK293 cells and cells overexpressing Necl-4 and CD137 (TNFRSF9) indicated that 1 μM of the test peptide was a suitable screening concentration. Under these conditions, the test peptide was screened for binding to human HEK293 cells individually expressing 5484 full-length human plasma membrane and secreted proteins. As a result, nine primary hits including Necl-4 and CD137 were identified.

[0319] Each primary hit was re-expressed together with two control receptors (TGFBR2 and EGFR) and retested using 1 μM BCY13582 test peptide, 1 μM BCY13582 test peptide in the presence of 100 μM BCY11863, and other positive and negative control treatments (Figure 4). After removing non-specific, non-reproducible, and non-significant hits, three specific interactions for the test peptides remained. These were nectin-4 in non-tethered and tethered forms and CD137 - the primary targets.

[0320] No specific off-target interactions for BCY13582 were identified, indicating high specificity for its primary target.

[0321] (Antitumor activity of BCY11863 in a syngeneic nectin-4 overexpressing MC38 tumor model (MC38#13) when administered at 5 mg / kg at 20.0, 24 h and 10 mg / kg at 0 h twice a week) Six - to eight - week - old female C57BL / 6J - hCD137 mice [B - hTNFRSF9(CD137) mice; Biocytogen] were subcutaneously implanted with 1×10 6 MC38#13 cells (MC38 cells modified to overexpress mouse nectin - 4). When the mean tumor volume reached approximately 95 mm 3 ³, the mice were randomly assigned to treatment groups (n = 6 / cohort) and treated with a weekly dose of vehicle (25 mM histidine, 10% sucrose, pH 7) or 10 mg / kg of BCY11863 at two different dosing schedules for two dosing cycles (5 mg / kg at 0 h and 24 h on D0 and D7 or 10 mg / kg of BCY11863 at 0 h on D0 and D7). All treatments were administered intravenously (IV). Tumor growth was monitored from the start of treatment until day 15.

[0322] BCY11863 produced significant antitumor activity on both dosing schedules. However, when the complete responses were analyzed on day 15 after treatment initiation, the dosing schedule of 5 mg / kg administered at 0 h and 24 h was superior to the 10 mg / kg dose administered at 0 h (Figure 23). Administration of 5 mg / kg of BCY11863 at 0 h and 24 h on D0 and D7 resulted in complete tumor responses in 4 out of 6 cases, whereas administration of 10 mg / kg of BCY11863 at 0 h on D0 and D7 resulted in complete tumor responses in 1 out of 6 cases. These data, combined with the BCY11863 mouse plasma PK data, indicate that almost complete antitumor responses are achieved in the MC38#13 tumor model when BCY11863 plasma exposure is maintained at levels provided by 5 mg / kg doses at 0 h and 24 h in a once-weekly cycle.

[0323] (21. Antitumor Activity of BCY11863 in a Syngeneic Neclotin-4 Overexpressing MC38 Tumor Model (MC38#13)) Female C57BL / 6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] at 6 - 8 weeks of age were subcutaneously implanted with 1 × 10 6 MC38#13 cells (MC38 cells modified to overexpress mouse nectin-4) at three weekly doses of 3, 10, and 30 mg / kg, dosed once a week, once every two weeks, and once a day. When the average tumor volume reached approximately 107 mm 3 The mice were randomly assigned to treatment groups (n = 6 / cohort) and treated with 21 daily doses of vehicle (25 mM histidine, 10% sucrose, pH 7). BCY11863 treatment was performed at three different total dose levels (total weekly doses of 3, 10, and 30 mg / kg) divided into three different schedules (QD: daily; BIW: twice a week, or QW: once a week). Different BCY11863 treatment cohorts received either 21 daily doses (0.43, 1.4, or 4.3 mg / kg), 6 twice-weekly doses (1.5, 5, or 15 mg / kg), or 3 once-weekly doses (3, 10, or 30 mg / kg). All treatments were administered intravenously (IV). When the tumor reached 2000 mm3 Tumor growth was monitored until a volume of more than 3 was reached or until 31 days after the start of treatment. Complete responders (animals without palpable tumors) were observed until D52.

[0324] BCY11863 produced significant antitumor activity in many dosing schedules, and the BIW dosing schedule, especially the BIW dosing at 5 mg / kg, was the most effective schedule. This is shown by the number of complete responder animals on day 52. On day 52 after the start of treatment, 15 / 18 mice treated BIW with BCY11863 were complete responders, 12 / 18 mice treated QD with BCY11863 were complete responders, and 6 / 18 mice treated QW with BCY11863 were complete responders. The BIW dosing at 5 mg / kg resulted in a 100% complete response rate with 6 / 6 CR (Figure 24). These data combined with the BCY11863 mouse plasma PK data indicate that continuous BCY11863 plasma exposure is not required for the antitumor response to BCY11863 in the MC38#13 tumor model.

[0325] (22. In Vivo Efficacy Study of EphA2 Heterotandem Bicyclic Complex) Six- to eight-week-old female C57BL / 6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] were subcutaneously implanted with 1 × 10 6 MC38 cells. When the average tumor volume reached approximately 76 mm 3 the mice were randomly assigned to treatment groups (n = 6 / cohort) and treated with a once-daily dose of vehicle (25 mM histidine, 10% sucrose, pH 7). BCY12491 treatment was performed at two different dose levels (5 and 15 mg / kg) and two different dosing schedules (QD: once daily; Q3D: once every 3 days). The animals received either 22 QD doses or 8 Q3D doses intraperitoneally (ip). The tumor was 2000 mm 3Tumor growth was monitored until a volume exceeding [volume] was reached or until 73 days after the start of treatment. After 73 days, five complete responder animals were re-challenged with MC38 tumor cell transplantation together with five untreated C57BL / 6J-hCD137 mice. Tumor growth was monitored for 20 days.

[0326] BCY12491 produced significant anti-tumor activity at all doses and dosing schedules used in the study. By day 41 after the start of treatment, 2 out of 6 BCY12491 5 mg / kg Q3D-treated animals became complete responders (CR; no palpable tumor remained), 3 out of 6 BCY12491 5 mg / kg QD-treated animals became CR, 4 out of 6 BCY12491 15 mg / kg Q3D-treated animals became CR, and all (6 / 6) of the BCY12491 15 mg / kg QD-treated animals became CR. These data combined with BCY12491 mouse plasma PK data indicate that continuous BCY12491 plasma exposure is not required for the maximum anti-tumor response to BCY12491 in the MC38 tumor model. Furthermore, complete responder animals showed rejection and no tumor growth upon re-challenge with MC38 tumor cell transplantation, whereas untreated mice transplanted with the same tumor cells simultaneously established tumor growth with 100% engraftment rate by day 22 after tumor cell transplantation. This indicates the development of immunogenic memory by BCY12491 treatment resulting in a complete tumor response (Figure 27).

[0327] The dependence of BCY12491 activity on various immune cell populations was determined when treating MC38 tumor-bearing C57BL / 6J-hCD137 mice with depleted CD8+ T cells or NK1.1+ NK cells with BCY12491. Female C57BL / 6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] at 6 - 8 weeks of age were given 1×10 6Individual MC38#13 cells (a clone of MC38 modified to overexpress nectin-4) were subcutaneously implanted. Three days after cell implantation, mice received ip injection of vehicle (PBS), 100 μg of anti-CD8 depletion antibody (rat IgG2b, clone 2.42) or anti-NK depletion antibody (mouse IgG2a, clone PK136) (or combinations thereof) or corresponding isotype control antibodies (rat IgG2b isotype control or mouse IgG2a isotype control). Mice received additional doses of depletion antibody (or isotype control) 5 and 10 days after the first antibody administration. Cell depletion was confirmed by flow cytometry 4 and 12 days after the first depletion antibody administration. When the tumor volume reached approximately 111 mm 3 ³ (5 days after the first depletion antibody administration), mice began to receive vehicle or BCY12491 intravenously (iv) at 15 mg / kg twice a week (BIW). Mice received a total of 4 doses of BCY12491. Tumor growth was monitored until day 28 or until the tumor volume exceeded 2000 mm 3 ³.

[0328] Treatment with BCY12491 resulted in a significant decrease in tumor growth rate and an increase in survival in MC38#13 tumor-bearing mice treated with vehicle or isotype control antibody. The benefit of BCY12491 treatment on tumor growth rate and survival was lost in CD8-depleted mice. Depletion of NK1.1+ cells did not affect the antitumor activity of BCY12491 treatment and the subsequent survival benefit. This data indicates that the activity of BCY12491 in the MC38#13 tumor model is dependent on CD8+ T cells but not on NK1.1+ NK cells (Figure 28).

[0329] The antitumor activities of BCY12730 and BCY12723 were shown together with BCY12491 activity. Six- to eight-week-old female C57BL / 6J-hCD137 mice [B-hTNFRSF9 (CD137) mice; Biocytogen] were subcutaneously implanted with 1×10 6 ⁶ individual MC38 cells. When the average tumor volume reached approximately 92 mm 3When the mice reached [a certain condition], they were randomly assigned to the treatment groups (n = 6 / cohort) and intravenously treated with the Q3D dose of vehicle (25 mM histidine, 10% sucrose, pH 7), 15 mg / kg of BCY12730, BCY12723, or BCY12491 (7 Q3D doses). Tumor growth was monitored for 28 days or until the tumor exceeded 2000 mm 3 Tumor growth was monitored until the tumor exceeded 2000 mm or for 28 days. BCY12491, BCY12730, and BCY12723 showed significant antitumor activity resulting in complete responses in 4 out of 6 BCY12491 - treated animals, 3 out of 6 BCY12730 - treated animals, and 2 out of 6 BCY12723 - treated animals (Figure 29).

[0330] The antitumor activities of BCY13048 and BCY13050 were shown together with BCY12491 activity. Female C57BL / 6J - hCD137 mice [B - hTNFRSF9(CD137) mice; Biocytogen] at 6 - 8 weeks of age were subcutaneously implanted with 1×10 6 MC38 cells. When the average tumor volume reached approximately 76 mm 3 the mice were randomly assigned to the treatment groups (n = 6 / cohort) and intravenously treated with the twice - weekly (BIW) dose of vehicle (25 mM histidine, 10% sucrose, pH 7), 5 mg / kg of BCY13048, BCY13050, or BCY12491 (6 BIW doses). Tumor growth was monitored for 28 days or until the tumor exceeded 2000 mm 3 Tumor growth was monitored until the tumor exceeded 2000 mm or for 28 days. BCY12491, BCY13048, and BCY13050 showed significant antitumor activity resulting in complete responses in 2 out of 6 BCY12491 - treated animals, 5 out of 6 BCY13048 - treated animals, and 3 out of 6 BCY13050 - treated animals (Figure 30). This application provides an invention in the following aspects. (Aspect 1) (a) A first peptide ligand that binds to a component present on a cancer cell; via a linker, (b) Two or more second peptide ligands that bind to a component present on an immune cell; Conjugated to: Comprising, 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 a covalent bond with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, a heterotandem bicyclic peptide complex. (Aspect 2) The heterotandem bicyclic peptide complex according to Aspect 1, wherein the immune cell is selected from leukocytes; lymphocytes (e.g., T lymphocytes or T cells, B cells, or natural killer cells); CD8 or CD4; CD8; dendritic cells, follicular dendritic cells, and granulocytes. (Aspect 3) The heterotandem bicyclic peptide complex according to Aspect 1 or Aspect 2, wherein the reactive group is selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine residues. (Aspect 4) The heterotandem bicyclic peptide complex according to any one of Aspects 1 to 3, wherein the component present on the immune cell is CD137. (Aspect 5) The heterotandem bicyclic peptide complex according to any one of Aspects 1 to 4, wherein the two or more second peptide ligands comprise CD137-binding bicyclic peptide ligands. (Aspect 6) The CD137-binding bicyclic peptide ligand is (Chemical formula 1) TIFF0007704732000160.tif240170 (where [MerPro] i 、C i 、C ii 、C iii and [Cysam] iii represents the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, where Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, Aad represents α-L-aminoadipic acid, MerPro represents 3-mercaptopropionic acid, Cysam represents cysteamine, and NMeAla represents N-methyl-alanine) The heterotandem bicyclic peptide complex according to embodiment 5, comprising an amino acid sequence selected from: or a pharmaceutically acceptable salt thereof. (Embodiment 7) The CD137-binding bicyclic peptide ligand is (Formula 2) TIFF0007704732000161.tif10170 (where C i 、C ii and C iii each represent the first, second, and third cysteine residues, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, and Nle represents norleucine) The heterotandem bicyclic peptide complex according to embodiment 5 or embodiment 6, comprising an amino acid sequence that is: or a pharmaceutically acceptable salt thereof. (Embodiment 8) The CD137-binding bicyclic peptide ligand contains N- and / or C-terminal modifications, and Ac-A-(SEQ ID NO: 5)-Dap (referred to herein as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (referred to herein as BCY7741); Ac-(SEQ ID NO: 6)-Dap (referred to herein as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (referred to herein as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (referred to herein as BCY8045); Ac-(SEQ ID NO: 8)-A (referred to herein as BCY8919); Ac-(SEQ ID NO: 9)-A (referred to herein as BCY8920); Ac-(SEQ ID NO: 10)-A (referred to herein as BCY8927); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); Ac-(SEQ ID NO: 60)-Dap(PYA) (referred to herein as BCY11144); Ac-A-(SEQ ID NO: 61)-K (referred to herein as BCY11613); Ac-(SEQ ID NO: 62)-Dap(PYA) (referred to herein as BCY12023); Ac-(SEQ ID NO: 63) (referred to herein as BCY12149); Ac-(SEQ ID NO: 64) (referred to herein as BCY12143); Ac-(SEQ ID NO: 65) (referred to herein as BCY12147); Ac-(SEQ ID NO: 66) (referred to herein as BCY12145); Ac-(SEQ ID NO: 67) (referred to herein as BCY12146); Ac-(SEQ ID NO: 68) (referred to herein as BCY12150); Ac-(SEQ ID NO: 69) (referred to herein as BCY12352); Ac-(SEQ ID NO: 72)-[1,2-diaminoethane] (referred to herein as BCY12358); [Palmitic acid]-[yGlu]-[yGlu]-(SEQ ID NO: 73) (referred to herein as BCY12360); Ac-(SEQ ID NO: 75) (referred to herein as BCY12381); Ac-(SEQ ID NO: 76) (referred to herein as BCY12382); Ac-(SEQ ID NO: 77)-K (referred to herein as BCY12357); Ac-(SEQ ID NO: 78)-[dA] (referred to herein as BCY13095); [Ac]-(SEQ ID NO: 78)-K (referred to herein as BCY13389); Ac-(SEQ ID NO: 79)-[dA] (referred to herein as BCY13096); and Ac-(SEQ ID NO: 80) (referred to herein as BCY13097); (wherein Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid), or a pharmaceutically acceptable salt thereof : A heterotandem bicyclic peptide complex according to embodiment 6 or embodiment 7, comprising. (Embodiment 9) The CD137-binding bicyclic peptide ligand comprises N- and / or C-terminal modifications, and Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); (wherein Ac represents an acetyl group), or a pharmaceutically acceptable salt thereof : A heterotandem bicyclic peptide complex according to any one of embodiments 6 to 8, comprising. (Embodiment 10) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 3, wherein the component present on the immune cell is OX40. (Embodiment 11) The heterotandem bicyclic peptide complex according to any one of aspects 1 to 3 and 10, wherein the two or more second peptide ligands include an OX40-binding bicyclic peptide ligand. (Aspect 12) The OX40-binding bicyclic peptide ligand has the amino acid sequence: (Chemical formula 3) TIFF0007704732000162.tif131170TIFF0007704732000163.tif247170TIFF0007704732000164.tif109170 For example: (Chemical formula 4) TIFF0007704732000165.tif9170 (where C i 、C ii and C iii each represent a first, second, and third cysteine residue), or a modified derivative, or a pharmaceutically acceptable salt thereof: The heterotandem bicyclic peptide complex according to aspect 11. (Aspect 13) The OX40-binding bicyclic peptide ligand further includes N- and / or C-terminal modifications, and A-(SEQ ID NO: 82)-A-[Sar6]-[KBiot] (referred to herein as BCY10551); A-(SEQ ID NO: 82)-A (referred to herein as BCY10371); A-(SEQ ID NO: 84)-A-[Sar6]-[KBiot] (referred to herein as BCY10552); [Biot]-G-[Sar5]-A-(SEQ ID NO: 84)-A (referred to herein as BCY10479); A-(SEQ ID NO: 84)-A (referred to herein as BCY10378); [Biot]-G-[Sar5]-A-(SEQ ID NO: 85)-A (referred to herein as BCY11371); A-(SEQ ID NO: 85)-A (referred to herein as BCY10743); [Biot]-G-[Sar5]-A-(SEQ ID NO: 87)-A (referred to herein as BCY10482); A-(SEQ ID NO: 87)-A-[Sar6]-[KBiot] (referred to herein as BCY10549); A-(SEQ ID NO: 87)-A-K(Pya) (referred to herein as BCY11607); Ac-A-(SEQ ID NO: 87)-A-K(Pya) (referred to herein as BCY12708); A-(SEQ ID NO: 87)-A (referred to herein as BCY10351); A-(SEQ ID NO: 88)-A-[Sar6]-[KBiot] (referred to herein as BCY11501); A-(SEQ ID NO: 88)-A (referred to herein as BCY10729); A-(SEQ ID NO: 89)-A-[Sar6]-[KBiot] (referred to herein as BCY10550); A-(SEQ ID NO: 89)-A (referred to herein as BCY10361); A-(SEQ ID NO: 90)-A-[Sar6]-[KBiot] (referred to as BCY10794 in this specification); A-(SEQ ID NO: 90)-A (referred to as BCY10349 in this specification); [Biot]-G-[Sar5]-A-(SEQ ID NO: 91)-A (referred to as BCY11369 in this specification); A-(SEQ ID NO: 91)-A (referred to as BCY10331 in this specification); A-(SEQ ID NO: 92)-A (referred to as BCY10375 in this specification); A-(SEQ ID NO: 93)-A (referred to as BCY10364 in this specification); A-(SEQ ID NO: 94)-A (referred to as BCY10365 in this specification); A-(SEQ ID NO: 95)-A (referred to as BCY10366 in this specification); A-(SEQ ID NO: 96)-A (referred to as BCY10367 in this specification); A-(SEQ ID NO: 97)-A (referred to as BCY10368 in this specification); A-(SEQ ID NO: 98)-A (referred to as BCY10369 in this specification); A-(SEQ ID NO: 99)-A (referred to as BCY10374 in this specification); A-(SEQ ID NO: 100)-A (referred to as BCY10376 in this specification); A-(SEQ ID NO: 101)-A (referred to as BCY10737 in this specification); A-(SEQ ID NO: 102)-A (referred to as BCY10738 in this specification); A-(SEQ ID NO: 103)-A (referred to as BCY10739 in this specification); A-(SEQ ID NO: 104)-A (referred to as BCY10740 in this specification); A-(SEQ ID NO: 105)-A (referred to as BCY10741 in this specification); A-(SEQ ID NO: 106)-A (referred to as BCY10742 in this specification); A-(SEQ ID NO: 107)-A (referred to as BCY10380 in this specification); A-(SEQ ID NO: 108)-A (referred to as BCY10370 in this specification); A-(SEQ ID NO: 109)-A (referred to as BCY10372 in this specification); A-(SEQ ID NO: 110)-A (referred to as BCY10373 in this specification); A-(SEQ ID NO: 111)-A (referred to as BCY10379 in this specification); A-(SEQ ID NO: 112)-A (referred to as BCY10377 in this specification); A-(SEQ ID NO: 113)-A (referred to as BCY10744 in this specification); A-(SEQ ID NO: 114)-A (referred to as BCY10343 in this specification); A-(SEQ ID NO: 115)-A (referred to as BCY10350 in this specification); A-(SEQ ID NO: 116)-A (referred to as BCY10352 in this specification); A-(SEQ ID NO: 117)-A (referred to as BCY10353 in this specification); A-(SEQ ID NO: 118)-A (referred to as BCY10354 in this specification); A-(SEQ ID NO: 119)-A (referred to as BCY10730 in this specification); A-(SEQ ID NO: 120)-A (referred to as BCY10731 in this specification); A-(SEQ ID NO: 121)-A (referred to as BCY10339 in this specification); A-(SEQ ID NO: 122)-A (referred to as BCY10340 in this specification); A-(SEQ ID NO: 123)-A (referred to as BCY10342 in this specification); A-(SEQ ID NO: 124)-A (referred to as BCY10345 in this specification); A-(SEQ ID NO: 125)-A (referred to as BCY10347 in this specification); A-(SEQ ID NO: 126)-A (referred to as BCY10348 in this specification); A-(SEQ ID NO: 127)-A (referred to as BCY10720 in this specification); A-(SEQ ID NO: 128)-A (referred to as BCY10721 in this specification); A-(SEQ ID NO: 129)-A (referred to as BCY10722 in this specification); A-(SEQ ID NO: 130)-A (referred to as BCY10723 in this specification); A-(SEQ ID NO: 131)-A (referred to as BCY10724 in this specification); A-(SEQ ID NO: 132)-A (referred to as BCY10725 in this specification); A-(SEQ ID NO: 133)-A (referred to as BCY10726 in this specification); A-(SEQ ID NO: 134)-A (referred to as BCY10727 in this specification); A-(SEQ ID NO: 135)-A (referred to as BCY10728 in this specification); A-(SEQ ID NO: 136)-A (referred to as BCY10360 in this specification); A-(SEQ ID NO: 137)-A (referred to as BCY10363 in this specification); A-(SEQ ID NO: 138)-A (referred to as BCY10732 in this specification); A-(SEQ ID NO: 139)-A (referred to as BCY10733 in this specification); A-(SEQ ID NO: 140)-A (referred to as BCY10734 in this specification); A-(SEQ ID NO: 141)-A (referred to as BCY10735 in this specification); A-(SEQ ID NO: 142)-A (referred to as BCY10736 in this specification); A-(SEQ ID NO: 143)-A (referred to as BCY10336 in this specification); A-(SEQ ID NO: 144)-A (referred to as BCY10337 in this specification); A-(SEQ ID NO: 145)-A (referred to as BCY10338 in this specification); A-(SEQ ID NO: 146)-A (referred to as BCY10346 in this specification); A-(SEQ ID NO: 147)-A (referred to as BCY10357 in this specification); A-(SEQ ID NO: 148)-A (referred to as BCY10362 in this specification); A-(SEQ ID NO: 149)-A (referred to as BCY10332 in this specification); A-(SEQ ID NO: 150)-A (referred to as BCY10717 in this specification); A-(SEQ ID NO: 151)-A (referred to as BCY10718 in this specification); A-(SEQ ID NO: 152)-A (referred to as BCY10334 in this specification); and A-(SEQ ID NO: 153)-A (referred to as BCY10719 in this specification); For example: A-(SEQ ID NO: 87)-A-K(Pya) (referred to as BCY11607 in this specification); (where Pya represents a 4-pentynoyl moiety) : The heterotandem bicyclic peptide complex according to embodiment 12, comprising an amino acid sequence selected from (Embodiment 14) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 13, 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. (Embodiment 15) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 14, wherein the component present on the cancer cells is nectin-4. (Embodiment 16) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 15, wherein the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand. (Embodiment 17) the nectin-4 binding bicyclic peptide ligand is (Formula 5) TIFF0007704732000166.tif136170 (wherein, [MerPro] i 、C i 、C ii 、C iii , and [Cysam] iii represent first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, Sar 10 represents 10 sarcosine units, B-Ala represents β-alanine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine) : the heterotandem bicyclic peptide complex according to embodiment 16, comprising an amino acid sequence selected from or a pharmaceutically acceptable salt thereof. (Embodiment 18) the nectin-4 binding bicyclic peptide ligand optionally includes an N-terminal modification, and SEQ ID NO: 1 (referred to herein as BCY8116); [PYA]-[B-Ala]-[Sar10 -(SEQ ID NO: 1) (referred to herein as BCY8846); [PYA]-(SEQ ID NO: 1) (referred to herein as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO: 1) (referred to herein as BCY11016); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 2) (referred to herein as BCY11942); Ac-(SEQ ID NO: 3) (referred to herein as BCY8831); SEQ ID NO: 4 (referred to herein as BCY11414); [PYA]-[B-Ala]-(SEQ ID NO: 14) (referred to herein as BCY11143); palmitic acid-yGlu-yGlu-(SEQ ID NO: 14) (referred to herein as BCY12371); Ac-(SEQ ID NO: 14) (referred to herein as BCY12024); Ac-(SEQ ID NO: 16) (referred to herein as BCY12364); Ac-(SEQ ID NO: 18) (referred to herein as BCY12366); and Ac-(SEQ ID NO: 19) (referred to herein as BCY12367); (wherein, PYA represents 4-pentynoic acid, B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units), or a pharmaceutically acceptable salt thereof : the heterotandem bicyclic peptide complex according to embodiment 17, comprising. (Embodiment 19) The heterotandem bicyclic peptide complex according to embodiment 17 or 18, wherein the nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO: 1 (referred to herein as BCY8116). (Embodiment 20) The heterotandem bicyclic peptide complex according to any one of embodiments 15 to 19, selected from any one of the complexes listed in Tables A and B, such as BCY11027, BCY11863, and BCY11864. (Embodiment 21) The heterotandem bicyclic peptide complex according to any one of embodiments 15 to 19, selected from any one of the complexes listed in Table E, such as BCY12967. (Embodiment 22) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 14, wherein the component present on the cancer cell is EphA2. (Embodiment 23) The EphA2 binding bicyclic peptide ligand is (Formula 6) TIFF0007704732000167.tif249170TIFF0007704732000168.tif16170 (where [MerPro] i 、C i 、C ii 、C iii , and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, HyP represents trans-4-hydroxy-L-proline, HArg represents homoarginine, PYA represents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, 1Nal represents 1-naphthylalanine, hGlu represents homoglutamic acid, Thi represents thienyl-alanine, 4ThiAz represents β-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 3Thi represents, and palmitoyl-Glu-LysN 3 [PYA] is (Formula 7) TIFF0007704732000169.tif134170: represents, and [K(PYA-(palmitoyl-Glu-LysN 3 )] is (Formula 8) TIFF0007704732000170.tif146170 : represents, Nle represents norleucine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine) : The heterotandem bicyclic peptide complex according to embodiment 22, comprising an amino acid sequence selected from the above or a pharmaceutically acceptable salt thereof. (Embodiment 24) The EphA2 binding bicyclic peptide ligand is (Formula 9) TIFF0007704732000171.tif9170 (where C i 、C ii , and C iii represent the first (i), second (ii), and third (iii) cysteine groups, HyP represents trans-4-hydroxy-L-proline, and HArg represents homoarginine) : The heterotandem bicyclic peptide complex according to embodiment 23, comprising the amino acid sequence or a pharmaceutically acceptable salt thereof. (Embodiment 25) The EphA2-binding bicyclic peptide ligand is (Formula 10) TIFF0007704732000172.tif9170 (where C i 、C ii , and C iii represent the first (i), second (ii), and third (iii) cysteine groups, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine) : The heterotandem bicyclic peptide complex according to embodiment 23, comprising the amino acid sequence or a pharmaceutically acceptable salt thereof. (Embodiment 26) The EphA2-binding bicyclic peptide ligand optionally contains an N-terminal modification and A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); [B-Ala]-[Sar 10 -A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY6099); [PYA]-A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY11813); Ac-A-[HArg]-D-(SEQ ID NO: 24)-[K(PYA)] (referred to herein as BCY11814); Ac-A-[HArg]-D-(SEQ ID NO: 24)-K (referred to herein as BCY12734); [NMeAla]-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY13121); [Ac]-(SEQ ID NO: 24)-L[dH]G[dK] (referred to herein as BCY13125); [PYA]-[B-Ala]-[Sar 10 -VGP-(SEQ ID NO: 25) (referred to herein as BCY8941); Ac-A-[HArg]-D-(SEQ ID NO: 26) (referred to herein as BCY11815); Ac-A-[HArg]-D-(SEQ ID NO: 27) (referred to herein as BCY11816); Ac-A-[HArg]-D-(SEQ ID NO: 28) (referred to herein as BCY11817); Ac-A-[HArg]-D-(SEQ ID NO: 29) (referred to herein as BCY12735); (Palmitoyl-Glu-LysN 3 )[PYA]A[HArg]D-(SEQ ID NO: 29) (hereinafter known as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO: 30) (referred to herein as BCY12736); Ac-A-[HArg]-D-(SEQ ID NO: 31) (referred to as BCY12737 herein); A-[HArg]-D-(SEQ ID NO: 32) (referred to as BCY12738 herein); A-[HArg]-E-(SEQ ID NO: 32) (referred to as BCY12739 herein); A-[HArg]-D-(SEQ ID NO: 33) (referred to as BCY12854 herein); A-[HArg]-D-(SEQ ID NO: 34) (referred to as BCY12855 herein); A-[HArg]-D-(SEQ ID NO: 35) (referred to as BCY12856 herein); A-[HArg]-D-(SEQ ID NO: 35)-[dA] (referred to as BCY12857 herein); (SEQ ID NO: 35)-[dA] (referred to as BCY12861 herein); [NMeAla]-[HArg]-D-(SEQ ID NO: 35) (referred to as BCY13122 herein); [dA]-ED-(SEQ ID NO: 35) (referred to as BCY13126 herein); [dA]-[dA]-D-(SEQ ID NO: 35) (referred to as BCY13127 herein); AD-(SEQ ID NO: 35) (referred to as BCY13128 herein); A-[HArg]-D-(SEQ ID NO: 36) (referred to as BCY12858 herein); A-[HArg]-D-(SEQ ID NO: 37) (referred to as BCY12859 herein); Ac-(SEQ ID NO: 37)-[dK] (referred to as BCY13120 herein); A-[HArg]-D-(SEQ ID NO: 38) (referred to as BCY12862 herein); A-[HArg]-D-(SEQ ID NO: 39) (referred to as BCY12863 herein); [dA]-[HArg]-D-(SEQ ID NO: 39)-[dA] (referred to as BCY12864 herein); (SEQ ID NO: 40)-[dA] (referred to as BCY12865 herein); A-[HArg]-D-(SEQ ID NO: 41) (referred to as BCY12866 herein); A-[HArg]-D-(SEQ ID NO: 42) (referred to as BCY13116 herein); A-[HArg]-D-(SEQ ID NO: 43) (referred to as BCY13117 herein); A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); [dA]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13123); [d1Nal]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13124); A-[HArg]-D-(SEQ ID NO: 47) (referred to herein as BCY13130); A-[HArg]-D-(SEQ ID NO: 48) (referred to herein as BCY13131); A-[HArg]-D-(SEQ ID NO: 49) (referred to herein as BCY13132); A-[HArg]-D-(SEQ ID NO: 50) (referred to herein as BCY13134); A-[HArg]-D-(SEQ ID NO: 51) (referred to herein as BCY13135); (SEQ ID NO: 154)-[dK] (referred to herein as BCY13129); A[HArg]D-(SEQ ID NO: 155) (referred to herein as BCY13133); A[HArg]D-(SEQ ID NO: 156) (referred to herein as BCY13917); A[HArg]D-(SEQ ID NO: 157) (referred to herein as BCY13918); A[HArg]D-(SEQ ID NO: 158) (referred to herein as BCY13919); A[HArg]D-(SEQ ID NO: 159) (referred to herein as BCY13920); A[HArg]D-(SEQ ID NO: 160) (referred to herein as BCY13922); A[HArg]D-(SEQ ID NO: 161) (referred to herein as BCY13923); A[HArg]D-(SEQ ID NO: 162) (referred to herein as BCY14047); A[HArg]D-(SEQ ID NO: 163) (referred to herein as BCY14048); and A[HArg]D-(SEQ ID NO: 164) (referred to herein as BCY14313); (wherein, PYA represents 4-pentynoic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, HArg represents homoarginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, palmitoyl-Glu-LysN 3 [PYA] is (Chemical Formula 11) TIFF0007704732000173.tif136170 : (represented by), or a pharmaceutically acceptable salt thereof : comprising the heterotandem bicyclic peptide complex according to any one of aspects 23 to 25. (Aspect 27) The EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal modification, and A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); (where HArg represents homoarginine), or a pharmaceutically acceptable salt thereof : comprising the heterotandem bicyclic peptide complex according to any one of aspects 23 to 26. (Aspect 28) The EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal modification, and A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); (where HArg represents homoarginine), or a pharmaceutically acceptable salt thereof : comprising the heterotandem bicyclic peptide complex according to any one of aspects 23 to 26. (Aspect 29) The heterotandem bicyclic peptide complex according to any one of aspects 22 to 28, selected from any one of the complexes listed in Table C, such as BCY12491, BCY12730, BCY13048, BCY13050, BCY13053, and BCY13272. (Aspect 30) The heterotandem bicyclic peptide complex according to aspect 29, which is BCY12491. (Aspect 31) The heterotandem bicyclic peptide complex according to aspect 29, which is BCY13272. (Aspect 32) The heterotandem bicyclic peptide complex according to any one of aspects 1 to 13, wherein the component present on the cancer cell is PD-L1. (Aspect 33) The PD-L1-binding bicyclic peptide ligand (Formula 12) TIFF0007704732000174.tif55170 (where C i 、C ii , and C iii each represent the first, second, and third cysteine residues, PYA represents 4-pentynoic acid, and HArg represents homoarginine) : The heterotandem bicyclic peptide complex according to aspect 32, comprising an amino acid sequence selected from, or a pharmaceutically acceptable salt thereof. (Aspect 34) The PD-L1-binding bicyclic peptide ligand optionally comprises N-terminal and / or C-terminal modifications, and [PYA]-[B-Ala]-[Sar 10 -SDK-(SEQ ID NO: 52) (referred to herein as BCY10043); Ac-D-[HArg]-(SEQ ID NO: 52)-PSH (referred to herein as BCY11865); Ac-SDK-(SEQ ID NO: 53) (referred to herein as BCY11013); Ac-SDK-(SEQ ID NO: 53)-PSH (referred to herein as BCY10861); Ac-D-[HArg]-(SEQ ID NO: 54)-PSH (referred to herein as BCY11866); Ac-D-[HArg]-(SEQ ID NO: 55)-PSH (referred to herein as BCY11867); Ac-D-[HArg]-(SEQ ID NO: 56)-PSH (referred to herein as BCY11868); Ac-D-[HArg]-(SEQ ID NO: 57)-PSH (referred to herein as BCY11869); Ac-SD-[HArg]-(SEQ ID NO: 58)-PSHK (referred to herein as BCY12479); and Ac-SD-[HArg]-(SEQ ID NO: 59)-PSHK (referred to herein as BCY12477); (wherein PYA represents 4-pentynoic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, and HArg represents homoarginine), or a pharmaceutically acceptable salt thereof : The heterotandem bicyclic peptide complex according to embodiment 33, comprising. (Embodiment 35) The heterotandem bicyclic peptide complex according to any one of embodiments 32 to 34, selected from any one of the complexes listed in Table D. (Embodiment 36) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 5, wherein the two or more second peptide ligands comprise one CD137-binding bicyclic peptide ligand and one OX40-binding bicyclic peptide. (Embodiment 37) The heterotandem bicyclic peptide complex according to embodiment 36, which is a complex listed in Table F. (Embodiment 38) The heterotandem bicyclic peptide complex according to embodiment 1, selected from any one of the complexes listed in Tables G and H. (Embodiment 39) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 38, wherein the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA). (Embodiment 40) The heterotandem bicyclic peptide complex according to any one of embodiments 1 to 39, wherein the pharmaceutically acceptable salt is selected from free acid or sodium, potassium, calcium, ammonium salts. (Embodiment 41) A pharmaceutical composition comprising the heterotandem bicyclic peptide complex according to any one of Aspects 1 to 40 in combination with one or more pharmaceutically acceptable excipients. (Aspect 42) The heterotandem bicyclic peptide complex according to any one of Aspects 1 to 40 for use in the prevention, suppression, or treatment of cancer. (Aspect 43) A method for treating cancer, the method comprising administering the heterotandem bicyclic peptide complex according to any one of Aspects 1 to 40 at a dosing frequency that does not maintain a plasma concentration of the complex that exceeds the in vitro EC 50 of the complex.

Claims

1. (a) A first peptide ligand that binds to a component present on cancer cells selected from EphA2, nectin-4, and PD-L1; via a linker, (b) Conjugated to two or more second peptide ligands that bind to a component present on immune cells that are CD137 or OX40: A heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein each of the peptide ligands comprises a polypeptide containing at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms a covalent bond with the reactive groups of the polypeptide, resulting in the formation of at least two polypeptide loops on the molecular scaffold, (i) The two or more second peptide ligands are: - An amino acid sequence selected from the following: C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 11); (SEQ ID NO: 11)-A (referred to herein as BCY14601); C i IEEGQYC ii FADPY[Nle]C iii (Accession No.: 5); C i [tBuAla]PE[D - Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO: 6); C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (SEQ ID NO: 7); C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO: 8); C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (SEQ ID NO: 9); C i [tBuAla]P[K(PYA)][D - Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO: 10); C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (SEQ ID NO: 12); C i [tBuAla]PE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO: 60); C i IEE[dK(PYA)]QYC ii FADPY[Nle]C iii (Accession No.: 61); C i [tBuAla]EE(dK)PYC ii FADPY[Nle]C iii (SEQ ID NO: 62); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 63); C i [tBuAla]EE[dK(PYA)]PYC ii FADPY[Nle]C iii (Sequence number: 64); C i [tBuAla]PE[dK(PYA)]PYC ii FANPY[Nle]C iii (SEQ ID NO: 65); C i [tBuAla]PE[dK(PYA)]PYC ii FAEPY[Nle]C iii (Accession No.: 66); C i [tBuAla]PE[dK(PYA)]PYC ii FA[Aad]PY[Nle]C iii (SEQ ID NO: 67); C i [tBuAla]PE[dK(PYA)]PYC ii FAQPY[Nle]C iii (SEQ ID NO: 68); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle][Cysam] iii (SEQ ID NO: 69); [MerPro] i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 70; referred to herein as BCY12353); [MerPro] i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle][Cysam] iii (SEQ ID NO: 71; referred to herein as BCY12354); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 72); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 73); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 74; referred to herein as BCY12372); C i [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeAla]Y[Nle]C iii (SEQ ID NO: 75); C i [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeDAla]Y[Nle]C iii (SEQ ID NO: 76); C i [tBuAla]P[K(PYA)][dA]PYC ii FADPY[Nle]C iii (SEQ ID NO: 77); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (Accession No.: 78); C i [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 79); C i [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 80); and [MerPro] i [tBuAla]EE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO: 81; referred to herein as BCY13137); (Here, [MerPro] i , C i , C ii , C iii , and [Cysam] iii represent first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, Aad represents α-L-aminoadipic acid, MerPro represents 3-mercaptopropionic acid, Cysam represents cysteamine, and NMeAla represents N-methyl-alanine), or a pharmaceutically acceptable salt thereof; A CD137-binding bicyclic peptide ligand comprising a polypeptide containing the same, or - An amino acid sequence selected from the following: C i ILWC ii LPEPHDEC iii (Array No.: 82); C i A K / S N / E C ii DPFWYQFYC iii (Array number: 83); C i AKNC ii DPFWYQFYC iii (Sequence number: 84); C i ASEC ii DPFWYQFYC iii (Array number: 85); C i L / N YSPC ii WHPLN D / K C iii (Array number: 86); C i LYSPC ii WHPLNDC iii (Accession No.: 87); C i NYSPC ii WHPLNKC iii (Sequence number: 88); C i WYEYDC ii NNWERC iii (Sequence number: 89); C i VIRYSPC ii SHYLNC iii (Accession No.: 90); C i DYSPWWHPC ii NHIC iii (Accession No.: 91); C i DAC ii LYPDYYVC iii (Accession No.: 92); C i RLWC ii IPAPTDDC iii (Array number: 93); C i TMWC ii IPAKGDWC iii (Sequence number: 94); C i MLWC ii LPAPTDEC iii (Sequence number: 95); C i ILWC ii LPEPPDEC iii (Accession number: 96); C i LLWC ii IPNPDDNC iii (Array number: 97); C i WLWC ii VPNPDDTC iii (Sequence number: 98); C i VLWC ii TPYPGDDC iii (Accession number: 99); C i ALWC ii IPDPQDEC iii (Array number: 100); C i TLWC ii IPDASDSC iii (Array number: 101); C i QLWC ii IPDADDDC iii (Array number: 102); C i QLWC ii VPEPGDSC iii (Accession No.: 103); C i ALWC ii IPEESDDC iii (Array number: 104); C i VLWC ii IPEPQDKC iii (Accession No.: 105); C i TLWC ii IPDPDDSC iii (Array number: 106); C i RLWC ii VPKAEDYC iii (Accession No.: 107); C i TKPC ii IAYYNQSC iii (Accession No.: 108); C i MNPC ii IAYYQQEC iii (Accession No.: 109); C i TNAC ii VAYYHQAC iii (Accession No.: 110); C i SDPC ii ISYYNQAC iii (Sequence number: 111); C i DPPC ii DPFWYAFYC iii (Sequence number: 112); C i PDDC ii DPFWYNFYC iii (Sequence number: 113); C i RYSPC ii YHPHNC iii (Array number: 114); C i LYSPC ii NHPLNSC iii (Accession No.: 115); C i EDNYC ii FMWTPYC iii (Sequence number: 116); C i LDSPC ii WHPLNDC iii (Array number: 117); C i RFSPC ii SHPLNQC iii (Array number: 118); C i KYSPC ii WHPLNLC iii (Sequence number: 119); C i RYSPC ii WHPLNNC iii (Array number: 120); C i EWISC ii PGEPHRWWC iii (Sequence number: 121); C i VWEAC ii PEHPDQWWC iii (Sequence number: 122); C i STWHC ii FWNLQEGKC iii (Sequence number: 123); C i EWKAC ii EHDRERWWC iii (Array number: 124); C i RTWQC ii FYEWQNGHC iii (Accession number: 125); C i KTWDC ii FWASQVSEC iii (Sequence number: 126); C i STWQC ii FYDLQEGHC iii (Sequence number: 127); C i TTWEC ii FYDLQEGHC iii (Sequence number: 128); C i ETWEC ii FWRLQAGEC iii (Sequence number: 129); C i RTWQC ii FWDLQEGLC iii (Sequence number: 130); C i STWQC ii FWDSQLGAC iii (Array number: 131); C i ETWEC ii FWEWQVGSC iii (Sequence number: 132); C i TTWEC ii FWDLQEGLC iii (Accession No.: 133); C i HTWDC ii FYQWQDGHC iii (Sequence number: 134); C i TTWEC ii FYSLQDGHC iii (Sequence number: 135); C i NEDMYC ii FMWMEC iii (Accession No.: 136); C i LYEYDC ii YTWRRC iii (Accession number: 137); C i RYEYDC ii HTWQRC iii (Sequence number: 138); C i WYEYDC ii TTWERC iii (Sequence number: 139); C i WYEYDC ii RTWTRC iii (Sequence number: 140); C i LYEYDC ii HTWTRC iii (Accession number: 141); C i WYEYDC ii RTWTFC iii (Sequence number: 142); C i HGGVWC ii IPNINDSC iii (Accession No.: 143); C i DSPVRC ii YWNTQKGC iii (Accession No.: 144); C i GSPVPC ii YWNTRKGC iii (Accession No.: 145); C i APFEFNC ii YTWRPC iii (Sequence number: 146); C i RVLYSPC ii YHWLNC iii (Sequence number: 147); C i SIMYSPC ii EHPHNHC iii (Sequence number: 148); C i DKWEPDHLC ii YWWC iii (Sequence number: 149); C i DAWPETHVC ii YWWC iii (Sequence number: 150); C i DEYTPEHLC ii YWWC iii (Sequence number: 151); C i WINYSISPC ii YVGEC iii (Accession No.: 152); and C i RYEYPEHLC ii YTWQC iii (Accession No.: 153); (Here, C i , C ii , and C iii each represent a first, second, and third cysteine residue), or a modified derivative or a pharmaceutically acceptable salt thereof; Comprising a polypeptide containing the same, an OX40-binding bicyclic peptide ligand, and (ii) The first peptide ligand is: - An amino acid sequence selected from the following: C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1; referred to herein as BCY8116); C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 3); C i PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 4; referred to herein as BCY11414); C i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (Sequence number: 14); [MerPro] i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 15; referred to herein as BCY12363); C i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO: 16); [MerPro] i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO: 17; referred to herein as BCY12365); C i P[1Nal][dK]C ii M[HArg]HWSTP[HyP]WC iii (Sequence number: 18); C i P[1Nal][dK]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO: 19); C i P[1Nal][dE]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 20; referred to herein as BCY12368); C i P[1Nal][dA]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 21; referred to herein as BCY12369); C i P[1Nal][dE]C ii L[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 22; referred to herein as BCY12370); and C i P[1Nal][dE]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO: 23; referred to herein as BCY12384); (Here, [MerPro] i C i C ii C iii and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, Sar 10 represents 10 sarcosine units, B-Ala represents β-alanine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine), or a pharmaceutically acceptable salt thereof; A nectin-4-binding bicyclic peptide ligand comprising a polypeptide containing the same, or - An amino acid sequence selected from the following: C i [HyP]LVNPLC ii LEP[d1Nal]WTC iii (Accession No.: 44); C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (Accession number: 24); C i LWDPTPC ii ANLHL[HArg]C iii (Accession number: 25); C i [HyP]LVNPLC ii L[K(PYA)]P[dD]W[HArg]C iii (Accession No.: 26); C i [HyP][K(PYA)]VNPLC ii LHP[dD]W[HArg]C iii (Sequence number: 27); C i [HyP]LVNPLC ii [K(PYA)]HP[dD]W[HArg]C iii (Sequence number: 28); C i [HyP]LVNPLC ii LKP[dD]W[HArg]C iii (Accession No.: 29); C i [HyP]KVNPLC ii LHP[dD]W[HArg]C iii (Accession number: 30); C i [HyP]LVNPLC ii KHP[dD]W[HArg]C iii (Accession number: 31); C i [HyP]LVNPLC ii LHP[dE]W[HArg]C iii (Accession number: 32); C i [HyP]LVNPLC ii LEP[dD]W[HArg]C iii (Accession No.: 33); C i [HyP]LVNPLC ii LHP[dD]WTC iii (Sequence number: 34); C i [HyP]LVNPLC ii LEP[dD]WTC iii (Accession No.: 35); C i [HyP]LVNPLC ii LEP[dA]WTC iii (Accession No.: 36); C i [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO: 37; referred to herein as BCY12860); C i [HyP][Cba]VNPLC ii LHP[dD]W[HArg]C iii (Accession number: 38); C i [HyP][Cba]VNPLC ii LEP[dD]WTC iii (Accession No.: 39); C i [HyP][Cba]VNPLC ii L[3,3-DPA]P[dD]WTC iii (Accession No.: 40); C i [HyP]LVNPLC ii L[3,3-DPA]P[dD]W[HArg]C iii (Accession No.: 41); C i [HyP]LVNPLC ii LHP[d1Nal]W[HArg]C iii (Sequence number: 42); C i [HyP]LVNPLC ii L[1Nal]P[dD]W[HArg]C iii (Accession No.: 43); C i [HyP]LVNPLC ii L[1Nal]P[dD]WTC iii (Accession number: 45; referred to herein as BCY13119); C i [HyP][Cba]VNPLC ii LEP[dA]WTC iii (Accession No.: 46); C i [HyP][hGlu]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO: 47); C i [HyP]LVNPLC ii [hGlu]HP[dD]W[HArg]C iii (Sequence number: 48); C i [HyP]LVNPLC ii L[hGlu]P[dD]W[HArg]C iii (SEQ ID NO: 49); C i [HyP]LVNPLC ii LHP[dNle]W[HArg]C iii (Accession number: 50); C i [HyP]LVNPLC ii L[Nle]P[dD]W[HArg]C iii (Accession No.: 51); [MerPro] i [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (Accession No.: 154); C i [HyP]LVNPLC ii LHP[dD]W[HArg][Cysam] iii (SEQ ID NO: 155); C i [HyP]LVNPLC ii L[His3Me]P[dD]W[HArg]C iii (SEQ ID NO: 156); C i [HyP]LVNPLC ii L[His1Me]P[dD]W[HArg]C iii (SEQ ID NO: 157); C i [HyP]LVNPLC ii L[4ThiAz]P[dD]W[HArg]C iii (Accession No.: 158); C i [HyP]LVNPLC ii LFP[dD]W[HArg]C iii (Accession No.: 159); C i [HyP]LVNPLC ii L[Thi]P[dD]W[HArg]C iii (Accession No.: 160); C i [HyP]LVNPLC ii L[3Thi]P[dD]W[HArg]C iii (Accession No.: 161); C i [HyP]LVNPLC ii LNP[dD]W[HArg]C iii (Accession No.: 162); C i [HyP]LVNPLC ii LQP[dD]W[HArg]C iii (Accession No.: 163); and C i [HyP]LVNPLC ii L[K(PYA-(Palmitoyl-Glu-LysN 3 )]P[dD]W[HArg]C iii (SEQ ID NO: 164); (Here, [MerPro] i , C i , C ii , C iii , and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, HyP represents trans-4-hydroxy-L-proline, HArg represents homoarginine, PYA represents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, 1Nal represents 1-naphthylalanine, hGlu represents homoglutamic acid, Thi represents thienyl-alanine, 4ThiAz represents β-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 3Thi represents 3-thienylalanine, palmitoyl-Glu-LysN 3 [PYA] is 【Chemical Formula 1】 represents [K(PYA-(palmitoyl-Glu-LysN 3 )] 【Chemical 2】 representing, Nle represents norleucine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine), or a pharmaceutically acceptable salt thereof; An EphA2-binding bicyclic peptide ligand comprising a polypeptide containing the same, or - An amino acid sequence selected from the following: C i SAGWLTMC ii QKLHLC iii (Accession No.: 52); C i SAGWLTMC ii Q[K(PYA)]LHLC iii (Accession No.: 53); C i SKGWLTMC ii Q[K(Ac)]LHLC iii (Accession number: 54); C i SAGWLTKC ii Q[K(Ac)]LHLC iii (SEQ ID NO: 55); C i SAGWLTMC ii K[K(Ac)]LHLC iii (Accession number: 56); C i SAGWLTMC ii Q[K(Ac)]LKLC iii (Accession No.: 57); C i SAGWLTMC ii Q[HArg]LHLC iii (SEQ ID NO: 58); and C i SAGWLTMC ii [HArg]QLNLC iii (Accession No.: 59); (Here, C i , C ii , and C iii represent the first, second, and third cysteine residues, respectively, PYA represents 4-pentynoic acid, and HArg represents homoarginine), or a pharmaceutically acceptable salt thereof; A PD-L1-binding bicyclic peptide ligand comprising a polypeptide containing the same, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof.

2. The two or more second peptide ligands are an amino acid sequence selected from the following: C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 11); (SEQ ID NO: 11)-A (referred to herein as BCY14601); C i IEEGQYC ii FADPY[Nle]C iii (SEQ ID NO: 5); C i [tBuAla]PE[D - Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO: 6); C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (Accession No.: 7); C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO: 8); C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (SEQ ID NO: 9); C i [tBuAla]P[K(PYA)][D - Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO: 10); C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (SEQ ID NO: 12); C i [tBuAla]PE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO: 60); C i IEE[dK(PYA)]QYC ii FADPY[Nle]C iii (Accession No.: 61); C i [tBuAla]EE(dK)PYC ii FADPY[Nle]C iii (SEQ ID NO: 62); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 63); C i [tBuAla]EE[dK(PYA)]PYC ii FADPY[Nle]C iii (Accession No.: 64); C i [tBuAla]PE[dK(PYA)]PYC ii FANPY[Nle]C iii (SEQ ID NO: 65); C i [tBuAla]PE[dK(PYA)]PYC ii FAEPY[Nle]C iii (SEQ ID NO: 66); C i [tBuAla]PE[dK(PYA)]PYC ii FA[Aad]PY[Nle]C iii (Accession No.: 67); C i [tBuAla]PE[dK(PYA)]PYC ii FAQPY[Nle]C iii (Accession No.: 68); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle][Cysam] iii (SEQ ID NO: 69); [MerPro] i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 70; referred to herein as BCY12353); [MerPro] i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle][Cysam] iii (SEQ ID NO: 71; referred to herein as BCY12354); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 72); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 73); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 74; referred to herein as BCY12372); C i [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeAla]Y[Nle]C iii (SEQ ID NO: 75); C i [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeDAla]Y[Nle]C iii (SEQ ID NO: 76); C i [tBuAla]P[K(PYA)][dA]PYC ii FADPY[Nle]C iii (SEQ ID NO: 77); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 78); C i [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 79); C i [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 80); and [MerPro] i [tBuAla]EE[dK]PYC ii FADPY[Nle]C iii (Sequence number: 81; referred to as BCY13137 in this specification); (Here, [MerPro] i , C i , C ii , C iii , and [Cysam] iii represent first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentenoic acid, Aad represents α-L-aminoadipic acid, MerPro represents 3-mercaptopropionic acid, Cysam represents cysteamine, NMeAla represents N-methyl-alanine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to claim 1, comprising a CD137-binding bicyclic peptide ligand containing the same.

3. The CD137-binding bicyclic peptide ligand has the following amino acid sequence: C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 11); (Here, C i , C ii , and C iii represent the first, second, and third cysteine residues, respectively, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, Nle represents norleucine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to claim 2, containing the same.

4. The CD137-binding bicyclic peptide ligand comprises a polypeptide containing an N- and / or C-terminal modification, and the polypeptide is: Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); Ac-A-(SEQ ID NO: 5)-Dap (referred to herein as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (referred to herein as BCY7741); Ac-(SEQ ID NO: 6)-Dap (referred to herein as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (referred to herein as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (referred to herein as BCY8045); Ac-(SEQ ID NO: 8)-A (referred to herein as BCY8919); Ac-(SEQ ID NO: 9)-A (referred to herein as BCY8920); Ac-(SEQ ID NO: 10)-A (referred to herein as BCY8927); Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); Ac-(SEQ ID NO: 60)-Dap(PYA) (referred to herein as BCY11144); Ac-A-(SEQ ID NO: 61)-K (referred to herein as BCY11613); Ac-(SEQ ID NO: 62)-Dap(PYA) (referred to herein as BCY12023); Ac-(SEQ ID NO: 63) (referred to herein as BCY12149); Ac-(SEQ ID NO: 64) (referred to herein as BCY12143); Ac-(SEQ ID NO: 65) (referred to herein as BCY12147); Ac-(SEQ ID NO: 66) (referred to herein as BCY12145); Ac-(SEQ ID NO: 67) (referred to herein as BCY12146); Ac-(SEQ ID NO: 68) (referred to herein as BCY12150); Ac-(SEQ ID NO: 69) (referred to herein as BCY12352); Ac-(SEQ ID NO: 72)-[1,2-diaminoethane] (referred to herein as BCY12358); [Palmitic acid]-[yGlu]-[yGlu]-(SEQ ID NO: 73) (referred to herein as BCY12360); Ac-(SEQ ID NO: 75) (referred to herein as BCY12381); Ac-(SEQ ID NO: 76) (referred to herein as BCY12382); Ac-(SEQ ID NO: 77)-K (referred to herein as BCY12357); Ac-(SEQ ID NO: 78)-[dA] (referred to herein as BCY13095); [Ac]-(SEQ ID NO: 78)-K (referred to herein as BCY13389); Ac-(SEQ ID NO: 79)-[dA] (referred to herein as BCY13096); or Ac-(SEQ ID NO: 80) (referred to herein as BCY13097); (wherein Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to claim 2 or 3, comprising

5. The CD137-binding bicyclic peptide ligand comprises a polypeptide comprising an N- and / or C-terminal modification, and the polypeptide is: Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); (wherein Ac represents an acetyl group), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 2 to 4.

6. The two or more second peptide ligands comprise an amino acid sequence selected from: C i LYSPC ii WHPLNDC iii (Accession No.: 87); C i ILWC ii LPEPHDEC iii (Sequence number: 82); C i A K / S N / E C ii DPFWYQFYC iii (Array number: 83); C i AKNC ii DPFWYQFYC iii (Sequence number: 84); C i ASEC ii DPFWYQFYC iii (Array number: 85); C i L / N YSPC ii WHPLN D / K C iii (Array number: 86); C i NYSPC ii WHPLNKC iii (Sequence number: 88); C i WYEYDC ii NNWERC iii (Sequence number: 89); C i VIRYSPC ii SHYLNC iii (Accession No.: 90); C i DYSPWWHPC ii NHIC iii (Accession No.: 91); C i DAC ii LYPDYYVC iii (Accession number: 92); C i RLWC ii IPAPTDDC iii (Accession number: 93); C i TMWC ii IPAKGDWC iii (Sequence number: 94); C i MLWC ii LPAPTDEC iii (Array number: 95); C i ILWC ii LPEPPDEC iii (Accession number: 96); C i LLWC ii IPNPDDNC iii (Array number: 97); C i WLWC ii VPNPDDTC iii (Sequence number: 98); C i VLWC ii TPYPGDDC iii (Accession No.: 99); C i ALWC ii IPDPQDEC iii (Array number: 100); C i TLWC ii IPDASDSC iii (Array number: 101); C i QLWC ii IPDADDDC iii (Array number: 102); C i QLWC ii VPEPGDSC iii (Accession No.: 103); C i ALWC ii IPEESDDC iii (Array number: 104); C i VLWC ii IPEPQDKC iii (Accession No.: 105); C i TLWC ii IPDPDDSC iii (Sequence number: 106); C i RLWC ii VPKAEDYC iii (Accession No.: 107); C i TKPC ii IAYYNQSC iii (Accession number: 108); C i MNPC ii IAYYQQEC iii (Accession No.: 109); C i TNAC ii VAYYHQAC iii (Accession No.: 110); C i SDPC ii ISYYNQAC iii (Sequence number: 111); C i DPPC ii DPFWYAFYC iii (Sequence number: 112); C i PDDC ii DPFWYNFYC iii (Array number: 113); C i RYSPC ii YHPHNC iii (Array number: 114); C i LYSPC ii NHPLNSC iii (Accession No.: 115); C i EDNYC ii FMWTPYC iii (Sequence number: 116); C i LDSPC ii WHPLNDC iii (Array number: 117); C i RFSPC ii SHPLNQC iii (Array number: 118); C i KYSPC ii WHPLNLC iii (Sequence number: 119); C i RYSPC ii WHPLNNC iii (Sequence number: 120); C i EWISC ii PGEPHRWWC iii (Sequence number: 121); C i VWEAC ii PEHPDQWWC iii (Sequence number: 122); C i STWHC ii FWNLQEGKC iii (Sequence number: 123); C i EWKAC ii EHDRERWWC iii (Sequence number: 124); C i RTWQC ii FYEWQNGHC iii (Sequence number: 125); C i KTWDC ii FWASQVSEC iii (Sequence number: 126); C i STWQC ii FYDLQEGHC iii (Sequence number: 127); C i TTWEC ii FYDLQEGHC iii (Sequence number: 128); C i ETWEC ii FWRLQAGEC iii (Sequence number: 129); C i RTWQC ii FWDLQEGLC iii (Sequence number: 130); C i STWQC ii FWDSQLGAC iii (Array number: 131); C i ETWEC ii FWEWQVGSC iii (Sequence number: 132); C i TTWEC ii FWDLQEGLC iii (Sequence number: 133); C i HTWDC ii FYQWQDGHC iii (Sequence number: 134); C i TTWEC ii FYSLQDGHC iii (Sequence number: 135); C i NEDMYC ii FMWMEC iii (Accession number: 136); C i LYEYDC ii YTWRRC iii (Accession number: 137); C i RYEYDC ii HTWQRC iii (Sequence number: 138); C i WYEYDC ii TTWERC iii (Sequence number: 139); C i WYEYDC ii RTWTRC iii (Sequence number: 140); C i LYEYDC ii HTWTRC iii (Sequence number: 141); C i WYEYDC ii RTWTFC iii (Sequence number: 142); C i HGGVWC ii IPNINDSC iii (Accession No.: 143); C i DSPVRC ii YWNTQKGC iii (Accession No.: 144); C i GSPVPC ii YWNTRKGC iii (Accession No.: 145); C i APFEFNC ii YTWRPC iii (Sequence number: 146); C i RVLYSPC ii YHWLNC iii (Sequence number: 147); C i SIMYSPC ii EHPHNHC iii (Sequence number: 148); C i DKWEPDHLC ii YWWC iii (Sequence number: 149); C i DAWPETHVC ii YWWC iii (Sequence number: 150); C i DEYTPEHLC ii YWWC iii (Sequence number: 151); C i WINYSISPC ii YVGEC iii (Accession No.: 152); and C i RYEYPEHLC ii YTWQC iii (Sequence number: 153); (Here, C i , C ii , and C iii each represent a first, second, and third cysteine residue), or a modified derivative or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to claim 1, comprising an OX40-binding bicyclic peptide ligand.

7. The OX40-binding bicyclic peptide ligand further comprises a polypeptide comprising an N- and / or C-terminal modification, and the polypeptide is: A-(SEQ ID NO: 87)-A-K(Pya) (referred to herein as BCY11607); A-(SEQ ID NO: 82)-A-[Sar6]-[KBiot] (referred to herein as BCY10551); A-(SEQ ID NO: 82)-A (referred to herein as BCY10371); A-(SEQ ID NO: 84)-A-[Sar6]-[KBiot] (referred to herein as BCY10552); [Biot]-G-[Sar5]-A-(SEQ ID NO: 84)-A (referred to herein as BCY10479); A-(SEQ ID NO: 84)-A (referred to herein as BCY10378); [Biot]-G-[Sar5]-A-(SEQ ID NO: 85)-A (referred to herein as BCY11371); A-(SEQ ID NO: 85)-A (referred to as BCY10743 in this specification); [Biot]-G-[Sar5]-A-(SEQ ID NO: 87)-A (referred to as BCY10482 in this specification); A-(SEQ ID NO: 87)-A-[Sar6]-[KBiot] (referred to as BCY10549 in this specification); Ac-A-(SEQ ID NO: 87)-A-K(Pya) (referred to as BCY12708 in this specification); A-(SEQ ID NO: 87)-A (referred to as BCY10351 in this specification); A-(SEQ ID NO: 88)-A-[Sar6]-[KBiot] (referred to as BCY11501 in this specification); A-(SEQ ID NO: 88)-A (referred to as BCY10729 in this specification); A-(SEQ ID NO: 89)-A-[Sar6]-[KBiot] (referred to as BCY10550 in this specification); A-(SEQ ID NO: 89)-A (referred to as BCY10361 in this specification); A-(SEQ ID NO: 90)-A-[Sar6]-[KBiot] (referred to as BCY10794 in this specification); A-(SEQ ID NO: 90)-A (referred to as BCY10349 in this specification); [Biot]-G-[Sar5]-A-(SEQ ID NO: 91)-A (referred to as BCY11369 in this specification); A-(SEQ ID NO: 91)-A (referred to as BCY10331 in this specification); A-(SEQ ID NO: 92)-A (referred to as BCY10375 in this specification); A-(SEQ ID NO: 93)-A (referred to as BCY10364 in this specification); A-(SEQ ID NO: 94)-A (referred to as BCY10365 in this specification); A-(SEQ ID NO: 95)-A (referred to as BCY10366 in this specification); A-(SEQ ID NO: 96)-A (referred to as BCY10367 in this specification); A-(SEQ ID NO: 97)-A (referred to as BCY10368 in this specification); A-(SEQ ID NO: 98)-A (referred to as BCY10369 in this specification); A-(SEQ ID NO: 99)-A (referred to as BCY10374 in this specification); A-(SEQ ID NO: 100)-A (referred to as BCY10376 in this specification); A-(SEQ ID NO: 101)-A (referred to as BCY10737 in this specification); A-(SEQ ID NO: 102)-A (referred to as BCY10738 in this specification); A-(SEQ ID NO: 103)-A (referred to as BCY10739 in this specification); A-(SEQ ID NO: 104)-A (referred to as BCY10740 in this specification); A-(SEQ ID NO: 105)-A (referred to as BCY10741 in this specification); A-(SEQ ID NO: 106)-A (referred to as BCY10742 in this specification); A-(SEQ ID NO: 107)-A (referred to as BCY10380 in this specification); A-(SEQ ID NO: 108)-A (referred to as BCY10370 in this specification); A-(SEQ ID NO: 109)-A (referred to as BCY10372 in this specification); A-(SEQ ID NO: 110)-A (referred to as BCY10373 in this specification); A-(SEQ ID NO: 111)-A (referred to as BCY10379 in this specification); A-(SEQ ID NO: 112)-A (referred to as BCY10377 in this specification); A-(SEQ ID NO: 113)-A (referred to as BCY10744 in this specification); A-(SEQ ID NO: 114)-A (referred to as BCY10343 in this specification); A-(SEQ ID NO: 115)-A (referred to as BCY10350 in this specification); A-(SEQ ID NO: 116)-A (referred to as BCY10352 in this specification); A-(SEQ ID NO: 117)-A (referred to as BCY10353 in this specification); A-(SEQ ID NO: 118)-A (referred to as BCY10354 in this specification); A-(SEQ ID NO: 119)-A (referred to as BCY10730 in this specification); A-(SEQ ID NO: 120)-A (referred to as BCY10731 in this specification); A-(SEQ ID NO: 121)-A (referred to as BCY10339 in this specification); A-(SEQ ID NO: 122)-A (referred to as BCY10340 in this specification); A-(SEQ ID NO: 123)-A (referred to as BCY10342 in this specification); A-(SEQ ID NO: 124)-A (referred to as BCY10345 in this specification); A-(SEQ ID NO: 125)-A (referred to as BCY10347 in this specification); A-(SEQ ID NO: 126)-A (referred to as BCY10348 in this specification); A-(SEQ ID NO: 127)-A (referred to as BCY10720 in this specification); A-(SEQ ID NO: 128)-A (referred to as BCY10721 in this specification); A-(SEQ ID NO: 129)-A (referred to herein as BCY10722); A-(SEQ ID NO: 130)-A (referred to herein as BCY10723); A-(SEQ ID NO: 131)-A (referred to herein as BCY10724); A-(SEQ ID NO: 132)-A (referred to herein as BCY10725); A-(SEQ ID NO: 133)-A (referred to herein as BCY10726); A-(SEQ ID NO: 134)-A (referred to herein as BCY10727); A-(SEQ ID NO: 135)-A (referred to herein as BCY10728); A-(SEQ ID NO: 136)-A (referred to herein as BCY10360); A-(SEQ ID NO: 137)-A (referred to herein as BCY10363); A-(SEQ ID NO: 138)-A (referred to herein as BCY10732); A-(SEQ ID NO: 139)-A (referred to herein as BCY10733); A-(SEQ ID NO: 140)-A (referred to herein as BCY10734); A-(SEQ ID NO: 141)-A (referred to herein as BCY10735); A-(SEQ ID NO: 142)-A (referred to herein as BCY10736); A-(SEQ ID NO: 143)-A (referred to herein as BCY10336); A-(SEQ ID NO: 144)-A (referred to herein as BCY10337); A-(SEQ ID NO: 145)-A (referred to herein as BCY10338); A-(SEQ ID NO: 146)-A (referred to herein as BCY10346); A-(SEQ ID NO: 147)-A (referred to herein as BCY10357); A-(SEQ ID NO: 148)-A (referred to herein as BCY10362); A-(SEQ ID NO: 149)-A (referred to herein as BCY10332); A-(SEQ ID NO: 150)-A (referred to herein as BCY10717); A-(SEQ ID NO: 151)-A (referred to herein as BCY10718); A-(SEQ ID NO: 152)-A (referred to herein as BCY10334); and A-(SEQ ID NO: 153)-A (referred to herein as BCY10719); (wherein Pya represents a 4-pentynoyl moiety) The heterotandem bicyclic peptide complex according to claim 6, selected from

8. The OX40-binding bicyclic peptide ligand comprises a polypeptide further comprising an N- and / or C-terminal modification, and the polypeptide is: A-(SEQ ID NO: 87)-A-K(Pya) (referred to herein as BCY11607); (wherein Pya represents a 4-pentynoyl moiety) The heterotandem bicyclic peptide complex according to claim 7.

9. The first peptide ligand comprises an amino acid sequence selected from the following: C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1; referred to herein as BCY8116); C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 3); C i PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 4; referred to herein as BCY11414); C i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (Sequence number: 14); [MerPro] i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 15; referred to herein as BCY12363); C i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO: 16); [MerPro] i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO: 17; referred to herein as BCY12365); C i P[1Nal][dK]C ii M[HArg]HWSTP[HyP]WC iii (Sequence number: 18); C i P[1Nal][dK]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO: 19); C i P[1Nal][dE]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 20; referred to herein as BCY12368); C i P[1Nal][dA]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 21; referred to herein as BCY12369); C i P[1Nal][dE]C ii L[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 22; referred to herein as BCY12370); and C i P[1Nal][dE]C ii M[HArg]EWSTP[HyP]WC iii (Sequence number: 23; in this specification, BCY12384); (Here, [MerPro] i , C i , C ii , C iii , and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, Sar 10 represents 10 sarcosine units, B-Ala represents β-alanine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 1 to 8, comprising a nectin-4-binding bicyclic peptide ligand.

10. The nectin-4-binding bicyclic peptide ligand is as follows: SEQ ID NO: 1 (referred to herein as BCY8116); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 1) (referred to herein as BCY8846); [PYA]-(SEQ ID NO: 1) (referred to herein as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO: 1) (referred to herein as BCY11016); [PYA]-[B-Ala]-[Sar 10 -(SEQ ID NO: 2) (referred to herein as BCY11942); Ac-(SEQ ID NO: 3) (referred to herein as BCY8831); SEQ ID NO: 4 (referred to herein as BCY11414); [PYA]-[B-Ala]-(SEQ ID NO: 14) (referred to herein as BCY11143); Palmitic acid-yGlu-yGlu-(SEQ ID NO: 14) (referred to herein as BCY12371); Ac-(SEQ ID NO: 14) (referred to herein as BCY12024); Ac-(SEQ ID NO: 16) (referred to herein as BCY12364); Ac-(SEQ ID NO: 18) (referred to herein as BCY12366); or Ac-(SEQ ID NO: 19) (referred to herein as BCY12367); (Here, PYA represents 4-pentenoic acid, B-Ala represents β-alanine, and Sar 10 represents 10 sarcosine units), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to claim 9, comprising a polypeptide.

11. The heterotandem bicyclic peptide complex according to claim 9 or claim 10, wherein the nectin-4-binding bicyclic peptide ligand comprises SEQ ID NO: 1 (referred to herein as BCY8116).

12. As follows: - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-bound BCY8116 conjugated to two BCY8928 ligands with dLys(PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate BCY8116 with dLys(PYA)4 to two BCY12143 ligands; - Trimesic acid with N-terminal PYA - [Peg 10 3 BCY11015, in which two BCY8928 ligands are bound with dLys(PYA)4 via a linker;​ - Trimesic acid with N-terminal PYA - [Peg 10 3 BCY11015, which is bound to two BCY11014 ligands via a linker with C-terminal Dap(PYA);​ - TCA-[Peg with N-terminal PYA 10 3 BCY11015, which is conjugated to two BCY8928 ligands via a linker with dLys(PYA)4;​ - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker is used to bind BCY8116 to two BCY11014 ligands at the C-terminus with Dap(PYA); - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker-bound BCY8116 conjugated to two BCY7744 ligands with dLys(PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker was used to conjugate dLys(PYA)4 to two BCY12149 ligands to form BCY8116; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to bind BCY8116 to two BCY12147 ligands with dLys(PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY12352 ligands with dLys(PYA)4-conjugated BCY8116; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker was used to conjugate dLys(PYA)4 to two BCY12145 ligands to form BCY8116; - N-(Acid-PEG 3 )-N-bis(PEG 3 )-linked to two BCY8928 ligands via an azide linker and conjugated to BCY12024 with dLys(PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to bind dLys(PYA)4 to two BCY12353 ligands to form BCY8116; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-linked BCY8116 bound to two BCY12354 ligands with dLys(PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-bound BCY12371 conjugated with dLys(PYA)4 to two BCY8928 ligands; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12384 with dLys(PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY12381 ligands with dLys(PYA)4-conjugated BCY8116; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker was used to conjugate dLys(PYA)4 to two BCY12382 ligands to form BCY8116; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker to which BCY8116 is attached with dLys(PYA)4 to BCY8928 and with dLys(PYA)4 to BCY13389; - At the N-terminus, a N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys(PYA)4 to two BCY14601 ligands to form BCY8116; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker was used to conjugate dLys(PYA)4 to BCY14601 and dLys(PYA)4 to BCY8928, and BCY8116 was conjugated; - Tet-[Peg with an N-terminal PYA 10 4 BCY11016 bound to three BCY7744 ligands with dLys(PYA)4 via a linker; or​ - Tet-[Peg with N-terminal PYA 10 4 BCY11016, which is conjugated to three BCY8928 ligands with dLys(PYA)4 via a linker;​ Or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 9 to 11.

13. BCY11027: [Chemical Formula 3] , BCY11863: 【Chemical Formula 4】 And BCY11864: 【Chemical Formula 5】 And a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 9 to 12, selected from

14. BCY12967: 【Chemical Formula 6】 or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 9 to 11.

15. The amino acid sequence from which the first peptide ligand is selected: C i [HyP]LVNPLC ii LEP[d1Nal]WTC iii (Accession No.: 44); C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (Accession number: 24); C i LWDPTPC ii ANLHL[HArg]C iii (Accession No.: 25); C i [HyP]LVNPLC ii L[K(PYA)]P[dD]W[HArg]C iii (Sequence number: 26); C i [HyP][K(PYA)]VNPLC ii LHP[dD]W[HArg]C iii (Accession No.: 27); C i [HyP]LVNPLC ii [K(PYA)]HP[dD]W[HArg]C iii (SEQ ID NO: 28); C i [HyP]LVNPLC ii LKP[dD]W[HArg]C iii (Accession No.: 29); C i [HyP]KVNPLC ii LHP[dD]W[HArg]C iii (Accession No.: 30); C i [HyP]LVNPLC ii KHP[dD]W[HArg]C iii (Accession No.: 31); C i [HyP]LVNPLC ii LHP[dE]W[HArg]C iii (Accession number: 32); C i [HyP]LVNPLC ii LEP[dD]W[HArg]C iii (Accession No.: 33); C i [HyP]LVNPLC ii LHP[dD]WTC iii (Accession number: 34); C i [HyP]LVNPLC ii LEP[dD]WTC iii (Accession No.: 35); C i [HyP]LVNPLC ii LEP[dA]WTC iii (Accession No.: 36); C i [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (Accession number: 37; referred to herein as BCY12860); C i [HyP][Cba]VNPLC ii LHP[dD]W[HArg]C iii (Accession number: 38); C i [HyP][Cba]VNPLC ii LEP[dD]WTC iii (Accession No.: 39); C i [HyP][Cba]VNPLC ii L[3,3-DPA]P[dD]WTC iii (Accession number: 40); C i [HyP]LVNPLC ii L[3,3-DPA]P[dD]W[HArg]C iii (Accession No.: 41); C i [HyP]LVNPLC ii LHP[d1Nal]W[HArg]C iii (Accession No.: 42); C i [HyP]LVNPLC ii L[1Nal]P[dD]W[HArg]C iii (SEQ ID NO: 43); C i [HyP]LVNPLC ii L[1Nal]P[dD]WTC iii (Accession No.: 45; referred to herein as BCY13119); C i [HyP][Cba]VNPLC ii LEP[dA]WTC iii (Accession No.: 46); C i [HyP][hGlu]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO: 47); C i [HyP]LVNPLC ii [hGlu]HP[dD]W[HArg]C iii (SEQ ID NO: 48); C i [HyP]LVNPLC ii L[hGlu]P[dD]W[HArg]C iii (Sequence number: 49); C i [HyP]LVNPLC ii LHP[dNle]W[HArg]C iii (Accession number: 50); C i [HyP]LVNPLC ii L[Nle]P[dD]W[HArg]C iii (Accession number: 51); [MerPro] i [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (Accession No.: 154); C i [HyP]LVNPLC ii LHP[dD]W[HArg][Cysam] iii (SEQ ID NO: 155); C i [HyP]LVNPLC ii L[His3Me]P[dD]W[HArg]C iii (SEQ ID NO: 156); C i [HyP]LVNPLC ii L[His1Me]P[dD]W[HArg]C iii (SEQ ID NO: 157); C i [HyP]LVNPLC ii L[4ThiAz]P[dD]W[HArg]C iii (Accession No.: 158); C i [HyP]LVNPLC ii LFP[dD]W[HArg]C iii (Accession No.: 159); C i [HyP]LVNPLC ii L[Thi]P[dD]W[HArg]C iii (Accession No.: 160); C i [HyP]LVNPLC ii L[3Thi]P[dD]W[HArg]C iii (Accession No.: 161); C i [HyP]LVNPLC ii LNP[dD]W[HArg]C iii (Accession number: 162); C i [HyP]LVNPLC ii LQP[dD]W[HArg]C iii (Sequence number: 163); and C i [HyP]LVNPLC ii L[K(PYA-(Palmitoyl-Glu-LysN 3 )]P[dD]W[HArg]C iii (SEQ ID NO: 164); (Here, [MerPro] i , C i , C ii , C iii , and [Cysam] iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam, HyP represents trans-4-hydroxy-L-proline, HArg represents homoarginine, PYA represents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, 1Nal represents 1-naphthylalanine, hGlu represents homoglutamic acid, Thi represents thienyl-alanine, 4ThiAz represents β-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 3Thi represents 3-thienylalanine, palmitoyl-Glu-LysN 3 [PYA] is 【Chemical Formula 7】 : represents [K(PYA-(palmitoyl-Glu-LysN 3 )] 【Chemical Formula 8】 : (where Nle represents norleucine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 1 to 8, comprising an EphA2-binding bicyclic peptide ligand containing

16. The EphA2-binding bicyclic peptide ligand is as follows: C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (Accession No.: 24); (Here, C i , C ii , and C iii represent the first (i), second (ii), and third (iii) cysteine groups, HyP represents trans-4-hydroxy-L-proline, and HArg represents homoarginine) The heterotandem bicyclic peptide complex according to claim 15, comprising an amino acid sequence or a pharmaceutically acceptable salt thereof.

17. The EphA2-binding bicyclic peptide ligand is as follows: C i [HyP]LVNPLC ii LEP[d1Nal]WTC iii (Accession No.: 44); (Here, C i , C ii , and C iii represent the first (i), second (ii), and third (iii) cysteine groups, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine) The heterotandem bicyclic peptide complex according to claim 15, comprising an amino acid sequence or a pharmaceutically acceptable salt thereof.

18. The EphA2-binding bicyclic peptide ligand is as follows: A-[HArg]-D-(SEQ ID NO: 24) (referred to as BCY9594 herein); [B-Ala]-[Sar 10 -A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY6099); [PYA]-A-[HArg]-D-(SEQ ID NO: 24) (referred to as BCY11813 herein); Ac-A-[HArg]-D-(SEQ ID NO: 24)-[K(PYA)] (referred to as BCY11814 herein); Ac-A-[HArg]-D-(SEQ ID NO: 24)-K (referred to as BCY12734 herein); [NMeAla]-[HArg]-D-(SEQ ID NO: 24) (referred to as BCY13121 herein); [Ac]-(SEQ ID NO: 24)-L[dH]G[dK] (referred to as BCY13125 herein); [PYA]-[B-Ala]-[Sar 10 -VGP-(SEQ ID NO: 25) (referred to herein as BCY8941); Ac-A-[HArg]-D-(SEQ ID NO: 26) (referred to as BCY11815 herein); Ac-A-[HArg]-D-(SEQ ID NO: 27) (referred to as BCY11816 herein); Ac-A-[HArg]-D-(SEQ ID NO: 28) (referred to as BCY11817 herein); Ac-A-[HArg]-D-(SEQ ID NO: 29) (referred to as BCY12735 herein); (Palmitoyl-Glu-LysN 3 )[PYA]A[HArg]D-(SEQ ID NO: 29) (hereinafter referred to as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO: 30) (referred to as BCY12736 in this specification); Ac-A-[HArg]-D-(SEQ ID NO: 31) (referred to as BCY12737 in this specification); A-[HArg]-D-(SEQ ID NO: 32) (referred to as BCY12738 in this specification); A-[HArg]-E-(SEQ ID NO: 32) (referred to as BCY12739 in this specification); A-[HArg]-D-(SEQ ID NO: 33) (referred to as BCY12854 in this specification); A-[HArg]-D-(SEQ ID NO: 34) (referred to as BCY12855 in this specification); A-[HArg]-D-(SEQ ID NO: 35) (referred to as BCY12856 in this specification); A-[HArg]-D-(SEQ ID NO: 35)-[dA] (referred to as BCY12857 in this specification); (SEQ ID NO: 35)-[dA] (referred to as BCY12861 in this specification); [NMeAla]-[HArg]-D-(SEQ ID NO: 35) (referred to as BCY13122 in this specification); [dA]-ED-(SEQ ID NO: 35) (referred to as BCY13126 in this specification); [dA]-[dA]-D-(SEQ ID NO: 35) (referred to as BCY13127 in this specification); AD-(SEQ ID NO: 35) (referred to as BCY13128 in this specification); A-[HArg]-D-(SEQ ID NO: 36) (referred to as BCY12858 in this specification); A-[HArg]-D-(SEQ ID NO: 37) (referred to as BCY12859 in this specification); Ac-(SEQ ID NO: 37)-[dK] (referred to as BCY13120 in this specification); A-[HArg]-D-(SEQ ID NO: 38) (referred to as BCY12862 in this specification); A-[HArg]-D-(SEQ ID NO: 39) (referred to as BCY12863 in this specification); [dA]-[HArg]-D-(SEQ ID NO: 39)-[dA] (referred to as BCY12864 in this specification); (SEQ ID NO: 40)-[dA] (referred to as BCY12865 in this specification); A-[HArg]-D-(SEQ ID NO: 41) (referred to as BCY12866 in this specification); A-[HArg]-D-(SEQ ID NO: 42) (referred to as BCY13116 in this specification); A-[HArg]-D-(SEQ ID NO: 43) (referred to as BCY13117 in this specification); A-[HArg]-D-(SEQ ID NO: 44) (referred to as BCY13118 in this specification); [dA]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to as BCY13123 in this specification); [d1Nal]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to as BCY13124 in this specification); A-[HArg]-D-(SEQ ID NO: 47) (referred to as BCY13130 in this specification); A-[HArg]-D-(SEQ ID NO: 48) (referred to as BCY13131 in this specification); A-[HArg]-D-(SEQ ID NO: 49) (referred to as BCY13132 in this specification); A-[HArg]-D-(SEQ ID NO: 50) (referred to as BCY13134 in this specification); A-[HArg]-D-(SEQ ID NO: 51) (referred to as BCY13135 in this specification); (SEQ ID NO: 154)-[dK] (referred to as BCY13129 in this specification); A[HArg]D-(SEQ ID NO: 155) (referred to as BCY13133 in this specification); A[HArg]D-(SEQ ID NO: 156) (referred to as BCY13917 in this specification); A[HArg]D-(SEQ ID NO: 157) (referred to as BCY13918 in this specification); A[HArg]D-(SEQ ID NO: 158) (referred to as BCY13919 in this specification); A[HArg]D-(SEQ ID NO: 159) (referred to as BCY13920 in this specification); A[HArg]D-(SEQ ID NO: 160) (referred to as BCY13922 in this specification); A[HArg]D-(SEQ ID NO: 161) (referred to as BCY13923 in this specification); A[HArg]D-(SEQ ID NO: 162) (referred to as BCY14047 in this specification); A[HArg]D-(SEQ ID NO: 163) (referred to as BCY14048 in this specification); or A[HArg]D-(SEQ ID NO: 164) (referred to as BCY14313 in this specification); (Here, PYA represents 4-pentenoic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, HArg represents homoarginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, palmitoyl-Glu-LysN 3 [PYA] is, 【Chemical Formula 9】 : represents), or a pharmaceutically acceptable salt thereof A heterodimeric bicyclic peptide complex according to any one of claims 15 to 17, comprising a polypeptide comprising:

19. The EphA2-binding bicyclic peptide ligand is as follows: A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); (wherein, HArg represents homoarginine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 15 to 18, comprising a polypeptide containing the same.

20. The EphA2-binding bicyclic peptide ligand is as follows: A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); (wherein, HArg represents homoarginine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to any one of claims 15 to 18, comprising a polypeptide containing the same.

21. As follows: - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY8928 ligands bound with dLys (PYA)4 to form BCY9594; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker was used to conjugate BCY9594 with two BCY12143 ligands via dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind BCY9594 to two BCY12149 ligands with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-mediated conjugation of dLys (PYA)4 to two BCY12147 ligands in BCY9594; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to bind BCY9594 to two BCY12145 ligands with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-mediated BCY9594 conjugated to two BCY12150 ligands with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY12352 ligands with dLys (PYA)4 to BCY9594; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-bound BCY9594 conjugated to two BCY12353 ligands with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY12354 ligands bound with dLys (PYA)4 to give BCY9594; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-bound BCY9594 conjugated to two BCY12360 ligands with dLys (PYA)4; - C-terminal Lys with N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was conjugated to two BCY8928 ligands with dLys (PYA)4 to give BCY12734; - N-(acid-PEG at Lys8 3 )-N-bis(PEG 3 )-BCY12735 conjugated to two BCY8928 ligands via an azide linker with dLys (PYA)4; - N-(acid-PEG at Lys2 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12736 with dLys (PYA)4; - N-(acid-PEG at Lys7 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to BCY12737; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY8928 ligands bound with dLys (PYA)4 to give BCY12738; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY12739; - BAPG-(Peg at the N-terminus 5 ) 2 BCY9594, in which two BCY8928 ligands are bound with dLys (PYA)4 via a linker; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12854 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker is used to bind dLys (PYA)4 to two BCY8928 ligands to form BCY12855; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12856 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY12857; - N-terminally, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY12858; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker is used to bind two BCY8928 ligands with dLys (PYA)4 to BCY12859; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY8928 ligands conjugated with dLys (PYA)4; BCY12860 - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to BCY12861; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to form BCY12862; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker to which two BCY8928 ligands are bound with dLys(PYA)4 to BCY12863; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY8928 ligands bound with dLys (PYA)4 to give BCY12864; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY12865; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12866 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY12353 ligands to form BCY12856; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY13137 ligands to form BCY9594; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY13137 ligands to BCY12856; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to BCY13116; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY13117; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to BCY13118; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY13119 with dLys (PYA)4; - N-(acid-PEG with C-terminal dLys 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to BCY13120; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to bind two BCY8928 ligands to BCY13121 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY13122; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY13123; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY8928 ligands with dLys (PYA)4 conjugated BCY13124; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY13126 with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to form BCY13127; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY13128; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-linked BCY13130 conjugated with dLys (PYA)4 to two BCY8928 ligands; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY13131 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY13132; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to two BCY8928 ligands conjugated with dLys (PYA)4; BCY13134 - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY13135 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY12353 ligands to BCY12865 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY12353 ligands to BCY12860 with dLys (PYA)4; - N-(Acid-PEG with C-terminal dLys 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands with dLys (PYA)4 to BCY13125; - At the C-terminus, dLys is used to conjugate N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to conjugate two BCY8928 ligands to BCY13129 with dLys (PYA)4; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY13133 with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to form BCY13917; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to which two BCY8928 ligands are bound with dLys (PYA)4 via a linker; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to two BCY8928 ligands to form BCY13919; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands with dLys (PYA)4 to BCY13920; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker to which two BCY8928 ligands are bound with dLys (PYA)4 to form BCY13922; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker-bound BCY13923 conjugated to two BCY8928 ligands with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to form BCY14047; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY14048 with dLys (PYA)4; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker-bound BCY14313 conjugated with dLys(PYA)4 to two BCY8928 ligands; - N-(acid-PEG at Lys 8 3 )-N-bis(PEG 3 -azide) linker to bind two BCY8928 ligands with dLys (PYA)4 to BCY14327; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker was used to conjugate dLys (PYA)4 to BCY8928 and dLys (PYA)4 to BCY13389 to form BCY9594; - N-terminally N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker to which dLys(PYA)4 and BCY13118 conjugated with dLys(PYA)4 are attached to BCY8928 and BCY13389; - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker to bind BCY14601 with dLys(PYA)4 and BCY13118 bound to BCY14601 with dLys(PYA)4; or - At the N-terminus, N-(acid-PEG 3 )-N-bis(PEG 3 )-azide linker was used to conjugate dLys(PYA)4 to BCY8928 and dLys(PYA)4 to BCY14601 to obtain BCY13118; Or a pharmaceutically acceptable salt thereof, The heterotandem bicyclic peptide complex according to any one of claims 15 to 20, comprising the same.

22. BCY12491: 【Chemical 10】 , BCY12730: 【Chemical 11】 , BCY13048: 【Chemical Formula 12】 , BCY13050: 【Chemical 13】 , BCY13053: 【Chemical Formula 14】 and BCY13272: 【Chemical Formula 15】 and a pharmaceutically acceptable salt thereof, The heterotandem bicyclic peptide complex according to any one of claims 15 to 21, selected from the same.

23. BCY12491: 【Chemical 16】 Or BCY13272: 【Chemical 17】 Or a pharmaceutically acceptable salt thereof, The heterotandem bicyclic peptide complex according to claim 22, which is the same.

24. The first peptide ligand is an amino acid sequence selected from the following: C i SAGWLTMC ii QKLHLC iii (Accession number: 52); C i SAGWLTMC ii Q[K(PYA)]LHLC iii (Accession No.: 53); C i SKGWLTMC ii Q[K(Ac)]LHLC iii (Accession number: 54); C i SAGWLTKC ii Q[K(Ac)]LHLC iii (Accession No.: 55); C i SAGWLTMC ii K[K(Ac)]LHLC iii (Accession number: 56); C i SAGWLTMC ii Q[K(Ac)]LKLC iii (Accession No.: 57); C i SAGWLTMC ii Q[HArg]LHLC iii (Accession No.: 58); and C i SAGWLTMC ii [HArg]QLNLC iii (Accession number: 59); (Here, C i , C ii , and C iii represent the first, second, and third cysteine residues, respectively, PYA represents 4-pentynoic acid, and HArg represents homoarginine), or a pharmaceutically acceptable salt thereof, The heterotandem bicyclic peptide complex according to any one of claims 1 to 8, comprising a PD-L1-binding bicyclic peptide ligand containing the same.

25. The PD-L1-binding bicyclic peptide ligand is as follows: [PYA]-[B-Ala]-[Sar 10 -SDK-(SEQ ID NO: 52) (referred to herein as BCY10043); Ac-D-[HArg]-(SEQ ID NO: 52)-PSH (referred to herein as BCY11865); Ac-SDK-(SEQ ID NO: 53) (referred to herein as BCY11013); Ac-SDK-(SEQ ID NO: 53)-PSH (referred to herein as BCY10861); Ac-D-[HArg]-(SEQ ID NO: 54)-PSH (referred to herein as BCY11866); Ac-D-[HArg]-(SEQ ID NO: 55)-PSH (referred to herein as BCY11867); Ac-D-[HArg]-(SEQ ID NO: 56)-PSH (referred to herein as BCY11868); Ac-D-[HArg]-(SEQ ID NO: 57)-PSH (referred to as BCY11869 herein); Ac-SD-[HArg]-(SEQ ID NO: 58)-PSHK (referred to as BCY12479 herein); or Ac-SD-[HArg]-(SEQ ID NO: 59)-PSHK (referred to as BCY12477 herein); (Here, PYA represents 4-pentenoic acid, B-Ala represents β-alanine, Sar 10 represents 10 sarcosine units, and HArg represents homoarginine), or a pharmaceutically acceptable salt thereof The heterotandem bicyclic peptide complex according to claim 24, comprising a polypeptide containing any of the above.

26. The following - Lys(PYA)9 with TCA-[Peg 10 3 BCY10861 conjugated to two BCY8928 ligands with dLys4 via a linker;​ - N-(Acid-PEG with C-terminal Lys 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12479 with dLys(PYA)4; - N-(acid-PEG with C-terminal Lys 3 )-N-bis(PEG 3 -azide) linker was used to conjugate two BCY8928 ligands to BCY12477 with dLys(PYA)4; or a pharmaceutically acceptable salt thereof, The heterotandem bicyclic peptide complex according to claim 24 or 25, comprising any of the above.

27. BCY11780: 【Chemical Formula 18】 The heterotandem bicyclic peptide complex according to any one of claims 24 to 26, which is or a pharmaceutically acceptable salt thereof.

28. The heterotandem bicyclic peptide complex according to claim 1 or 2, wherein the two or more second peptide ligands comprise one CD137-binding bicyclic peptide ligand and one OX40-binding bicyclic peptide.

29. BCY12733: 【Chemical 19】 The heterotandem bicyclic peptide complex according to claim 28, which is or a pharmaceutically acceptable salt thereof.

30. BCY14415: 【Chemical 20】 , BCY14416: 【Chemical 21】 , BCY14417: 【Chemical 22】 , BCY14418: 【Chemical 23】 , BCY13582: 【Chemical 24】 , BCY13583: 【Chemical 25】 and BCY13628: 【Chemical 26】 The heterotandem bicyclic peptide complex according to claim 1, selected from the above and pharmaceutically acceptable salts thereof.

31. The heterotandem bicyclic peptide complex according to any one of claims 1 to 30, wherein the immune cells are selected from leukocytes; lymphocytes; CD8 or CD4; CD8; dendritic cells, follicular dendritic cells, and granulocytes.

32. The heterotandem bicyclic peptide complex according to any one of claims 1 to 31, wherein the immune cells are selected from T lymphocytes, T cells, B cells, or natural killer cells.

33. The heterotandem bicyclic peptide complex according to any one of claims 1 to 32, 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.

34. The heterotandem bicyclic peptide complex according to any one of claims 1 to 33, wherein the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

35. The heterotandem bicyclic peptide complex according to any one of claims 1 to 34, wherein the pharmaceutically acceptable salt is selected from sodium, potassium, calcium, and ammonium salts.

36. The heterotandem bicyclic peptide complex according to any one of claims 1 to 35, further comprising an effector group selected from a cytotoxic agent, a radioactive chelating agent, and a chromophore.

37. A pharmaceutical composition comprising the heterotandem bicyclic peptide complex according to any one of claims 1 to 36.

38. Use of the heterotandem bicyclic peptide complex according to any one of claims 1 to 36 in the manufacture of a medicament for the prevention, suppression, or treatment of cancer.

39. A pharmaceutical composition for treating cancer, comprising the heterotandem bicyclic peptide complex according to any one of claims 1 to 36, wherein the heterotandem bicyclic peptide complex is used such that it is administered at a dosing frequency that does not maintain a plasma concentration of the complex exceeding in vitro EC 50 The pharmaceutical composition as described above.

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