Heterotandem bicyclic peptide conjugates

Abstract

To provide a ligand binding to nectin-4 present on cancer cells, or a pharmaceutically acceptable salt thereof.SOLUTION: The present invention relates to heterotandem bicyclic peptide complexes comprising a first peptide ligand that binds to a component present on a cancer cell conjugated via a linker to a second peptide ligand that binds to a component present on an immune cell. The invention also relates to the use of said heterotandem bicyclic peptide complexes in the prevention, inhibition or treatment of cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a method for producing a cancer cell-associated compound comprising administering to a patient a first peptide ligand comprising: The conjugate is then conjugated to a second peptide ligand that binds to a component present on immune cells. The present invention also relates to heterotandem bicyclic peptide conjugates, including those conjugated with the tandem bicyclic peptide conjugates. and the use of said heterotandem bicyclic peptide complex in the prevention, suppression, or treatment of cancer. do. [Background technology]

[0002] BACKGROUND OF THE INVENTION Cyclic peptides can bind to protein targets with high affinity and target specificity, Therefore, they are an attractive class of molecules for therapeutic development. The antimicrobial peptides include, for example, vancomycin, the immunosuppressant cyclosporine, or the anti-inflammatory drug Like the cancer drug octreotide, it is already being used successfully in the clinic (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24. The excellent binding properties are due to the The conformational flexibility of the ring structure as well as the relatively large interaction surface formed between the target and the ring Generally, macrocycles are used in combination with the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 (Wu et al. (2007), Science 330, 1066-71), Arg-Gly binding to integrin αVb3 -Asp motif cyclic peptide (355 Å 2 )(Xiong et al. (2002), Science 296(5565), 151-5), or cyclic peptide inhibitors that bind to urokinase-type plasminogen activator Upain-1 (603Å 2 (2007), J Struct Biol 160(1), 1-10), hundreds of It bonds to a surface of square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides. This results in a lower entropy loss when binding to the target, resulting in higher binding The reduced flexibility also leads to the fixation of target-specific conformations, and the linear peptide This effect is due to the fact that when the ring is opened, it binds to other MM peptides. Potent and selective matrix metalloproteinase 8 (MMP-8) loses its selectivity for P This is exemplified by specific inhibitors (Cherney et al. (1998), J Med Chem 41(11), 1749- 51) The advantageous binding properties achieved by macrocyclization have been demonstrated in, for example, vancomycin, nicotinamide, and nicotinamide. In polycyclic peptides with multiple peptide rings, such as actinomycin and actinomycin, Even more pronounced.

[0004] Various research teams have previously synthesized polypeptides containing cysteine ​​residues into synthetic molecular structures. (Kemp and McNamara, 1985, J. Org. Chem; Timmerman et al., 2005, Meloen and coworkers reported that tris(bromomethyl)benzene and related molecules of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces It was used for rapid and quantitative cyclization (Timmerman et al., 2005, ChemBioChem). a compound (wherein the compound reacts with a cysteine-containing polypeptide by, for example, tris(bromomethyl) (i) by linking to a molecular scaffold such as benzene) The method is disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] Generating and screening large libraries of bicyclic peptides against targets of interest A phage display-based combinatorial approach has been developed to (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). This results in a linear peptide containing three cysteine ​​residues and two random six-amino acid regions. A combinatorial library of peptides (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage. The cysteine ​​side chain was covalently attached to a small molecule (tris-(bromomethyl)benzene) It was further cyclized. 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; , (b) a second peptide ligand that binds to a component present on an immune cell; Conjugated to Contains: wherein each of the peptide ligands is separated by at least two loop sequences. A polypeptide containing at least three reactive groups and a compound that forms a covalent bond with the reactive groups of the polypeptide. and a molecular scaffold that results in at least two polypeptide loops. A heterotandem bicyclic peptide complex formed on the molecular scaffold, wherein the heterotandem bicyclic peptide complex comprises first and second peptide ligands: [Table 1] TIFF2026027327000002.tif237170TIFF2026027327000003.tif237170TIFF2026027 327000004.tif237170TIFF2026027327000005.tif236170TIFF2026027327000006.ti f237170TIFF2026027327000007.tif236170TIFF2026027327000008.tif237170TIFF2026027327000009.tif237170 (where 1Nal represents 1-naphthylalanine, HArg represents homoarginine, and HyP represents hydrochloride. B-Ala stands for β-alanine, PYA stands for 4-pentynoic acid, and 3,3-DPA stands for 3,3-diphenylalanine, Cba stands for β-cyclobutylalanine, and hGlu stands for homoglucan. NMeAla stands for N-methyl-alanine, tBuAla stands for thiamin, Nle stands for norleucine, NMeAla stands for N-methyl-alanine, tBuAla stands for thiamin ... represents t-butyl-alanine, Aad represents α-L-aminoadipic acid, and Ac represents an acetyl group. and Dap represents diaminopropionic acid), or a pharmaceutically acceptable salt thereof. A heterotandem bicyclic peptide conjugate is provided, comprising:

[0007] According to a further aspect of the present invention, there is provided a heterotandem bicyclic peptide as defined herein. Pharmaceutical compositions are provided that include the conjugates in combination with one or more pharmaceutically acceptable excipients. .

[0008] According to a further aspect of the present invention there is provided a method for preventing, suppressing or treating cancer. There is provided a heterotandem bicyclic peptide conjugate as defined herein. DETAILED DESCRIPTION OF THE INVENTION

[0009] (Detailed Description of the Invention) (First peptide ligand) References herein to the term "cancer cells" refer to cells that are known to be involved in cancer. Cancer cells are formed when genes involved in regulating cell division are damaged. Carcinogenesis is the sudden loss of genetic material in normal cells that disrupts the normal balance between growth and cell death. This occurs through mutations and epimutations, resulting in uncontrolled cell division and The evolution of these cells by natural selection occurs in an uncontrolled and often rapid manner. Uncontrolled cell proliferation can result in benign or malignant tumors (cancer). Benign tumors are tumors that spread to other parts of the body. Malignant tumors do not invade other organs or spread to distant sites. can spread (metastasize) to other organs and become life-threatening.

[0010] In one embodiment, the cancer cells are selected from the group consisting of HT1080, A549, SC-OV-3, PC3, H1376, NCI-H292, LnC ap, MC38, 4T1-D02, and RKO tumor cells.

[0011] In one embodiment, the component present on cancer cells is nectin-4.

[0012] Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which includes four members. Nectins are involved in the polarity of epithelial, endothelial, immune, and neuronal cells during development and adulthood. Cells play an important role in various biological processes such as proliferation, differentiation, and migration. These are adhesion molecules that are involved in several pathological processes in humans. are the primary receptors for poliovirus, herpes simplex virus, and measles virus Mutations in the genes encoding nectin-1 (PVRL1) or nectin-4 (PVRL4) are associated with other disorders. Nectin-4 is expressed during fetal development and causes ectodermal dysplasia syndrome associated with glaucoma. In adult tissues, its expression is more restricted than that of other members of the family. -4 is primarily responsible for 50%, 49%, and 86% of breast, ovarian, and lung cancers, respectively. It is a tumor-associated antigen on poorly performing tumors. Its expression is not detected in corresponding normal tissues. In breast tumors, nectin-4 is mainly expressed in triple-negative and ERBB2+ cancers. The detection of soluble forms of nectin-4 in the serum of patients with these cancers is associated with prognosis. Serum nectin-4 levels increase during metastatic progression and decrease after treatment. These results indicate that Nectin-4 may 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 combination that targets nectin-4. It is an anti-cancer drug conjugate (ADC) currently in clinical research for the treatment of patients with solid tumors. It is being studied.

[0013] In one embodiment, the first peptide ligand is a Nectin-4 binding bicyclic peptide ligand. Includes

[0014] Suitable examples of Nectin-4 binding bicyclic peptide ligands are those whose peptides are incorporated herein by reference. This is disclosed in PCT patent application PCT / GB2019 / 051740, which is incorporated herein by reference.

[0015] In one embodiment, the Nectin-4 binding bicyclic peptide has the sequence number described herein. The peptide is selected from any of peptides Nos. 52 to 66.

[0016] In an alternative embodiment, the component present on cancer cells is EphA2.

[0017] Eph receptor tyrosine kinases (Ephs) are kinases that phosphorylate proteins on tyrosine residues. It belongs to the large receptor tyrosine kinase (RTK) family, which is a type of enzyme. Phospholipase ligands (ephrins) regulate cell positioning and tissue organization (Poliakov et al., 2004). Dev Cell 7, 465-80). Functional and biochemical Eph responses are associated with higher ligand oligomerization. This occurs in the state (Stein et al. (1998) Genes Dev 12, 667-678).

[0018] Among other patterning functions, various Ephs and ephrins play roles in vascular development. It has been shown that knockout of EphB4 and ephrin-B2 remodels the capillary bed. This results in a lack of vascularization (Poliakov et al., supra) and embryonic lethality. Sustained expression of Eph receptors and ephrins is also observed in newly formed adult microvessels. (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0019] The deregulated reappearance of some ephrins and their receptors in adults also contributes to tumor invasion, It has been observed that it contributes to metastasis and neovascularization (Nakamoto et al., 2002). c Res Tech 59, 58-67; Brantley-Sieders et al., supra). In addition, several Eph phagocytic Milli members have been found to be overexpressed in tumor cells derived from a variety of human tumors. (Brantley-Sieders et al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0020] EPH receptor A2 (ephrin type-A receptor 2) is encoded by the EPHA2 gene in humans It is a protein that is

[0021] EphA2 is a ubiquitous protein that is frequently correlated with disease progression, metastasis, and poor prognosis in many human cancers, e.g., For example: 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), stomach 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. (2002) 009) Hepatol Res. 39, 1169-1177), and glioblastoma (Wykosky et al. (2005) Mol Cancer Re s. 3, 541-551; Li et al. (2010) Tumour Biol. 31, 477-488).

[0022] Although the full role of EphA2 in cancer progression has not yet been clarified, it is thought to be involved in tumor cell proliferation, survival, and There is evidence of an interplay between these two factors at many stages of cancer progression, including survival, invasion, and angiogenesis. Downregulation of EphA2 expression suppresses tumor cancer cell proliferation (Binda et al. (2012) Cancer Cell 22, 765-780), whereas 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, 2-1 1; Lin et al. (2007) Cancer 109, 332-40), as well as metastatic progression (Brantley-Sieders et al. Reference (2005) FASEB J. 19, 1884-1886).

[0023] Antibody-drug conjugates with EphA2 inhibit tumor growth in rat and mouse xenograft models. It has been shown to significantly reduce tumor growth (Jackson et al., 2008, Cancer Research 68, 9367-9374), and similar approaches have been attempted in humans, but treatment has been associated with a high incidence of treatment-related adverse events. The drug had to be discontinued due to the following reasons (Annunziata et al. (2013) Invest New drugs 31, 77-84).

[0024] In one embodiment, the first peptide ligand is an EphA2-binding bicyclic peptide ligand. Includes.

[0025] Suitable examples of EphA2-binding bicyclic peptide ligands are those peptides described herein by reference. Disclosed in WO 2019 / 122860, WO 2019 / 122861, and WO 2019 / 122863, which are incorporated herein by reference. It has been done.

[0026] In one embodiment, the EphA2-binding bicyclic peptide ligand comprises a sequence described herein. The peptide is selected from any of the peptides in columns 10 to 51.

[0027] In an alternative embodiment, the component present on cancer cells is PD-L1.

[0028] Programmed cell death 1 ligand 1 (PD-L1) is expressed on mouse chromosome 19 and human chromosome 9. It is a 290 amino acid type I transmembrane protein encoded by the CD274 gene. Chronic infections, such as chronic viral infections (including, for example, HIV, HBV, HCV, and HTLV, among others) chronic bacterial infections (e.g., including, among others, Helicobacter pylori) and chronic parasitic infections (including Schistosoma mansoni). PD-L1 expression is expressed by T cells, B cells, and macrophages. several cells, including dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, muscle cells, and placenta. It has been detected in several tissues and cell types.

[0029] PD-L1 expression is also involved in suppressing anti-tumor immune activity. Tumors are recognized by host T cells. Although tumors express antigens that can be recognized, immunological clearance of tumors is rare. Part of the defect is due to immunosuppression by the tumor microenvironment. PD-L1 expression, which is a component of this suppressive environment, acts in concert with other immunosuppressive signals. PD-L1 expression is found in breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, and nerve. In vivo studies have been performed in a wide variety of solid tumors, including glioma, thyroid, thymic epithelial, and head and neck tumors. (Brown JA et al., 2003 Immunol. 170:1257-66; Dong H et al., 2003 Immunol. 170:1257-66) 2002 Nat. Med. 8:793-800; Hamanishi J et al., 2007 Proc. Natl. Acad. Sci. USA 1 04:3360-65; Strome SE et al., 2003 Cancer Res. 63:6501-5; Inman BA et al., 200 7 Cancer 109:1499-505; Konishi J et al., 2004 Clin. Cancer Res. 10:5094-100; Na Kanishi 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. S ci. USA 101: 17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). Furthermore, PD expression of programmed cell death protein 1 (also known as PD-1 and CD279), the receptor for IL-1 This expression is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C). (2003 Immunol. 171:4574-81). Most importantly, tumor PD-L1 expression is associated with disease progression. Studies linking PD-L1 expression to outcome have shown that PD-L1 expression is associated with renal, ovarian, bladder, breast, gastric, and ovarian cancers. It has been shown to be strongly correlated with poor prognosis in pancreatic cancer (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al., 2007 Cancer 1 09:1499-505; 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 1 01:17174-79; Wu C et al., 2006 Acta Histochem. 108:19-24). Furthermore, these studies Therefore, higher levels of PD-L1 expression in tumors are associated with advanced tumor stage and deeper tissue structures. It has been suggested that this may facilitate invasion of the tissue.

[0030] The PD-1 pathway may also play a role in hematologic malignancies. PD-L1 binds to a number of bone marrow It is expressed in tumor cells but not in normal plasma cells (Liu J et al., 2007 Blood 1 10:296-304). PD-L1 is expressed in some primary T-cell lymphomas, especially anaplastic large cell T-lymphoma. PD-1 is expressed in the angioimmunoblastic lymphoma (ANLL) and inflammatory bowel disease (Anthropoietin-1). PD-L1 is highly expressed on T cells in 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 regional Hodgkin lymphoma, T cells associated with lymphocytes or histiocytes (L&H) cells express PD-1 Microbial analysis using gene readout induced by PD-1 ligation Array analysis reveals that tumor-associated T cells express PD-1 signals in situ in Hodgkin lymphoma (Chemnitz JM et al., 2007 Blood 110:3226-33) PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma. (Shimauchi T et al., 2007 Int. J. Cancer 121: 2585-90). These tumor cells It is hyporesponsive to CR signals.

[0031] Studies in animal models have shown that PD-L1 on tumors blocks T cell activation and tumor cell lysis. It has been shown that tumor-specific T cell death can be aggravated by T cell proliferation and proliferation, and in some cases, can lead to increased tumor-specific T cell death (Dong H et al. (2002 Nat. Med. 8:793-800; Hirano F et al., 2005 Cancer Res. 65:1089-96) Tumor-associated APCs can also utilize the PD-1:PD-L1 pathway to regulate antitumor T cell responses. PD-L1 expression on tumor-associated myeloid DC populations is upregulated by tumor-environment factors. (Curiel TJ et al., 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in the nodes express IDO, which potently 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).

[0032] In one embodiment, the first peptide ligand is a PD-L1-binding bicyclic peptide ligand. Includes.

[0033] Suitable examples of PD-L1 binding bicyclic peptide ligands are those peptides described herein by reference. and GB Patent Applications Nos. 1905631.6 and 1904622.6, which are incorporated herein by reference.

[0034] In one embodiment, the PD-L1 binding bicyclic peptide is selected from the group consisting of SEQ ID NOs: 1 to 5 as described herein. The peptide is selected from any of the nine peptides.

[0035] In an alternative embodiment, the component present on cancer cells is prostate specific membrane antigen (PSM). A).

[0036] Prostate-specific membrane antigen (PSMA) (glutamic acid carboxypeptidase II (GCPII), N-acetylglucosamine) L-aspartyl-L-glutamic acid peptidase I (NAALADase I), and NAAG peptidase In humans, folate hydrolase 1 (FOLH1) is encoded by the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750 amino acids and weighs approximately 84 kDa.

[0037] Human PSMA is highly expressed in the prostate, approximately 100 times more than in most other tissues. In some prostate cancers, PSMA is the second most highly upregulated gene product, and This high expression level of PSMA is 8-12 times higher than that in cancerous prostate cells. It is being developed as a potential biomarker for the treatment and imaging of several cancers. In human prostate cancer, tumors with higher expression are associated with faster progression times and larger tumors. It is associated with a high percentage of patients suffering from recurrence.

[0038] In one embodiment, the first peptide ligand is a PSMA-binding bicyclic peptide ligand. include.

[0039] Suitable examples of PSMA-binding bicyclic peptide ligands are those peptides incorporated herein by reference. GB Patent Applications Nos. 1820325.7 and 1912723.2 and PCT Patent Application No. PCT / EP2019 / 06, which are incorporated herein by reference. This is disclosed in US Pat. No. 6273.

[0040] (Second peptide ligand) Reference herein to the term "immune cell" includes any cell within the immune system. Suitable examples include lymphocytes (e.g., T lymphocytes or T cells, B cells, or natural In one embodiment, the T cells are CD8 or CD4 T cells. In a further embodiment, the T cells are CD8. Other examples of immune cells include dendritic cells. These include follicles, follicular dendritic cells, and granulocytes.

[0041] In one embodiment, the component present on immune cells is CD137.

[0042] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Death factor receptor superfamily member 9 (TNFRSF9), 4-IBB, and lymphocyte activation CD137 can be expressed by activated T cells, but is mostly CD137 expression is more pronounced on CD8+ T cells than on CD4+ T cells. Furthermore, CD137 expression is more pronounced on dendritic cells, filtrates, and erythrocytes. It is found in follicular dendritic cells, natural killer cells, granulocytes, and cells of the blood vessel wall at sites of inflammation. One characterized activity of CD137 is its costimulatory activity on activated T cells. Cross-linking of enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. , can enhance immune activity and eliminate tumors in mice.

[0043] CD137 is a T cell costimulatory receptor that is induced upon TCR activation (Nam et al., Curr. Can cer Drug Targets, 5:357-363(2005); Waits et al., Annu. Rev. Immunol., 23:23-68( In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is expressed on CD4+CD25+ regulatory T cells. It is also expressed in T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, binds to B cells, monocytes / macrophages, and dendritic cells. It has been described in relation to antigen-presenting cells containing the IL-1 receptor (Watts et al., Annu. Rev. Immunol. 23:23- Upon interaction with its ligand, CD137 mediates TCR-induced T cell proliferation, cytotoxicity, and immune responses. This results in increased cytokine production, functional maturation, and prolonged CD8+ T cell survival (Nam et al., Curr. Cancer Drug Targets, 5:357-363(2005); Watts et al., Annu. Rev. Immunol, 23:23-6 8(2005)).

[0044] via CD137 by either CD137L or an agonistic monoclonal antibody (mAb) against CD137 signaling leads to increased TCR-induced T cell proliferation, cytokine production, and functional maturation, as well as These effects are due to (1) the activation of NF-κB, c-Jun NH2-terminal kinase, and (2) the activation of CD8+ T cells. enzymes / stress-activated protein kinases (JNK / SAPK), and p38 mitogen-activated protein (2) activation of the protein kinase (MAPK) signaling pathway, and (3) anti-apoptotic and pericellular responses Regulation of phase-related gene expression: resulting from

[0045] Experiments performed in both CD137-deficient and CD137L-deficient mice demonstrated a fully The importance of CD137 costimulation in the generation of effective T cell responses was further demonstrated.

[0046] IL-2 and IL-15 activated NK cells express CD137, and ligation of CD137 by agonistic mAbs stimulates NK cell proliferation and IFN-γ secretion, but not their cytolytic activity.

[0047] Furthermore, CD137-stimulated NK cells promote the proliferation of activated T cells in vitro.

[0048] According to its costimulatory function, agonistic mAbs against CD137 have been shown to inhibit the proliferation of cardiac and skin allografts. promotes rejection, eradicates established tumors, expands primary antiviral CD8+ T cell responses, and stimulates T cell These studies have demonstrated that CD137 signaling increases tumor cytolytic activity. This supports the view that IL-1 promotes T cell function, which may enhance immunity against tumors and infections.

[0049] In one embodiment, the second peptide ligand is a CD137-binding bicyclic peptide ligand. Includes.

[0050] Suitable examples of CD137-binding bicyclic peptide ligands are those peptides described herein by reference. This is disclosed in WO 2019 / 025811, which is incorporated herein by reference.

[0051] In one embodiment, the CD137-binding bicyclic peptide is SEQ ID NO: 67 described herein. Selected from any of ~84 peptides.

[0052] (Linker) The first peptide ligand can be linked to the second peptide ligand via any suitable linker. It will be understood that the linker can be conjugated to The design involves two bicyclic peptides that bind to both target receptors either singly or simultaneously. and presented in a manner that allows unhindered binding to their respective targets. Furthermore, the linker allows for simultaneous binding to both targets while The linker should maintain an appropriate distance between target cells to produce the desired functional result. The properties of the polymer may be modified to increase length, stiffness, or solubility to optimize the desired functional outcome. The linker can be adjusted to allow for the attachment of multiple bicycles to the same target. Increasing the valency of any binding peptide can also be designed to This may serve to increase the affinity of the heterotandem for the target cell or to enhance the target receptor. It may serve to induce oligomerization of one or both of the bodies.

[0053] In one embodiment, the linker has the following sequences: -PEG5- and TCA-[PEG 10 ]3 is selected do.

[0054] Structural representations of these linkers are detailed below: [ka] .

[0055] (heterotandem complex) In one specific embodiment, the first peptide ligand comprises a TATA scaffold. and a second peptide ligand comprising a PD-L1-binding bicyclic peptide ligand linked to a TATA a CD137-binding bicyclic peptide ligand attached to a scaffold, The complex is selected from the complexes listed in Table A: Table A (PD-L1:CD137; 1:1) [Table 2]

[0056] In one embodiment, the heterotandem bicyclic peptide conjugate comprises BCY12375 and BCY12021 : is selected from.

[0057] In one specific embodiment, the first peptide ligand comprises a TATA scaffold. and a second peptide ligand comprising TATA a CD137-binding bicyclic peptide ligand attached to a scaffold, The complex is selected from the complexes listed in Table B: Table B (EphA2:CD137; 1:1) [Table 3] TIFF2026027327000013.tif224170

[0058] In one embodiment, the heterotandem bicyclic peptide conjugate is selected from the group consisting of BCY13035, BCY13040, BCY13253, BCY13254, BCY13340, and BCY13342.

[0059] In one specific embodiment, the first peptide ligand comprises a TATA scaffold. and a second peptide ligand comprising: The heterotypic structure comprises a CD137-binding bicyclic peptide ligand attached to a TATA scaffold. The endothelial conjugate is selected from the conjugates listed in Table C: Table C (Nectin-4:CD137; 1:1) [Table 4]

[0060] In one embodiment, the heterotandem bicyclic peptide conjugate is selected from the group consisting of BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661, and BCY12969.

[0061] Unless otherwise defined, all technical and scientific terms used herein are understood to be within the skill of the art. , e.g., peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. have the same meaning as generally understood by practitioners in the field of science and technology. are used in methods of molecular biology, genetics, and biochemistry (incorporated herein by reference). Included in the article by Sambrook et al., Molecular Cloning: A Laboratory Manual A Laboratory Manual), 3rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Sp Ring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology in Molecular Biology (1999) 4th ed., John Wiley & Sons).

[0062] (Nomenclature) (Molecular format) N- or C-terminal extensions to the bicyclic core sequence may be added to the left or right side of the sequence, separated by a hyphen. It is added to the right side. For example, an N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO: X) It is expressed as:

[0063] (reverse peptide sequence) In view of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, The peptide sequences disclosed in also have utility in their retro-inverso forms. For example, if the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and The stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids). (These amino acids become L-amino acids, and L-amino acids become D-amino acids.) or a reference to an amino acid either as the one-letter or three-letter code for that amino acid Unless otherwise specified, amino acids are intended to be represented herein as L-amino acids. When such an amino acid is intended to be represented as a D-amino acid, the amino acid , for example, [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc., are prefixed with a lowercase d in square brackets. will be done.

[0064] (peptide ligand) The peptide ligands referred to herein are those that are covalently attached to a molecular scaffold. Typically, such peptides are covalently bonded to a scaffold. and two or more reactive groups (i.e., cysteine ​​residues) that can form a symmetric peptide. The sequence between the reactive groups is called a loop sequence because it forms a loop when it binds to the backbone. In this case, the peptide comprises a sequence inherent in and / or cysteamine, and a scaffold comprising at least three reactive groups selected from the group consisting of a hydroxypropyl methylcellulose, ... Form at least two loops on the fold.

[0065] (reactive group) The molecular scaffolds of the present invention can be attached to polypeptides via functional or reactive groups on the polypeptide. These may be attached to peptides, which are typically found in polypeptide polymers. Such reactive groups are formed from the side chains of certain amino acids. side chain, or N-terminal amino group, or any other suitable reactive group, e.g., penicillamine Details of suitable reactive groups can be found in WO 2009 / 098450.

[0066] Examples of reactive groups in natural amino acids are the thiol group of cysteine, the amino group of lysine, and the amino group of asparagine. Carboxyl group of guanine or glutamic acid, guanidinium group of arginine, tyrosine The phenolic group of α-amino acids is the phenolic group of α-amino acids, and the hydroxyl group of serine. -Provides a wide range of reactive groups including carbonyl, alkyne, vinyl, or aryl halide groups The amino and carboxyl groups at the ends of the polypeptide can also be used as molecular scaffolds. It can serve as a reactive group to form a covalent bond with the bond / molecular core.

[0067] The polypeptides of the present invention contain at least three reactive groups. The more reactive groups used, the more Loops can be formed in the molecular scaffold.

[0068] In a preferred embodiment, a polypeptide is produced that has three reactive groups. Reaction of peptides with molecular scaffolds / cores with three-fold symmetry results in the formation of single 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, but It does not code for the isomeric state of the molecule formed upon reaction of the dode with the molecular core. Where isomers can be formed, the assignment of nucleic acids to product isomers is unambiguously defined. If multiple product isomers are formed, the nucleic acid may be used in a screening or selection process. No information can be given about the properties of the isolated product isomers. Single product isomers This information is also advantageous when specific members of the libraries of the invention are synthesized. In the case of a mixture of isomers, the chemical reaction between the polypeptide and the molecular scaffold results in Instead, a single product isomer is produced.

[0069] In another embodiment, a polypeptide having four reactive groups is produced. The reaction of tides with molecular scaffolds / molecular cores with tetrahedral symmetry gives two products Isomers are produced when two different product isomers are encoded by the same nucleic acid. However, it is possible to chemically synthesize both isomers, separate the two isomers, and then target both isomers with the target ligand. The properties of the isolated isomers can be determined by testing for binding to the do.

[0070] In one embodiment of the invention, at least one of the reactive groups of the polypeptide is The use of orthogonal reactive groups allows the orthogonal reactive groups to be attached to specific portions of the molecular core. Using a linking strategy involving orthogonal reactive groups, the resulting product can be directed to the desired position. In other words, the number of product isomers can be limited by at least three bonds. Attach at least three reactive groups that are separate or different from the reactive groups selected for the remaining ones. By selecting for one or more of the following, specific positions on the molecular scaffold can be obtained. This effectively achieves a specific order of attachment or orientation of specific reactive groups on a polypeptide to the desired position. This can be done.

[0071] In another embodiment, the reactive group of the polypeptide of the invention reacts with a molecular linker, thereby In this case, the linker is a molecular scaffold and a polypeptide in the final conjugated state. The peptides can react with the molecular scaffold to intercalate.

[0072] In some embodiments, the identity of the members of a library or set of polypeptides is The amino acids can be replaced with any natural or unnatural amino acid. Only the loop sequence is replaced. functional groups for cross-linking the polypeptide to the molecular core so that it is exchangeable; These replaceable amino acids are excluded. The replaceable polypeptide sequences are random. Either a regular sequence, a regular sequence, or a sequence with random and regular amino acids The position of these amino acids determines the loop size, so the amino acids with reactive groups Each of the amino acids is at a defined position within the polypeptide.

[0073] In one embodiment, the polypeptide having three reactive groups has the sequence (X) l Y(X) m Y(X) n Y(X) o where Y represents an amino acid having a reactive group and X represents a random amino acid. , m and n are the intervening polypeptide segments (which may be the same or different) represents a number of 3 to 6 that defines the length of the adjacent polypeptide segments; Represents a number between 0 and 20 that specifies the length.

[0074] An alternative to thiol-mediated conjugation is via covalent interactions. Alternatively, these techniques can be used to attach molecular scaffolds to peptides. The present invention provides a method for preparing a small molecule of interest that is different from the molecular scaffold and further comprises: Thus, after selection or isolation, modification or attachment of said further moiety to the polypeptide may be performed. can be used - in this embodiment, obviously, the bond is covalent These methods involve the use of complementary reactive groups. Proteins with unnatural amino acids having the required chemically reactive groups in combination with small molecules and by producing peptide-displaying phage or by selecting molecules after a selection / isolation step. When produced, unnatural amino acids are incorporated into chemically or recombinantly synthesized polynucleotides. Incorporation into peptides can be used instead of (or in combination with) thiol-mediated methods. Further details can be found in WO 2009 / 098450 or Heinis et al., Nat Ch em Biol 2009, 5(7), 502-7.

[0075] In one embodiment, the reactive group is cysteine, 3-mercaptopropionic acid, and / or cysteamine residues.

[0076] (Pharmaceutically acceptable salts) Salt forms are within the scope of the present invention and reference to a peptide ligand includes the salt form of that ligand. It will be understood that

[0077] The salts of the present invention can be prepared by conventional chemical methods, e.g., Pharmaceutical Salts: Properties, Selection, and Uses ical Salts: Properties, Selection, and Use), P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002 Depending on the method, they can be synthesized from a parent compound containing a basic or acidic moiety. Such salts are prepared by dissolving the free acid or base form of these compounds in water with an appropriate base or acid. or in an organic solvent, or in a mixture of the two. can be done.

[0078] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, and the like. ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoin Acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphorsulfonic acid, (+) -(1S)-Camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, Enoic acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptan D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid, α-oxoglutaric acid, glycolic acid, hippuric acid, halogenated water hydrobromic acid (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, malo (±)-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, Acetic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglycerin glutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, Sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenate and valeric acid, and acylated amino acids and cation exchange resins. Examples of the salt include mono- and di-salts formed with the acid.

[0079] One particular group of salts is 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, naphtha Sulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactate It comprises salts formed from biotic acid. One particular salt is the hydrochloride salt. Another particular salt is , acetate.

[0080] The compound is anionic or has a functional group that can be anionic (e.g., —CO OH is -COO - In the case where the salt is formed with an organic or inorganic base, a suitable cation is formed. Examples of suitable inorganic cations include Li + , Na + , and K + Alkali such as Metal ions, Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Others Examples of suitable organic cations include, but are not limited to, the cations: Ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R 2 + , NHR3 + , NR4 + Some suitable substituted ammonium salts include, but are not limited to: Examples of nium ions include methylamine, ethylamine, diethylamine, and propylamine. amine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, Ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzene amine, choline, meglumine, and tromethamine, as well as lysine and arginine Examples of common quaternary ammonium ions include those derived from amino acids. H3)4 + is.

[0081] When the compounds of the invention contain amine functions, they can be prepared, for example, by methods well known to those skilled in the art. By reaction with an alkylating agent according to the formula: Quaternary ammonium compounds are within the scope of the present invention.

[0082] (Modified derivative) Modified derivatives of the peptide ligands defined herein are considered to be within the scope of the present invention. It will be understood that examples of such suitable modified derivatives include N-terminal and / or C-terminal End modification; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., one or more Substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of the amino acid residue with another non-natural isosteric or isoelectronic amino acid; addition of a spacer group; Substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more L-amino acid residues by one or more D-amino acid residues; substitution; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; Replacement of a bond with a surrogate bond; modification of peptide backbone length; on the α-carbon of one or more amino acid residues substitution of hydrogen with another chemical group, cysteine, lysine, glutamic acid / aspartic acid, and suitable amines of the amino acids, such as tyrosine, to functionalize the amino acids, thiol, etc. Directly suitable for modification and functionalization with phenol-, carboxylic acid-, and phenol-reactive reagents. Amino acids that introduce cross-linking activity, e.g., alkyne or azide-bearing moieties, respectively, are used. Introduction or substitution of amino acids with azide or alkyne groups that allow functionalization of The modifications include one or more of the following:

[0083] In one embodiment, the modified derivatives include N-terminal and / or C-terminal modifications. In embodiments, the modified derivatives include N-terminal modifications using suitable amino reaction chemistries, and and / or C-terminal modification using suitable carboxy reaction chemistry. The N- or C-terminal modifications may include, but are not limited to, cytotoxic agents, radioactive chelators, or It involves the addition of effector groups, including chromophores.

[0084] In a further embodiment, the modified derivative comprises an N-terminal modification. In this embodiment, the N-terminal modification comprises an N-terminal acetyl group. A phenyl group (referred to herein as C i The group called acetic anhydride or other suitable hydroxyl group is reacted with acetic anhydride or other suitable hydroxyl group during peptide synthesis. The resulting molecule is capped with a reagent and acetylated at the N-terminus. This offers the advantage of eliminating potential recognition points for aminopeptidases, and allows for the synthesis of bicyclic peptides. Avoid possible decomposition.

[0085] In an alternative embodiment, the N-terminal modification is for conjugation of an effector group and Includes the addition of a molecular spacer group that promotes retention of the potency of the bicyclic peptide against its target. .

[0086] In a further embodiment, the modified derivative comprises a C-terminal modification. In this embodiment, the C-terminal modification comprises an amide group. In the specification, C iii The C- This embodiment results in a terminally amidated molecule. This offers the advantage of eliminating potential recognition points and reducing the proteolytic potential of bicyclic peptides. To lower.

[0087] In one embodiment, the modified derivatives include one or more unnatural amino acid residues of one or more amino acid residues. In this embodiment, the cleavage site is substituted with a group that is recognized by a degradative protease. have isosteric / isoelectronic side chains that do not disrupt or have any adverse effects on targeting efficacy Unnatural amino acids may also be selected.

[0088] Alternatively, proteolytic hydrolysis of nearby peptide bonds may result in conformational and steric Non-natural amino acids with constrained amino acid side chains may also be used to hinder. In particular, these include proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisopropyl methyl esters, butyric acid, Aib), and cycloamino, a simple derivative of amino-cyclopropylcarboxylic acid. Regarding acids.

[0089] In one embodiment, the modified derivative comprises the addition of a spacer group. In the modified derivative, the N-terminal cysteine ​​(C i ) and / or a C-terminal cysteine ​​(C iii ) to This includes the addition of a spacer group.

[0090] In one embodiment, the modified derivatives comprise one or more oxidation-resistant amino acid residues of one or more oxidation-sensitive amino acid residues. In a further embodiment, the modified derivative comprises a substitution with a tripeptide amino acid residue. This embodiment includes the replacement of the naphthylalanine residue with a naphthylalanine or alanine residue. Offers the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligands do.

[0091] In one embodiment, the modified derivatives comprise one or more hydrophobic amino acids of one or more charged amino acid residues. In an alternative embodiment, the modified derivatives include substitution with one or more hydrophobic acid residues. The substitution of one or more amino acid residues with one or more charged amino acid residues. The correct balance of 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 therefore the available free radicals in plasma. The concentration of the isolated fraction is affected by the charge of the amino acid residues, while the charged amino acid residues (especially arginine) affect the concentration of the isolated fraction. This combination may affect the interaction of the phospholipid membranes on the cell surface with the This may affect the half-life, volume of distribution, and exposure of peptide drugs, potentially affecting clinical outcomes. Furthermore, the amount of charged and hydrophobic amino acids can be adjusted depending on the endpoint. The correct combination and number of residues reduces irritation at the injection site (if the peptide drug is administered subcutaneously). It can be reduced.

[0092] In one embodiment, the modified derivatives are one or more D-amino acid residues of one or more L-amino acid residues. This embodiment involves substitution by steric hindrance and stabilization of the β-turn conformation. The tendency of D-amino acids to form complexes is thought to enhance proteolytic stability (Tugyi et al. Reference (2005) PNAS, 102(2), 413-418).

[0093] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine. This embodiment has the advantage of eliminating potential proteolytic attack sites. do.

[0094] Each of the above modifications may serve to purposefully improve the potency or stability of the peptide. It should be noted that further improvement in potency based on modifications is achieved by the following mechanism: Can: -Utilizes the hydrophobic effect, resulting in a lower dissociation rate, so that higher affinity is achieved Incorporating hydrophobic moieties; - Utilizes long-range ionic interactions, resulting in faster association rates and higher affinity Incorporating charged groups (e.g., Schreiber et al., Rapid Electrostatic Assisted Protein Synthesis) Rapid, electrostatically assisted association of proteins (1996), Nature St. ruct. Biol. 3, 427-31); and For example, amino acid side chains should be correctly oriented so that entropy loss is minimized upon target binding. The torsion angles of the backbone are tightly constrained so that entropy loss is minimized upon target binding. By restricting the degree of cyclization and for the same reason introducing further cyclization into the molecule, Incorporating additional constraints into peptides (For a review, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 31 85-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0095] (Isotopic Variation) The present invention relates to a compound in which one or more atoms have the same atomic number but different atomic masses or nuclei that are commonly found in nature. is replaced by an atom having an atomic mass or mass number different from the mass number of the present invention. All known pharmaceutically acceptable (radio)isotope-labeled peptide ligands, as well as related The present invention relates to a compound having a metal chelating group attached thereto that can carry a (radioactive) isotope. Peptide ligands (called "effectors"), as well as specific functional groups associated with them (radioactive Peptides of the invention covalently substituted with isotopes or isotopically labeled functional groups Contains a ligand.

[0096] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention are isotopes of hydrogen, e.g. , 2 H(D) and 3 H(T), an isotope of carbon, e.g. 11 C. 13 C and 14 C, an isotope of chlorine, e.g. 3 6 Cl, isotopes of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I, and 131 I, Isotopes of nitrogen, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O, and 18 O, Lin Isotopes of, e.g., 32 P, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, Gari Isotopes of uranium, e.g., 67 Ga or 68 Ga, isotopes of yttrium, e.g.90 Y and Ru Tethium isotopes, e.g. 177 Lu, as well as isotopes of bismuth, e.g., 213 Contains Bi.

[0097] Certain isotope-labeled peptide ligands of the invention, e.g., incorporate a radioisotope. The present invention relates to the use of Nectin-4 in drug and / or substrate tissue distribution studies and in the detection of Nectin-4 targets on diseased tissues. The peptide ligands of the present invention are useful for clinically evaluating the presence and / or absence of a target. The marker is a complex between a labeled compound and another molecule, peptide, protein, enzyme, or receptor. These compounds have valuable diagnostic properties in that they can be used to detect or identify the formation of Further detection or identification methods may include, for example, radioisotopes, enzymes, fluorescent substances, etc. substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and Compounds labeled with labeling agents such as fluorophores (e.g., fluorophores and luciferase) can be used. The isotope tritium, i.e. 3 H(T) and carbon-14, i.e., 14 C uses its built-in It is particularly useful for this purpose given the ease and means of detection available. do.

[0098] Deuterium, i.e., 2 Substitution with heavier isotopes such as H(D) results in greater metabolic stability , for example, as a result of increased in vivo half-life or reduced dosage requirements. may provide additional therapeutic benefits and may therefore be preferred in some circumstances. do.

[0099] 11 C. 18 F, 15 O, and13 Substitution with positron-emitting isotopes such as N is useful for investigating target occupancy. It may be useful in positron emission topography (PET) studies for

[0100] Isotopically labeled compounds of the peptide ligands of the present invention are typically prepared using conventional techniques known to those skilled in the art. or by using appropriate isotopically labeled reagents in place of previously utilized unlabeled reagents. These compounds can be prepared by processes similar to those described in the accompanying examples.

[0101] (Molecular scaffolds) Molecular scaffolds are described, for example, in WO 2009 / 098450 and the references cited therein. It is described in the literature, in particular in WO 2004 / 077062 and WO 2006 / 078161.

[0102] As described in the aforementioned documents, molecular scaffolds are made of low molecular weight molecules, such as small organic molecules. It may be a child.

[0103] In one embodiment, the molecular scaffold may be a polymer. In this case, the molecular scaffold is composed of amino acids, nucleotides, or carbohydrates. It is a polymer.

[0104] In one embodiment, the molecular scaffold reacts with a functional group of the polypeptide to form a covalent bond. It contains a reactive group capable of forming a bond.

[0105] The molecular scaffold contains chemical groups that form bonds with the peptide, e.g., amines, thiols, etc. alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, Alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and halides It may contain acyl halogenides.

[0106] In one embodiment, the molecular scaffold is a hexahydro-1,3,5-triazine, particularly 1,3,5-triacryloylhexahydro-1,3,5-triazine ("TATA"), or its It may comprise or consist of derivatives.

[0107] The molecular scaffolds of the present invention are used to encode polypeptides of the encoded libraries of the present invention. The functional groups of the hydroxyl groups contain chemical groups that allow them to form covalent bonds with the molecular scaffold. The chemical groups include amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, and the like. Acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides The functional groups are selected from a wide range of groups including alkyl halides, alkyl halides, and acyl halides.

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

[0109] Examples include bromomethylbenzene or iodoacetamide. Other scaffolds used for selective coupling to cysteines in proteins include The cold reactive groups are maleimides, αβ-unsaturated carbonyl-containing compounds, and α-halomethylcarbonyls. The maleic acid compounds that can be used as molecular scaffolds in the present invention are carboxyl-containing compounds. Examples of imides include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)amine ) benzene, tris-(maleimido)benzene. αβ-unsaturated carbonyl-containing compounds An example of such a compound is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (T ATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). α An example of a -halomethylcarbonyl-containing compound is N,N',N''-(benzene-1,3,5-triyl)tris( 2-bromoacetamide). Selenocysteine ​​also has the same reactivity as cysteine. Cysteine ​​is a naturally occurring amino acid and can be used in the same reactions. Whenever reference is made to selenosides, unless the context suggests otherwise, Stains may be used instead.

[0110] (synthesis) The peptides of the present invention may be synthetically produced by standard techniques and then subjected to in vitro molecular spectroscopy. This can be done using standard chemistry. This allows for rapid production of soluble materials for further downstream experimentation or validation. Such a method is disclosed in Timmerman et al. (supra). This can be achieved using conventional chemistries such as those used in

[0111] Thus, the present invention also provides a polypeptide selected as described herein. or the preparation of a conjugate, wherein the preparation is carried out as described below. In one embodiment, these steps include any further steps such as: The method is performed on the final polypeptide conjugate produced by the method described above.

[0112] Optionally, the amino acid residues in the polypeptide of interest are selected to form a conjugate or complex. may be replaced when manufacturing.

[0113] The peptide can be extended, for example, to incorporate additional loops and thus introduce multiple specificities. You can also enter.

[0114] To extend the peptide, it is simply performed using standard solid or solution phase chemistry: Orthogonally protected lysines (and analogs) are used at their N- or C-termini or within loops. Standard (bio)conjugation techniques can be used to chemically extend the active Alternatively, additions may be made by introducing a modified or activatable N- or C-terminus, e.g., (D awson et al., 1994, Protein Synthesis by Native Chemical Ligation (Synthesis) (Thesis of Proteins by Native Chemical Ligation). Science 266:776-779) by fragment condensation or native chemical ligation, as described in, for example, Chang et al. Proc Natl Acad Sci U S A. 1994 Dec 20; 91(26):12544-8 or Hikari et al. Literature, Bioorganic & Medicinal Chemistry Letters, Volume 18, Issue 22, November 15, 2008, This may also be done enzymatically using subtiligase as described in the US Pat. No. 6,600,000 (pp. 6000-6003).

[0115] Alternatively, the peptides may be further conjugated via disulfide bonds. This allows the first and second peptides to react with each other in the reducing environment of the cell. In this case, the molecular scaffold The nucleotide sequence (e.g., TATA) is selected during the chemical synthesis of the first peptide to react with the three cysteine ​​groups. then, additional cysteines or thiols can be added to the first peptide. can be added to the N- or C-terminus of the cysteine ​​or thiol, so that the cysteine ​​or thiol is reacts only with free cysteines or thiols of the peptide to form disulfide-bonded bicyclic A peptide-peptide conjugate of the formula:

[0116] A similar technique can be used to synthesize two bicyclic bispecific macrocycles, potentially giving rise to tetraspecific molecules. Applies equally to the synthesis / coupling of molecules.

[0117] Furthermore, the addition of other functional groups or effector groups can be accomplished using appropriate chemistry via N- or C- Coupling at the terminal end or through a side chain may be achieved in the same manner. In this manner, the coupling is performed in such a way as not to block the activity of either entity. do.

[0118] (Pharmaceutical composition) According to a further aspect of the present invention, the peptide ligand as defined herein is administered to one or more pharmaceutical agents. Pharmaceutical compositions containing the compounds in combination with pharmaceutically acceptable excipients are provided.

[0119] Typically, the peptide ligand is administered in purified form together with a pharmacologically appropriate excipient or carrier. Typically, these excipients or carriers are saline and / or buffered solutions. The medium includes an aqueous or alcoholic / aqueous solution, an emulsion, or a suspension. Oral vehicles include sodium chloride solution, Ringer's dextrose, and dextrose. , and sodium chloride, and lactated Ringer's. Adjuvants, such as carboxymethylcellulose, may be used to keep the polypeptide complex in suspension. from thickeners such as cellulose acetate, polyvinylpyrrolidone, gelatin, and alginate may be selected.

[0120] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as Ringer's Also included are those based on dextrose. Preservatives and other additives, such as antimicrobials, Biological agents, antioxidants, chelating agents, and inert gases may also be present (Mack, 1982; Remington's Pharmaceutical Sciences, 16th ed.

[0121] The peptide ligands of the present invention may be administered as separate compositions or in combination with other agents. These may include antibodies, antibody fragments, and various immunotherapeutic agents, e.g. , cyclosporine, methotrexate, adriamycin, or cisplatin, and immunosuppressants Pharmaceutical compositions can be prepared by combining various protein ligands of the present invention with the toxins. "Cocktails" of cytotoxic or other drugs, or drugs that are pooled or pooled prior to administration polypeptides selected with different targeting ligands, whether or not they are It may even include a combination of selected polypeptides according to the invention having different specificities. Cut.

[0122] The route of administration of the pharmaceutical composition according to the present invention may be any of those generally known to those skilled in the art. For therapy, the peptide ligands of the invention may be administered to any patient according to standard techniques. Administration can be parenteral, intravenous, intramuscular, intraperitoneal, transdermal, or pulmonary route. any method, including via a catheter or, equally appropriately, by direct injection using a catheter. Preferably, the pharmaceutical composition according to the present invention is The dosage and frequency of administration depend on the age, sex, and condition of the patient, as well as other medications. The use of these drugs is determined by the concurrent administration of other drugs, contraindications, and other parameters considered by the clinician. do.

[0123] The peptide ligands of the present invention are lyophilized prior to storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and is compatible with freezing methods known in the art. Lyophilization and reconstitution techniques are available. Lyophilization and reconstitution can result in varying degrees of activity. This may result in losses and the level may need to be adjusted upward to compensate. It will be understood by those skilled in the art.

[0124] Compositions containing the peptide ligands of the present invention or a cocktail thereof are useful for prophylactic and / or therapeutic purposes. In certain therapeutic applications, the concentration of selected cells can be increased. At least partial inhibition, suppression, modulation, killing, or some other measurable parameter of the group. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose." The amount required to achieve this will depend on the severity of the disease and the general state of the patient's own immune system. The dose is determined by the amount of selected peptide ligands, generally 0.005 to 5.0 mg per kilogram of body weight. The range is 0.05 to 2.0 mg / kg / day, with doses of 0.05 to 2.0 mg / kg / day being more commonly used. Compositions containing the present peptide ligands or cocktails thereof may also exhibit similar or slightly less It may be administered in a dosage that is not too high.

[0125] Compositions containing peptide ligands according to the invention can be used in prophylactic and therapeutic settings. , to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in mammals Furthermore, the peptide ligands described herein can be selected ex vivo or in vitro. selectively to kill or deplete a target cell population from a heterogeneous collection of cells. The blood from the mammal can be filtered or otherwise effectively removed. The peptide ligand can be combined in vitro and then administered to mammals according to standard techniques. Killing or otherwise removing unwanted cells from the blood for return to the animal .

[0126] (therapeutic use) According to a further aspect of the present invention there is provided a method for preventing, suppressing or treating cancer. There is provided a heterotandem bicyclic peptide conjugate as defined herein.

[0127] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include tumors of epithelial origin (adenocarcinomas, various types of adenomas and carcinomas, including squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), e.g. Examples include bladder and urinary tract, breast, gastrointestinal tract (esophagus, stomach (gastric), small intestine, colon, rectum). , and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidneys, lungs (e.g. , adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g. Cancer of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovaries, fallopian tubes, Peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid gland (e.g., thyroid follicles) cancer), adrenal gland, prostate, skin, and adnexal cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratinocyte carcinoma) acanthoma, dysplastic nevi); hematologic malignancies (i.e., leukemia, lymphoma) and premalignant hematologic disorders borderline malignancies, including hematologic malignancies of the lymphoid lineage and related diseases (e.g., acute lymphoblastic leukemia, lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large cell lymphoma Follicular B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma , hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multifocal myeloma, and post-transplant lymphoproliferative disorders), and hematologic malignancies and related diseases of the myeloid lineage (e.g., For example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), Hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia); mesenchymal Tumors of origin, e.g., sarcomas of the soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, Chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma , synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumor, benign and malignant histiocytoma, and protruding Dermatofibrosarcoma; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma) cysts, meningiomas, ependymomas, pineal tumors, and schwannomas); endocrine tumors (e.g., pituitary tumors) tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid gland ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, spermatozoa) epithelioma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and pediatric and embryonal tumors (e.g., , medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or Congenital or other syndromes that predispose to tumors (e.g., xeroderma pigmentosum) These include, but are not limited to:

[0128] In further embodiments, the cancer is, for example, non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BLL), or lymphoma (BL), multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia Leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hematopoietic malignancies selected from: Johns Hopkins lymphoma (HL) and chronic myeloid leukemia (CML) be selected.

[0129] Reference herein to the term "prevention" refers to the administration of a protective composition prior to the induction of disease. "Suppression" includes administration of a composition after an inductive event but before the clinical appearance of the disease. "Treatment" includes administration of a protective composition after disease symptoms have become manifest.

[0130] Screening for the efficacy of peptide ligands in protecting against or treating disease Animal model systems are available that can be used to study the effects of steroids on the immune system. Allows for the development of polypeptide ligands that can cross-react with human and animal targets This is facilitated by the present invention.

[0131] The invention will now be further described with reference to the following examples. [Example]

[0132] (Example) In general, the heterotandem bicyclic peptide conjugates of the present invention can be prepared according to the following general method. It can be prepared as follows: [ka]

[0133] A mixture of bicycle 1 (1.0 equiv.) and NHS-PEG5-N3 (1.6 equiv.) was dissolved in MeCN / HO (1:1) to form a solution The pH of the mixture is adjusted to 8 by dropwise addition of NaHCO3 (0.1 M). The reaction mixture is stirred at 30 °C for 2 h, after which The residue was then purified by preparative HPLC to give the intermediate Body 2 is obtained.

[0134] A mixture of intermediate 2 (1.0 equiv.) and bicycle 2 (1.0 equiv.) was dissolved in t-BuOH / HO (1:1), followed by Add CuSO (1.0 equiv.), VcNa (2.3 equiv.), and THPTA (1.0 equiv.). Finally, add 0.2 M NH CO3 is added to adjust the pH to 8. The reaction mixture is stirred at 40°C under N2 atmosphere for 16 hours. The reaction mixture was directly purified by preparative HPLC.

[0135] More detailed studies of selected heterotandem bicyclic peptide complexes of the present invention are described herein. In the book, the following is provided:

[0136] Example 1: Synthesis of BCY12375 [ka] (Procedure for preparation of palmitic acid-PEG10-N3) [ka] Palmitic acid (100.0 mg, 282.89 μmol, 1.0 equiv.), compound 2 (150.0 mg, 284.84 μmol, 1.0 equiv.), A mixture of DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 equiv.) and DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 equiv.) was dissolved in DMF (2 mL). The reaction mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 1 was completely consumed. One major peak with the desired m / z (MW: 765.03, observed m / z: 765.22) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and give a residue. The residue was then purified by preparative HPLC (neutral conditions). 9.41 μmol, 35.14% yield, 96.27% purity) was obtained as a white solid.

[0137] (Procedure for preparation of palmitic acid-PEG10-BCY12023) [ka] Compound 3 (50.0 mg, 22.07 μmol, 1.0 equivalent), compound 2 (17.0 mg, 22.22 μmol, 1.0 equivalent), and A mixture of 10.0 mg of t-BuOH / HO (1:1, 1 mL, pre-degassed and N The solution was dissolved in CuSO4 (0.4 M, 56.0 μL, 1.0 equiv.) and VcNa (1 0.0 mg, 50.48 μmol, 2.3 equiv) was added under N2. The pH of this solution was adjusted with 0.2 M NH4HCO3 (1:1 tB The pH was adjusted to 8 by dropwise addition of HCl (in HClOH / H2O), resulting in a pale yellow solution. The mixture was stirred at 40°C under ambient temperature for 2 hours. LC-MS confirmed that palmitic acid-PEG10-N3 was converted back to the desired m / z. One major peak with m / z (calculated MW: 3030.60, observed m / z: 1010.35 ([M / 3+H] + 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 palmitic acid-PEG10-BCY1202 3 (43.0 mg, 13.97 μmol, 63.30% yield, 98.46% purity) was obtained as a white solid.

[0138] (Procedure for preparation of palmitic acid-PEG10-BCY12023-PEG5-N3) [ka] A mixture of compound 5 (43.0 mg, 14.19 μmol, 1.0 equivalent) and compound 6 (10.0 mg, 23.13 μmol, 1.6 equivalent) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and the pH of this solution was then adjusted to 0.05 by dropwise addition of NaHCO3 (0.1 M). The reaction mixture was stirred at 30°C for 2 hours. LC-MS showed that compound 5 was completely consumed. One major peak with the desired m / z (MW: 3347.94, observed m / z: 1673.7 ([(M / 2+H + ]), 1115.9([(M / 3+H + The reaction mixture was concentrated under reduced pressure. The solvent was removed to give a residue, which was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-BCY12023-PEG5-N3 (16.0 mg, 4.43 μmol, 31.25% yield, 92.78%) Purity) was obtained as a white solid.

[0139] (Procedure for preparation of BCY12375) [ka] Compound 7 (8.0 mg, 2.39 μmol, 1.0 equiv.), Compound 8 (6.5 mg, 2.39 μmol, 1.0 equiv.), and TH A mixture of PTA (1.1 mg, 2.53 μmol, 1.0 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and flushed with N three times) The solution was dissolved in 0.4 M CuSO4 (0.4 M, 6.0 μL, 1.0 equiv.) and VcNa (1.0 mg, 5 0.05 μmol, 2.1 equiv) was added under N2. The pH of this solution was adjusted to 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O). The temperature was adjusted to 8 by dropwise addition of HCl, and the solution turned pale yellow. The reaction mixture was heated at 40°C under a N2 atmosphere. The mixture was stirred at rt for 16 h. LC-MS showed that compound 7 was recovered, with one major peak having the desired m / z. (calculated MW: 6064.08, observed m / z: 1516.4 ([M / 4+H] + ), 1212.8([M / 5+H] + )) is checked The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA condition) to give BCY12375 (6.2 mg, 0.99 μmol, 41.62%). Yield, 97.27% purity) was obtained as a white solid.

[0140] Example 2: Synthesis of BCY12021 [ka] (Procedure for preparation of palmitic acid-PEG10-BCY11144) [ka] Compound 3 (160.0 mg, 69.45 μmol, 1.0 equivalent), Compound 4 (56.0 mg, 72.20 μmol, 1.0 equivalent), A mixture of 1000 mg of HCl and THPTA (35.0 mg, 80.55 μmol, 1.1 equiv.) was dissolved in t-BuOH / HO (1:1, 2 mL, pre-degassed). , purged with N2 three times), followed by addition of CuSO4 (0.4 M, 56.0 μL, 1.0 equiv.) and VcN a (30.0 mg, 151.43 μmol, 2.2 equiv.) was added under N2. The pH of this solution was adjusted to 1:1 with 0.2 M NH4HCO3 (1:1 The temperature was adjusted to 8 by dropwise addition of 100 mL of t-BuOH / H2O (in t-BuOH / H2O), and the solution turned pale yellow. The mixture was stirred under atmospheric pressure at 40° C. for 16 hours. LC-MS showed one major peak with the desired m / z ( Calculated MW: 3068.70, observed m / z: 1533.81 ([M / 2+H] + ), 1023.43([M / 3+H] + )) is checked 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 palmitic acid-PEG10-BCY11144 (150.0 m g, 46.83 μmol, 67.42% yield, 95.80% purity) was obtained as a white solid.

[0141] (Procedure for the preparation of palmitic acid-PEG10-BCY11144-PEG5-N3) [ka] Compound 5 (47.0 mg, 15.32 μmol, 1.0 equivalent), compound 6 (7.0 mg, 16.19 μmol, 1.0 equivalent), and A mixture of DIEA (3.0 mg, 22.97 μmol, 4.0 μL, 1.5 equiv.) was dissolved in DMF (1 mL). The mixture was stirred at 30° C. for 2 h. LC-MS showed that compound 5 was completely consumed and had the desired m / z. One major peak (MW: 3386.03, observed m / z: 1693.21 ([M / 2+H]+ ), 1129.13([M / 3+ H] + The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was then purified by preparative HPLC (neutral conditions). G10-BCY11144-PEG5-N3 (20.0 mg, 5.72 μmol, 37.33% yield, 96.79% purity) was obtained as a white solid. was obtained as.

[0142] (Procedure for preparation of BCY12021) [ka] Compound 7 (10.0 mg, 2.95 μmol, 1.0 equivalent), compound 8 (8.2 mg, 3.02 μmol, 1.0 equivalent), and T A mixture of HPTA (1.5 mg, 3.45 μmol, 1.1 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and filled with N) was diluted with 3 mL of t-BuOH / HO (1:1, 1 mL, pre-degassed and filled with N). The solution was dissolved in 100 ml of 10 ... , 7.57 μmol, 2.5 equiv) was added under N2. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / H2 The solution was adjusted to 8 by dropwise addition of 4 (in 0), and the solution turned pale yellow. The mixture was stirred at 0° C. for 16 hours. LC-MS showed one major peak with the desired m / z (calculated M W: 6102.17, observed m / z: 1525.17 ([M / 4+H] + ), 1221.3([M / 5+H] + )) was shown. The mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA conditions). Purification by HPLC afforded BCY12021 (6.6 mg, 1.02 μmol, 34.62% yield, 94.54% purity) as a white solid. It was obtained as a form.

[0143] Example 3: Synthesis of BCY11468 [ka] (Procedure for preparation of COM113) [ka] Compound 1 (50.0 mg, 124.4 μmol, 1.0 equivalent), EDCI (95.4 mg, 497.7 μmol, 4.0 equivalent), HOBt ( 2 mL of a mixture of 55.5 mg, 410.6 μmol, 3.3 equiv.) and DMAP (15.2 mg, 124.4 μmol, 1.0 equiv.) The mixture was dissolved in DMF, and then DIEA (134.9 mg, 1.04 mmol, 181.8 μL, 8.4 equivalents) was added and the mixture was homogenized. A clear solution was formed. Next, compound 2 (200.0 mg, 379.8 μmol, 3.0 mL) dissolved in DMF (2 mL) was added. 5 equivalents) was added dropwise to this solution. The reaction mixture was stirred at 30°C for 16 hours. Product 1 was completely consumed, and one major peak with the desired m / z (MW: 1891.19, observed m / z: 945.8600([M / 2+H + ]) and 612.4400 ([(M-3H2O) / 3+H + ])) was shown to be detected. The reaction mixture was directly purified by preparative HPLC (TFA conditions), and after lyophilization, COM113 (161m g, 85.67 μmol, 68% yield) was obtained as a yellow oil.

[0144] (Procedure for preparation of COM113-BCY8928) [ka] COM113 (50.0 mg, 26.44 μmol, 1.0 equiv.) and BCY8928 (53.0 mg, 23.9 μmol, 0.9 equiv.) The solution was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then added CuSO4 (0.4 M, 66.1 μL, 1.0 equivalent), VcNa (10 0.5 mg, 53.0 μmol, 2.0 equiv.), and THPTA (23.0 mg, 52.93 μmol, 2.0 equiv.). Afterwards, 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C under N2 atmosphere for 16 hours. LC-MS showed that the compound had the desired m / z. One major peak (calculated MW: 4108.77, observed m / z: 1369.97 ([M / 3+H] + )) is shown The reaction mixture was purified by preparative HPLC (TFA conditions) to give compound 2 (14.0 mg, 3.21 μmol , 12.14% yield, 94.16% purity) was obtained as a white solid.

[0145] (Procedure for the preparation of palmitic acid NHS ester) [ka] Palmitic acid (500 mg, 1.95 mmol, 586.85 μL, 1.0 equiv.), 1-hydroxypyrrolidine-2,5- A solution of dione (250 mg, 2.17 mmol, 1.11 equiv) in DCM (5 mL) was added to EDCI (747.60 mg, 3.90 mmol, 2.0 The mixture was stirred at 30°C for 16 hours. TLC showed that reactant 1 was completely consumed. The reaction was clean and showed one new spot forming. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The desired product was purified by chromatography (SiO2, DCM:MeOH = 0-100:1). Palmitic acid NHS ester (0.68 g, 1.92 mmol, 98.65% yield) was obtained as a white solid. was made.

[0146] (Procedure for the preparation of palmitic acid-propargylalanine) [ka] Compound 3 (120 mg, 339.47 μmol, 1.0 equivalent) and Compound 4 (57.60 mg, 509.20 μmol, 1.5 equivalent) In a DMF (6 mL) solution, DIEA (131.62 mg, 1.02 mmol, 177.39 μL, 3.0 equiv.) and DMAP (41.47 mg, The mixture was stirred at 40° C. for 16 hours. LC-MS showed that the reaction When the compound 3 is completely consumed, one major peak with the desired m / z or desired mass is detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification was performed by preparative HPLC (TFA conditions). Palmitic acid-propargylalanine (90 mg, 256.03 μmol, 75.42% yield) was obtained as a white solid.

[0147] (Procedure for the preparation of COM113-BCY8928-palmitic acid) [ka] Compound 2 (14.0 mg, 3.41 μmol, 1.0 equivalent) and compound 3 (1.1 mg, 3.13 μmol, 0.9 equivalent) were added together. The solution was first dissolved in 2 mL of t-BuOH / H2O (1:1), and then added CuSO4 (0.4 M, 10.0 μL, 1.1 equivalents), VcNa (2. HCl (2.0 mg, 10.1 μmol, 2.9 equiv.), and THPTA (2.0 mg, 4.6 μmol, 1.3 equiv.). The pH was adjusted to 8 by adding 0.2 M NH4HCO3. All solvents were degassed and refilled with N2 three times. The reaction mixture was stirred under N2 atmosphere at 35 °C for 16 h. LC-MS confirmed the desired m / z. One major peak (calculated MW: 4460.29, observed m / z: 1486.92 ([M / 3+H] + ) , 1115.58([M / 4+H] + ), 895.83([M / 5+H] + The reaction mixture was subjected to preparative HPLC (TFA conditions). Compound 4 (5.9 mg, 1.28 μmol, 37.66% yield, 97.0% purity) was obtained as a white solid. obtained as an object.

[0148] (Procedure for preparation of BCY11468) [ka] Compound 4 (5.9 mg, 1.32 μmol, 1.0 equiv.) and BCY11016 (3.0 mg, 1.29 μmol, 1 equiv.) were first mixed in 2 The solution was dissolved in 1 mL of t-BuOH / HO (1:1), followed by adding CuSO (0.4 M, 8.0 μL, 2.4 equiv.), VcNa (2.0 mg , 7.6 eq.), and THPTA (2.0 mg, 3.5 eq.) were added. Finally, 1 M NH4HCO3 was added to give p The H was adjusted to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was stirred under N2 atmosphere. The mixture was stirred at 30° C. for 16 hours. LC-MS showed one major peak with the desired m / z (calculated Observed MW: 6783.93, observed m / z: 1131.7 ([M / 6+H] + The reaction mixture was analyzed by preparative HPLC ( Purification by TFA (2.2 mg, 0.312 μmol, 23.57% yield, 96.16% purity) gave BCY11468. was obtained as a white solid.

[0149] Example 4: Synthesis of BCY11618 [ka] (Procedure for preparation of BCY8920-PEG5-N3) [ka] BCY8920 (50.0 mg, 23.39 μmol, 1.0 equivalent), compound 2 (10.2 mg, 23.51 μmol, 1.01 equivalent), A mixture of HCl and NaHCO (2.0 mg, 24.8 μmol, 1.0 equiv.) was dissolved in MeCN / HO (1:1, 2 mL). C-MS showed complete consumption of BCY8920 and one major peak with the desired m / z (calculated Observed MW: 2454.83, observed m / z: 1227.67 ([M / 2+H] + ) and 818.74([M / 3+H] + )) is detected The reaction mixture was stirred at 40°C for 2 hours until the reaction mixture was Concentration at 25°C removes the solvent to give a residue, which is then purified by preparative HPLC (TFA conditions). BCY8920-PEG5-N3 (25 mg, 9.70 μmol, 41.47% yield, 95.26% purity) was obtained as a white solid. This was obtained.

[0150] (Procedure for preparation of BCY11143-dK(palmitic acid)) [ka] BCY11143 (30.0mg, 12.84μmol, 1.0eq), Compound 5 (5.0mg, 14.12μmol, 1.1eq), DI A mixture of EA (1.7 mg, 12.84 μmol, 2.2 μL, 1.0 equiv.) and DMAP (1.6 mg, 12.84 μmol, 1.0 equiv.) The mixture was dissolved in DMF. The reaction mixture was stirred at 40°C under N2 atmosphere for 2 hours. One major peak with the desired m / z (calculated MW: 2575.14, observed m / z: 128 7.68([M / 2+H + The reaction mixture was filtered and concentrated under reduced pressure. The residue was then purified by preparative HPLC (TFA conditions). Lumitic acid (18.3 mg, 6.95 μmol, 54.17% yield, 97.86% purity) was obtained as a white solid. It was.

[0151] (Procedure for preparation of BCY11618) [ka] Compound 3 (5 mg, 2.04 μmol, 1.0 equivalent), compound 6 (5.8 mg, 2.3 μmol, 1.1 equivalent), and THPTA A mixture of (0.9 mg, 2.07 μmol, 1.0 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and flushed three times with N) was The solution was dissolved in CuSO4 (0.4 M, 5.1 μL, 1.0 equiv.) and VcNa (0.4 M, 5.1 μL, 1.0 equiv.). 1 μL, 1.0 equiv.) was added under N2. The pH of this solution was adjusted with a drop of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 40°C for 6 hours under a N2 atmosphere. LC-MS showed that compound 3 was completely consumed and one major peak with the desired m / z was observed. MW: 5029.97, observed m / z: 1257.8 ([M / 4+H] + ) and 1006.6([M / 5+H] + )) The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA condition) to give BCY11618 (5.3 mg, 1.0 μmol, 49.15 % yield, 95% purity) was obtained as a white solid.

[0152] Example 5: Synthesis of BCY11776 [ka] (Procedure for preparation of BCY8116-Peg5-N3) [ka] BCY8116 (50.0 mg, 23.39 μmol, 1.0 equivalent), compound 2 (10.2 mg, 23.51 μmol, 1.01 equivalent), A mixture of HCl and NaHCO (2.0 mg, 24.8 μmol, 1.0 equiv.) was dissolved in MeCN / HO (1:1, 2 mL). C-MS showed complete consumption of BCY8116 and one major peak with the desired m / z (calculated Observed MW: 2454.83, observed m / z: 1227.67 ([M / 2+H + ]), 818.74([M / 3+H + ])) is detected. The reaction mixture was stirred at 25°C for 1 hour until the reaction mixture was cooled to room temperature. Concentration removed the solvent to give a residue which was then purified by preparative HPLC (TFA conditions). Compound 3 (25.0 mg, 9.70 μmol, 41.47% yield, 95.26% purity) was obtained as a white solid. Ta.

[0153] (Procedure for the preparation of compound BCY11144-dK (palmitic acid)) [ka] BCY11144 (50.0mg, 21.7μmol, 1.0eq), Compound 5 (8.5mg, 23.87μmol, 1.1eq), DIE A mixture of A (2.81 mg, 21.7 μmol, 4.0 μL, 1.0 equivalent) and DMAP (2.7 mg, 21.7 μmol, 1.0 equivalent) The product was dissolved in DMF. The reaction mixture was stirred under N2 atmosphere at 25 °C for 2 hours. LC-MS showed , compound 3 was completely consumed, and one major peak with the desired m / z (calculated MW: 2542 0.08, observed m / z: 1271.7 ([M / 2+H + The reaction mixture was filtered. The residue was then purified by preparative HPLC (TFA conditions). Compound 6 (18.3 mg, 6.95 μmol, 54.17% yield, 96.68% purity) was obtained as a white solid. Obtained.

[0154] (Procedure for preparation of BCY11776) [ka] Compound 3 (10 mg, 4.0 μmol, 1.0 equivalent), compound 6 (11.2 mg, 4.4 μmol, 1.1 equivalent), and THPT A mixture of A (1.8 mg, 1.0 equiv.) in t-BuOH / HO (1:1, 1 mL, previously degassed and purged with N three times) of HCl), followed by adding CuSO4 (0.4 M, 5.1 μL, 1 equiv.) and VcNa (0.4 M, 5.1 μL, 1 equiv.) The pH of this solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O) under N2. The solution then turned pale yellow. The reaction mixture was stirred under N2 atmosphere at 40 °C for 6 hours. MS showed complete consumption of compound 3 with one major peak with the desired m / z (calculated MW: 5031.9, observed m / z: 1258.52 ([M / 4+H + ]), 1006.7([M / 5+H + ])) is detected The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Purification by preparative HPLC (TFA conditions) gave BCY11776 (12.5 mg, 2.4 μmol, 60.11% yield, 96.6%). Purity) was obtained as a white solid.

[0155] Example 6: Synthesis of BCY11860 [ka] (Procedure for preparation of BCY8920-Peg5-BCY11143) [ka] BCY8920-PEG5-N3 (20.0 mg, 8.15 μmol, 1.0 equivalent), compound 2 (21.0 mg, 8.96 μmol, 1.1 equivalent) A mixture of 1:1 t-BuOH / HO (1 mL, previously degassed) and THPTA (0.4 M, 21.0 μL, 1.0 equiv.) was dissolved in 1 mL of t-BuOH / HO (1:1, previously degassed) , purged with N2 three times), followed by addition of CuSO4 (0.4 M, 21.0 μL, 1.0 equiv.) and VcN a (0.4 M, 21.0 μL, 1.0 equiv.) was added under N2. The pH of this solution was adjusted with 0.2 M NH4HCO3 (1:1 t-BuO The temperature was adjusted to 8 by dropwise addition of HCl (H / H2O), resulting in a pale yellow solution. The reaction mixture was then cooled under a N2 atmosphere. The mixture was stirred at 40° C. for 4 hours under reduced pressure. LC-MS showed that compound 1 was completely consumed and had the desired m / z. One major peak (calculated MW: 4791.56, observed m / z: 1597.28 ([M / 3+H] + ), 1198. 18([M / 4+H] + The reaction mixture was filtered and concentrated under reduced pressure. The crude product was purified by preparative HPLC (TFA conditions) to give BCY8920-Peg5-BCY1 1143 (22.5 mg, 4.25 μmol, 52.13% yield, 90.44% purity) was obtained as a white solid.

[0156] (Procedure for preparation of BCY11860) [ka] Compound 3 (5.0 mg, 1.04 μmol, 1.0 equiv.), Compound 4 (1.08 mg, 1.15 μmol, 1.1 equiv.), and D Mixture of IEA (0.4 M, 1.04 μmol, 3.0 μL, 1.0 equivalent) and DMAP (0.2 mg, 1.04 μmol, 1.0 equivalent) The product was dissolved in DMF (1.0 mL). The reaction mixture was stirred at 30° C. for 2 hours. LC-MS showed that the compound 3 was completely consumed, and one major peak with the desired m / z (MW: 5617.56, observed m / z : 1404.56([(M / 4+H + The reaction mixture was concentrated under reduced pressure to give The solvent was removed to give a residue, which was then purified by preparative HPLC (neutral conditions). BCY11860 (2.9 mg, 0.48 μmol, 45.86% yield, 92.70% purity) was obtained as a white solid. .

[0157] Example 7: Synthesis of BCY12020 [ka] (Procedure for preparation of palmitic acid-PEG10-N3) [ka] Palmitic acid-NHS (100.0 mg, 282.89 μmol, 1.0 equiv.), compound 2 (150.0 mg, 284.84 μmol , 1.0 equiv.) and DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 equiv.) in DMF (2 mL). The reaction mixture was stirred at 30° C. for 2 hours. LC-MS showed that compound 1 was completely consumed. One major peak with the desired m / z (MW: 765.03, observed m / z: 765.22) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and give a residue. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-N3 (79.0 mg) , 99.41 μmol, 35.14% yield, 96.27% purity) was obtained as a white solid.

[0158] (Procedure for preparation of palmitic acid-PEG10-BCY11144) [ka] Compound 3 (160.0 mg, 69.45 μmol, 1.0 equivalent), Compound 2 (56.0 mg, 72.20 μmol, 1.0 equivalent), A mixture of 1000 mg of HCl and THPTA (35.0 mg, 80.55 μmol, 1.1 equiv.) was dissolved in t-BuOH / HO (1:1, 2 mL, pre-degassed). , purged with N2 three times), followed by addition of CuSO4 (0.4 M, 56.0 μL, 1.0 equiv.) and VcN a (30.0 mg, 151.43 μmol, 2.2 equiv.) was added under N2. The pH of this solution was adjusted to 1:1 with 0.2 M NH4HCO3 (1:1 The temperature was adjusted to 8 by dropwise addition of 100 mL of t-BuOH / H2O (in t-BuOH / H2O), and the solution turned pale yellow. The mixture was stirred under atmospheric pressure at 40° C. for 16 hours. LC-MS showed one major peak with the desired m / z ( Calculated MW: 3068.70, observed m / z: 1533.81 ([M / 2+H] + ), 1023.43([M / 3+H] + )) is shown The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPPE. Purification by LC (TFA conditions) gave palmitic acid-PEG10-BCY11144 (150.0 mg, 46.83 μmol, 67 The product was obtained as a white solid (0.42% yield, 95.80% purity).

[0159] (Procedure for preparation of palmitic acid-PEG10-BCY11144-PEG5-N3) [ka] Compound 5 (47.0 mg, 15.32 μmol, 1.0 equivalent), compound 6 (7.0 mg, 16.19 μmol, 1.1 equivalent), and A mixture of DIEA (3.0 mg, 22.97 μmol, 4.0 μL, 1.5 equiv.) was dissolved in DMF (1 mL). The mixture was stirred at 30° C. for 2 h. LC-MS showed that compound 5 was completely consumed and had the desired m / z. One major peak (MW: 3386.03, observed m / z: 1693.21 ([M / 2+H] + ), 1129.13([M / 3+ H] + The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was then purified by preparative HPLC (neutral conditions). 10-BCY11144-PEG5-N3 (20.0 mg, 5.72 μmol, 37.33% yield, 96.79% purity) was obtained as a white solid. This was obtained.

[0160] (Procedure for preparation of BCY12020) [ka] Compound 7 (50.0 mg, 14.77 μmol, 1.0 equivalent), compound 8 (35.0 mg, 15.06 μmol, 1.0 equivalent), and A mixture of 10.0 mg of t-BuOH / HO (1:1, 2 mL, pre-degassed and N The solution was dissolved in CuSO4 (0.4 M, 38.0 μL, 1.0 equiv.) and VcNa (6 0.5 mg, 32.81 μmol, 2.2 equiv.) was added under N2. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-Bu The temperature was adjusted to 8 by dropwise addition of HCl (in OH / H2O), resulting in a pale yellow solution. The reaction mixture was then cooled under a N2 atmosphere. The mixture was stirred at 40° C. for 16 hours under reduced pressure. LC-MS showed one major peak with the desired m / z (calculated Detected MW: 5709.68, observed m / z: 1902.80 ([M / 3+H] + ), 1427.56([M / 4+H] + )) was shown The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (TFA Purification by the same conditions yielded BCY12020 (54.8 mg, 9.49 μmol, 64.24% yield, 98.83% purity). It was obtained as a colored solid.

[0161] Example 8: Synthesis of BCY12661 [ka] (Procedure for the preparation of compound 2) [ka] The peptide was synthesized using standard Fmoc chemistry. DCM was added to chlorotrityl resin (1 mmol, 0 Into a reaction vessel containing Fmoc-Lys(N3)-OH (1 equiv., 395.4 mg, 1 mmol) and Fmoc-Lys(N3)-OH (1 equiv., 395.4 mg, 1 mmol) DIEA (4.0 equiv.) was added dropwise and mixed for 2 hours. OH (2 mL) was added and mixed for 30 min. The resin was drained and washed 5 times with DMF. Fmoc deprotection This was achieved by adding 20% ​​piperidine / DMF and mixing for 30 minutes. The resin was drained and washed with DMF for 5 minutes. For chain elongation, the Fmoc-amino acid solution was added and mixed for 30 seconds, then washed twice. Activation buffer (containing HBTU and DIEA in DMF) was added and the mixture was stirred under continuous N2 bubbling. The mixture was stirred for 1 hour. Deprotection and coupling were repeated until the peptide was complete. Ta. [Table 5]

[0162] After the last amino acid coupling, the resin was washed three times with MeOH and then dried under vacuum. 10 ml of cleavage cocktail (95% TFA / 2.5% TIS / 2.5% H2O) was added to the cleavage mixture containing the side-chain protected peptide. The resin was filtered off and the filtrate was concentrated to give a solution. The solvent was removed. The crude peptide was lyophilized to give the final product, Compound 2 (azide palmitate). 200 mg (97.78% purity, 37.06% yield) of methylpropional (methacrylate) was obtained. Calculated MW: 539.72, observed MW: 539.72. m / z: 540.4([M+H] + ).

[0163] (Procedure for the preparation of BCY12023-palmitic acid azide) [ka] Compound 1 (40.0 mg, 17.66 μmol, 1.0 equivalent), compound 2 (9.5 mg, 17.66 μmol, 1.0 equivalent), and A mixture of 100 mL of t-BuOH / HO (1:1, pre-degassed and N2 The solution was dissolved in CuSO4 (0.4 M, 45.0 μL, 1.0 equiv.) and VcNa (8. 0 mg, 35.33 μmol, 2.0 equiv) was added under N. The pH of this solution was adjusted with 0.2 M NH4HCO3 (1:1 t-BuO The temperature was adjusted to 8 by dropwise addition of HCl (H / H2O), resulting in a pale yellow solution. The reaction mixture was then cooled under a N2 atmosphere. The mixture was stirred at 40° C. for 4 hours under reduced pressure. LC-MS showed that compound 1 was completely consumed and had the desired m / z. One major peak (calculated MW: 2804.30, observed m / z: 1402.8 ([M / 2+H] + ), 935.9( [M / 3+H] + The reaction mixture was filtered and concentrated under reduced pressure to give the residue The crude product was purified by preparative HPLC (TFA conditions) to give BCY12023-palmitic acid The azide (35.0 mg, 12.11 μmol, 68.54% yield, 97.00% purity) was obtained as a white solid. .

[0164] (Procedure for preparation of BCY12023-Palmitic acid-PEG5-N3) [ka] A mixture of compound 3 (35.0 mg, 12.48 μmol, 1.0 equivalent) and compound 4 (5.4 mg, 12.48 μmol, 1.0 equivalent) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and the pH of this solution was then adjusted to 0.05 by dropwise addition of NaHCO3 (0.1 M). The reaction mixture was stirred at 30°C for 2 hours. LC-MS showed that compound 3 was completely consumed. One major peak with the desired m / z (MW: 3121.63, observed m / z: 1561.2 ([(M / 2+H + The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was then purified by preparative HPLC (neutral conditions). Mitic acid-PEG5-N3 (11.4 mg, 3.46 μmol, 27.71% yield, 94.70% purity) was obtained as a white solid. This was obtained.

[0165] (Procedure for preparation of BCY12661) [ka] Compound 5 (11.4 mg, 3.65 μmol, 1.0 equivalent), compound 6 (8.3 mg, 3.65 μmol, 1.0 equivalent), and T A mixture of HPTA (1.6 mg, 3.65 μmol, 1.0 equiv.) in t-BuOH / HO (1:1, 1 mL, pre-degassed and filled with N) was diluted with 3 mL of t-BuOH / HO (1:1, 1 mL, pre-degassed and filled with N). The solution was dissolved in 100 mL of 10 ... , 7.30 μmol, 2.0 equiv) was added under N2. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / H2 The solution was adjusted to 8 by dropwise addition of 4 (in 0), and the solution turned pale yellow. The mixture was stirred at 0° C. for 4 hours. LC-MS showed that compound 3 was completely consumed and one residue with the desired m / z was obtained. Major peak (calculated MW: 5374.21, observed m / z: 1344.5 ([M / 4+H] + )) is detected The reaction mixture was filtered and concentrated under reduced pressure to give a residue. was purified by preparative HPLC (TFA conditions) to give BCY12661 (9.8 mg, 19.63 μmol, 48.73% yield, 97%). 0.60% purity) was obtained as a white solid.

[0166] Example 9: Synthesis of BCY12969 [ka] General Procedure for Preparation of Compound 1 [ka] The peptide was synthesized using standard Fmoc chemistry. DCM was added to chlorotrityl resin (1 mmol, 0 A reaction vessel containing Fmoc-γGlu(OtBu)-OH (0.425 mg, 1 mmol, 1 equiv.) and Fmoc-γGlu(OtBu)-OH (0.91 g, 1.1 mmol / g). The mixture was stirred while bubbling with N2. DIEA (4.0 equiv.) was added dropwise and the mixture was stirred for 2 After stirring for 30 minutes, MeOH (4.6 mL) was added and mixed for 30 minutes. The resin was drained and resuspended in DMF. The resin was washed five times with 20% piperidine / DMF and mixed for 30 minutes. The resin was then rinsed with DMF five times. The Fmoc-amino acid solution was added to the resin and mixed for 30 seconds. The activator and DIEA were added, and N2 was bubbled through the mixture for 1 hour. The deprotection and coupling steps were repeated using: Note: [Table 6]

[0167] After coupling of palmitic acid, the resin was washed three times with MeOH and then dried under vacuum. The peptide was cleaved from the resin at room temperature by the addition of 20% HFIP / 80% DCM, and the mixture was stirred for 1 h. This procedure was repeated once more, after which the resin was filtered and the filtrate was concentrated to give a solvent The crude peptide was lyophilized to give the final product (280 mg, 84.80% purity, 44.67% Yield: 1.0g. Calculated MW: 626.8, observed m / z: 627.4 ([M+H] + ).

[0168] General procedure for the preparation of compound 3 [ka] To a solution of compound 2 (15.8 mg, 25.1 μmol, 1.1 equivalents) in DMF (0.5 mL) was added EDCI (4.4 mg, 22.8 μmol, 1.0 equiv.) was added and stirred for 10 min. Then, HOSu (2.9 mg, 25.1 μmol, 1.1 equiv.) and DIE A (8.8 mg, 68.5 μmol, 11.9 μL, 3 eq) was added to the mixture. The mixture was stirred at 25° C. for 16 h. Then, BCY12358 (50.0 mg, 22.8 μmol, 1.0 equiv.) in DMF (0.5 mL) was added to the mixture. This was stirred at 25° C. for another 4 hours. LC-MS showed that BCY12358 was completely consumed and the desired m / z One major peak with m / z (calculated MW: 2798.42, observed m / z: 1399.6 [M / 2+H] + ) The reaction mixture was subjected to preparative HPLC (A: 0.075% TFA in H2O, B: ACN). Further purification gave compound 3 (21.9 mg, 7.83 μmol, 34.3% yield) as a white solid. Ta.

[0169] General procedure for the preparation of compound 5 [ka] Compound 4 (20.0 mg, 8.03 μmol, 1.0 equiv.), compound 3 (22 A mixture of 1.5 mg, 8.03 μmol, 1.0 equiv. (1.5 mg, 8.03 μmol, 1.0 equiv.) and THPTA (4.0 mg, 9.21 μmol, 1.15 equiv.) was degassed. , purged with N2 three times, then added CuSO4 (0.4 M, 20.1 μL, 1.0 equiv.), VcNa (0.4 M, 40.2 μL, 2. 0 equiv.), and NH4HCO3 (0.2 M, 80.4 μL, 2.0 equiv.) were added to the mixture. The mixture was cooled to room temperature under a N2 atmosphere. The mixture was stirred at 30° C. under ambient temperature for 2 hours. LC-MS confirmed that compound 4 was completely consumed and had the desired m / z. One major peak (calculated MW: 5288.25, observed m / z: 1322.3 [M / 4+H] + , 1763.8 [M / 3+H] + ) was detected. EDTA (0.5 M, 20.0 μL) was added to the reaction mixture. The reaction mixture was concentrated under reduced pressure to give crude compound 5 (42.0 mg, crude) as a gray solid. was obtained as and used in the next step without further purification.

[0170] General procedure for the preparation of BCY12969 [ka] To a solution of compound 5 (42.0 mg, 8.22 μmol, 1.0 equiv.) in DCM (0.25 mL) was added TFA (3.37 μmol, 0.25 mL , 458.6 equivalents) was added dropwise. The mixture was stirred at 30° C. for 1 hour. LC-MS confirmed that compound 5 was completely synthesized. The resulting chromatogram was analyzed to determine the m / z of the chromatogram, and one major peak with the desired m / z (calculated MW: 5176.04, observed m / z: 1035.7[M / 5+H] + , 1294.9[M / 4+H] + , 1726.8[M / 3+H] +) was detected. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC (A: 0.075% HCl in HO). Purification by HCl (B: TFA, C: ACN) gave BCY12969 (2.6 mg, 0.48 μmol, 5.85% yield, 92.4% purity). was obtained as a white solid.

[0171] Example 10: Synthesis of BCY13035 [ka] (Procedure for preparation of BCY12860-PEG5-N3) [ka] BCY12860 (40.0 mg, 19.40 μmol, 1.0 equivalent), Compound 2 (10.0 mg, 21.34 μmol, 1.1 equivalent) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by dropwise addition of NaHCO3 (0.1 M). The reaction mixture was stirred at 25° C. for 1 hour. LC-MS showed that the compound had the desired m / z. The reaction mixture was concentrated under reduced pressure to remove the solvent and give a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY12860-PEG5-N3 (39.7 mg, 15.02 μg) mol, 77.41% yield, 90.0% purity) was obtained as a white solid. MW: 2378.78, observed m / z: 1190.1 ([(M / 2+H + ]), 793.5([(M / 3+H + ]).

[0172] (Procedure for preparation of BCY13035) [ka] Compound 3 (39.7 mg, 16.69 μmol, 1.0 equivalent), BCY8928 (41.0 mg, 18.36 μmol, 1.1 equivalent), and A mixture of t-BuOH / HO (1:1, 1 mL, pre-degassed and flushed with N three times) was added to the flask. The solution was dissolved in 0.4M purged solution of CuSO4 (0.4M, 55 μL, 1.3 equiv.) and VcNa (0.4M, 109 2.6 equiv.) was added under N2. The pH of this solution was adjusted with a drop of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O). The reaction mixture was heated at 40°C for 2 hours under a N2 atmosphere. LC-MS showed that compound 3 was completely consumed and one major peak with the desired m / z was observed. 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 BCY13035 (42.0 mg, 8.85 μmol , 53.04% yield, 96.41% purity) was obtained as a white solid. Calculated MW: 4596.37. Observed m / z: 1532.9 ([M / 3+H] + ), 1149.9([M / 4+H] + ).

[0173] Example 11: Synthesis of BCY13040 [ka] (Procedure for preparation of BCY12865-PEG5-N3) [ka] BCY12865 (30.0 mg, 13.99 μmol, 1.0 equivalent) and Compound 1 (6.1 mg, 14.11 μmol, 1.01 equivalent) was dissolved in 1 mL of MeCN / H2O (1:1), and then 1 M NaHCO3 was added to adjust the pH to 8. The mixture was stirred at 25° C. for 2 h. LC-MS showed that BCY12865 was completely consumed and the desired m / z was obtained. The reaction mixture was subjected to preparative HPLC (TFA conditions). Further purification afforded compound 2 (15.6 mg, 6.32 μmol, 45.19% yield, 99.76% purity) as a white solid. Calculated MW: 2461.87, observed m / z: 1231.5 ([M / 2+H] + ) and 821. 3([M / 3+H]+).

[0174] (Procedure for preparation of BCY13040) [ka] Compound 2 (15.6 mg, 6.34 μmol, 1.0 equiv.) and BCY8928 (14.5 mg, 6.54 μmol, 1.03 equiv.) First, it was dissolved in 2 mL of t-BuOH / H2O (1:1), and then added CuSO4 (0.4 M, 16 μL, 1.01 equivalents), VcNa (3 0.0 mg, 15.14 μmol, 2.39 equiv.), and THPTA (3 mg, 6.90 μmol, 1.09 equiv.). 1M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 40°C under N2 atmosphere for 16 hours. LC-MS showed that compound 2 had completely disappeared. The reaction mixture was analyzed and showed one major peak with the desired m / z. was purified by preparative HPLC (TFA conditions) to give BCY13040 (15.8 mg, 3.31 μmol, 52.27% yield, 98%). 0.1% purity) was obtained as a white solid. Calculated MW: 4679.45, observed m / z: 156 0.8([M / 3+H] + ), 1170.9([M / 4+H] + ), 936.6([M / 5+H] + ).

[0175] Example 12: Synthesis of BCY13253 [ka] (Procedure for preparation of BCY13119-PEG5-N3) [ka] BCY13119 (35.0 mg, 17.20 μmol, 1.0 equivalent), Compound 2 (7.8 mg, 18.06 μmol, 1.05 equivalent) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by dropwise addition of NaHCO3 (0.1 M). The reaction mixture was stirred at 25° C. for 1 hour. LC-MS showed that the compound had the desired m / z. One major peak (MW: 2352.74, observed m / z: 1177.4 ([(M / 2+H + ])) is detected The reaction mixture was concentrated under reduced pressure to remove the solvent and give a residue. The residue was purified by preparative HPLC (neutral conditions). BCY13119-PEG5-N3 (25.7 mg, 9.97 μmol, 58 The product was obtained as a white solid (0.0% yield, 91.3% purity).

[0176] (Procedure for preparation of BCY13253) [ka] Compound 3 (25.7mg, 10.92μmol, 1.0eq), compound 2 (26.6mg, 12.02μmol, 1.1eq), and A mixture of 100 mL of t-BuOH / HO (1:1, pre-degassed and N2 The solution was dissolved in CuSO4 (0.4 M, 33.0 μL, 1.2 equiv.) and VcNa (5. 2 mg, 26.21 μmol, 2.4 equiv.) was added under N. The pH of this solution was adjusted to 0.2 M NH.sub.4HCO.sub.3 (1:1 t-BuO The temperature was adjusted to 8 by dropwise addition of HCl (H / H2O), resulting in a pale yellow solution. The reaction mixture was then cooled under a N2 atmosphere. The mixture was stirred at 25° C. for 2 hours under reduced pressure. LC-MS showed that compound 3 was completely consumed and had the desired m / z. One major peak (calculated MW: 4570.32, observed m / z: 1143.4 ([M / 4+H] + ), 914.9( [M / 5+H] + The reaction mixture was filtered and concentrated under reduced pressure to give the residue The crude product was purified by preparative HPLC (TFA condition) to give BCY13253 (17.5 mg, 3.67 μmol, 33.58% yield, 95.8% purity) was obtained as a white solid.

[0177] Example 13: Synthesis of BCY13254 [ka] (Procedure for preparation of BCY13120-PEG5-N3) [ka] A mixture of BCY13120 (40.0 mg, 17.92 μmol, 1.0 equivalent) and compound 2 (8.5 mg, 19.72 μmol, 1.1 equivalent) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and the pH of this solution was then adjusted to 0.05 by dropwise addition of NaHCO3 (0.1 M). The temperature was adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. LC-MS showed that BCY13120 had completely disappeared. The resulting eluate was concentrated to give one major peak with the desired m / z (MW: 2548.99, observed m / z: 1275.3 ([( M / 2+H + The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was then purified by preparative HPLC (neutral conditions). 5-N3 (27.3 mg, 10.46 μmol, 58.38% yield, 97.7% purity) was obtained as a white solid.

[0178] (Procedure for preparation of BCY13254) [ka] Compound 3 (27.3 mg, 10.71 μmol, 1.0 equivalent), compound 2 (26.1 mg, 11.78 μmol, 1.1 equivalent), and A mixture of t-BuOH / HO (1:1, 1 mL, pre-degassed and N2 The solution was dissolved in CuSO4 (0.4 M, 33.0 μL, 1.2 equiv.) and VcNa (5.2 mg, 2 6.24 μmol, 2.4 equiv.) was added under N. The pH of this solution was adjusted to 0.2 M NH.sub.4HCO.sub.3 (1:1 t-BuOH / H.sub.2O). The reaction mixture was stirred for 25 minutes under a N2 atmosphere. The mixture was stirred at 5° C. for 2 hours. LC-MS showed that compound 3 was completely consumed and one compound with the desired m / z was obtained. Major peak (calculated MW: 4766.58, observed m / z: 1192.5 ([M / 4+H] + ), 954.1([M / 5+ H] + The reaction mixture was filtered and concentrated under reduced pressure to give a residue The crude product was purified by preparative HPLC (TFA condition) to give BCY13254 (36.5 mg, 7.49 μmol). l, 69.92% yield, 97.8% purity) was obtained as a white solid.

[0179] Example 14: Synthesis of BCY13340 [ka] (Procedure for preparation of BCY12865-PEG5-N3) [ka] BCY12865 (50 mg, 23.32 μmol, 1.0 equiv.) and Compound 1 (10.5 mg, 24.28 μmol, 1.04 equiv.) The solution was dissolved in 2 mL of MeCN / H2O (1:1) and the pH was adjusted to 8 by adding 1 M NaHCO3. The mixture was stirred at 25° C. for 2 hours. LC-MS showed that BCY12865 was completely consumed and the desired m / z One major peak (calculated MW: 2461.87, observed m / z: 1231.6 ([M / 2+H] + ) and 82 1.4([M / 3+H] + The reaction mixture was purified by preparative HPLC (TFA conditions). Upon preparation, compound 2 (31.5 mg, 12.62 μmol, 54.14% yield, 98.66% purity) was obtained as a white solid. This was obtained.

[0180] (Procedure for preparation of BCY13340) [ka] Compound 2 (31.5 mg, 12.80 μmol, 1.0 equivalent), BCY12353 (27 mg, 12.92 μmol, 1.0 equivalent), and A mixture of t-BuOH / HO (1:1, 2 mL, pre-degassed and N2 The solution was dissolved in CuSO4 (0.4 M, 32 μL, 1.0 equiv.) and VcNa (5.1 m g, 25.74 μmol, 2.0 equiv) was added under N2. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / The solution was adjusted to 8 by dropwise addition of HCl (in H2O), and the solution turned pale yellow. The mixture was stirred at 40° C. for 1 hour. LC-MS showed that compound 2 was completely consumed and compound 1 with the desired m / z was obtained. One major peak (calculated MW: 4551.32, observed m / z: 1517.7 ([M / 3+H] + ) and 1138. 6([M / 4+H] + The reaction mixture was filtered and concentrated under reduced pressure to give The residue was obtained. The crude product was purified by preparative HPLC (TFA conditions) to give BCY13340 (34.7 mg, 7. 62 μmol, 59.59% yield, 89.59% purity) was obtained as a white solid.

[0181] Example 15: Synthesis of BCY13342 [ka] (Procedure for preparation of BCY12860-PEG5-N3) [ka] BCY12860 (28.0 mg, 13.58 μmol, 1.0 equivalent) and Compound 2 (6.5 mg, 14.94 μmol, 1.1 equivalent) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by dropwise addition of NaHCO3 (0.1 M). The reaction mixture was stirred at 25° C. for 1 hour. LC-MS confirmed that BCY12860 was completely synthesized. One major peak with the desired m / z (MW: 2378.78, observed m / z: 1190.2 ( [(M / 2+H + The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was then purified by preparative HPLC (neutral conditions). EG5-N3 (20.7 mg, 8.41 μmol, 61.95% yield, 96.7% purity) was obtained as a white solid.

[0182] (Procedure for preparation of BCY13342) [ka] Compound 3 (20.7mg, 8.70μmol, 1.0eq), compound 4 (19.0mg, 9.14μmol, 1.05eq), and A mixture of t-BuOH / HO (1:1, 1 mL, pre-degassed and N2 The solution was dissolved in CuSO4 (0.4 M, 28.3 μL, 1.3 equiv.) and VcNa (4. 5 mg, 22.62 μmol, 2.6 equiv.) was added under N. The pH of this solution was adjusted to 0.2 M NH.sub.4HCO.sub.3 (1:1 t-BuO The temperature was adjusted to 8 by dropwise addition of HCl (H / H2O), resulting in a pale yellow solution. The reaction mixture was then cooled under a N2 atmosphere. The mixture was stirred at 25° C. for 2 hours under reduced pressure. LC-MS showed that compound 3 was completely consumed and had the desired m / z. One major peak (calculated MW: 4468.24, observed m / z: 1118.6 ([M / 4+H] + )) detected The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The product was purified by preparative HPLC (TFA conditions) to give BCY13342 (21.7 mg, 4.60 μmol, 52.85% yield). Yield: 94.7% purity) was obtained as a white solid.

[0183] (Analysis data) The following heterotandem bicyclic peptide complexes of the present invention were analyzed using mass spectrometry and HPLC. The HPLC settings were as follows: Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow rate: 1.0ml / min Column: Gemini-NX C18 5um 110A 150 * 4.6mm Instrument: Agilent 1200 HPLC-BE(1-614)

[0184] The gradients used are shown in the table below: [Table 7] and the data was generated as follows: [Table 8] TIFF2026027327000080.tif239170TIFF2026027327000081.tif227170TIFF2026027327 000082.tif243170TIFF2026027327000083.tif239170TIFF2026027327000084.tif92170

[0185] (biological data) (1. CD137 Reporter Assay Co-culture with Tumor Cells) R1 medium by adding 1% FBS to RPMI-1640 (a component of Promega kit CS196005) Prepare a culture medium called R1. Serial dilutions of the test article in R1 are placed in a sterile 96-well plate. Prepare 25 μL of test article or R1 (as background control) per well using a white thin film. Add tumor cells to designated wells in cell culture plates. * The cells were collected and concentrated at 400,000 cells / mL. Add 25 (twenty-five) μL / well of tumor cells to a white cell culture plate. Jurkat cells (Promega kit CS196005, 0.5 mL) were thawed in a water bath and then added to 5 mL of Add 25 (twenty-five) µL / well of pre-warmed R1 medium to the white Jurkat cells. Add to plate. Incubate cells and test article at 37°C, 5% CO2 for 6 hours. At the end of the incubation, 75 μL / well of Bio-Glo™ reagent (Promega) was added and incubated for 10 minutes. Afterwards, the luminescence was read using a plate reader (Clariostar, BMG). The fold change compared to the cell line used in the co-culture was calculated and expressed as log(agonist) in GraphPad Prism. Plotted as a function of response, EC 50 Determine the induction factor (nM) and the induction fold over background (Max). Determine.

[0186] The tumor cell type used in the co-culture is NCI-H292, which has been shown to express Nectin-4. The tumor cell type used in the co-culture for EphA2 is PC3. For PD-L1 The tumor cell type used in the co-culture is RKO.

[0187] Nectin-4 / CD137 hybridization in a CD137 reporter co-culture assay using NCI-H292 cells A summary of the fold induction induced by the tandem peptides is shown in Table 1. The average EC 50and a pre- Page 10 ... Compare with the control BCY10000. Table 1: Nectin-4 / CD137 heterotandem bicyclic peptides in CD137 reporter assays Induction factor induced by the complex [Table 9]

[0188] EphA2 / CD137 heterotandem in a CD137 reporter co-culture assay using PC3 cells A summary of the fold induction induced by the peptides is shown in Table 2. All compounds were 0.54n Average EC of M 50 and compared to plate control BCY9173, which has an Emax of 42-fold over background. Compare. Table 2: EphA2 / CD137 heterotandem bicyclic peptide conjugates in CD137 reporter assays Induction factor guided by the body [Table 10]

[0189] PD-L1 / CD137 heterotandem in a CD137 reporter co-culture assay using RKO cells A summary of the fold induction elicited by the peptides is shown in Table 3. Table 3: PD-L1 / CD137 heterotandem bicyclic peptide conjugates in CD137 reporter assays Induction factor guided by the body [Table 11]

[0190] 2. Pharmacokinetics of CD137 heterotandem bicyclic peptide complexes in SD rats Male SD rats were administered 2 mg / kg of each compound formulated in 25 mM histidine HCl, 10% sucrose pH 7. Each heterotandem bicyclic peptide complex was administered. Serial blood samples (approximately 80 μL blood / time point) were collected. All blood samples were anticoagulated with 2 μL K2-EDTA (0.5 M). Immediately transfer to a pre-chilled microcentrifuge tube containing 0.5 mL of blood and place on wet ice. The fluid samples were immediately processed for plasma by centrifugation at 3000 g at approximately 4°C. The precipitant was immediately added to the plasma, mixed thoroughly, and centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was transferred to a pre-labeled polypropylene microcentrifuge tube, followed by The samples were quickly frozen on dry ice. If necessary, the samples were stored below 70°C until analysis. 7. 5 μL of the supernatant sample was analyzed by LC-MS / MS analysis using an Orbitrap Q Exactive in positive ion mode. The plasma concentration versus time data was analyzed by Phoenix Wiley. by a non-compartmental approach using the Nonlin 6.3 software program. Analysis was performed. C0, Cl, Vdss, T 1 / 2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) , and plasma concentration versus time profile graphs were reported. The pharmacokinetic parameters of the experiment were: As shown in Table 4: Table 4: Pharmacokinetic parameters in SD rats [Table 12] The present application provides the following aspects of the invention. (Aspect 1) (a) a first peptide ligand that binds to a component present on a cancer cell; , (b) a second peptide ligand that binds to a component present on an immune cell; Conjugated to Contains: wherein each of the peptide ligands is separated by at least two loop sequences. A polypeptide containing at least three reactive groups and a compound that forms a covalent bond with the reactive groups of the polypeptide. and a molecular scaffold that results in at least two polypeptide loops. A heterotandem bicyclic peptide complex formed on the molecular scaffold, wherein the heterotandem bicyclic peptide complex comprises first and second peptide ligands: (Table 1) TIFF2026027327000089.tif236170TIFF2026027327000090.tif236170TIFF202602732700009 1.tif236170TIFF2026027327000092.tif236170TIFF2026027327000093.tif236170TIFF2026 027327000094.tif236170TIFF2026027327000095.tif236170TIFF2026027327000096.tif236170TIFF2026027327000097.tif236170 (where 1Nal represents 1-naphthylalanine, HArg represents homoarginine, and HyP represents hydrochloride). B-Ala stands for β-alanine, PYA stands for 4-pentynoic acid, and 3,3-DPA stands for 3,3-diphenylalanine, Cba stands for β-cyclobutylalanine, and hGlu stands for homoglucan. NMeAla stands for N-methyl-alanine, tBuAla stands for thiamin, Nle stands for norleucine, NMeAla stands for N-methyl-alanine, tBuAla stands for thiamin ... represents t-butyl-alanine, Aad represents α-L-aminoadipic acid, and Ac represents an acetyl group. and Dap represents diaminopropionic acid), or a pharmaceutically acceptable salt thereof. The heterotandem bicyclic peptide complex, characterized in that it comprises: (Aspect 2) The immune cells may be white blood cells; lymphocytes (e.g., T lymphocytes or T cells, B cells, or nasal cells). Choose from: CD8 or CD4; CD8; dendritic cells, follicular dendritic cells, and granulocytes 2. The heterotandem bicyclic peptide conjugate of embodiment 1, wherein (Aspect 3) In accordance with embodiment 1 or embodiment 2, the second peptide ligand comprises a CD137-binding bicyclic peptide ligand. 2. A heterotandem bicyclic peptide complex as described in (Aspect 4) The CD137-binding bicyclic peptide is selected from any of the peptides of SEQ ID NOs: 67 to 84. 4. The heterotandem bicyclic peptide conjugate according to embodiment 3. (Aspect 5) 1. The method of claim 1, wherein the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand. 5. The heterotandem bicyclic peptide complex according to any one of claims 1 to 4. (Aspect 6) The Nectin-4 binding bicyclic peptide is selected from any of the peptides of SEQ ID NOs: 52 to 66. 6. The heterotandem bicyclic peptide conjugate of embodiment 5, (Aspect 7) Conjugates listed in Table C, e.g., BCY11468, BCY11618, BCY11776, BCY11860, BCY 12020, BCY12661, and BCY12969. Telotandem bicyclic peptide conjugates. (Aspect 8) 5. The method of claim 1, wherein the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand. The heterotandem bicyclic peptide conjugate of any one of claims 1 to 4. (Aspect 9) The EphA2-binding bicyclic peptide is selected from any of the peptides set forth in SEQ ID NOs: 10 to 51. 9. The heterotandem bicyclic peptide conjugate according to embodiment 8. (Aspect 10) Conjugates listed in Table B, e.g., BCY13035, BCY13040, BCY13253, BCY13254, BCY 10. The heterotandem of embodiment 8 or embodiment 9, wherein the heterotandem is selected from any one of BCY13340 and BCY13342. Bicyclic peptide conjugates. (Aspect 11) 5. The method of claim 1, wherein the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand. The heterotandem bicyclic peptide conjugate of any one of claims 1 to 4. (Aspect 12) the PD-L1-binding bicyclic peptide is selected from any of the peptides set forth in SEQ ID NOs: 1 to 9; 12. The heterotandem bicyclic peptide conjugate according to embodiment 11. (Aspect 13) A conjugate selected from any one of the conjugates listed in Table A, e.g., BCY12375 and BCY12021. 13. The heterotandem bicyclic peptide conjugate according to embodiment 11 or embodiment 12, (Aspect 14) The molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)tripropanol. 14. The heterotandem of any one of embodiments 1 to 13, wherein the heterotandem is selected from the group consisting of tetra-2-ene-1-one (TATA), ... Bicyclic peptide conjugates. (Aspect 15) The pharmaceutically acceptable salts may be the free acid or salts of sodium, potassium, calcium, ammonium, 15. The heterotandem bicyclic peptide of any one of embodiments 1 to 14, selected from the group consisting of ammonium salts. Chid complex. (Aspect 16) The heterotandem bicyclic peptide conjugate according to any one of aspects 1 to 15 is administered in combination with one or more pharmaceutical agents. in combination with a suitably acceptable excipient. (Aspect 17) 16. The method of claim 1, wherein the compound of claim 1 is a compound selected from the group consisting of benzodiazepines, ... Heterotandem bicyclic peptide conjugates.

Claims

1. A ligand that binds to Nectin-4 present on cancer cells or a pharmaceutically acceptable salt thereof. a polypeptide containing at least three reactive groups separated by at least two sequences and a molecular scaffold that forms a covalent bond with the reactive group of the polypeptide, As a result, a polypeptide comprising the at least two sequences, the reactive group, and the molecular scaffold is obtained. A small loop is formed; The polypeptide may be modified by replacing one or more of its cysteine ​​residues C with 3-mercaptopropionic acid, cysteine modified by substitution with theamine or penicillamine, CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC It is characterized by: where 1Nal represents 1-naphthylalanine, HArg represents homoarginine, and HyP represents represents hydroxyproline, and The molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl) tripropane. The ligand or a pharmaceutically acceptable salt thereof is 2-(2-ene-1-one)-2-ene-1-one (TATA).

2. The polypeptide has one or more of its cysteine ​​residues C substituted by penicillamine. and modified by, CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC 2. The ligand of claim 1, wherein:

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