Heterotandem bicyclic peptide complex

Abstract

This invention provides a cyclic peptide that can bind to protein targets with high affinity and target specificity. [Solution] The present invention relates to a heterotandem bicyclic peptide complex comprising a first peptide ligand that binds to a component present on cancer cells, conjugated via a linker to a second peptide ligand that binds to a component present on immune cells. The present invention also relates to the use of the heterotandem bicyclic peptide complex in the prevention, suppression, or treatment of cancer.

Description

[Technical Field]

[0001] (Field of invention) This invention relates to a first peptide ligand that binds to a component present on cancer cells, and a linker —a second peptide ligand that binds to components present on immune cells via — The present invention relates to heterotandem bicyclic peptide complexes, including those that have been ligated. Regarding the use of the 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, it is an attractive molecular class for the development of therapeutic drugs. In fact, several cyclic peptides Tide is used, for example, in antimicrobial peptides such as vancomycin, immunosuppressants such as cyclosporine, or antimicrobial agents. Like the cancer drug octreotide, it has already been successfully used in clinics (Driggers et al.). (Reference: (2008), Nat Rev Drug Discov 7(7), 608-24). Excellent binding properties are found in peptides and labels. Not only the relatively large interaction surface formed between the target and the annular structure, but also the three-dimensional flexibility of the annular structure This is also due to the decrease in [unclear]. Typically, macrocyclic molecules are cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ; Wu et al. (2007), Science 330, 1066-71), Arg-Gly binding to integrin αVb3 -Cyclic peptide with an Asp motif (355Å) 2 )(Xiong et al. (2002), Science 296(5565), 151-5), or cyclic peptide inhibitors that bind to urokinase-type plasminogen activators. Upaine-1 (603 Å) 2 ; As in the literature by Zhao et al. (2007), J Struct Biol 160(1), 1-10), hundreds It binds to the surface of a square angstrom.

[0003] Due to their cyclic configuration, macrocyclic peptide molecules are less flexible than linear peptides. Furthermore, the entropy loss when binding to the target becomes smaller, resulting in higher binding. Affinity is generated. The decrease in flexibility also leads to the fixation of the target-specific three-dimensional structure, and linear particles Compared to butyl, it increases binding specificity. This effect occurs when the ring opens, and other MMs The potent and selective nature of matrix metalloproteinase 8 (MMP-8), which loses its selectivity for P. This has been demonstrated by specific inhibitors (Cherney et al. (1998), J Med Chem 41(11), 1749- 51). The advantageous binding properties achieved by macrocyclization include, for example, vancomycin and nasal cyclamin. In polycyclic peptides having multiple peptide rings, such as n and actinomycin It is even more pronounced.

[0004] Various research teams have previously synthesized polypeptides containing cysteine ​​residues into molecular structures. Connecting (Kemp and McNamara's work (1985), J. Org. Chem; Timmerman et al.'s work (2005), ChemBioChem). Meloen and collaborators have identified tris(bromomethyl)benzene and related molecules. Multiple peptide loops on a synthetic scaffold for structural mimicry of a protein surface Used for rapid and quantitative cyclization (Timmerman et al. (2005), ChemBioChem). Candidate drug formulation. Compound (where the compound is a cysteine-containing polypeptide, for example, tris(bromomethicone)) (A method of fabrication) by linking molecules such as benzene to a molecular scaffold. The law is disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] Construct and screen large libraries of bicyclic peptides targeting specific targets. A combinatorial approach based on phage display is being developed to achieve this. (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly described. Belt contains a linear peptide containing three cysteine ​​residues and two random 6-amino acid regions. A combinatorial library of cys(Cys-(Xaa)6-Cys-(Xaa)6-Cys) is presented on the phage. Therefore, the cysteine ​​side chain is covalently bonded to a low molecular weight (tris-(bromomethyl)benzene). It was made more cyclical. [Overview of the project]

[0006] (Summary of the invention) According to a first aspect of the present invention, (a) A first peptide ligand that binds to components present on cancer cells via a linker , (b) A second peptide ligand that binds to components present on immune cells; something conjugated : includes, Here, each of the peptide ligands is separated by at least two loop sequences. A polypeptide comprising at least three reactive groups and a compound forming a covalent bond with the reactive groups of the polypeptide. It contains a molecular scaffold, and as a result, at least two polypeptide loops A heterotandem bicyclic peptide complex formed on the molecular scaffold, The heterotandem bicyclic peptide complex consists of the following first and second peptide ligands: [Table 1] TIFF2026143628000002.tif237170TIFF2026143628000003.tif237170TIFF2026143 628000004.tif237170TIFF2026143628000005.tif236170TIFF2026143628000006.ti f237170TIFF2026143628000007.tif236170TIFF2026143628000008.tif237170TIFF2026143628000009.tif237170(where 1Nal represents 1-naphthylalanine, HArg represents homoarginine, and HyP represents hydro Xyproline is represented, B-Ala is represented by β-alanine, PYA is represented by 4-pentic acid, and 3,3-DPA is 3,3-diphenylalanine is represented by Cba, β-cyclobutylalanine is represented by hGlu, and homoglu is represented by tBuAla represents tamic acid, Nle represents norleucine, NMeAla represents N-methyl-alanine, and tBuAla represents t-butyl-alanine, Aad represents α-L-aminoadipic acid, and Ac represents an acetyl group. (where Dap represents diaminopropionic acid), or a pharmaceutically acceptable salt thereof. A heterotandem bicyclic peptide complex is provided, characterized by containing [a specific compound].

[0007] According to a further aspect of the present invention, heterotandem bicyclic peptides as defined herein A pharmaceutical composition is provided that contains the complex in combination with one or more excipients that are acceptable as pharmaceuticals. .

[0008] According to a further aspect of the present invention, this invention is for use in the prevention, suppression, or treatment of cancer. A heterotandem bicyclic peptide complex as defined in the specification is provided. [Modes for carrying out the invention]

[0009] (Detailed description of the invention) (First peptide ligand) The term "cancer cells" as used herein refers to cells that are known to be involved in cancer. Includes any cell. Cancer cells are formed when genes involved in regulating cell division are damaged. It is revealed. Carcinogenesis is the sudden change in the genetic material of normal cells that disrupts the normal balance between proliferation and cell death. It results from mutations and epimutations. As a result, uncontrolled cell division and in vivo The evolution of these cells occurs through natural selection, which is uncontrolled and often rapid. Uncontrolled cell proliferation can lead to benign or malignant tumors (cancer). Benign tumors can spread to other parts of the body. It does not invade other tissues. Malignant tumors invade other organs and distant sites. It can spread (metastasize) and become life-threatening.

[0010] In one embodiment, cancer cells include HT1080, A549, SC-OV-3, PC3, H1376, NCI-H292, and LnC. Selected from 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 component of the nectin family of proteins, which includes four members. It is a child. Nectin is involved in the development and development of epithelial, endothelial, immune, and neuronal polarity during development and adulthood. Cells that play a vital role in various biological processes such as proliferation, differentiation, and migration. These are adhesion molecules. They are involved in several pathological processes in humans. These are the primary receptors for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding nectin-1 (PVRL1) or nectin-4 (PVRL4) can lead to other abnormalities. It causes ectodermal dysplasia syndromes associated with this condition. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of the family. Nectin -4 is mainly due to the fact that in 50%, 49%, and 86% of breast cancer, ovarian cancer, and lung cancer, respectively, It is a tumor-associated antigen found on tumors with poor post-inflammatory dialysis. Its expression is not detectable in the corresponding normal tissue. In breast tumors, nectin-4 is mainly expressed in triple-negative and ERBB2+ cancers. In the serum of patients with these cancers, the detection of soluble nectin-4 is indicative of prognosis. It is associated with poor performance. Serum nectin-4 levels increase during the metastatic progression phase and decrease after treatment. These results suggest that nectin-4 could be a reliable target for cancer treatment. This is instigating. Therefore, several anti-nectin-4 antibodies have been described in the prior art. In particular, enfortumab vedotin (ASG-22ME) is an antibody-drug targeting nectin-4. It is an adjugate (ADC) and is currently being used in clinical research for the treatment of patients with solid tumors. It has been thoroughly investigated.

[0013] In one embodiment, the first peptide ligand is a nectin-4 linked bicyclic peptide ligator. Includes nd.

[0014] A suitable example of a nectin-4 linked bicyclic peptide ligand is the peptide as described by reference. This is disclosed in PCT patent application PCT / GB2019 / 051740, which is incorporated into the details.

[0015] In one embodiment, the nectin-4 linked bicyclic peptide is the sequence number described herein. Selected from one of peptides number 52-66.

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

[0017] Eph receptor tyrosine kinase (Eph) is a kinase that phosphorylates proteins at tyrosine residues. It is a large receptor tyrosine kinase (RTK) belonging to the RTK group. Eph and its membrane-bound form Eph Phosphate ligands (ephrin) regulate cell arrangement and tissue composition (Poliakov et al. (2004)). Dev Cell 7, 465-80). Functional and biochemical Eph responses are associated with higher ligand oligomerization. It occurs under certain conditions (Stein et al. (1998) Genes Dev 12, 667-678).

[0018] Among other patterning functions, various Eph and ephrines play a role in angiogenesis. It has been shown that knockout of EphB4 and ephrin-B2 reconstructs the capillary bed. This results in a lack of ability to form blood vessels (Poliakov et al., see above) and embryonic lethality. Sustained expression of Eph receptors and ephrins was also observed in newly formed adult microvessels. (References: Brantley-Sieders et al. (2004), Curr Pharm Des 10, 3431-42; Adams (2003)) J Anat 202, 105-12).

[0019] The disregulated reappearance of several ephrins and their receptors in adults is also linked to tumor invasion. It has been observed to contribute to metastasis and neoangiogenesis (Nakamoto et al. (2002) Micros). c Res Tech 59, 58-67; Brantley-Sieders et al. (see above). Furthermore, several Ephfa MillieMember has been shown to be overexpressed in tumor cells derived from various human tumors. (Brantley-Sieders et al., as mentioned above); 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 in humans by the EPHA2 gene. It is a protein that is produced.

[0021] EphA2 is associated with disease progression, metastasis, and poor prognosis in many human cancers, for example. 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), pre Prostate carcinoma (Walker-Daniels et al. (1999) Prostate 41, 275-280), liver cancer (Yang et al. (2 009) Hepatol Res. 39, 1169-1177), and glioblastoma (Wykosky et al. (2005) Mol Cancer Re Upregulation is described in Li et al.'s paper (2010) Tumour Biol. 31, 477-488.

[0022] The full role of EphA2 in cancer progression is not yet clear, but it is involved in the proliferation of tumor cells and the life cycle of tumor cells. Evidence exists of interactions at many stages of cancer progression, including preservation, invasion, and angiogenesis. Downregulation of EphA2 expression suppresses the proliferation of tumor cancer cells (Binda et al. (2012) Cancer Cell 22, 765-780), on the other hand, EphA2 blockade is associated with VEGF-induced cell migration (Hess et al. (2001) Cancer Res. 61, 3250-3255), budding and angiogenesis (Cheng et al. (2002) Mol Cancer Res. 1, 2-1 1; Lin et al. (2007) Cancer 109, 332-40), and metastatic progression (Brantley-Sieders et al.) It inhibits the study (2005, FASEB J. 19, 1884-1886).

[0023] Antibody-drug conjugates with EphA2 showed tumors in rat and mouse xenograft models. It has been shown to significantly reduce ulcer growth (Jackson et al. (2008) Cancer Research). 68, 9367-9374), similar methods have been attempted in humans, but the treatment has been associated with treatment-related adverse events. It had to be discontinued due to the cause (Annunziata et al. (2013) Invest New Drugs 31, 77-84).

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

[0025] A suitable example of an EphA2-linked bicyclic peptide ligand is the peptide as specified herein by reference. This is incorporated within and disclosed in WO 2019 / 122860, WO 2019 / 122861, and WO 2019 / 122863. It is being done.

[0026] In one embodiment, the EphA2-linked bicyclic peptide ligand is the compound described herein. A peptide is selected from among those in column numbers 10 to 51.

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

[0028] Programmed cell death ligand 1 (PD-L1) is found on mouse chromosome 19 and human chromosome 9. PD-L1 expression is a 290-amino acid type I transmembrane protein encoded by the CD274 gene. This includes chronic infections, such as chronic viral infections (e.g., particularly HIV, HBV, HCV, and HTLV). (e.g., chronic bacterial infections, particularly those involving Helicobacter pylori) (m), as well as chronic parasitic infections (e.g., including Schistosoma mansoni) It is involved in evading the immune response. PD-L1 expression is involved in T cells, B cells, and macrophages. , 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 the suppression of antitumor immune activity. Tumors are recognized by host T cells. Although it expresses antigens that can be recognized, immunological clearance of the tumor is rare. Some of the problems are due to immunosuppression caused by the tumor microenvironment. In many tumors PD-L1 expression is a component of this suppressive environment and works in coordination with other immunosuppressive signals. It is used. PD-L1 expression is found in the breast, lungs, colon, ovaries, melanoma, bladder, liver, salivary glands, stomach, and nerves. In various solid tumors, including gliomas, thyroid tumors, thymic epithelium, head tumors, and neck tumors, Insidiomycosis As shown in the literature by Brown JA et al., 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Literature by Hamanishi J et al., 2007 Proc. Natl. Acad. Sci. USA 1 04:3360-65; Literature by Strome SE et al., 2003 Cancer Res. 63:6501-5; Literature by Inman BA et al., 200 7 Cancer 109:1499-505; Konishi J et al., 2004 Clin. Cancer Res. 10:5094-100; Na References by Kanishi J et al., 2007 Cancer Immunol. Immunother. 56:1173-82; References by Nomi T et al., 2007 Clin. Cancer Res. 13:2151-57; Literature by 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 - The receptor for programmed cell death protein 1 (also known as PD-1 and CD279) Currently, it is upregulated by tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C (References, 2003 Immunol. 171:4574-81). Most importantly, PD-L1 expression in tumors is associated with disease. Studies linking PD-L1 expression to outcomes have shown that PD-L1 expression is associated with kidney cancer, ovarian cancer, bladder cancer, breast cancer, gastric cancer, and It has been shown to be strongly correlated with an unfavorable prognosis in pancreatic cancer (Hamanishi J et al.). References, 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Literature by Inman BA et al., 2007 Cancer 1 09:1499-505; Konishi J et al., 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J References by Nomi T et al., 2007 Cancer Immunol. Immunother. 56:1173-82; References by Nomi T et al., 2007 Clin Cancer Res. 13:2151-57; Literature by 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 lead to advancement of tumor stages and deeper tissue formation. It has been suggested that this may facilitate penetration into structures.

[0030] The PD-1 pathway can also play a role in hematological malignancies. PD-L1 is involved in numerous bone marrow processes. 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 present in some primary T-cell lymphomas, particularly anaplastic large cell T lymphomas. This is shown (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is an angioimmunoblastic ligament. Highly expressed in T cells of cymphocarcinoma, PD-L1 is expressed in the associated follicular dendritic cell network. (Dorfman DM et al., 2006 Am. J. Surg. Pathol. 30:802-10). Nodular lymphocytes are dominant. In PD-1 Hodgkin lymphoma, T cells associated with lymphocytes or histiocytes (L&H) cells are PD-1 It expresses the gene. Microprocessors use the readout of the gene induced by PD-1 ligation. Array analysis revealed that tumor-associated T cells in Hodgkin lymphoma were PD-1 sigma It has been suggested that it responds to nal (Chemnitz JM et al., 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed in CD4 T cells of HTLV-1-mediated adult T-cell leukemia and lymphoma. (Shimauchi T et al., 2007 Int. J. Cancer 121: 2585-90). These tumor cells are T It exhibits low responsiveness to CR signals.

[0031] Studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis. It has been shown to be harmful and, in some cases, lead to increased tumor-specific T cell death (Dong H et al.). (References: Hirano F et al., 2002 Nat. Med. 8:793-800; Hirano F et al., 2005 Cancer Res. 65:1089-96) Tumor-associated APCs also utilize the PD-1:PD-L1 pathway to regulate the antitumor T cell response. Yes, it is possible. PD-L1 expression in tumor-associated bone marrow dendritic cells is upregulated by tumor environmental factors. (Curiel TJ et al., 2003 Nat. Med. 9:562-67). B16 melanoma tumor inflow area lymph Plasmacytoid dendritic cells (DCs) within the nodule express IDO, which strongly activates the repressive 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-linked bicyclic peptide ligand. Includes.

[0033] A suitable example of a PD-L1-linked bicyclic peptide ligand is one in which the peptide is specified by reference herein. This is incorporated within, as disclosed in GB Patent Applications 1905631.6 and 1904622.6.

[0034] In one embodiment, the PD-L1-linked bicyclic peptide is one of the sequence numbers 1-2 described herein. Selected from one of the nine peptides.

[0035] In an alternative embodiment, the components present on cancer cells are prostate-specific membrane antigens (PSM). A) is correct.

[0036] Prostate-specific membrane antigen (PSMA) (glutamate carboxypeptidase II (GCPII), N-acetyl L-L-aspartyl-L-glutamate peptidase I (NAALADase I), and NAAG peptidase (also known as folate hydrolase 1) is a gene that, in humans, is controlled by the FOLH1 (folate hydrolase 1) gene. It is an enzyme that is produced. Human GCPII contains 750 amino acids and has a weight of approximately 84 kDa.

[0037] Human PSMA is highly expressed in the prostate gland, at approximately 100 times more than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, and non It is 8 to 12 times higher than the levels in cancerous prostate cells. Due to this high expression, PSMA It is being developed as a potential biomarker for the treatment and imaging of several cancers. In human prostate cancer, tumors with higher expression tend to have a faster progression time and are larger. It is associated with a certain percentage of patients suffering from relapses.

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

[0039] A suitable example of a PSMA-conjugated bicyclic peptide ligand is a peptide whose peptide is as specified herein by reference. This includes GB patent applications 1820325.7 and 1912723.2 and PCT patent application PCT / EP2019 / 06. This is disclosed in document number 6273.

[0040] (Second peptide ligand) The term "immune cell" as used herein includes any cell within the immune system. Suitable examples include lymphocytes (e.g., T lymphocytes or T cells, B cells, or natural cells). Examples include white blood cells such as killer cells. In one embodiment, T cells are CD8 or CD4. In a further embodiment, the T cell is CD8. Other examples of immune cells include dendritic cells. Examples include vesicles, 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. It is also known as the tumor necrosis receptor. Death factor receptor superfamily member 9 (TNFRSF9), 4-IBB, and lymphocyte activation It is induced by (ILA). CD137 can be expressed by activated T cells, but mostly CD137 expression can be expressed more in CD8+ T cells than in CD4+ T cells. Furthermore, CD137 expression is associated with dendritic cells and filtration. It is found in sporangial dendritic cells, natural killer cells, granulocytes, and cells in the blood vessel wall at the site of inflammation. One characteristic activity of CD137 is its co-stimulatory activity towards activated T cells. Cross-linking enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. Furthermore, it This can enhance immune activity and eliminate tumors in mice.

[0043] CD137 is a T cell costimulatory receptor 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 ( 2005). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is CD4+CD25+ regulated. It is also expressed in T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, is used to stimulate B cells, monocytes / macrophages, and dendritic cells. Regarding antigen-presenting cells, this is described (Watts et al., Annu. Rev. Immunol, 23:23- 68 (2005). When interacting with its ligand, CD137 increases TCR-induced T cell proliferation, cytoplasmic regeneration. This leads to tokine 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] CD137-mediated by either CD137L or an operative monoclonal antibody (mAb) against CD137. Signaling leads to increased TCR-induced T cell proliferation, cytokine production, and functional maturation, and This also leads to an extension of CD8+ T cell survival. These effects are due to (1) NF-κB, c-Jun NH2-terminal nucleotides. JNK / SAPK enzymes, stress-activated protein kinases, and p38 mitogen-activated proteins. (2) Activation of the protein kinase (MAPK) signaling pathway, and anti-apoptotic and pericellular processes. Regulation of period-related gene expression: This occurs as a result.

[0045] Experiments conducted in both CD137-deficient mice and CD137L-deficient mice showed that they were completely suitable. The importance of CD137 co-stimulation in the development of a distinct T cell response was further demonstrated.

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

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

[0048] According to its co-stimulatory function, agonist mAbs for CD137 are used in allografts of the heart and skin. It promotes rejection, eradicates colonized tumors, expands the primary antiviral CD8+ T cell response, and T cells These studies have shown that CD137 signaling increases cell lysis. I support the view that it promotes T cell function, which can enhance immunity against ulcers and infections.

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

[0050] A suitable example of a CD137-linked bicyclic peptide ligand is a peptide whose peptide is as specified herein by reference. This is disclosed in WO 2019 / 025811, which is incorporated within the document.

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

[0052] (Linker) The first peptide ligand is linked to the second peptide ligand via any suitable linker. It will be understood that it can be conjugated to. Typically, the linker The design involves two bicyclic peptides binding to their target receptors individually or simultaneously. Therefore, they are presented in a way that allows them to be combined without being interfered with by each of their targets. Furthermore, the linker allows simultaneous binding to both targets. Therefore, an appropriate distance should be maintained between target cells to produce the desired functional outcome. The properties of - increase length, stiffness, or solubility to optimize the desired functional outcome. It can be adjusted to allow multiple birings to bond to the same target. It can also be designed to do so. Increasing the valency of any of the bound peptides is a target It may play a role in increasing the affinity of heterotandem to target cells, or in target receptor It may help induce oligomerization of one or both parts of the body.

[0053] In one embodiment, the linker has the following sequence: -PEG5- and TCA-[PEG 10 Selected from ]3 ru.

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

[0055] (Heterotandem complex) In one specific embodiment, the first peptide ligand is a TATA scaffold. It contains a PD-L1-linked bicyclic peptide ligand bound to the second peptide ligand, and the second peptide ligand is TATA The scaffold contains a CD137-linked bicyclic peptide ligand, and the heterotande 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 complex consists of BCY12375 and BCY12021 Selected from:

[0057] In one specific embodiment, the first peptide ligand is a TATA scaffold. It contains an EphA2-linked bicyclic peptide ligand bound to the second peptide ligand, and the second peptide ligand is TATA The scaffold contains a CD137-linked bicyclic peptide ligand, and the heterotande The complex is selected from the complexes listed in Table B: Table B (EphA2:CD137; 1:1) [Table 3] TIFF2026143628000013.tif224170

[0058] In one embodiment, the heterotandem bicyclic peptide complex consists of BCY13035, BCY13040, Selected from BCY13253, BCY13254, BCY13340, and BCY13342.

[0059] In one specific embodiment, the first peptide ligand is a TATA scaffold. It contains a nectin-4 linked bicyclic peptide ligand, and the second peptide ligand is The TATA scaffold contains a CD137-linked bicyclic peptide ligand, and the heterota The ndem complex is selected from the complexes listed in Table C: Table C (Nectin-4:CD137; 1:1) [Table 4]

[0060] In one embodiment, the heterotandem bicyclic peptide complex consists of BCY11468, BCY11618, Selected from BCY11776, BCY11860, BCY12020, BCY12661, and BCY12969.

[0061] Unless otherwise defined, all technical and scientific terms used herein refer to the relevant fields. For example, peptide chemistry, cell culture, and phage display, nucleic acid chemistry, and biochemistry It has the same meaning as that generally understood by experts in the field of study. Standard technique. However, it is used in molecular biology, genetics, and biochemistry methods (as incorporated herein by reference). This is included in Sambrook et al.'s literature, Molecular Cloning: Laboratory Manual. A Laboratory Manual), 3rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Sp Ring Harbor, NY; Ausubel et al.'s work, Short Protocols in Molecular Biology (See *in Molecular Biology* (1999), 4th edition, John Wiley & Sons).

[0062] (Nomenclature) (Molecular format) N- or C-terminal extensions to a biring core sequence are separated by a hyphen, either on the left or right side of the sequence. It is added to the right side. For example, the N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(Sequence ID X) It is expressed as follows.

[0063] (Reverse peptide sequence) In consideration of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, this specification The peptide sequences disclosed here are also useful in their retro-inverso form. It is expected that this will be found. For example, the sequence may be reversed (i.e., the N-terminus becomes the C-terminus, and the C- The end becomes the N-terminus), and its stereochemistry is similarly reversed (i.e., the D-amino acid becomes the L-amino acid). (It becomes an acid, and L-amino acids become D-amino acids). To avoid misunderstanding, the official name is... Or, any reference to an amino acid, either as a one-letter or three-letter abbreviation, is Unless otherwise specified, in this specification, terms that are intended to be expressed as L-amino acids are... When such amino acids are intended to be represented as D-amino acids, the amino acids For example, [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc., with a lowercase 'd' placed before the square brackets. It will be done.

[0064] (Peptide ligand) The peptide ligands referred to herein are covalently bound to the molecular scaffold. This refers to peptides that are covalently bonded to a scaffold. Typically, such peptides are covalently bonded to a scaffold. Two or more reactive groups (i.e., cysteine ​​residues) that can form a peptide, and the peptide The interaction between the reactive groups is called a loop sequence because it forms a loop when it binds to the ferrule. It contains the sequence inherent in it. In this case, the peptide is cysteine, 3-mercaptopropion It comprises at least three reactive groups selected from acids and / or cysteamines, and scaffold Form at least two loops on the fold.

[0065] (reactive group) The molecular scaffold of the present invention allows the polypeptide to be transmitted via functional groups or reactive groups on the polypeptide. They may be bound to butyl. These are typically found in polypeptide polymers. It is formed from the side chains of specific amino acids. Such reactive groups include cysteine ​​side chains and lysine. The side chain, or the N-terminal amino group, or any other suitable reactive group, for example, penicillamine. It may be present. Details of suitable reactive groups can be found in WO 2009 / 098450.

[0066] Examples of reactive groups in natural amino acids include the thiol group of cysteine, the amino group of lysine, and asparagus. Carboxyl group of ginic acid or glutamic acid, guanidium group of arginine, tyrosine It is the phenol group of or the hydroxyl group of serine. Non-natural amino acids include azide and ketone. - Provides a broad range of reaction groups including carbonyl, alkyne, vinyl, or aryl halide groups. It is possible. The amino and carboxyl groups at the terminals of the polypeptide are also molecular scaffolding. It can act as a reactive group that forms a covalent bond with the rud / molecular core.

[0067] The polypeptide of the present invention contains at least three reactive groups. The polypeptide contains 4 or It can also contain the above reactive groups. The more reactive groups used, the more Loops can be formed within the molecular scaffold.

[0068] In a preferred embodiment, a polypeptide having three reactive groups is produced. Single generation occurs through the reaction of peptides with molecular scaffolds / molecular cores having 3 rotational symmetry. Isomers are produced. The production of a single product isomer is preferable for several reasons. Nucleic acids in compound libraries encode only the primary sequence of polypeptides, but the polypeptides It does not encode the isomerized molecule formed during the reaction between the do and the molecular core. Only one product. If isomers can be formed, the assignment of nucleic acids to the product isomers is clearly defined. When multiple product isomers are formed, nucleic acids are screened or selected during the selection process. Information regarding the properties of isolated product isomers cannot be provided. Single product isomer This information is also advantageous when specific members of the library of the present invention are synthesized. In this case, a chemical reaction between the polypeptide and the molecular scaffold results in a mixture of isomers. Instead, a single product isomer is produced.

[0069] In another embodiment, a polypeptide having four reactive groups is produced. The reaction of tide with a molecular scaffold / molecular core having tetrahedral symmetry yields two products. Isomers are produced. When two different product isomers are encoded by the same nucleic acid... Even if you chemically synthesize both isomers, separate the two isomers, and target both isomers, By testing the bonding with the ion, the properties of the isolated isomer can be determined. ru.

[0070] In one embodiment of the present invention, at least one of the reaction groups of the polypeptide is the remaining reaction It is orthogonal to the group. The use of orthogonal reactive groups involves placing the orthogonal reactive group in a specific part of the molecular core. It becomes possible to direct the reaction to a specific position. Using a linking strategy involving orthogonal reactive groups, the resulting organism The number of compound isomers can be limited. In other words, at least three of the bonds For the remaining group, at least three separate or different reactants are bonded to the selected reactants. By selecting one or more of these, a specific position on the molecular scaffold can be selected. To effectively achieve the binding or orientation of specific reaction groups of polypeptides in a specific order to a given position. It is possible.

[0071] In another embodiment, the reactive group of the polypeptide of the present invention reacts with a molecular linker, In this case, the linker is the molecular scaffold in the final bonded state and the polymer It can react with molecular scaffolds, allowing it to enter between the butyl molecules.

[0072] In some embodiments, members of a polypeptide library or set Mino acids can be replaced with any natural or non-natural amino acids. Only loop sequences can be replaced. A polypeptide having a functional group for crosslinking the molecular core so that it can be interchanged is These are excluded from the interchangeable amino acids. The interchangeable polypeptide sequences are random. One of the following: a random sequence, a fixed sequence, or a sequence having random amino acids and fixed amino acids. It has. The positions of these amino acids determine the loop size, so the amino acids that have a reactive group All of the no acids are located in specific positions within the polypeptide.

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

[0074] Using alternatives to thiol-mediated conjugations, via covalent interactions This allows molecular scaffolds to be attached to peptides. Alternatively, these techniques Further parts (e.g., molecular scaffolds and different target small molecules) may be used in the present invention. Therefore, after selection or isolation, in the further modification or attachment of the portion to the polypeptide It can be used - in this representation, obviously, the bond is a covalent bond. It is not necessary and can include non-covalent bonds. These methods have complementary reactive groups. Proteins containing unnatural amino acids that have the necessary chemical reaction groups when combined with small molecules, and This is done by producing phages that present peptides, or by the molecules after the selection / isolation step. During production, non-natural amino acids are chemically or recombinantly synthesized poly By incorporating it into a peptide, instead of (or in combination with) thiol-mediated methods It can be used. For further details, see WO 2009 / 098450 or the literature by Heinis et al., Nat Ch This can be found in em Biol 2009, 5(7), 502-7.

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

[0076] (Salt that is acceptable as a medicine) The salt form is within the scope of the present invention, and references to peptide ligands include the salt form of said ligand. It will be understood that...

[0077] The salts of the present invention are obtained by conventional chemical methods, e.g., pharmaceutical salts: properties, selection, and use (Pharmaceut ical Salts: Properties, Selection, and Use), P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, dated August 2002. Depending on the method, it can be synthesized from a parent compound containing a basic or acidic moiety. Such salts, when combined with a suitable base or acid in water, convert the free acid or base form of these compounds into a suitable base or acid. Alternatively, it may be prepared by reacting it in an organic solvent or in a mixture of the two. It is possible.

[0078] Acid addition salts (mono-salts 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, and ascorbic acid. Rubic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoin Acids, 4-acetamidobenzoic acid, butanoic acid, (+)camphoric acid, camphorsulfonic acid, (+) -(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, Econic acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-Hydroxyethanesulfonic acid, Formic acid, Fumaric acid, Mucoic acid, Gentisic acid, Glucohept Glutamic acid, D-gluconic acid, glucuronic acid (for example, D-glucuronic acid, etc.), glutamic acid (for example (For example, L-glutamic acid, α-oxoglutaric acid, glycolic acid, hippuric acid, halogenated water) Hydrogen acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L- Lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malo Mandelic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene- 1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitrate, oleic acid, or Triacid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvate, L-pyrog Rumamine, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, Sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylen Selected from the group consisting of acids, valeric acid, acylated amino acids, and cation exchange resins. Examples include monosalts or disalts formed with an acid.

[0079] One particular group of salts includes 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 lenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactob ionic acid. One particular salt is a hydrochloride. Another particular salt is an acetate.

[0080] When a compound is anionic or has a functional group that can be anionic (e.g., -CO OH can be -COO - ), a salt may be formed with an organic or inorganic base to generate a suitable cation . Examples of suitable inorganic cations include Li + , Na + , and K + alkali metals such as metal ions, Ca 2+ and Mg 2+ alkaline earth metal cations such as, and Al 3+ or Zn + other such as cations, but are not limited thereto. Examples of suitable organic cations include, ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NH3R + , NH2R 2 + , NHR3 + , NR4 + ), but are not limited thereto. Some suitable substituted ammoni um ions include, for example, those derived from methylamine, ethylamine, diethylamine, propylamine , dicyclohexylamine, triethylamine, butylamine, ethylenediamine, Ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzyl Luamine, choline, meglumine, and tromethamine, as well as lysine and arginine, etc. Examples include those derived from amino acids. A common example of a quaternary ammonium ion is N(C) H3)4 + That is the case.

[0081] If the compounds of the present invention contain amine functions, these can be used, for example, by methods well known to those skilled in the art. Reaction with an alkylating agent may form a quaternary ammonium salt. Quaternary ammonium compounds are within the scope of the present invention.

[0082] (Modified derivative) Modified derivatives of peptide ligands as defined herein are within the scope of the present invention. This will be understood. Examples of such preferred modified derivatives include the N-terminus and / or C-terminus. 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 a polar amino acid residue with one or more isoelectronically distributed or isoelectronically distributed amino acids; one or more nonpolar amino acids Substitution of the no-acid residue with other unnatural isoparticulate or isoelectronic amino acids; addition of a spacer group; 1 Substitution of the above oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; one or more A Substitution of amino acid residues with alanine, or substitution of one or more L-amino acid residues with one or more D-amino acid residues Substitution; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; one or more peptide Substitution of bond substitutes; modification of peptide backbone length; on the α-carbon of one or more amino acid residues Substitution of hydrogen by another chemical group, cysteine, lysine, glutamic acid / aspartic acid, and suitable amines and thios that functionalize amino acids such as tyrosine. Modification with carboxylic acids and phenol-reactive reagents, and straight is suitable for functionalization. The amino acids that introduce reactivity, for example, the moiety having an alkyne or azide, respectively. Introduction or substitution of amino acids having azide or alkyne groups that enable functionalization: Select from One or more modifiers can be selected.

[0083] In one embodiment, the modified derivative includes N-terminal and / or C-terminal modifications. In one embodiment, the modified derivative is N-terminally modified using a suitable amino reaction chemistry, and This includes C-terminal modification using a suitable carboxyl reaction chemistry. Further embodiments include The N-terminal or C-terminal modification may, but is not limited to, a cytotoxic agent, a radiochelating agent, or Includes the addition of an effector group, which contains a chromophore.

[0084] In further embodiments, the modified derivative includes an N-terminal modification. In this embodiment, the N-terminal modification includes an N-terminal acetyl group. C group (in this specification) i The group called (a group called) is used in peptide synthesis with acetic anhydride or other suitable This yields a molecule that is capped with a reagent and has its N-terminus acetylated. This embodiment is It offers the advantage of removing potential recognition sites of aminopeptidases, and bicyclic peptides Avoid the possibility of disintegration.

[0085] In an alternative embodiment, the N-terminal modification is used for the conjugation of effector groups and This includes the addition of a molecular spacer group to promote the retention of the bicyclic peptide's efficacy against its target. .

[0086] In further embodiments, the modified derivative includes C-terminal modification. In this embodiment, the C-terminal modification includes an amide group. In the specification, C iii The group called C- is synthesized as an amide during peptide synthesis. This results in a molecule with amidated ends. This embodiment is a latent carboxypeptidase. This offers the advantage of removing specific recognition points and increases the potential for proteolytic degradation of bicyclic peptides. To reduce.

[0087] In one embodiment, the modified derivative is one or more unnatural amino acid residues of one or more amino acid residues. This includes substitution by a group. In this embodiment, it is recognized by a degradable protease. It also has isoelectron-distributed / isoelectron-side chains that do not have any adverse effects on target efficacy. Non-natural amino acids may be selected.

[0088] Alternatively, proteolytic hydrolysis of nearby peptide bonds can affect the three-dimensional structure. Non-natural amino acids having constrained amino acid side chains may be used to prevent interference. In particular, these include proline analogs, bulky side chains, and Cα-disubstituted derivatives (e.g., aminoiso). Butyric acid (Aib), and cycloamino acids, which are simple derivatives of amino-cyclopropylcarboxylic acid. Regarding acids.

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

[0090] In one embodiment, the modified derivative is one or more oxidation-resistant amino acid residues of one or more oxidation-sensitive amino acid residues. This includes substitution with an anti-amino acid residue. In a further embodiment, the modified derivative is a triple This embodiment includes substitution of a tophan residue with a naphthylalanine or alanine residue. This offers the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand. do.

[0091] In one embodiment, the modified derivative is one or more hydrophobic amino acids of one or more charged amino acid residues. This includes substitution with acid residues. In an alternative embodiment, the modified derivative is one or more hydrophobic acid residues. Includes substitution of amino acid residues with one or more charged amino acid residues. Charged amino acid residues and hydrophobic The correct balance of amino acid residues is an important characteristic of bicyclic peptide ligands. For example, Hydrophobic amino acid residues affect the degree of plasma protein binding, and therefore the available free-flowing amino acids in plasma. It affects the concentration of the separated fraction, while charged amino acid residues (especially arginine) affect the peptide. This could affect the interaction with the phospholipid membrane on the cell surface. This combination of the two This may affect the half-life, volume of distribution, and exposure of peptide drugs, and may have clinical implications. It can be adjusted according to the point. Furthermore, charged amino acid residues and hydrophobic amino acids The correct combination and number of residues minimizes irritation at the injection site (when peptide drugs are administered subcutaneously). It can be reduced.

[0092] In one embodiment, the modified derivative is one or more L-amino acid residues and one or more D-amino acid residues This includes substitution by . This embodiment stabilizes the β-turn stereostructure by steric hindrance. The tendency of D-amino acids is thought to enhance the stability of protein degradation (Tugyi et al.) Literature (2005) PNAS, 102(2), 413-418).

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

[0094] It should be noted that each of the above-described modifications serves to intentionally improve the efficacy or stability of the peptide. Further improvement of efficacy based on modification can be achieved by the following mechanisms: - utilizing hydrophobic effects to incorporate hydrophobic sites that result in lower dissociation rates, such that higher affinity is achieved; - utilizing long-range ionic interactions to incorporate charged groups that result in faster association rates and higher affinity (for example, refer to the document by Schreiber et al., Rapid electrostatically assisted association of proteins (Rapid, electrostatically assisted association of proteins) (1996), Nature St ruct. Biol. 3, 427-431); and - for example, incorporating additional constraint into the peptide by properly constraining amino acid side chains such that entropy loss is minimized upon target binding, constraining backbone torsion angles such that entropy loss is minimized upon target binding, and introducing further intramolecular cyclization for the same reason, to provide additional constraint to the peptide (for a review, see the document by Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 31 85-3203, and the document by Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-4418).

[0095] (Isotopic Variation) The present invention relates to all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present invention, wherein one or more atoms have the same atomic number but are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature, as well as the peptides of the present invention attached with a metal chelating group capable of holding a related (radio)isotope tide ligands (called "effectors"), and peptide ligands of the present invention wherein specific functional groups are covalently replaced with related (radio)isotopes or isotope-labeled functional groups.

[0096] Examples of suitable isotopes for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, such as , 2 H(D) and 3 H(T), isotopes of carbon, for example, 11 C, 13 C and 14 C, isotopes of chlorine, for example, 3 6 Cl, isotopes of fluorine, for example, 18 F, isotopes of iodine, for example, 123 I, 125 I, and 131 I, isotopes of nitrogen, for example, 13 N and 15 N, isotopes of oxygen, for example, 15 O, 17 O, and 18 O, phosphorus isotopes, for example, 32 P, isotopes of sulfur, for example, 35 S, isotopes of copper, for example, 64 Cu, gallium isotopes, for example, 67 Ga or 68 Ga, isotopes of yttrium, for example,​​​​90 Y, and also Ru Tethium isotopes, for example, 177 Lu, and bismuth isotopes, for example, 213 Includes Bi.

[0097] The present invention incorporates a specific isotope-labeled peptide ligand, for example, a radioactive isotope. This is used in studies of the tissue distribution of drugs and / or substrates, as well as in studies of nectin-4 substrates on affected tissues. Useful for clinically evaluating the presence and / or absence of a target. The Peptidoligan of the present invention The term refers to a complex between a labeled compound and other molecules, peptides, proteins, enzymes, or receptors. It has valuable diagnostic properties in that it can be used to detect or identify the formation of Furthermore, detection or identification methods may include, for example, radioactive isotopes, enzymes, and fluorescent substances. Quality, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and Compounds labeled with labeling agents such as luciferase can be used. The sex isotope tritium, that is, 3 H(T) and carbon-14, that is, 14 C has its built-in Considering the ease and the means of detection available, it is particularly useful for this purpose. ru.

[0098] Deuterium, that is, 2 Substitution with heavier isotopes such as H(D) leads to greater metabolic stability. For example, specific results obtained as a result of an increased in vivo half-life or a reduced required dosage. It may offer therapeutic benefits, and therefore, in some situations, it may be preferable. ru.

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

[0100] The isotope-labeled compound of the peptide ligand of the present invention is usually prepared by conventional techniques known to those skilled in the art or by using an appropriate isotope-labeled reagent in place of a previously used unlabeled reagent by a process similar to that described in the appended examples.

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

[0102] As described in the aforementioned documents, the molecular scaffold is a low molecular weight such as a small organic molecule and may be a molecule.

[0103] In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment , the molecular scaffold is composed of amino acids, nucleotides, or carbohydrates and is a macromolecule.

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

[0105] The molecular scaffold has a chemical group that forms a bond with a peptide, such as amine, thio ol, alcohol, ketone, aldehyde, nitrile, carboxylic acid, ester, alkene, alkyne, azide, anhydride, succinimide, maleimide, alkyl halide, and ha May contain locyl acyls.

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

[0107] The molecular scaffold of the present invention is a polypeptide of the encoded library of the present invention. It contains a chemical group that allows the functional group to form a covalent bond with the molecular scaffold. The chemical group is an amine, thiol, alcohol, ketone, aldehyde, nitrile, or carbohydrate. Bonic acid, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides A wide range of functional groups are selected, including alkyl halides and acyl halides.

[0108] It can be used on a molecular scaffold to react with the thiol group of cysteine. The scaffold reaction groups that can be formed are alkyl halides (or halogenoalkanes or (It has also been named haloalkane.)

[0109] Examples include bromomethylbenzene or iodoacetamide. Other scaffolds used to selectively couple cysteine ​​in proteins The reactive groups are maleimide, αβ-unsaturated carbonyl-containing compounds, and α-halomethyl compounds. It is a rubonyl-containing compound. It can be used as a molecular scaffold in the present invention. Examples of imides include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl) Examples include benzene and tris-(maleimide)benzene. αβ-unsaturated carbonyl-containing compounds An example of an object is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)trippropa-2-en-1-on(T ATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). α - An example of a compound containing hamethylcarbonyl is N,N',N''-(benzene-1,3,5-triyl)tris( It is 2-bromoacetamide. Selenocysteine ​​also has similar reactivity to cysteine. It is a natural amino acid and can be used in the same reaction. Therefore, cysteine ​​is said Whenever it is applied, unless the context suggests otherwise, generally, serenopsis It is permissible to use stain as a substitute.

[0110] (synthesis) The peptides of the present invention are synthesized using standard techniques, and then molecularly synthesized in vitro. It can be reacted with cafold. Standard chemistry is used to carry this out. This allows for rapid analysis of soluble materials for further downstream experiments or verification. This enables large-scale preparation. Such a method is disclosed in the literature by Timmerman et al. (above). This can be achieved using conventional chemistry, such as the methods described.

[0111] Therefore, the present invention also includes polypeptides selected as described herein. Or relating to the manufacture of conjugates, where said manufacture is described below. This includes any further steps. In one embodiment, these steps are performed by chemical synthesis. This is performed on the polypeptide conjugate of the final product.

[0112] Optionally, amino acid residues in the target polypeptide can be used to form a conjugate or complex. It is also used as a substitute when manufacturing.

[0113] By extending the peptide, for example, by incorporating another loop, multiple specificities can be introduced. You can also enter.

[0114] To extend the peptide, it is simply done using standard solid-phase or liquid-phase chemistry. Using orthogonally protected lysine (and its analogues), at its N-terminus or C-terminus or within the loop. It may be chemically extended using standard (bio)conjugation techniques. An N- or C-terminus that has been modified or is activatable may be introduced. Alternatively, the addition may be, for example, (D awson et al., 1994, Protein synthesis by native chemical ligation (Synth (Described in "Hesis of Proteins by Native Chemical Ligation" (Science 266:776-779)) This is done by fragment condensation or native chemical ligation, or for example (Chang et al.) The literature, Proc Natl Acad Sci US 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 performed enzymatically using the subtilis gauze described on pages 6000-6003.

[0115] Alternatively, the peptide may undergo further conjugation via disulfide bonds. They may be elongated or modified. This is because the first and second peptides interact with each other in the reducing environment of the cell. It has the additional advantage of allowing dissociation into molecular scaffolds. D (for example, TATA) reacts with three cysteine ​​groups in the chemical synthesis of the first peptide. It can be added in between; then, further cysteine ​​or thiol can be added to the first peptide. It can be added to the N or C-terminus, and as a result, this cysteine ​​or thiol becomes second The peptide reacts only with free cysteine ​​or thiol to form a disulfide-bonded bicyclic molecule. A peptide-peptide conjugate was formed.

[0116] A similar technique potentially generates quadruple-specific molecules, specifically two bicyclic bispecific macrocyclic compounds. This applies equally to molecular synthesis / coupling.

[0117] Furthermore, the addition of other functional groups or effector groups can be done using appropriate chemistry, either N- or C- This may be achieved in the same manner by coupling at the terminal or via the side chain. In this case, the coupling is performed in a manner that does not block the activity of either entity. ru.

[0118] (Pharmaceutical composition) According to a further aspect of the present invention, one or more peptide ligands as defined herein are used in a drug A pharmaceutical composition is provided that contains a combination of excipients that are acceptable as pharmaceuticals.

[0119] Typically, this peptide ligand is purified together with a pharmacologically appropriate excipient or carrier. They are used in a manner. Typically, these excipients or carriers are used in saline and / or buffered solutions. Includes aqueous or alcohol / aqueous solutions, emulsions, or suspensions containing a medium. Oral vehicles include sodium chloride solution, Ringer's dextrose, and dextrose. Examples include sodium chloride and lactated ringer. Physiologically acceptable preferred Adjuvants are used if necessary to keep the polypeptide complex suspended, such as carboxymethyl From cellulose, polyvinylpyrrolidone, gelatin, and thickeners such as alginates It may be selected.

[0120] Intravenous vehicles include fluid and nutritional supplements and electrolyte supplements, such as Ringarde Examples include those based on chistrose. Also, preservatives and other additives, such as antimicrobial agents. Biopharmaceuticals, antioxidants, chelating agents, and inert gases may be present (Mack's literature (1982)). Remington's Pharmaceutical Sciences, 16th edition.

[0121] The peptide ligand of the present invention can be administered separately as a composition or in combination with other drugs. These may be used. These include antibodies, antibody fragments, and various immunotherapy drugs, for example. , silcosporine, methotrexate, adriamycin, or cisplatin, and immunotherapy Toxins can be cited. The pharmaceutical composition is a variety of protein ligands of the present invention. A "cocktail" of cytotoxic agents or other drugs, or pooled before administration. Polypeptides selected using different target ligands, whether or not they have been selected, This may even include combinations of selected polypeptides according to the present invention that have different specificities. Cut.

[0122] The route of administration of the pharmaceutical composition according to the present invention is any route that is generally known to those skilled in the art. For therapeutic purposes, the peptide ligand of the present invention can be used in any patient according to standard techniques. It can be administered via parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, or pulmonary route. Any method involving intermediation, or similarly, any method including direct injection using a catheter. It can be in an appropriate form. Preferably, the pharmaceutical composition according to the present invention is absorbable It is administered by injection. The dosage and frequency of administration depend on the patient's age, sex, and condition, as well as other medications. The timing of administration of substances, contraindications, and other parameters considered by the clinician are determined by these factors. ru.

[0123] The peptide ligand of the present invention is freeze-dried before storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective and is known in the art of freezing. Freeze-drying and reconstitution techniques can be used. Freeze-drying and reconstitution can be performed to varying degrees of activity. It may be necessary to adjust the level upwards to compensate for potential losses. This will be understood by those skilled in the art.

[0124] The composition containing the peptide ligand or cocktail thereof of the present invention is prophylactic and / or therapeutic. It can be administered for therapeutic purposes. In specific therapeutic applications, a collection of selected cells. At least partial inhibition, suppression, regulation, death, or any other measurable parameter of the group The amount sufficient to achieve the target is defined as the "therapeutic effective dose." The amount required to achieve this depends on the severity of the disease and the overall state of the patient's own immune system. The dosage is determined by the amount of peptide-ligan selected, which is generally between 0.005 and 5.0 mg per kilogram of body weight. The range is 0.05-2.0 mg / kg / kg, with doses of 0.05-2.0 mg / kg / kg being more commonly used. For preventative purposes. Compositions containing this peptide ligand or its cocktail are also similar or slightly less It may be administered in any dosage.

[0125] Compositions containing peptide ligands according to the present invention can be used in prophylactic and therapeutic settings. to assist in altering, inactivating, killing, or eliminating selective target cell populations in mammals. This can be done. Furthermore, peptide ligands described herein can be selected in vitro or in vitro. Selectively used to selectively kill or deplete target cell populations from heterogeneous cellular aggregates. It can be effectively removed by or in other ways. Selected mammalian blood It can be combined with a butyl ligand in vitro, and thereby, according to standard techniques, To return the organism to the dairy animal, the undesirable cells are killed or removed from the blood in another form. .

[0126] (therapeutic use) According to a further aspect of the present invention, this invention is for use in the prevention, suppression, or treatment of cancer. A heterotandem bicyclic peptide complex as defined in the specification is provided.

[0127] Examples of cancers (and their benign counterparts) that can be treated (or suppressed) include tumors of epithelial origin (adenocarcinoma). (including squamous cell carcinoma, transitional cell carcinoma, and other carcinomas, various types of adenomas and carcinomas), for example Examples include bladder and urinary tract, breast, gastrointestinal tract (esophagus, stomach (gastric), small intestine, colon, rectum). , including the anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidneys, lungs (for example) , adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g. Cancers of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovaries, fallopian tubes, Peritoneum, vagina, vulva, penis, cervix, uterine muscle, endometrium, thyroid gland (e.g., thyroid follicles) Cancers of the adrenal glands, prostate, skin, and adnexa (melanoma, basal cell carcinoma, squamous cell carcinoma, keratinization cancer) Acanthoma, dysplastic nevi; hematological malignancies (i.e., leukemia, lymphoma) and pre-malignant hematological disorders Borderline malignancies including hematological malignancies and related diseases of the lymphatic system (e.g., acute lymphoplasms) Lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphoma, for example, diffuse large cell lymphoma. B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma Tumors, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin lymphomas Hair cell leukemia, monoclonal gammaglobulinemia of unknown significance, plasmacytoma, multiple Myeloma and post-transplant lymphoproliferative disorders), as well as hematological malignancies and related diseases of the myeloid lineage (e.g.) For example, acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], Eosinophilia syndrome, myeloproliferative disorders, such as polycythemia vera, essential thrombocythemia, and primary eosinophilia. Myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemias; mesenchymal Tumors of origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, Chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma , synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytoma, and protruding Dermatofibrosarcoma; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma) Cystomas, meningiomas, ependymomas, pineal gland tumors, and Schwann cell tumors; endocrine tumors (e.g., pituitary gland tumors); Tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinomas. ); tumors of the eyeball and adnexa (e.g., retinoblastoma); tumors of the germ cells and trophoblasts (e.g., teratomas, spermatomas). Epithelioma, undifferentiated germ cell tumor, hydatidiform mole, and choriocarcinoma; as well as pediatric and embryonic tumors (e.g.) medulloblastoma, neuroblastoma, Wilms' tumor, and undifferentiated neuroectodermal tumors); or malignant patients Congenital or other syndromes that make a person more susceptible to tumors (e.g., xeroderma pigmentosum) These include, but are not limited to, the following:

[0128] In a further embodiment, cancer may be, for example, non-Hodgkin lymphoma (NHL), Burkitt's lymphoma. Lymphocytic leukemia (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), From hematopoietic malignancies selected from Dikin's lymphoma (HL) and chronic myeloid leukemia (CML): Selected.

[0129] The term "prevention" as used herein refers to the administration of a protective composition before the induction of disease. This includes administration. "Suppression" refers to the administration of the composition after an inducible event but before the clinical manifestation of the disease. This refers to the administration of protective compositions after disease symptoms have become apparent.

[0130] Screening the efficacy of peptide ligands in disease prevention or treatment. A set of animal models is available that can be used for this purpose. The use of the animal model set is: This enables the development of polypeptide ligands that can cross-react with human and animal targets. This is facilitated by the present invention.

[0131] The present invention will be further described below with reference to the following examples. [Examples]

[0132] (Examples) Generally, the heterotandem bicyclic peptide complex of the present invention is prepared according to the following general method. It can be prepared by: [ka]

[0133] A mixture of bicyclic 1 (1.0 equivalent) and NHS-PEG5-N3 (1.6 equivalents) was dissolved in MeCN / H2O (1:1), and the solution was prepared. Adjust the pH to 8 by adding NaHCO3 (0.1M) dropwise. Stir the reaction mixture at 30°C for 2 hours, then Then, the solution is concentrated under reduced pressure to remove the solvent. After that, the residue is purified by preparative HPLC, and an intermediate solution is obtained. Body 2 is obtained.

[0134] A mixture of intermediate 2 (1.0 equivalent) and bicyclic 2 (1.0 equivalent) is dissolved in t-BuOH / H2O (1:1), and then... Add CuSO4 (1.0 equivalent), VcNa (2.3 equivalents), and THPTA (1.0 equivalent). Finally, add 0.2M NH4H Add CO3 to adjust the pH to 8. Stir the reaction mixture under an N2 atmosphere at 40°C for 16 hours. The reaction mixture was directly purified by preparative HPLC.

[0135] More detailed experiments on the selected heterotandem bicyclic peptide complex of the present invention can be found in this specification. The following is provided in the book:

[0136] (Example 1: Synthesis of BCY12375) [ka] (Procedure for preparing palmitic acid-PEG10-N3) [ka] Palmitic acid (100.0 mg, 282.89 μmol, 1.0 equivalent), Compound 2 (150.0 mg, 284.84 μmol, 1.0 Dissolve a mixture of (equivalent) and DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 equivalents) in DMF (2 mL). The reaction mixture was stirred at 30°C for 2 hours. LC-MS confirmed that compound 1 was completely consumed. One major peak with the desired m / z (MW: 765.03, observed m / z: 765.22) was detected. It was shown that when the reaction mixture was concentrated under reduced pressure, the solvent was removed, leaving a residue. Subsequently, the residue was purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-N3 (79.0 mg, 9 9.41 μmol (35.14% yield, 96.27% purity) was obtained as a white solid.

[0137] (Procedure for the preparation of palmitate-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 THPTA (10.0 mg, 23.02 μmol, 1.0 equivalent) and t-BuOH / H2O (1:1, 1 mL, degassed beforehand, N Dissolve in (purged 3 times in step 2), then add CuSO4 (0.4M, 56.0μL, 1.0 equivalent) and VcNa (1 0.0 mg (50.48 μmol, 2.3 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 tB When the solution was adjusted to 8 by adding uOH / H2O dropwise, the solution turned pale yellow. The reaction mixture was then subjected to an N2 atmosphere. The mixture was stirred under air pressure at 40°C for 2 hours. By LC-MS, palmitic acid-PEG10-N3 was returned, and the desired m / One major peak with z (calculated MW: 3030.60, observed m / z: 1010.35 ([M / 3+H]) + It was shown that )) was detected. When the reaction mixture was filtered and concentrated under reduced pressure, the residue was obtained. The crude product was purified by preparative HPLC (TFA conditions), and palmitic acid-PEG10-BCY1202 was obtained. 3 (43.0 mg, 13.97 μmol, 63.30% yield, 98.46% purity) was obtained as a white solid.

[0138] (Procedure for the preparation of palmitic acid-PEG10-BCY12023-PEG5-N3) [ka] Compound 5 (43.0 mg, 14.19 μmol, 1.0 equivalent) and Compound 6 (10.0 mg, 23.13 μmol, 1.6 equivalents) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by adding NaHCO3 (0.1 M) dropwise. The mixture was adjusted to 8. The reaction mixture was stirred at 30°C for 2 hours. LC-MS confirmed that compound 5 was completely consumed. And there was 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 detection of ])) was shown. The reaction mixture was concentrated under reduced pressure. The solvent was then removed, leaving a residue. The residue 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%) The result was obtained as a white solid with a high purity.

[0139] (Procedure for preparing BCY12375) [ka] Compound 7 (8.0 mg, 2.39 μmol, 1.0 equivalent), Compound 8 (6.5 mg, 2.39 μmol, 1.0 equivalent), and TH A mixture of PTA (1.1 mg, 2.53 μmol, 1.0 equivalent) was mixed with t-BuOH / H2O (1:1, 1 mL, pre-degassed, and then diluted three times with N2). Dissolve in (purged), then add CuSO4 (0.4M, 6.0μL, 1.0 equivalent) and VcNa (1.0mg, 5 0.05 μmol (2.1 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (in a 1:1 t-BuOH / H2O solution). When the solution was adjusted to 8 by the dropwise addition of ), the solution turned pale yellow. The reaction mixture was stored at 40°C under an N2 atmosphere. The mixture was stirred for 16 hours. By LC-MS, compound 7 was returned, and one major peak with the desired m / z was observed. (Calculated MW: 6064.08, observed m / z: 1516.4 ([M / 4+H]) + ), 1212.8([M / 5+H] + )) is checked It was shown that it could be released. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. Purification of the crude product by preparative HPLC (TFA conditions) yielded BCY12375 (6.2 mg, 0.99 μmol, 41.62%). The product was obtained as a white solid in yield (97.27% purity).

[0140] (Example 2: Synthesis of BCY12021) [ka] (Procedure for the 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 THPTA (35.0 mg, 80.55 μmol, 1.1 equivalents) and t-BuOH / H2O (1:1, 2 mL, pre-degassed) (Purged three times with N2), then dissolved in CuSO4 (0.4M, 56.0μL, 1.0 equivalent) and VcN a (30.0 mg, 151.43 μmol, 2.2 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 When the solution was adjusted to 8 by the dropwise addition of t-BuOH / H2O, the solution turned pale yellow. The reaction mixture was then converted to N2 The mixture was stirred at 40°C for 16 hours under controlled conditions. LC-MS revealed 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 It was shown that it could be released. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. When the crude product is purified by preparative HPLC (TFA conditions), palmitic acid--PEG10-BCY11144 (150.0m) is obtained. (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 equivalents) was dissolved in DMF (1 mL). The mixture was stirred at 30°C for 2 hours. LC-MS revealed 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] + It was shown that )) was detected. When the reaction mixture was concentrated under reduced pressure, the solvent was removed. A residue was generated. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid --PE G10-BCY11144-PEG5-N3 (20.0 mg, 5.72 μmol, 37.33% yield, 96.79% purity) is a white solid. It was obtained as follows.

[0142] (Procedure for preparing 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 equivalents) and t-BuOH / H2O (1:1, 1 mL, pre-degassed and 3 mL with N2) Dissolve in (a solution that has been purged multiple times), then add CuSO4 (0.4M, 8.0μL, 1.0 equivalent) and VcNa (1.5mg) (7.57 μmol, 2.5 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / H2 When the solution was adjusted to 8 by adding (in O) dropwise, the solution turned pale yellow. The reaction mixture was stored under an N2 atmosphere for 4 The mixture was stirred at 0°C for 16 hours. LC-MS revealed 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. Reaction The mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was separated by preparative HPLC (TFA conditions). When purified by this method, BCY12021 (6.6 mg, 1.02 μmol, 34.62% yield, 94.54% purity) is obtained as a white solid. It was obtained as a physical object.

[0143] (Example 3: Synthesis of BCY11468) [ka] (Procedure for preparing COM113) [ka] Compound 1 (50.0 mg, 124.4 μmol, 1.0 equivalent), EDCI (95.4 mg, 497.7 μmol, 4.0 equivalent), HOBt ( A mixture of 55.5 mg (410.6 μmol, 3.3 equivalents) and DMAP (15.2 mg, 124.4 μmol, 1.0 equivalent) in 2 mL. Dissolve in DMF, then add DIEA (134.9 mg, 1.04 mmol, 181.8 μL, 8.4 equivalents) and mix until uniform. A high-quality solution was prepared. Next, compound 2 (200.0 mg, 379.8 μmol, 3.0) was dissolved in DMF (2 mL). (5 equivalents) was added dropwise to this solution. The reaction mixture was stirred at 30°C for 16 hours. By LC-MS, the chemical reaction was observed. When substance 1 is completely consumed, one major peak with the desired m / z (MW: 1891.19, observed m) is observed. / z: 945.8600([M / 2+H + ]) and 612.4400([(M-3H2O) / 3+H + It was shown that ])) was detected. The reaction mixture is purified directly by preparative HPLC (TFA conditions), and after freeze-drying, COM113 (161m (g, 85.67 μmol, 68% yield) was obtained as a yellow oily substance.

[0144] (Procedure for preparing COM113-BCY8928) [ka] COM113 (50.0 mg, 26.44 μmol, 1.0 equivalent) and BCY8928 (53.0 mg, 23.9 μmol, 0.9 equivalents) First, dissolve in 2 mL of t-BuOH / H2O (1:1), then add CuSO4 (0.4 M, 66.1 μL, 1.0 equivalent), VcNa (10 (0.5 mg, 53.0 μmol, 2.0 equivalents) and THPTA (23.0 mg, 52.93 μmol, 2.0 equivalents) were added. Later, 1M NH4HCO3 was added to adjust the pH to 8. All solvent was degassed and the mixture was purged three times with N2. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS was used to obtain the desired m / z. One major peak (calculated MW: 4108.77, observed m / z: 1369.97 ([M / 3+H]) + )) indicates The reaction mixture was purified by preparative HPLC (TFA conditions), yielding compound 2 (14.0 mg, 3.21 μmol). A white solid was obtained (12.14% yield, 94.16% purity).

[0145] (Procedure for the preparation of NHS palmitate) [ka] Palmitic acid (500 mg, 1.95 mmol, 586.85 μL, 1.0 equivalent), 1-hydroxypyrrolidine-2,5- In a 5 mL solution of DCM containing 250 mg of Zion (2.17 mmol, 1.11 equivalents), add EDCI (747.60 mg, 3.90 mmol, 2.0 An equivalent amount was added. The mixture was stirred at 30°C for 16 hours. TLC confirmed that reactant 1 was completely consumed. This showed that a new spot was formed. According to TLC, the reactants were clean. Yes, it was found. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was then subjected to column chromatography. The product was purified by matrixing (SiO2, DCM:MeOH = 0-100:1). The desired product was then dried. Then, NHS palmitate (0.68 g, 1.92 mmol, 98.65% yield) was obtained as a white solid. It was done.

[0146] (Procedure for preparing 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 equivalents) In a 6 mL DMF solution, DIEA (131.62 mg, 1.02 mmol, 177.39 μL, 3.0 equivalents) and DMAP (41.47 mg, 339.47 μmol (1.0 equivalent) was added. The mixture was stirred at 40°C for 16 hours. The reaction was analyzed by LC-MS. When substance 3 is completely consumed, one major peak with the desired m / z or desired mass is detected. This was shown. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. Purified by preparative HPLC (TFA conditions). Propargylalanine palmitate (90 mg, 256.03 A quantity of 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 equivalents) First, dissolve in 2 mL of t-BuOH / H2O (1:1), then add CuSO4 (0.4 M, 10.0 μL, 1.1 equivalents) and VcNa (2. (0 mg, 10.1 μmol, 2.9 equivalents) and THPTA (2.0 mg, 4.6 μmol, 1.3 equivalents) were added. Finally, Then, 0.2M NH4HCO3 was added to adjust the pH to 8. All the solvent was degassed, and the solution was perfused three times with N2. The reaction mixture was stirred at 35°C for 16 hours under an N2 atmosphere. The desired m / z was determined by LC-MS. It has 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 was shown. Preparative HPLC (TFA conditions) of the reaction mixture. Upon purification by this method, compound 4 (5.9 mg, 1.28 μmol, 37.66% yield, 97.0% purity) was obtained as a white solid. It was obtained as a physical object.

[0148] (Procedure for preparing BCY11468) [ka] Compound 4 (5.9 mg, 1.32 μmol, 1.0 equivalent) and BCY11016 (3.0 mg, 1.29 μmol, 1 equivalent) were first divided into two parts. Dissolve in mL of t-BuOH / H2O (1:1), then add CuSO4 (0.4M, 8.0 μL, 2.4 equivalents) and VcNa (2.0 mg). (7.6 equivalents), and THPTA (2.0 mg, 3.5 equivalents) were added. Finally, 1 M NH4HCO3 was added, p The H concentration was adjusted to 8. All solvents were degassed and the mixture was purged three times with N2. The reaction mixture was then stored under an N2 atmosphere. The mixture was stirred at 30°C for 16 hours. LC-MS was used to find one major peak with the desired m / z (calculated). MW: 6783.93, observed m / z: 1131.7 ([M / 6+H] + )) was shown. Preparative HPLC of the reaction mixture ( When purified under TFA conditions, BCY11468 (2.2 mg, 0.312 μmol, 23.57% yield, 96.16% purity) is obtained. It was obtained as a white solid.

[0149] (Example 4: Synthesis of BCY11618) [ka] (Procedure for preparing 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 NaHCO3 (2.0 mg, 24.8 μmol, 1.0 equivalent) was dissolved in MeCN / H2O (1:1, 2 mL). C-MS completely consumed the BCY8920, and one major peak with the desired m / z was calculated. 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 it was shown to be effective. Then, the reaction mixture was subjected to reduced pressure. When concentrated, the solvent is removed, leaving 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 found as a white solid. It was obtained.

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

[0151] (Procedure for preparing 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 equivalent) is prepared in t-BuOH / H2O (1:1, 1 mL), degassed beforehand, and then punctured three times with N2. Dissolve in (a mixture), then add CuSO4 (0.4M, 5.1μL, 1.0 equivalent) and VcNa (0.4M, 5.1 μL (1.0 equivalent) was added under N2 conditions. The pH of this solution was adjusted by adding a drop of 0.2M NH4HCO3 (in a 1:1 t-BuOH / H2O solution). When the concentration was adjusted to 8 by adding, the solution turned pale yellow. The reaction mixture was incubated at 40°C for 6 hours under an N2 atmosphere. The mixture was stirred. By LC-MS, compound 3 was completely consumed, and one major peak with the desired m / z was observed. -k (Calculated MW: 5029.97, Observed m / z: 1257.8 ([M / 4+H]) + ) and 1006.6([M / 5+H] + )) It was shown that [the substance] could be detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC (TFA conditions), and BCY11618 (5.3 mg, 1.0 μmol, 49.15) was obtained. The product (% yield, 95% purity) was obtained as a white solid.

[0152] (Example 5: Synthesis of BCY11776) [ka] (Procedure for preparing 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 NaHCO3 (2.0 mg, 24.8 μmol, 1.0 equivalent) was dissolved in MeCN / H2O (1:1, 2 mL). C-MS completely consumed BCY8116, and one major peak with the desired m / z was calculated. 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 following was indicated. Then, the reaction mixture was stirred under reduced pressure. Upon concentration, the solvent was removed, leaving 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 substance was dissolved in DMF. The reaction mixture was stirred at 25°C for 2 hours under an N2 atmosphere. By LC-MS... Compound 3 is completely consumed, and one major peak with the desired m / z (calculated MW: 2542) 0.08, observed m / z: 1271.7([M / 2+H + It was shown that ])) was detected. Reaction agent filter After passing through the solution and concentrating under reduced pressure, a residue is obtained, which is then purified by preparative HPLC (TFA conditions). Compound 6 (18.3 mg, 6.95 μmol, 54.17% yield, 96.68% purity) was prepared as a white solid. Obtained.

[0154] (Procedure for preparing 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 equivalent) was prepared by t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2). Dissolve in ( ), then add CuSO4 (0.4M, 5.1μL, 1 equivalent) and VcNa (0.4M, 5.1μL, 1 equivalent) The solution was added under N2 conditions. The pH of this solution was adjusted to 8 by the dropwise addition of 0.2 M NH4HCO3 (in a 1:1 t-BuOH / H2O solution). The solution then turned pale yellow. The reaction mixture was stirred at 40°C for 6 hours under an N2 atmosphere. LC- MS revealed that compound 3 was completely consumed, and one major peak with the desired m / z was found (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 obtain a residue. The crude product was divided. Purification by HPLC (TFA conditions) yielded BCY11776 (12.5 mg, 2.4 μmol, 60.11% yield, 96.6%). The result was obtained as a white solid with a high purity.

[0155] (Example 6: Synthesis of BCY11860) [ka] (Procedure for preparing 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 (amount), and THPTA (0.4M, 21.0μL, 1.0 equivalent) is prepared in t-BuOH / H2O (1:1, 1mL, pre-degassed). (Purged three times with N2), then dissolved in CuSO4 (0.4M, 21.0μL, 1.0 equivalent) and VcN a (0.4 M, 21.0 μL, 1.0 equivalent) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuO2). When the solution was adjusted to 8 by adding H / H2O dropwise, the solution turned pale yellow. The reaction mixture was then placed under an N2 atmosphere. The mixture was stirred at 40°C for 4 hours. LC-MS confirmed 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] + It was shown that )) was detected. When the reaction mixture was filtered and concentrated under reduced pressure, A residue was obtained. The crude product was purified by preparative HPLC (TFA conditions) and 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 preparing BCY11860) [ka] Compound 3 (5.0 mg, 1.04 μmol, 1.0 equivalent), Compound 4 (1.08 mg, 1.15 μmol, 1.1 equivalent), and D A 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 substance was dissolved in DMF (1.0 mL). The reaction mixture was stirred at 30°C for 2 hours. By LC-MS, the compound was identified. 3 is completely consumed, and there is one major peak with the desired m / z (MW: 5617.56, observed m / z : 1404.56([(M / 4+H + It was shown that ])) was detected. When the reaction mixture is concentrated under reduced pressure... The solvent was removed, leaving a residue. The residue 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 the preparation of palmitic acid-PEG10-N3) [ka] Palmitic acid-NHS (100.0 mg, 282.89 μmol, 1.0 equivalent), Compound 2 (150.0 mg, 284.84 μmol) A mixture of (1.0 equivalent) and DIEA (74.5 mg, 574.11 μmol, 100.0 μL, 2.0 equivalents) is added to DMF (2 mL). The compound was dissolved. The reaction mixture was stirred at 30°C for 2 hours. LC-MS confirmed that compound 1 was completely consumed. Then, one major peak with the desired m / z (MW: 765.03, observed m / z: 765.22) was detected. It was shown that when the reaction mixture is concentrated under reduced pressure, the solvent is removed and a residue is produced. The residue was then purified by preparative HPLC (neutral conditions). Palmitic acid-PEG10-N3 (79.0 mg) A white solid was obtained (99.41 μmol, 35.14% yield, 96.27% purity).

[0158] (Procedure for the preparation of palmitate-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 THPTA (35.0 mg, 80.55 μmol, 1.1 equivalents) and t-BuOH / H2O (1:1, 2 mL, pre-degassed) (Purged three times with N2), then dissolved in CuSO4 (0.4M, 56.0μL, 1.0 equivalent) and VcN a (30.0 mg, 151.43 μmol, 2.2 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 When the solution was adjusted to 8 by the dropwise addition of t-BuOH / H2O, the solution turned pale yellow. The reaction mixture was then converted to N2 The mixture was stirred at 40°C for 16 hours under the atmosphere. One main peak with the desired m / z was detected by LC-MS ( calculated MW: 3068.70, observed m / z: 1533.81 ([M / 2+H] + ), 1023.43 ([M / 3+H] + )) was observed. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was subjected to preparative HP LC (TFA conditions) for purification, so that palmitic acid-PEG10-BCY11144 (150.0 mg, 46.83 μmol, 67 .42% yield, 95.80% purity) was obtained as a white solid.

[0159] (Procedure for the preparation of palmitic acid-PEG10-BCY11144-PEG5-N3)

Chemical Structure

[0160] (Procedure for preparing 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 THPTA (10.0 mg, 23.02 μmol, 1.5 equivalents) and t-BuOH / H2O (1:1, 2 mL, degassed beforehand, N) Dissolve in (purged 3 times in step 2), then add CuSO4 (0.4M, 38.0μL, 1.0 equivalent) and VcNa (6 0.5 mg (32.81 μmol, 2.2 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-Bu). When the solution was adjusted to 8 by adding OH / H2O dropwise, the solution turned pale yellow. The reaction mixture was kept under an N2 atmosphere. The mixture was stirred at 40°C for 16 hours. LC-MS revealed a single major peak with the desired m / z (calculated). 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 obtain a residue. The crude product was separated by preparative HPLC (TFA). When purified under the specified conditions, BCY12020 (54.8 mg, 9.49 μmol, 64.24% yield, 98.83% purity) was obtained. It was obtained as a solid of color.

[0161] (Example 8: Synthesis of BCY12661) [ka] (Procedure for preparing Compound 2) [ka] Peptides were synthesized using standard Fmoc chemistry. DCM was mixed with chlorotrityl resin (1 mmol, 0 A reaction vessel containing 0.91 g, 1.10 mmol / g) and Fmoc-Lys(N3)-OH (1 equivalent, 395.4 mg, 1 mmol) N2 was added while bubbling. DIEA (4.0 equivalents) was added dropwise and mixed for 2 hours. After that, Me OH (2 mL) was added and mixed for 30 minutes. The resin was drained and washed 5 times with DMF. Fmoc deprotection was performed. This was done by adding 20% ​​piperidine / DMF and mixing for 30 minutes. The resin was drained and treated with DMF for 5 minutes. Washed several times. For chain extension, Fmoc-amino acid solution was added and mixed for 30 seconds, then Then, add the activation buffer (containing HBTU and DIEA in DMF) and perform continuous N2 bubbling. Then, the mixture was stirred for 1 hour. Deprotection and coupling were repeated until the peptide was complete. Ta. [Table 5]

[0162] After the final amino acid coupling, the resin was washed three times with MeOH and then dried under vacuum. I added 10 ml of a cleavage cocktail (95% TFA / 2.5% TIS / 2.5% H2O) containing a side-chain protective peptide. The resin was added to a flask at room temperature and stirred for 1 hour. The resin was filtered, and the filtrate was concentrated and dissolved. The medium was removed. When the crude peptide was freeze-dried, the final product compound 2 (adiphosphate palmitate) was obtained. (200 mg, 97.78% purity, 37.06% yield) was obtained. Calculated MW: 539.72, observed m / z: 540.4([M+H] + ).

[0163] (Procedure for the preparation of BCY12023-azide palmitate) [ka] A mixture of Compound 1 (40.0 mg, 17.66 μmol, 1.0 eq), Compound 2 (9.5 mg, 17.66 μmol, 1.0 eq), and THPTA (8.0 mg, 17.66 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, degassed beforehand and purged three times with N2), then CuSO4 (0.4 M, 45.0 μL, 1.0 eq) and VcNa (8. 0 mg, 35.33 μmol, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuO H / H2O), and the solution turned pale yellow. The reaction mixture was stirred at 40°C for 4 hours under a N2 atmosphere. LC-MS showed that Compound 1 was completely consumed, and one main peak with the desired m / z (calculated MW: 2804.30, observed m / z: 1402.8 ([M / 2+H] ) + , 935.9 ( [M / 3+H] + )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue . The crude product was purified by preparative HPLC (TFA conditions) to afford BCY12023-palmitic acid azide (35.0 mg, 12.11 μmol, 68.54% yield, 97.00% purity) as a white solid .

[0164] (Procedure for the preparation of BCY12023-palmitic acid-PEG5-N3)

Chemical Formula

[0165] (Procedure for preparing 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 equivalent) is prepared in t-BuOH / H2O (1:1, 1 mL, pre-degassed and sterilized with N2) for 3 minutes. Dissolve in (a solution that has been purged multiple times), then add CuSO4 (0.4M, 10.0μL, 1.1 equivalents) and VcNa (1.5mg) 7.30 μmol, 2.0 equivalents of t-BuOH were added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / H2 When the solution was adjusted to 8 by adding (in O) dropwise, the solution turned pale yellow. The reaction mixture was stored under an N2 atmosphere for 4 The mixture was stirred at 0°C for 4 hours. LC-MS confirmed that compound 3 was completely consumed and one compound with the desired m / z was obtained. Major peak (calculated MW: 5374.21, observed m / z: 1344.5 ([M / 4+H]) + )) is detected This was demonstrated. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. Crude product When purified by preparative HPLC (TFA conditions), BCY12661 (9.8 mg, 19.63 μmol, 48.73% yield, 97) was obtained. A 0.60% purity product was obtained as a white solid.

[0166] (Example 9: Synthesis of BCY12969) [ka] (General procedure for preparing compound 1) [ka] Peptides were synthesized using standard Fmoc chemistry. DCM was mixed with chlorotrityl resin (1 mmol, 0 A reaction vessel containing 0.91 g (1.1 mmol / g) and Fmoc-γGlu(OtBu)-OH (0.425 mg, 1 mmol, 1 equivalent) It was added and the mixture was stirred while bubbling with N2. DIEA (4.0 equivalents) was added dropwise, and the mixture was 2 The mixture was stirred for a certain amount of time. Then, 4.6 mL of MeOH was added and mixed for 30 minutes. The resin was drained and the mixture was heated in DMF. Washed 5 times. 20% piperidine / DMF was added to the resin and mixed for 30 minutes. Resin moisture The resin was cut and washed 5 times with DMF. Fmoc-amino acid solution was added to the resin and mixed for 30 seconds, then Then, the activator and DIEA were added, and N2 was passed through the mixture and bubbling for 1 hour. The following reagents The deprotection and coupling process was repeated using the following: Note: [Table 6]

[0167] After palmitic acid coupling, the resin was washed three times with MeOH, and then dried under vacuum. The peptide was cleaved from the resin at room temperature by adding 20% ​​HFIP / 80% DCM, and the mixture was left for 1 hour. Stirring. Repeat this procedure once more, then filter the resin, concentrate the filtrate, and extract the solvent. The crude peptide was removed. When the crude peptide was freeze-dried, the final product (280 mg, 84.80% purity, 44.67%) The yield was obtained. Calculated MW: 626.8, observed m / z: 627.4 ([M+H] + ).

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

[0169] (General procedure for preparing compound 5) [ka] Compound 4 (20.0 mg, 8.03 μmol, 1.0 equivalent) and Compound 3 (22) in t-BuOH (0.5 mL) and H2O (0.5 mL). A mixture of (0.5 mg, 8.03 μmol, 1.0 equivalent) and THPTA (4.0 mg, 9.21 μmol, 1.15 equivalents) was degassed. Purge three times with N2, then add CuSO4 (0.4M, 20.1μL, 1.0 equivalent), VcNa (0.4M, 40.2μL, 2. (0 equivalents) and NH4HCO3 (0.2M, 80.4 μL, 2.0 equivalents) were added to the mixture. The mixture was then heated in an N2 atmosphere. The mixture was stirred under gaseous conditions at 30°C 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] + It was shown that ) was detected. EDTA (0.5 M, 20.0 μL) was added to the reaction mixture. When the reaction mixture is concentrated under reduced pressure, the crude product compound 5 (42.0 mg, crude product) is a gray solid. The resulting product was used in the next step without further purification.

[0170] (General procedure for preparing BCY12969) [ka] To a solution of compound 5 (42.0 mg, 8.22 μmol, 1.0 equivalent) in DCM (0.25 mL), add TFA (3.37 μmol, 0.25 mL) (458.6 equivalents) was added dropwise. The mixture was stirred at 30°C for 1 hour. Compound 5 was completely removed by LC-MS. It was consumed, and there was 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] +It was shown that ) was detected. When the reaction mixture was concentrated under reduced pressure, a residue was obtained. The residue was separated by preparative HPLC (A: 0.075% in H2O). When purified using TFA (B: ACN), BCY12969 (2.6 mg, 0.48 μmol, 5.85% yield, 92.4% purity) is obtained. It was obtained as a white solid.

[0171] (Example 10: Synthesis of BCY13035) [ka] (Procedure for preparing BCY12860-PEG5-N3) [ka] BCY12860 (40.0 mg, 19.40 μmol, 1.0 equivalent), Compound 2 (10.0 mg, 21.34 μmol, 1.1 equivalents) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by adding NaHCO3 (0.1 M) dropwise. The reaction mixture was further adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. LC-MS was used to determine the desired m / z. A peak was observed. When the reaction mixture was concentrated under reduced pressure, the solvent was removed, leaving 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 ta m / z: 1190.1([(M / 2+H + ]), 793.5([(M / 3+H + ]).

[0172] (Procedure for preparing BCY13035) [ka] Compound 3 (39.7 mg, 16.69 μmol, 1.0 equivalent), BCY8928 (41.0 mg, 18.36 μmol, 1.1 equivalent), and Mixture of THPTA (0.4M, 55μL, 1.3 equivalents) with t-BuOH / H2O (1:1, 1mL, pre-degassed, then 3 times with N2). Dissolve in (purged), then add CuSO4 (0.4M, 55μL, 1.3 equivalents) and VcNa (0.4M, 10⁹) μL (2.6 equivalents) was added under N2 conditions. The pH of this solution was adjusted by adding a drop of 0.2M NH4HCO3 (in a 1:1 t-BuOH / H2O solution). When the concentration was adjusted to 8 by adding, the solution turned pale yellow. The reaction mixture was incubated at 40°C under an N2 atmosphere for 2 hours. The mixture was stirred. By LC-MS, compound 3 was completely consumed, and one major peak with the desired m / z was observed. It was shown that a trace was detected. When the reaction mixture was filtered and concentrated under reduced pressure, the residue was obtained. The crude product was purified by preparative HPLC (TFA conditions), yielding BCY13035 (42.0 mg, 8.85 μmol). A white solid was obtained (53.04% yield, 96.41% purity). 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 preparing 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) It 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 hours. LC-MS confirmed that BCY12865 was completely consumed and the desired m / z was achieved. It was shown that one major peak was detected. The reaction mixture was subjected to preparative HPLC (TFA conditions). Further purification yielded compound 2 (15.6 mg, 6.32 μmol, 45.19% yield, 99.76% purity) as a white solid. It was obtained as a physical object. Calculated MW: 2461.87, observed m / z: 1231.5 ([M / 2+H] + ) and 821. 3([M / 3+H]+).

[0174] (Procedure for preparing BCY13040) [ka] Compound 2 (15.6 mg, 6.34 μmol, 1.0 equivalent) and BCY8928 (14.5 mg, 6.54 μmol, 1.03 equivalents) First, dissolve in 2 mL of t-BuOH / H2O (1:1), then add CuSO4 (0.4 M, 16 μL, 1.01 equivalents) and VcNa (3 (0.0 mg, 15.14 μmol, 2.39 equivalents) and THPTA (3 mg, 6.90 μmol, 1.09 equivalents) were added. 1M NH4HCO3 was added to adjust the pH to 8. All solvent was degassed, and the mixture was purged three times with N2. The reaction mixture was stirred at 40°C for 16 hours under an N2 atmosphere. Compound 2 was completely eliminated by LC-MS. The reaction mixture was used, and it was shown that one major peak with the desired m / z was detected. When purified by preparative HPLC (TFA conditions), BCY13040 (15.8 mg, 3.31 μmol, 52.27% yield, 98%) was obtained. A 0.1% purity solution 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 preparing BCY13119-PEG5-N3) [ka] BCY13119 (35.0 mg, 17.20 μmol, 1.0 equivalent), Compound 2 (7.8 mg, 18.06 μmol, 1.05 equivalents) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by adding NaHCO3 (0.1 M) dropwise. The reaction mixture was further adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. LC-MS was used to determine the desired m / z. One major peak (MW: 2352.74, observed m / z: 1177.4 ([(M / 2+H + ])) is detected This was shown. When the reaction mixture was concentrated under reduced pressure, the solvent was removed, leaving a residue. The residue was purified by preparative HPLC (neutral conditions). BCY13119-PEG5-N3 (25.7 mg, 9.97 μmol, 58 A white solid was obtained (0.0% yield, 91.3% purity).

[0176] (Procedure for preparing BCY13253) [ka] Compound 3 (25.7mg, 10.92μmol, 1.0eq), compound 2 (26.6mg, 12.02μmol, 1.1eq), and A mixture of THPTA (5.7 mg, 13.11 μmol, 1.2 equivalents) and t-BuOH / H2O (1:1, 1 mL, degassed beforehand, N2 Dissolve in (purged three times), then add CuSO4 (0.4M, 33.0μL, 1.2 equivalents) and VcNa (5. 2 mg (26.21 μmol, 2.4 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuO2). When the solution was adjusted to 8 by adding H / H2O dropwise, the solution turned pale yellow. The reaction mixture was then placed under an N2 atmosphere. The mixture was stirred at 25°C for 2 hours. LC-MS confirmed 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] + It was shown that )) was detected. When the reaction mixture was filtered and concentrated under reduced pressure, the residue A residue was obtained. The crude product was purified by preparative HPLC (TFA conditions), and BCY13253 (17.5 mg, 3.67 mg) was obtained. A quantity of μmol (33.58% yield, 95.8% purity) was obtained as a white solid.

[0177] (Example 13: Synthesis of BCY13254) [ka] (Procedure for preparing 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 equivalents) The mixture was dissolved in MeCN / H2O (1:1, 1 mL), and then the pH of this solution was adjusted by adding NaHCO3 (0.1 M) dropwise. The mixture was adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. BCY13120 was completely eliminated by LC-MS. One major peak with the desired m / z was observed (MW: 2548.99, observed m / z: 1275.3) M / 2+H + It was shown that ])) was detected. When the reaction mixture was concentrated under reduced pressure, the solvent was removed. A residue was generated. The residue was then purified by preparative HPLC (neutral conditions). BCY13120-PEG 5-N3 (27.3 mg, 10.46 μmol, 58.38% yield, 97.7% purity) was obtained as a white solid.

[0178] (Procedure for preparing 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 THPTA (5.6 mg, 12.85 μmol, 1.2 equivalents) and t-BuOH / H2O (1:1, 1 mL, degassed beforehand, N2 It was dissolved in (a solution purged three times). CuSO4 (0.4M, 33.0μL, 1.2 equivalents) and VcNa (5.2mg, 2 6.24 μmol (2.4 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / H2O When the solution was adjusted to 8 by adding (amount in the middle) dropwise, the solution turned pale yellow. The reaction mixture was stored under an N2 atmosphere for 25 minutes. The mixture was stirred at °C for 2 hours. LC-MS confirmed 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] + It was shown that )) was detected. When the reaction mixture was filtered and concentrated under reduced pressure, the residue was The crude product was purified by preparative HPLC (TFA conditions) and BCY13254 (36.5 mg, 7.49 μmol) was obtained. 1 (69.92% yield, 97.8% purity) was obtained as a white solid.

[0179] (Example 14: Synthesis of BCY13340) [ka] (Procedure for preparing BCY12865-PEG5-N3) [ka] BCY12865 (50 mg, 23.32 μmol, 1.0 equivalent) and Compound 1 (10.5 mg, 24.28 μmol, 1.04 equivalents) It was dissolved in 2 mL of MeCN / H2O (1:1), and 1 M NaHCO3 was added to adjust the pH to 8. Then, it was mixed. The substance was stirred at 25°C for 2 hours. LC-MS revealed that BCY12865 was completely consumed and had 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 detection of )) was shown. The reaction mixture was purified by preparative HPLC (TFA conditions). When prepared, compound 2 (31.5 mg, 12.62 μmol, 54.14% yield, 98.66% purity) is a white solid. This was obtained.

[0180] (Procedure for preparing 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 THPTA (5.7 mg, 13.12 μmol, 1.0 equivalent) and t-BuOH / H2O (1:1, 2 mL, degassed beforehand, N2 Dissolve in (purged three times), then add CuSO4 (0.4M, 32μL, 1.0 equivalent) and VcNa (5.1M) (g, 25.74 μmol, 2.0 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuOH / When the solution was adjusted to 8 by dropwise addition (in H2O), the solution turned pale yellow. The reaction mixture was then subjected to an N2 atmosphere. The mixture was stirred at 40°C for 1 hour. LC-MS confirmed that compound 2 was completely consumed and compound 1 had the desired m / z. Two major peaks (calculated MW: 4551.32, observed m / z: 1517.7 ([M / 3+H]) + ) and 1138. 6([M / 4+H] + It was shown that )) was detected. When the reaction mixture was filtered and concentrated under reduced pressure, A residue was obtained. Purification of the crude product by preparative HPLC (TFA conditions) yielded BCY13340 (34.7 mg, 7 mg). 62 μmol (59.59% yield, 89.59% purity) was obtained as a white solid.

[0181] (Example 15: Synthesis of BCY13342) [ka] (Procedure for preparing 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 adding NaHCO3 (0.1 M) dropwise. The concentration was further adjusted to 8. The reaction mixture was stirred at 25°C for 1 hour. By LC-MS, BCY12860 was completely eliminated. One major peak is consumed and has the desired m / z (MW: 2378.78, observed m / z: 1190.2). [(M / 2+H + It was shown that ])) was detected. When the reaction mixture was concentrated under reduced pressure, the solvent was removed. The residue was removed, leaving a residue. The residue was then purified by preparative HPLC (neutral conditions). BCY12860-P EG5-N3 (20.7 mg, 8.41 μmol, 61.95% yield, 96.7% purity) was obtained as a white solid.

[0182] (Procedure for preparing BCY13342) [ka] Compound 3 (20.7 mg, 8.70 μmol, 1.0 equivalent), compound 4 (19.0 mg, 9.14 μmol, 1.05 equivalent), and A mixture of THPTA (5.0 mg, 11.31 μmol, 1.3 equivalents) and t-BuOH / H2O (1:1, 1 mL, degassed beforehand, N2 Dissolve in (purged three times), then add CuSO4 (0.4M, 28.3μL, 1.3 equivalents) and VcNa (4. 5 mg (22.62 μmol, 2.6 equivalents) was added under N2 conditions. The pH of this solution was adjusted to 0.2 M NH4HCO3 (1:1 t-BuO2). When the solution was adjusted to 8 by adding H / H2O dropwise, the solution turned pale yellow. The reaction mixture was then placed under an N2 atmosphere. The mixture was stirred at 25°C for 2 hours. LC-MS confirmed 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]) + )) was detected It was shown that the reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. Crude When the product was purified by preparative HPLC (TFA conditions), BCY13342 (21.7 mg, 4.60 μmol, 52.85% yield) was obtained. A white solid was obtained (with a purity of 94.7%).

[0183] (Analysis data) The following heterotandem bicyclic peptide complex of the present invention was analyzed using mass spectrometry and HPLC. Analysis was performed. 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 Equipment: Agilent 1200 HPLC-BE (1-614)

[0184] The gradients used are shown in the table below: [Table 7] It is described, and the data was created as follows: [Table 8] TIFF2026143628000080.tif239170TIFF2026143628000081.tif227170TIFF2026143628 000082.tif243170TIFF2026143628000083.tif239170TIFF2026143628000084.tif92170

[0185] (Biological data) (1. Co-culture of tumor cells with CD137 reporter assay) By adding 1% FBS to RPMI-1640 (a component of the Promega kit CS196005), R1 medium is obtained. Prepare a culture medium called R1. Serial dilutions of the test sample in R1 are placed in a sterile 96-well plate. Prepare the sample. Add 25 μL of the test sample or R1 (as a background control) per well to a white fine powder. Add to the designated wells in the cell culture plate. Tumor cells * The cells were collected and concentrated to 400,000 cells / mL. Resuspend in R1 medium at 25°C. Add 25(25) μL / well of tumor cells to a white cell culture plate. Add the following: Thaw Jurkat cells (Promega kit CS196005, 0.5 mL) in a water bath, and then add 5 ml of Add to pre-warmed R1 medium. Then, culture 25 (25) μL / well of Jurkat cells in white cell culture medium. Add to the plate. Incubate the cells and test sample at 37°C in 5% CO2 for 6 hours. 6 hours Finally, add 75 μL / well of Bio-Glo® reagent (Promega) and incubate for 10 minutes. Afterward, the luminescence is read using a plate reader (Clariostar, BMG). Cells only (Jurkat cells + The change ratio was calculated compared to the cell line used in co-culture, and log(agonist) was calculated using GraphPad Prism. Plotted as a response, EC 50 Determine the (nM) and the induction multiplier (Max) relative to the background. To determine.

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

[0187] In a CD137 reporter co-culture assay using NCI-H292 cells, nectin-4 / CD137 heterate Table 1 shows a summary of the induction ratios induced by rotandem peptides. The average EC of the mixture is 1.1 ± 0.5 nM. 50and pre- Compare with the control BCY10000. Table 1: Nectin-4 / CD137 heterotandem bicyclic peptide in CD137 reporter assay Induction magnification guided by the complex [Table 9]

[0188] In a CD137 reporter co-culture assay using PC3 cells, EphA2 / CD137 heterotandem Table 2 shows a summary of the induction ratios induced by the peptides. All compounds are 0.54n MJISEC 50 and compared to the plate control BCY9173, which has an Emax 42 times that of the background. To compare. Table 2: EphA2 / CD137 heterotandem bicyclic peptide complex in CD137 reporter assay Body-guided induction magnification [Table 10]

[0189] In a CD137 reporter co-culture assay using RKO cells, PD-L1 / CD137 heterotandem Table 3 summarizes the peptide-induced induced magnifications. Table 3: PD-L1 / CD137 heterotandem bicyclic peptide complex in CD137 reporter assay Body-guided induction magnification [Table 11]

[0190] (2. Pharmacokinetics of CD137 heterotandem bicyclic peptide complex in SD rats) Male SD rats were given 2 mg / kg of each formulation in 25 mM histidine HCl and 10% sucrose pH 7. Each heterotandem bicyclic peptide complex was administered. Continuous blood samples (approximately 80 μL of blood per time point) were taken. At that point, the blood sample was taken from the submandibular or saphenous vein. All blood samples were treated with 2 μL K2-EDTA (0.5M) as an anticoagulant. The blood was immediately transferred to a pre-cooled microcentrifuge tube and placed on wet ice. The liquid sample was immediately processed for plasma by centrifugation at approximately 4°C and 3000g. Internal standards were included. The precipitating agent was immediately added to the plasma, thoroughly mixed, and centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant is transferred to a pre-labeled polypropylene microcentrifuge tube, and then, The samples were rapidly frozen on dry ice. The samples were stored at temperatures below 70°C until analysis, if necessary. 7. A 5 μL supernatant sample was subjected to LC-MS / MS using Orbitrap Q Exactive in positive ion mode. The sample was injected directly for analysis, and the concentrations of the two rings were determined. Plasma concentration versus time data were obtained using Phoenix Wi. A non-compartmental approach using the nNonlin 6.3 software program Analyzed: C0, Cl, Vdss, T 1 / 2, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) The graphs of plasma concentration versus time profiles were reported. The pharmacokinetic parameters of the experiment were: As shown in Table 4: Table 4: Pharmacokinetic parameters in SD rats [Table 12] This application provides the invention in the following embodiments. (Aspect 1) (a) A first peptide ligand that binds to components present on cancer cells via a linker , (b) A second peptide ligand that binds to components present on immune cells; something conjugated : includes, Here, each of the peptide ligands is separated by at least two loop sequences. A polypeptide comprising at least three reactive groups and a compound forming a covalent bond with the reactive groups of the polypeptide. It contains a molecular scaffold, and as a result, at least two polypeptide loops A heterotandem bicyclic peptide complex formed on the molecular scaffold, The heterotandem bicyclic peptide complex consists of the following first and second peptide ligands: (Table 1) TIFF2026143628000089.tif236170TIFF2026143628000090.tif236170TIFF202614362800009 1.tif236170TIFF2026143628000092.tif236170TIFF2026143628000093.tif236170TIFF2026 143628000094.tif236170TIFF2026143628000095.tif236170TIFF2026143628000096.tif236170TIFF2026143628000097.tif236170(where 1Nal represents 1-naphthylalanine, HArg represents homoarginine, and HyP represents hydro Xyproline is represented, B-Ala is represented by β-alanine, PYA is represented by 4-pentic acid, and 3,3-DPA is 3,3-diphenylalanine is represented by Cba, β-cyclobutylalanine is represented by hGlu, and homoglu is represented by tBuAla represents tamic acid, Nle represents norleucine, NMeAla represents N-methyl-alanine, and tBuAla represents t-butyl-alanine, Aad represents α-L-aminoadipic acid, and Ac represents an acetyl group. (where Dap represents diaminopropionic acid), or a pharmaceutically acceptable salt thereof. The heterotandem bicyclic peptide complex, characterized by containing the above. (Aspect 2) The aforementioned immune cells are white blood cells; lymphocytes (for example, T lymphocytes or T cells, B cells, or n Natural killer cells); CD8 or CD4; CD8; dendritic cells, follicular dendritic cells, and granulocytes: selected from these. A heterotandem bicyclic peptide complex according to embodiment 1. (Aspect 3) Embodiment 1 or Embodiment 2, wherein the second peptide ligand comprises a CD137-linked bicyclic peptide ligand. A heterotandem bicyclic peptide complex as described in Model 2. (Aspect 4) The CD137-linked bicyclic peptide is selected from any of the peptides in Sequence ID No. 67 to 84. The heterotandem bicyclic peptide complex according to embodiment 3. (Aspect 5) Embodiment 1, in which the first peptide ligand comprises a nectin-4 linked bicyclic peptide ligand A heterotandem bicyclic peptide complex as described in any one of items ~4. (Aspect 6) The aforementioned nectin-4 linked bicyclic peptide is selected from any of the peptides of Sequence ID No. 52 to 66. A heterotandem bicyclic peptide complex according to embodiment 5. (Aspect 7) The units listed in Table C, for example, BCY11468, BCY11618, BCY11776, BCY11860, BCY A he selected from any one of 12020, BCY12661, and BCY12969, as described in Embodiment 5 or Embodiment 6. A telotandem bicyclic peptide complex. (Pattern 8) The first peptide ligand comprises an EphA2-linked bicyclic peptide ligand, according to embodiments 1 to 4. A heterotandem bicyclic peptide complex as described in any one of the items. (Aspect 9) The EphA2-linked bicyclic peptide is selected from any of the peptides in Sequence ID No. 10 to 51. , the heterotandem bicyclic peptide complex according to embodiment 8. (Aspect 10) The complexes listed in Table B, for example, BCY13035, BCY13040, BCY13253, BCY13254, BCY A heterotande according to embodiment 8 or embodiment 9, selected from either 13340 or BCY13342. A municyclic peptide complex. (Aspect 11) The first peptide ligand comprises a PD-L1-linked bicyclic peptide ligand, according to embodiments 1 to 4. A heterotandem bicyclic peptide complex as described in any one of the items. (Aspect 12) The PD-L1-linked bicyclic peptide is selected from any of the peptides of Sequence ID No. 1 to 9. A heterotandem bicyclic peptide complex according to embodiment 11. (Aspect 13) Select one of the complexes listed in Table A, for example, BCY12375 and BCY12021. A heterotandem bicyclic peptide complex according to embodiment 11 or embodiment 12. (Aspect 14) The aforementioned molecular scaffold is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)triple A heterotande selected from per-2-en-1-on (TATA) according to any one of embodiments 1 to 13. A municyclic peptide complex. (Aspect 15) The aforementioned salts that are acceptable as pharmaceuticals are free acids or sodium, potassium, calcium, and A heterotandem bicyclic peptide selected from mononium salts according to any one of embodiments 1 to 14. Tydo complex. (Aspect 16) A heterotandem bicyclic peptide complex according to any one of embodiments 1 to 15 is used with one or more pharmaceutical products. A pharmaceutical composition comprising a combination of such excipients. (Aspect 17) A description of any one of the embodiments 1 to 15 for use in the prevention, suppression, or treatment of cancer A heterotandem bicyclic peptide complex.

Claims

1. A ligand that binds to EphA2 present on cancer cells, or a pharmaceutically acceptable salt thereof. a polypeptide comprising 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, and As a result, the polypeptide consisting of the at least two sequences, the reactive group and the molecular scaffold A droop is formed; The polypeptide has one or more cysteine ​​residues C, 3-mercaptopropionic acid, cis Modified by substitution with theamine or penicillamine, C(HyP)LVNPLCLHP(D-Asp)W(HArg)C Characterized by: Here, HArg represents homoarginine, and HyP represents hydroxyproline, and The molecular scaffold is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)tripropane The ligand or a pharmaceutically acceptable salt thereof is a -2-en-1-one (TATA).

2. The polypeptide has one of its cysteine ​​residues C, 3-mercaptopropionic acid, cis Modified by substitution with theamine or penicillamine, C(HyP)LVNPLCLHP(D-Asp)W(HArg)C The ligand according to claim 1.

3. The polypeptide is subjected to the substitution of one or more cysteine ​​residues C with penicillamine. Modified by, C(HyP)LVNPLCLHP(D-Asp)W(HArg)C The ligand according to claim 1.

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