Methods for treating cancer

Abstract

This invention relates to a method for treating cancer in patients.

Description

[Technical Field]

[0001] This invention relates to the use of heterotonandem bicyclic peptide complexes comprising one or more CD137-binding peptide ligands, or pharmaceutically acceptable salts thereof, and immuno-oncology agents for the treatment of cancer. The invention also provides pharmaceutically acceptable compositions comprising heterotonandem bicyclic peptide complexes or pharmaceutically acceptable salts thereof, the heterotonandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands. [Previous Technology]

[0002] Cyclic peptides are a promising class of molecules for developing therapeutics due to their ability to bind to protein targets with high affinity and target specificity. In fact, several cyclic peptides have been successfully used clinically, such as the antibacterial peptide vancomycin, the immunosuppressant cyclosporine, and the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). This favorable binding property results from the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of the cyclic structure. Typically, macrocyclic compounds bind to surfaces of hundreds of square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide with an Arg-Gly-Asp motif bound to integrin αVb3 (355 Å2) (Xiong et al. (2002), Science 296 (5565), 151-5), or the cyclic peptide inhibitor upain-1 (603 Å2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10), which binds to urokinase-type plasminogen activator.

[0003] Due to their cyclic configuration, macrocycles are less flexible than linear peptides, resulting in less entropy loss and higher binding affinity when binding to a target. Reduced flexibility also allows for target-specific configuration locking, thereby increasing binding specificity compared to linear peptides. This effect has been demonstrated by potent and selective inhibitors of matrix metalloproteinase 8 (MMP-8), which loses its selectivity for other MMPs when its ring is open (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The advantageous binding properties achieved via macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

[0004] Different research groups have previously tethered peptides containing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues have used serotonin (bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings onto a synthetic backbone for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for generating candidate drug compounds are disclosed in WO 2004 / 077062 and WO 2006 / 078161, wherein these compounds are generated by linking a cysteine-containing peptide to a molecular backbone such as, for example, serotonin (bromomethyl)benzene.

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

[0006] It has now been found that heterotandem bicyclic peptide complexes containing one or more CD137-binding peptide ligands, or pharmaceutically acceptable salts thereof, cause a significant increase in tumor-infiltrating immune cells and immune responses. See, for example, the transcriptional analysis in Example 1, which showed a significant increase in several T-cell chemokine / cytokine immune cell scores and mRNA following treatment with each of BCY12491 and BT7480. Therefore, in one embodiment, the present invention provides a method for increasing an immune response in a cancer patient, comprising administering to the patient a therapeutically effective amount of a heterotandem bicyclic peptide complex containing one or more CD137-binding peptide ligands, or a pharmaceutically acceptable salt thereof.

[0007] It has also been found that, compared with each of the single-agent treatments, combinations of heterotandem bicyclic peptide complexes comprising one or more CD137-binding peptide ligands or pharmaceutically acceptable salts thereof with immuno-oncology agents significantly improve antitumor activity. See, for example, the combination therapy of BCY12491 with the PD-1 antagonist pembrolizumab in Example 2 produced more significant antitumor activity compared with treatment with each single agent. Therefore, in one embodiment, the present invention provides a method for treating a patient with cancer comprising administering to the patient a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or pharmaceutically acceptable salts thereof and an immuno-oncology agent.

Implementation Method

[0021] 1. Description of certain embodiments of the present invention:

[0022] It has been found that, compared with each of the single-agent treatments, heterotandem bicyclic peptide complexes containing one or more CD137-binding peptide ligands, or pharmaceutically acceptable salts thereof, cause a significant increase in tumor-infiltrating immune cells and immune responses, and that combinations of heterotandem bicyclic peptide complexes containing one or more CD137-binding peptide ligands, or pharmaceutically acceptable salts thereof, with immuno-oncology agents significantly improve antitumor activity. See, for example, data on treatment with each of BCY12491 and BT7480 in Example 1, and data on treatment with BCY12491 alone, PD-1 antagonist pembrolizumab alone, and combinations of BCY12491 and pembrolizumab in Example 2. Therefore, in one embodiment, this document provides a method or use of heterotandem bicyclic peptide complexes containing one or more CD137-binding peptide ligands, or pharmaceutically acceptable salts thereof, to increase immune responses in cancer patients. In another embodiment, this document provides a method or use for treating cancer in patients by combining a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof with an immuno-oncology agent.

[0023] In some embodiments, the present invention provides a method for increasing an immune response in a cancer patient, comprising administering to the patient a therapeutically effective amount of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides the use of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof for manufacturing a medicament for increasing an immune response in a cancer patient.

[0024] In some embodiments, the present invention provides a method for treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof, and an immuno-oncology agent. In some embodiments, the present invention provides the use of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof for manufacturing a medicament for treating a patient with cancer, wherein the medicament is used in combination with an immuno-oncology agent.

[0025] In some embodiments, the cancer is selected from the cancers described herein. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is associated with MT1-MMP. In some embodiments, the cancer is associated with binding protein-4. In some embodiments, the cancer is associated with EphA2. In some embodiments, the cancer is associated with PD-L1. In some embodiments, the cancer is associated with PSMA.

[0026] In some embodiments, as described herein, the heterotandem bicyclic peptide complex containing one or more CD137-binding peptide ligands is selected from heterotandem bicyclic peptide complexes containing one CD137-binding peptide ligand. In some embodiments, as described herein, the heterotandem bicyclic peptide complex containing one or more CD137-binding peptide ligands is selected from heterotandem bicyclic peptide complexes containing two or more CD137-binding peptide ligands.

[0027] In some embodiments, the heterotandem bicyclic peptide complex is BCY11863 (also known as BT7480) or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is BCY13272 (also known as BT7455) or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is BCY12491 or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is BCY11864 or a pharmaceutically acceptable salt thereof.

[0028] In some embodiments, the immuno-oncology agent is selected from the immuno-oncology agents described herein. In some embodiments, the immuno-oncology agent is a checkpoint inhibitor. In some embodiments, the immuno-oncology agent is a PD-1 antagonist. In some embodiments, the immuno-oncology agent is pembrolizumab. In some embodiments, the immuno-oncology agent is nivolumab.

[0029] In some embodiments, the present invention provides a method for increasing an immune response in a cancer patient, comprising administering to the patient a therapeutically effective amount of BT7480 or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides the use of BT7480 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing an immune response in a cancer patient. In some embodiments, the present invention provides a method for treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of BT7480 or a pharmaceutically acceptable salt thereof and an immuno-oncology agent. In some embodiments, the present invention provides the use of BT7480 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in a patient, wherein the medicament is used in combination with an immuno-oncology agent.

[0030] In some embodiments, the present invention provides a method for increasing an immune response in a cancer patient, comprising administering to the patient a therapeutically effective amount of BT7455 or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides the use of BT7455 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing an immune response in a cancer patient. In some embodiments, the present invention provides a method for treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of BT7455 or a pharmaceutically acceptable salt thereof and an immuno-oncology agent. In some embodiments, the present invention provides the use of BT7455 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in a patient, wherein the medicament is used in combination with an immuno-oncology agent.

[0031] In some embodiments, the heterotandem bicyclic peptide complex is administered at a dose of about 0.001-100 mg / kg. In some embodiments, the heterotandem bicyclic peptide complex is selected from those heterotandem bicyclic peptide complexes described herein, such as BT7480 or BT7455 or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered at a dose of about 0.001-0.01 mg / kg, about 0.01-0.1 mg / kg, about 0.1-1 mg / kg, about 1-10 mg / kg, about 10-25 mg / kg, about 25-50 mg / kg, or about 50-100 mg / kg. In some embodiments, the heterotandem bicyclic peptide complex is administered at a dose of about 0.1-75 mg / kg, about 1-50 mg / kg, about 5-25 mg / kg, or about 7.5-20 mg / kg. In some embodiments, the heterotandem bicyclic peptide complex is administered at doses of about 0.001 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 12.5 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg.

[0032] In some embodiments, the heterotandem bicyclic peptide complex is administered once, twice, three times, or four times a week. In some embodiments, the heterotandem bicyclic peptide complex is selected from those heterotandem bicyclic peptide complexes described herein, such as BT7480 or BT7455 or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered once daily. In some embodiments, the heterotandem bicyclic peptide complex is administered every two days. In some embodiments, the heterotandem bicyclic peptide complex is administered every three days. In some embodiments, the heterotandem bicyclic peptide complex is administered every four days. In some embodiments, the heterotandem bicyclic peptide complex is administered every five days. In some embodiments, the heterotandem bicyclic peptide complex is administered once a week. In some embodiments, the heterotandem bicyclic peptide complex is administered every 1.5 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered every two weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered every 2.5 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 3 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 4 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered once a month.

[0033] In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 1-4 weeks. In some embodiments, the heterotandem bicyclic peptide complex is selected from those heterotandem bicyclic peptide complexes described herein, such as BT7480 or BT7455 or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 5-8 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 9-12 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 13-20 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 21-28 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 4, 8, 12, 16, 20, 24, or 28 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of approximately 30 weeks or longer.

[0034] In some embodiments, the heterotandem bicyclic peptide complex is administered to the patient via intravenous bolus injection. In some embodiments, the heterotandem bicyclic peptide complex is selected from those heterotandem bicyclic peptide complexes described herein, such as BT7480 or BT7455 or a medically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered to the patient via intravenous infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over about 5-10 minutes. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over about 10-20 minutes. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over about 20-40 minutes. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over about 45, 50, or 55 minutes. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over about 1 hour. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over about 1-1.5 hours. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over approximately 1.5 to 2 hours. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over approximately 2 to 3 hours. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is administered over more than 3 hours.

[0035] The immuno-oncology agent is administered according to a dosing regimen recommended or approved by the FDA. In some embodiments, the immuno-oncology agent is administered at a dose of about 1-20 mg / kg. In some embodiments, the immuno-oncology agent is administered at a dose of about 1-5 mg / kg, about 6-10 mg / kg, about 11-15 mg / kg, or about 16-20 mg / kg. In some embodiments, the immuno-oncology agent is administered at a dose of about 1-10 mg / kg, about 5-15 mg / kg, or about 10-20 mg / kg. In some embodiments, the immuno-oncology agent is administered at a dose of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. In some embodiments, the immuno-oncology agent is administered at a dose of about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg / kg. In some embodiments, the immuno-oncology agent is administered once, twice, three times, or four times a week. In some embodiments, immuno-oncology agents are administered once daily. In some embodiments, immuno-oncology agents are administered every 2 days. In some embodiments, immuno-oncology agents are administered every 3 days. In some embodiments, immuno-oncology agents are administered every 4 days. In some embodiments, immuno-oncology agents are administered every 5 days. In some embodiments, immuno-oncology agents are administered once a week. In some embodiments, immuno-oncology agents are administered every 1.5 weeks. In some embodiments, immuno-oncology agents are administered every 2 weeks. In some embodiments, immuno-oncology agents are administered every 2.5 weeks. In some embodiments, immuno-oncology agents are administered every 3 weeks. In some embodiments, immuno-oncology agents are administered every 4 weeks. In some embodiments, immuno-oncology agents are administered once a month. In some embodiments, immuno-oncology agents are administered for a treatment period lasting approximately 1-4 weeks. In some embodiments, the immuno-oncology agent is administered for a treatment period of approximately 9-12 weeks, approximately 13-20 weeks, approximately 21-28 weeks, or approximately 29-36 weeks. In some embodiments, the immuno-oncology agent is administered for a treatment period of approximately 36 weeks or longer. In some embodiments, the immuno-oncology agent is administered to the patient via intravenous injection. In some embodiments, the immuno-oncology agent is administered to the patient via intravenous infusion. In some embodiments, the intravenous infusion of the immuno-oncology agent is administered over approximately 5-10 minutes. In some embodiments, the intravenous infusion of the immuno-oncology agent is administered over approximately 10-20 minutes or approximately 20-40 minutes. In some embodiments, the intravenous infusion of the immuno-oncology agent is administered over approximately 30, 40, 45, 50, 55, or 60 minutes.In some embodiments, intravenous infusion of immuno-oncology agents is performed over approximately 1-1.5 hours, approximately 1.5-2 hours, or approximately 2-3 hours.

[0036] In some embodiments, the pharmaceutically acceptable salt of the heterotandem bicyclic peptide complex or thereof is selected from heterotandem bicyclic peptide complex formulations as shown in the examples of the present invention. In some embodiments, the heterotandem bicyclic peptide complex is selected from those heterotandem bicyclic peptide complexes described herein, such as BT7480 or BT7455 or thereof, or their pharmaceutically acceptable salts. In some embodiments, the pharmaceutically acceptable salt of the heterotandem bicyclic peptide complex or thereof further comprises histidine. In some embodiments, the pharmaceutically acceptable salt of the heterotandem bicyclic peptide complex or thereof and histidine are at about pH 7. In some embodiments, the pharmaceutically acceptable salt of the heterotandem bicyclic peptide complex or thereof further comprises sucrose. In some embodiments, the pharmaceutically acceptable salt of the heterotandem bicyclic peptide complex or thereof further comprises about 10% w / v sucrose. In some embodiments, the pharmaceutically acceptable salt of the heterotandem bicyclic peptide complex or thereof further comprises water. In some embodiments, the present invention provides a medicament comprising a heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof, histidine, sucrose, and water, wherein the medicament is at approximately pH 7. An exemplary heterotandem bicyclic peptide complex is described.

[0037] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) one or more CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0038] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) one or more CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0039] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) a CD137-binding peptide ligand via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0040] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) a CD137-binding peptide ligand via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0041] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) two or more CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0042] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) two or more CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0043] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) two CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0044] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) two CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0045] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) three CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two ring sequences, and a molecular backbone that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

[0046] In some embodiments, the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) three CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two ring sequences, and a molecular backbone covalently bonded to the cysteine ​​residues of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone. First peptide ligand

[0047] The term "cancer cell" as used herein includes any cell known to be involved in cancer. Cancer cells form when the genes responsible for regulating cell division are damaged. Carcinogenesis is caused by mutations in the genetic material and epigenetic material of normal cells, which disrupt the normal balance between proliferation and cell death. This leads to uncontrolled cell division and the evolution of those cells through natural selection within the body. Uncontrolled and usually rapid cell proliferation can cause benign or malignant tumors (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors can invade other organs, spread to distant locations (metastasis), and become life-threatening.

[0048] In some embodiments, the cancer cell lines are selected from HT1080, A549, SC-OV-3, PC3, HT1376, NCI-H292, LnCap, MC38, MC38 #13, 4T1-D02, H322, HT29, T47D and RKO tumor cells.

[0049] In some embodiments, the component present on the cancer cells is binding protein-4.

[0050] Binding protein-4 is a surface molecule belonging to the binding protein protein family, which comprises four members. Binding proteins are cell adhesion molecules that play important roles in various biological processes, such as the polarity, proliferation, differentiation, and migration of epithelial cells, endothelial cells, immune cells, and neurons during development and adult lifespan. They are involved in several pathological processes in humans. They are the main receptors for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding binding protein-1 (PVRL1) or binding protein-4 (PVRL4) cause ectodermal dysplasia syndromes associated with other abnormalities. Binding protein-4 is expressed during fetal development. In adult tissues, its expression is more limited than that of other family members. Binding protein-4 is a tumor-associated antigen in 50%, 49%, and 86% of breast, ovarian, and lung cancers, respectively, primarily in tumors with poor prognosis. Its expression has not been detected in corresponding normal tissues. In breast tumors, binding protein-4 is primarily found in triple-negative and ERBB2+ carcinomas. Detection of the soluble form of binding protein-4 in the serum of patients with these cancers is associated with poor prognosis. Serum binding protein-4 levels increase during metastatic progression and decrease after treatment. These results suggest that binding protein-4 may be a reliable target for cancer treatment. Therefore, several anti-binding protein-4 antibodies have been described in the prior art. Specifically, enfortumab vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting binding protein-4 and is currently being investigated clinically for the treatment of patients with solid tumors.

[0051] In some embodiments, the first peptide ligand comprises a protein-4 binding bicyclic peptide ligand.

[0052] In some embodiments, the protein-4 binding bicyclic peptide coordination system is selected from the protein-4 binding bicyclic peptide ligands disclosed in WO 2019 / 243832, the contents of which are incorporated herein by reference in their entirety.

[0053] In some embodiments, the protein-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 1; referred to herein as BCY8116); CiP[1Nal][dD]CiiM[HArg]D[dW]STP[HyP][dW]Ciii (SEQ ID NO: 2); CiP[1Nal][dK](Sar10-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 3);CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 4; referred to herein as BCY11414); CiP[1Nal][dK]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 14); [MerPro]iP[1Nal][dK]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 15; referred to herein as BCY12363); CiP[1Nal][dK]CiiM[HArg]DWSTP[HyP]W[Cysam]iii (SEQ ID NO: 16); [MerPro]iP[1Nal][dK]CiiM[HArg]DWSTP[HyP]W[Cysam]iii (SEQ ID NO: 17; referred to herein as BCY12365); CiP[1Nal][dK]CiiM[HArg]HWSTP[HyP]WCiii (SEQ ID NO: 18); CiP[1Nal][dK]CiiM[HArg]EWSTP[HyP]WCiii (SEQ ID NO: 19); CiP[1Nal][dE]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 20; referred to herein as BCY12368); CiP[1Nal][dA]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 21; referred to herein as BCY12369); CiP[1Nal][dE]CiiL[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 19); 22 (referred to herein as BCY12370); and CiP[1Nal][dE]CiiM[HArg]EWSTP[HyP]WCiii (SEQ ID NO: 23; referred herein as BCY12384); wherein [MerPro]i, Ci, Cii, Ciiii and [Cysam]iii represent the first (i), second (ii) and third (iii) reactive groups selected from cysteine, MerPro and Cysam, 1Nal represents 1-naphthylalanine, HArg represents high-arginine, HyP represents trans-4-hydroxy-L-proline, Sar10 represents 10 sarcosine units, B-Ala represents β-alanine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteine, or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the binding protein-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 1; referred to herein as BCY8116); CiP[1Nal][dK](Sar10-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 3); and CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 4; referred herein as BCY11414); Ci, Cii, and Cii represent the first, second, and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; HArg represents high-arginine; HyP represents trans-4-hydroxy-L-proline; Sar10 represents 10 sarcosine units; and B-Ala represents β-alanine, or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the binding protein-4 binding bicyclic peptide ligand may include an N-terminal modification and comprises: SEQ ID NO: 1 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846); [PYA]-(SEQ ID NO: 1) (hereinafter referred to as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO: 1) (hereinafter referred to as BCY11016); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 2) (hereinafter referred to as BCY11942); Ac-(SEQ ID NO: 3) (hereinafter referred to as BCY8831); SEQ ID NO: 4 (hereinafter referred to as BCY11414); [PYA]-[B-Ala]-(SEQ ID NO: 14) (referred to herein as BCY11143); palmitic acid-yGlu-yGlu-(SEQ ID NO: 14) (referred to herein as BCY12371); Ac-(SEQ ID NO: 14) (referred to herein as BCY12024); Ac-(SEQ ID NO: 16) (referred to herein as BCY12364); Ac-(SEQ ID NO: 18) (referred to herein as BCY12366); and Ac-(SEQ ID NO: 19) (referred to herein as BCY12367); wherein PYA represents 4-pentynic acid, B-Ala represents β-alanine, and Sar10 represents 10 sarcosine units, or a pharmaceutically acceptable salt thereof.

[0056] In some embodiments, the binding protein-4 binding bicyclic peptide ligand may include an N-terminal modification and comprise: SEQ ID NO: 1 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 2) (hereinafter referred to as BCY11942); Ac-(SEQ ID NO: 3) (hereinafter referred to as BCY8831); and SEQ ID NO: 4 (hereinafter referred to as BCY11414); wherein PYA represents 4-pentynic acid, B-Ala represents β-alanine, and Sar10 represents 10 sarcosine units, or a pharmaceutically acceptable salt thereof.

[0057] In some embodiments, the binding protein-4 binding bicyclic peptide ligand comprises SEQ ID NO: 1 (referred to herein as BCY8116).

[0058] In some embodiments, the binding protein-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 1; hereinafter referred to as BCY8116); CiP[1Nal][dD]CiiM[HArg]D[dW]STP[HyP][dW]Ciii (SEQ ID NO: 2; hereinafter referred to as BCY11415); and CiP[1Nal][dK](Sar10-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 3); CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 4; hereinafter referred to as BCY11414); Ci, Cii, and Cii represent the first, second, and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; HArg represents high-arginine; HyP represents hydroxyproline; Sar10 represents 10 sarcosine units; and B-Ala represents β-alanine, or a pharmaceutically acceptable salt thereof.

[0059] In another embodiment, the binding protein-4 binding bicyclic peptide ligand may include an N-terminal modification and comprises: SEQ ID NO:1 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846); SEQ ID NO:2 (hereinafter referred to as BCY11415); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 2) (hereinafter referred to as BCY11942); Ac-(SEQ ID NO: 3) (hereinafter referred to as BCY8831); and SEQ ID NO: 4 (hereinafter referred to as BCY11414); wherein PYA represents 4-pentynic acid, B-Ala represents β-alanine, and Sar10 represents 10 sarcosine units, or a pharmaceutically acceptable salt thereof.

[0060] In some embodiments, the component present on the cancer cells is EphA2.

[0061] Eph receptor tyrosine kinase (Eph) belongs to a large group of receptor tyrosine kinases (RTKs), which are kinases that phosphorylate proteins on tyrosine residues. Eph and its membrane-bound ephrin ligands (ephrin) control cell localization and tissue structure (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur in a state of high ligand low polymerization (Stein et al. (1998) Genes Dev 12, 667-678).

[0062] Among other model functions, various Eph and hepatic ligands have been shown to play roles in vascular development. Gene knockout of EphB4 and hepatic ligand-B2 results in a lack of ability to remodel microvascular beds into blood vessels (Poliakov et al., see above) and embryonic lethality. Persistent expression of some Eph receptors and hepatic ligands has also been observed in newly formed adult microvessels (Brantley-Sieders et al. (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0063] Dysregulation of some hepatic glycoproteins and their receptors in adults has also been observed to contribute to tumor invasion, metastasis, and angiogenesis (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., see above). In addition, some Eph family members have been found to be overexpressed on tumor cells from various human tumors (Brantley-Sieders et al., see above); Marme (2002) Ann Hematol 81 Supplement 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0064] EPH receptor A2 (hepatic glycoprotein A receptor 2) is a protein encoded by the EPHA2 gene in humans.

[0065] EphA2 is upregulated in a variety of human cancers and is generally associated with disease progression, metastasis, and poor prognosis, such as breast (Zelinski et al. (2001) Cancer Res. 61, 2301-2306; Zhuang et al. (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al. (2011) PLoS One 6, e24426), lung (Brannan et al. (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al. (2003) Clin Cancer Res. 9, 613-618; Guo et al. (2013) J Thorac Oncol. 8, 301-308), stomach (Nakamura et al. (2005) Cancer Sci. 96, 42-47; Yuan et al. (2009)). Dig Dis Sci 54, 2410-2417), pancreas (Mudali et al. (2006) Clin Exp Metastasis 23, 357-365), prostate (Walker-Daniels et al. (1999) Prostate 41, 275-280), liver (Yang et al. (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al. (2005) Mol Cancer Res. 3, 541-551; Li et al. (2010) Tumor Biol. 31, 477-488).

[0066] The full role of EphA2 in cancer progression remains undefined, although there is evidence of interactions at numerous stages of cancer progression, including tumor cell growth, survival, invasion, and angiogenesis. EphA2 downregulation inhibits tumor cell spread (Binda et al. (2012) Cancer Cell 22, 765-780), while EphA2 blockade inhibits VEGF-induced cell migration (Hess et al. (2001) Cancer Res. 61, 3250-3255), cell germination and angiogenesis (Cheng et al. (2002) Mol Cancer Res. 1, 2-11; Lin et al. (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al. (2005) FASEB J. 19, 1884-1886).

[0067] Antibody-drug conjugates for EphA2 have been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al. (2008) Cancer Research 68, 9367-9374), and similar approaches have been attempted in humans, although treatment had to be discontinued due to treatment-related adverse events (Annunziata et al. (2013) Invest New drugs 31, 77-84).

[0068] In some embodiments, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

[0069] In some embodiments, the EphA2 binding bicyclic peptide coordination system is selected from the EphA2 binding bicyclic peptide ligands disclosed in WO 2019 / 122860, WO 2019 / 122861 and WO 2019 / 122863, the contents of each of which are incorporated herein by reference in their entirety.

[0070] One of the products of EphA2 is the active ingredient of EphA2. Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii(SEQ ID NO: 24); CiLWDPTPCiiANLHL[HArg]Ciii(SEQ ID NO: 25); Ci[HyP][K(PYA)]VNPLCiiLHP[dD]W[HArg]Ciii(SEQ ID NO: 27); Ci[HyP]LVNPLCii[K(PYA)]HP[dD]W[HArg]Ciii(SEQ ID NO: 28); NO: 29) Ci[HyP]KVNPLCiiLHP[dD]W[HArg]Ciii(SEQ ID NO: 30); 32) Ci[HyP]LVNPLCiiLEP[dD]W[HArg]Ciii(SEQ ID NO: 33); Ci[HyP]LVNPLCiiLHP[dD]WTCiii(SEQ ID NO: 34); Ci[HyP]LVNPLCiiLEP[dA]WTCiii(SEQ ID NO: 36); Ci[HyP]LVNPLCiiL[3,3-DPA]P[dD]WTCiii(SEQ ID NO: 37); Ci[HyP][Cba]VNPLCiiLHP[dD]W[HArg]Ciii(SEQ ID NO: 38) and Ci[HyP][Cba]VNPLCiiLEP[dD]WTCiii(SEQ ID NO: 39); 40)R Ci[HyP]LVNPLCiiL[3,3-DPA]P[dD]W[HArg]Ciii(SEQ ID NO: 41);Ci[HyP]LVNPLCiiLHP[d1Nal]W[HArg]Ciii (SEQ ID NO: 42); Ci[HyP]LVNPLCiiL[1Nal]P[dD]W[HArg]Ciii (SEQ ID NO: 43); Ci[HyP]LVNPLCiiLEP[d1Nal]WTCiii (SEQ ID NO: 44); Ci[HyP]LVNPLCiiL[1Nal]P[dD]WTCiii (SEQ ID NO: 45; referred to herein as BCY13119); Ci[HyP][Cba]VNPLCiiLEP[dA]WTCiii (SEQ ID NO: 46); Ci[HyP][hGlu]VNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 47); Ci[HyP]LVNPLCii[hGlu]HP[dD]W[HArg]Ciii (SEQ ID NO: 48); Ci[HyP]LVNPLCiiL[hGlu]P[dD]W[HArg]Ciii (SEQ ID NO: 49); Ci[HyP]LVNPLCiiLHP[dNle]W[HArg]Ciii (SEQ ID NO: 50); Ci[HyP]LVNPLCiiL[Nle]P[dD]W[HArg]Ciii (SEQ ID NO: 51); [MerPro]i[HyP]LVNPLCiiL[3,3-DPA]P[dD]WTCiii (SEQ ID NO: 154); Ci[HyP]LVNPLCiiLHP[dD]W[HArg][Cysam]iii (SEQ ID NO: 155); Ci[HyP]LVNPLCiiL[His3Me]P[dD]W[HArg]Ciii (SEQ ID NO: 156); Ci[HyP]LVNPLCiiL[His1Me]P[dD]W[HArg]Ciii (SEQ ID NO: 157); Ci[HyP]LVNPLCiiL[4ThiAz]P[dD]W[HArg]Ciii (SEQ ID NO: ၁၅၈); Ci[HyP]LVNPLCiiLFP[dD]W[HArg]Ciii (SEQ ID NO: 159); Ci[HyP]LVNPLCiiL[Thi]P[dD]W[HArg]Ciii (SEQ ID NO: 160); Ci[HyP]LVNPLCiiL[3Thi]P[dD]W[HArg]Ciii (SEQ ID NO: 161); It should be noted that there seems to be a non-standard or incorrect character "၁၅၈" in the original text which is retained as is during translation. If this is an error, it might need to be corrected in the source text for a more accurate translation.Ci[HyP]LVNPLCiiLNP[dD]W[HArg]Ciii(SEQ ID NO: 162); Ci[HyP]LVNPLCiiLQP[dD]W[HArg]Ciii(SEQ ID NO: 163); and Ci[HyP]LVNPLCiiL[K(PYA-(palmitoyl-Glu-LysN3))]P[dD]W[HArg]Ciii(SEQ ID NO: 164); Where [MerPro]i, Ci, Cii, Ciii, and [Cysam]iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam; HyP represents trans-4-hydroxy-L-proline; HArg represents high-arginine; PYA represents 4-pentynic acid; 3,3-DPA represents 3,3-diphenylalanine; Cba represents β-cyclobutylalanine; 1Nal represents 1-naphthylalanine; hGlu represents high-glutenic acid; and Thi represents... 2-Thienyl-alanine, 4ThiAz represents β-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histamine, His3Me represents N3-methyl-L-histamine, 3Thi represents 3-thienylalanine, palmityl-Glu-LysN3[PYA] represents [K(PYA-(palmityl-Glu-LysN3))] represents Nle represents leucine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteine, or a pharmaceutically acceptable salt thereof.

[0071] In some embodiments, the EphA2-binding bicyclic peptide ligand comprises the following amino acid sequence: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 24); wherein Ci, Cii and Ciii represent the first (i), second (ii) and third (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0072] In some embodiments, the EphA2-binding bicyclic peptide ligand comprises the following amino acid sequence: Ci[HyP]LVNPLCiiLEP[d1Nal]WTCiii (SEQ ID NO: 44); wherein Ci, Cii and Ciii represent the first (i), second (ii) and third (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and d1Nal represents 1-naphthylalanine, or a pharmaceutically acceptable salt thereof.

[0073] In some embodiments, the EphA2-binding bicyclic peptide ligand may include N-terminal and / or C-terminal modifications and comprises: A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY9594); [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY6099); [PYA]-A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY11813); Ac-A-[HArg]-D-(SEQ ID NO: 24)-[K(PYA)] (hereinafter referred to as BCY11814); Ac-A-[HArg]-D-(SEQ ID NO: 24)-K (hereinafter referred to as BCY12734); [NMeAla]-[HArg]-D-(SEQ ID NO: 24)-[SEQ ID NO: 24)-[K(PYA)] ... 24) (referred to herein as BCY13121); [Ac]-(SEQ ID NO: 24)-L[dH]G[dK] (referred to herein as BCY13125); [PYA]-[B-Ala]-[Sar10]-VGP-(SEQ ID NO: 25) (referred to herein as BCY8941); Ac-A-[HArg]-D-(SEQ ID NO: 26) (referred to herein as BCY11815); Ac-A-[HArg]-D-(SEQ ID NO: 27) (referred to herein as BCY11816); Ac-A-[HArg]-D-(SEQ ID NO: 28) (referred to herein as BCY11817); Ac-A-[HArg]-D-(SEQ ID NO: 29) (referred to herein as BCY12735); (palmitinyl-Glu-LysN3)[PYA]A[HArg]D-(SEQ ID NO: 29) (hereinafter referred to as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO: 30) (hereinafter referred to as BCY12736); Ac-A-[HArg]-D-(SEQ ID NO: 31) (hereinafter referred to as BCY12737); A-[HArg]-D-(SEQ ID NO: 32) (hereinafter referred to as BCY12738); A-[HArg]-E-(SEQ ID NO: 32) (hereinafter referred to as BCY12739); A-[HArg]-D-(SEQ ID NO: 33) (hereinafter referred to as BCY12854); A-[HArg]-D-(SEQ ID NO: 34) (hereinafter referred to as BCY12855);A-[HArg]-D-(SEQ ID NO: 35) (referred to herein as BCY12856); A-[HArg]-D-(SEQ ID NO: 35)-[dA] (referred to herein as BCY12857); (SEQ ID NO: 35)-[dA] (referred to herein as BCY12861); [NMeAla]-[HArg]-D-(SEQ ID NO: 35) (referred to herein as BCY13122); [dA]-ED-(SEQ ID NO: 35) (referred to herein as BCY13126); [dA]-[dA]-D-(SEQ ID NO: 35) (referred to herein as BCY13127); AD-(SEQ ID NO: 35) (referred to herein as BCY13128); A-[HArg]-D-(SEQ ID NO: 36) (referred to herein as BCY12858); A-[HArg]-D-(SEQ ID NO: 37) (referred to herein as BCY12859); Ac-(SEQ ID NO: 37)-[dK] (referred to herein as BCY13120); A-[HArg]-D-(SEQ ID NO: 38) (referred to herein as BCY12862); A-[HArg]-D-(SEQ ID NO: 39) (referred to herein as BCY12863); [dA]-[HArg]-D-(SEQ ID NO: 39)-[dA] (referred to herein as BCY12864); (SEQ ID NO: 40)-[dA] (referred to herein as BCY12865); A-[HArg]-D-(SEQ ID NO: 41) (referred to herein as BCY12866); A-[HArg]-D-(SEQ ID NO: 42) (referred to herein as BCY13116); A-[HArg]-D-(SEQ ID NO: 43) (referred to herein as BCY13117); A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); [dA]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13123); [d1Nal]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13124); A-[HArg]-D-(SEQ ID NO: 47) (referred to herein as BCY13130); A-[HArg]-D-(SEQ ID NO: 48) (referred to herein as BCY13131);A-[HArg]-D-(SEQ ID NO: 49) (referred to herein as BCY13132); A-[HArg]-D-(SEQ ID NO: 50) (referred to herein as BCY13134); A-[HArg]-D-(SEQ ID NO: 51) (referred to herein as BCY13135); (SEQ ID NO: 154)-[dK] (referred to herein as BCY13129); A[HArg]D-(SEQ ID NO: 155) (referred to herein as BCY13133); A[HArg]D-(SEQ ID NO: 156) (referred to herein as BCY13917); A[HArg]D-(SEQ ID NO: 157) (referred to herein as BCY13918); A[HArg]D-(SEQ ID NO: 158) (referred to herein as BCY13919); A[HArg]D-(SEQ ID NO: 159) (referred to herein as BCY13920); A[HArg]D-(SEQ ID NO: 160) (referred to herein as BCY13922); A[HArg]D-(SEQ ID NO: 161) (referred to herein as BCY13923); A[HArg]D-(SEQ ID NO: 162) (referred to herein as BCY14047); A[HArg]D-(SEQ ID NO: 163) (referred to herein as BCY14048); and A[HArg]D-(SEQ ID NO: 164) (referred to herein as BCY14313); Where PYA represents 4-pentynic acid, B-Ala represents β-alanine, Sar10 represents 10 sarcosine units, HArg represents high-arginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, and palmityl-Glu-LysN3[PYA] represents: , or a pharmaceutically acceptable salt thereof.

[0074] In some embodiments, the EphA2-binding bicyclic peptide ligand may include N-terminal and / or C-terminal modifications and includes: A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); wherein HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0075] In some embodiments, the EphA2-binding bicyclic peptide ligand may include N-terminal and / or C-terminal modifications and includes: A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); wherein HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0076] In some embodiments, the EphA2 binding bicyclic peptide ligand comprises the amino acid sequence: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 24); and CiLWDPTPCiiANLHL[HArg]Ciii (SEQ ID NO: 25); wherein Ci, Cii and Ciii represent the first, second and third cysteine ​​residues, respectively, HyP represents hydroxyproline, dD represents aspartic acid in the D configuration, and HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0077] In some embodiments, the EphA2-binding bicyclic peptide ligand comprises the amino acid sequence: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 24); wherein Ci, Cii and Ciii represent the first, second and third cysteine ​​residues, respectively, HyP represents hydroxyproline, dD represents aspartic acid in the D configuration, and HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0078] In some embodiments, the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification and includes: A-HArg-D-(SEQ ID NO: 24) (hereinafter referred to as BCY9594); [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY6099); [PYA]-[B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY6169); and [PYA]-[B-Ala]-[Sar10]-VGP-(SEQ ID NO: 25) (hereinafter referred to as BCY8941); Where HArg represents high arginine, PYA represents 4-pentynic acid, Sar10 represents 10 sarcosine units, and B-Ala represents β-alanine, or a medically acceptable salt thereof.

[0079] In some embodiments, the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification and includes: A-HArg-D-(SEQ ID NO: 24) (hereinafter referred to as BCY9594). Wherein HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, the component present on the cancer cells is PD-L1.

[0081] Planned cell death ligand 1 (PD-L1) is a 290-amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in immune response escape, which is associated with chronic infections such as chronic viral infections (including HIV, HBV, HCV, and HTLV), chronic bacterial infections (including Helicobacter pylori), and chronic parasitic infections (including Schistosoma mansoni). PD-L1 expression has been detected in a wide range of tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, myocytes, and the placenta.

[0082] PD-L1 expression is also involved in the suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immune clearance of tumors is rare. This failure is partly attributed to the immunosuppression of the tumor microenvironment. PD-L1 on many tumors is a component of this suppressive environment and works synergistically with other immunosuppressive signals. PD-L1 has been visualized in situ in a wide range of solid tumors, including the breast, lung, colon, ovary, melanoma, bladder, liver, salivary glands, stomach, glioma, thyroid, thymic epithelium, head, and neck (Brown JA et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003 Cancer Res. 63:6501-5; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother). 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). Furthermore, the expression of PD-L1 receptor planned cell death protein 1 (also known as PD-1 and CD279) is upregulated in tumor-infiltrating lymphocytes, and this also contributes to tumor immunosuppression (Blank C et al. 2003 Immunol. 171:4574-81).Most importantly, studies on the correlation between PD-L1 expression in tumors and disease outcomes have shown that PD-L1 expression is largely associated with poor prognosis in renal cell carcinoma, ovarian cancer, bladder cancer, breast cancer, gastric cancer, and pancreatic cancer (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). Furthermore, these studies have shown that higher PD-L1 expression levels on tumors can promote tumor progression and invasion into deeper tissue structures.

[0083] The PD-1 pathway also plays a role in hematologic malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al. 2007 Blood 110:296-304). PD-L1 is expressed on some primary T-cell lymphomas, especially degenerative large cell T-lymphomas (Brown JA et al., 2003 Immunol. 170:1257-66). PD-1 is highly expressed on T cells in angioimmunoblastic lymphomas, and PD-L1 is expressed on associated follicular dendritic cell networks (Dorfman DM et al. 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-dominated Hodgkin lymphoma, PD-1 is expressed on T cells associated with lymphocytic or histiocytic (L&H) cells. Microarray analysis using gene readouts induced by PD-1 conjugation showed that tumor-associated T cells respond to in situ PD-1 signaling in Hodgkin's lymphoma (Chemnitz JM et al. 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma (Shimauchi T et al. 2007 Int. J. Cancer 121: 2585-90). These tumor cells are hyporesponsive to TCR signaling.

[0084] Studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis, and in some cases induces increased tumor-specific T cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96). Tumor-associated APCs can also utilize the PD-1:PD-L1 pathway to control anti-tumor T cell responses. PD-L1 expression in tumor-associated bone marrow DC populations is upregulated by tumor environmental factors (Curiel TJ et al. 2003 Nat. Med. 9:562-67). Plasma-like dendritic cells (DCs) in the tumor-draining lymph nodes of B16 melanoma exhibit IDO, which largely activates the inhibitory activity of regulatory T cells. The inhibitory activity of regulatory T cells treated with IDO requires contact between the cells and DCs expressing IDO (Sharma MD et al. 2007 Clin. Invest. 117:2570-82).

[0085] In some embodiments, the first peptide ligand comprises a PD-L1 binding bicyclic peptide ligand.

[0086] In some embodiments, the PD-L1 binding bicyclic peptide coordination system is selected from the PD-L1 binding bicyclic peptide ligands disclosed in WO 2020 / 128526 and WO 2020 / 128527, the contents of each of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, the PD-L1 binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: CiSAGWLTMCiiQKLHLCiii (SEQ ID NO: 52); CiSAGWLTMCiiQ[K(PYA)]LHLCiii (SEQ ID NO: 53); CiSKGWLTMCiiQ[K(Ac)]LHLCiii (SEQ ID NO: 54); CiSAGWLTKCiiQ[K(Ac)]LHLCiii (SEQ ID NO: 55); CiSAGWLTMCiiK[K(Ac)]LHLCiii (SEQ ID NO: 56); CiSAGWLTMCiiQ[K(Ac)]LKLCiii (SEQ ID NO: 57); CiSAGWLTMCiiQ[HArg]LHLCiii (SEQ ID NO: 58); and CiSAGWLTMCii[HArg]QLNLCiii (SEQ ID NO: 59); Ci, Cii, and Cii represent the first, second, and third cysteine ​​residues, respectively; PYA represents 4-pentynic acid; and HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0088] In some embodiments, the PD-L1 binding bicyclic peptide ligand may include N-terminal and / or C-terminal modifications and comprises: [PYA]-[B-Ala]-[Sar10]-SDK-(SEQ ID NO: 52) (hereinafter referred to as BCY10043); Ac-D-[HArg]-(SEQ ID NO: 52)-PSH (hereinafter referred to as BCY11865); Ac-SDK-(SEQ ID NO: 53) (hereinafter referred to as BCY11013); Ac-SDK-(SEQ ID NO: 53)-PSH (hereinafter referred to as BCY10861); Ac-D-[HArg]-(SEQ ID NO: 54)-PSH (hereinafter referred to as BCY11866); Ac-D-[HArg]-(SEQ ID NO: 55)-PSH (hereinafter referred to as BCY11867); Ac-D-[HArg]-(SEQ ID NO: 56)-PSH (referred to herein as BCY11868); Ac-D-[HArg]-(SEQ ID NO: 57)-PSH (referred to herein as BCY11869); Ac-SD-[HArg]-(SEQ ID NO: 58)-PSHK (referred to herein as BCY12479); and Ac-SD-[HArg]-(SEQ ID NO: 59)-PSHK (referred to herein as BCY12477); wherein PYA represents 4-pentynic acid, B-Ala represents β-alanine, Sar10 represents 10 sarcosine units, and HArg represents high-arginine, or a pharmaceutically acceptable salt thereof.

[0089] In some embodiments, the PD-L1 binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: Ci[HArg]DWCiiHWTFSHGHPCiii (SEQ ID NO: 82); CiSAGWLTMCiiQKLHLCiii (SEQ ID NO: 52); and CiSAGWLTMCiiQ[K(PYA)]LHLCiii (SEQ ID NO: 53); wherein Ci, Cii, and Cii represent the first, second, and third cysteine ​​residues, respectively, HArg represents high-arginine, and PYA represents 4-pentynyl acid, or a pharmaceutically acceptable salt thereof.

[0090] In some embodiments, the PD-L1 binding bicyclic peptide ligand comprises N-terminal and / or C-terminal modifications and includes: [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 82) (hereinafter referred to as BCY8938); [PYA]-[B-Ala]-[Sar10]-SDK-(SEQ ID NO: 52) (hereinafter referred to as BCY10043); NH2-SDK-(SEQ ID NO: 52)-[Sar10]-[K(PYA)] (hereinafter referred to as BCY10044); NH2-SDK-(SEQ ID NO: 53) (hereinafter referred to as BCY10045); and Ac-SDK-(SEQ ID NO: 53)-PSH (hereinafter referred to as BCY10861); Where PYA represents 4-pentynic acid, B-Ala represents β-alanine, and Sar10 represents 10 sarcosine units or a medically acceptable salt thereof.

[0091] In some embodiments, the component present on the cancer cells is prostate-specific membrane antigen (PSMA).

[0092] Prostate-specific membrane antigen (PSMA) (also known as glutamic acid carboxypeptidase II (GCPII), N-acetylglucosyl-L-glutamic acid peptidase I (NAALAD enzyme I), and NAAG peptidase) is an enzyme encoded by the FOLH1 (folate hydroxylase 1) gene in humans. Human GCPII contains 750 amino acids and weighs approximately 84 kDa.

[0093] Human PSMA is highly expressed in the prostate, approximately one hundred times more so than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, with levels 8 to 12 times higher than in non-cancerous prostate cells. Due to this high expression, PSMA has been developed as a potential biomarker for therapies and imaging in some cancers. In human prostate cancer, tumors with higher expression are associated with faster progression time and a greater percentage of patients experiencing recurrence.

[0094] In some embodiments, the first peptide ligand comprises a PSMA-binding bicyclic peptide ligand.

[0095] In some embodiments, the PSMA-binding bicyclic peptide coordination system is selected from the PSMA-binding bicyclic peptide ligands disclosed in WO 2019 / 243455 and WO 2020 / 120980, the contents of each of which are incorporated herein by reference in their entirety.

[0096] In some embodiments, the component present on the cancer cells is a membrane type 1 metalloproteinase (MT1-MMP).

[0097] In some embodiments, the first peptide ligand comprises an MT1-MMP-binding bicyclic peptide ligand.

[0098] In some embodiments, the MT1-MMP binding bicyclic peptide coordination system is selected from the MT1-MMP binding bicyclic peptide ligands disclosed in WO 2016 / 067035, WO 2017 / 191460 and WO 2018 / 115204, the contents of each of which are incorporated herein by reference in their entirety. CD137 binding peptide ligand

[0099] CD137 is a member of the tumor necrosis factor (TNF) receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF9) and 4-1BB, and it is induced by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but to a greater extent on CD8+ than on CD4+ T cells. Furthermore, CD137 expression has been found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. One characteristic activity of CD137 is its co-stimulatory activity on activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In addition, it can enhance immune activity to eliminate tumors in mice.

[0100] CD137 is a T cell co-stimulatory receptor induced upon TCR activation (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu. Rev. Immunol., 23:23-68 (2005)). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described on antigen-presenting cells including B cells, monocytes / macrophages, and dendritic cells (Watts et al., Annu. Rev. Immunol., 23:23-68 (2005)). When interacting with its ligands, CD137 induces TCR-induced T cell proliferation, cytokine production, functional maturation, and prolonged CD8+ T cell survival (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et al., Annu. Rev. Immunol, 23:23-68 (2005)).

[0101] Communication via CD137 through CD137L or anti-CD137 activating monoclonal antibodies (mAbs) leads to increased TCR-induced T cell proliferation, cytokine production and functional maturation, and prolonged CD8+ T cell survival. These effects are caused by: (1) activation of the NF-κB, c-Jun NH2-terminal kinase / suppressed protein kinase (JNK / SAPK) and p38 mitogen-activated protein kinase (MAPK) communication pathways and (2) control of anti-apoptotic and cell cycle-related gene expression.

[0102] Experiments in both CD137 and CD137L deficient mice have further demonstrated the importance of CD137 co-stimulation in generating fully competent T cell responses.

[0103] IL-2 and IL-15 activated NK cells express CD137, and NK cell proliferation and IFN-γ secretion are stimulated by binding CD137 with effector mAbs, but without stimulating their cytolytic activity.

[0104] In addition, CD137 stimulates NK cells to promote the expansion of activated T cells in vitro.

[0105] Based on its co-stimulatory function, anti-CD137 agonist mAbs have been shown to promote allogeneic transplant rejection in the heart and skin, eradicate established tumors, extend primary antiviral CD8+ T cell responses, and increase T cell cytolytic potential. These studies support the view that CD137 signaling promotes T cell function, which may enhance immunity against tumors and infections.

[0106] In some embodiments, when the heterotandem bicyclic peptide complex contains two or more CD137-binding peptide ligands, two or more of the CD137-binding peptide ligands have the same peptide sequence. In some embodiments, when the heterotandem bicyclic peptide complex contains two or more CD137-binding peptide ligands, two or more of the CD137-binding peptide ligands have different peptide sequences. In some embodiments, when the heterotandem bicyclic peptide complex contains two or more CD137-binding peptide ligands, two or more of the CD137-binding peptide ligands are the same. In some embodiments, when the heterotandem bicyclic peptide complex contains two or more CD137-binding peptide ligands, two or more of the CD137-binding peptide ligands are different.

[0107] In some embodiments, when the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the CD137-binding peptide ligand is a CD137-binding bicyclic peptide ligand. In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, two or more of these CD137-binding peptide ligands are CD137-binding bicyclic peptide ligands.

[0108] In some embodiments, the CD137-binding bicyclic peptide coordination system is selected from those CD137-binding bicyclic peptide ligands disclosed in WO 2019 / 025811. In some embodiments, when the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the CD137-binding peptide ligand is selected from those CD137-binding bicyclic peptide ligands disclosed in WO 2019 / 025811. In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, two or more of these CD137-binding bicyclic peptide ligands are independently selected from those CD137-binding bicyclic peptide ligands disclosed in WO 2019 / 025811. The contents of WO 2019 / 025811 are incorporated herein by reference in their entirety.

[0109] On behalf of CD137, photo ID137 is on Facebook: CiIEEGDPQYiNiFA 5); Ci[tBuAla]PE[D-Ala]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 6); CiIEEGQYCiiF[D-Ala]DPY[Nle]Ciii(SEQ ID NO: 7)? Ci[tBuAla]PK[D-Ala]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 8);C[tBuAla]PE[D-Lys]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 9)! Ci[tBuAla]P[K(PYA)][D-Ala]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 10); Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 11)? CiIEE[D-Lys(PYA)]QYCiiFADPY(Nle)Ciii(SEQ ID NO: 12);C[tBuAla]PE[dK]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 60);CIEE[dK(NiSEQC]]DPQYC 61); Ci[tBuA]EE(dK)PYCiiFADPY[Nle]Ciii(SEQ ID NO: 62);C[tBuA]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 63)? Ci[tBuAla]EE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 64); Ci[tBuAla]PE[dK(NEW)]PYCiiFANPY[Nle]Ciii(SEQ ID NO: 65); Ci[tBuAla]PE[dK(PYA)]PYCiiFAEPY[Nle]Ciii(SEQ ID NO: 66);C[tBuAla]PE[dK(PYA)]PYCiiFA[Aad]PY[Nle]Ciii(SEQ ID NO: 67)? Ci[tBuAla]PE[dK(NEW)]PYCiiFAQPY[Nle]Ciii(SEQ ID NO: 68)!Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle][Cysam]iii(SEQ ID NO: 69); [MerPro]i[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 70; name in text BCY12353); [MerPro]i[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle][Cysam]iii(SEQ ID NO: 71; name in text BCY12354); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 72); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 73); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 74; name in text BCY12372); Ci[tBuAla]PE[dK(PYA)]PYCiiFAD[NMeAla]Y[Nle]Ciii(SEQ ID NO: 75); Ci[tBuAla]PE[dK(PYA)]PYCiiFAD[NMeDAla]Y[Nle]Ciii(SEQ ID NO: 76); Ci[tBuAla]P[K(PYA)][dA]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 77); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 78); Ci[tBuAla]PE[dK(Me,PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 79); Ci[tBuAla]PE[dK(Me,PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 80); and [MerPro]i[tBuAla]EE[dK]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 81; referred to as BCY13137 in this text);Wherein [MerPro]i, Ci, Cii, Ciii, and [Cysam]iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam; Nle represents leucine; tBuAla represents tert-butyl-alanine; PYA represents 4-pentynic acid; Aad represents α-L-aminoadipic acid; MerPro represents 3-mercaptopropionic acid; Cysam represents cysteine; and NMeAla represents N-methyl-alanine, or a pharmaceutically acceptable salt thereof.

[0110] In some embodiments, the CD137-binding bicyclic peptide ligand comprises the following amino acid sequences: CiIEEGQYCiiFADPY[Nle]Ciii (SEQ ID NO: 5); Ci[tBuAla]PE[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 6); CiIEEGQYCiiF[D-Ala]DPY[Nle]Ciii (SEQ ID NO: 7); Ci[tBuAla]PK[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 8); Ci[tBuAla]PE[D-Lys]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 9); Ci[tBuAla]P[K(PYA)][D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 10); Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 11); and CiIEE[D-Lys(PYA)]QYCiiFADPY(Nle)Ciii (SEQ ID NO: 12); wherein Ci, Ci and Ciii represent the first, second and third cysteine ​​residues, respectively, Nle represents leucine, tBuAla represents tert-butyl-alanine, and PYA represents 4-pentyneic acid, or a pharmaceutically acceptable salt thereof.

[0111] In some embodiments, the CD137-binding bicyclic peptide ligand comprises the amino acid sequence: Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 11); wherein Ci, Ci and Ciii represent the first, second and third cysteine ​​residues, respectively, tBuAla represents tert-butyl-alanine, PYA represents 4-pentynyl acid, and Nle represents leucine, or a pharmaceutically acceptable salt thereof.

[0112] In some embodiments, the CD137-binding bicyclic peptide ligand comprises N-terminal and C-terminal modifications and includes: Ac-A-(SEQ ID NO: 5)-Dap (hereinafter referred to as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (hereinafter referred to as BCY7741); Ac-(SEQ ID NO: 6)-Dap (hereinafter referred to as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (hereinafter referred to as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (hereinafter referred to as BCY8045); Ac-(SEQ ID NO: 8)-A (hereinafter referred to as BCY8919); Ac-(SEQ ID NO: 9)-A (hereinafter referred to as BCY8920); Ac-(SEQ ID NO: 10)-A (hereinafter referred to as BCY8927); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY14601); Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); Ac-(SEQ ID NO: 60)-Dap(PYA) (referred to herein as BCY11144); Ac-A-(SEQ ID NO: 61)-K (referred to herein as BCY11613); Ac-(SEQ ID NO: 62)-Dap(PYA) (referred to herein as BCY12023); Ac-(SEQ ID NO: 63) (referred to herein as BCY12149); Ac-(SEQ ID NO: 64) (referred to herein as BCY12143); Ac-(SEQ ID NO: 65) (referred to herein as BCY12147); Ac-(SEQ ID NO: 65) (referred to herein as BCY12147); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY14601); Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); Ac-(SEQ ID NO: 60)-Dap(PYA) (referred to herein as BCY11144); Ac-A-(SEQ ID NO: 61)-K (referred to herein as BCY11613); Ac-(SEQ ID NO: 62)-Dap(PYA) (referred to herein as BCY12023); Ac-(SEQ ID NO: 63)-K (referred to herein as BCY12149); Ac-(SEQ ID NO: 64)-K (referred to herein as BCY12143); Ac-(SEQ ID NO: 66) (referred to as BCY12145 in this article); Ac-(SEQ ID NO: 67) (referred to as BCY12146 in this article); Ac-(SEQ ID NO: 68) (referred to as BCY12150 in this article); Ac-(SEQ ID NO: 69) (referred to as BCY12352 in this article); Ac-(SEQ ID NO: 72)-[1,2-diaminoethane] (referred to as BCY12358 in this article); [palmitic acid]-[yGlu]-[yGlu]-(SEQ ID NO: 73) (referred to as BCY12360 in this article); Ac-(SEQ ID NO: 75) (referred to as BCY12381 in this article);Ac-(SEQ ID NO: 76) (referred to herein as BCY12382); Ac-(SEQ ID NO: 77)-K (referred to herein as BCY12357); Ac-(SEQ ID NO: 78)-[dA] (referred to herein as BCY13095); [Ac]-(SEQ ID NO: 78)-K (referred to herein as BCY13389); Ac-(SEQ ID NO: 79)-[dA] (referred to herein as BCY13096); and Ac-(SEQ ID NO: 80) (referred to herein as BCY13097); wherein Ac represents acetyl, Dap represents diaminopropionic acid, and PYA represents 4-pentynic acid, or a pharmaceutically acceptable salt thereof.

[0113] In some embodiments, the CD137-binding bicyclic peptide ligand comprises N-terminal and C-terminal modifications and includes: Ac-A-(SEQ ID NO: 5)-Dap (hereinafter referred to as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (hereinafter referred to as BCY7741); Ac-(SEQ ID NO: 6)-Dap (hereinafter referred to as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (hereinafter referred to as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (hereinafter referred to as BCY8045); Ac-(SEQ ID NO: 8)-A (hereinafter referred to as BCY8919); Ac-(SEQ ID NO: 9)-A (hereinafter referred to as BCY8920); Ac-(SEQ ID NO: 10)-A (hereinafter referred to as BCY8927); Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); and Ac-A-(SEQ ID NO: 12)-A (referred to herein as BCY7744); wherein Ac represents acetyl, Dap represents diaminopropionic acid, and PYA represents 4-pentynic acid, or a pharmaceutically acceptable salt thereof.

[0114] In some embodiments, the CD137-binding bicyclic peptide ligand comprises N-terminal and C-terminal modifications and includes: Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); wherein Ac represents acetyl or a pharmaceutically acceptable salt thereof.

[0115] In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, each of the two or more CD137-binding peptide ligands has the same peptide sequence and the peptide sequence comprises Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928), wherein Ac represents acetyl or a pharmaceutically acceptable salt thereof.

[0116] In some embodiments, when the heterotandem bicyclic peptide complex comprises two CD137-binding peptide ligands, both CD137-binding peptide ligands have the same peptide sequence comprising Ac-(SEQ ID NO: 11)-A (hereinafter referred to as BCY8928), wherein Ac represents acetyl or a pharmaceutically acceptable salt thereof. The linker comprises a heterotandem bicyclic peptide complex containing two or more CD137-binding peptide ligands.

[0117] It will be understood that the first peptide ligand can bind to two or more second peptide ligands via any suitable linker. Typically, the linker is designed such that the three bicyclic peptides are presented in a manner that allows them to bind to their respective targets individually or simultaneously with two target receptors without hindrance. Furthermore, the linker should allow simultaneous binding to two targets while maintaining an appropriate distance between the target cells, which will produce the desired functional outcome. The properties of the linker can be tuned to increase length, stiffness, or solubility to optimize the desired functional outcome. The linker can also be designed to allow more than one bicyclic linker to the same target. Increasing the valence of any binding peptide can be used to increase the affinity of the heterotandem linker for the target cells or may help induce oligomerization of one or both of the target receptors.

[0118] In some embodiments, the linker is a branched chain linker to allow a first peptide to be located at one end and two or more second peptides to be located at the other end.

[0119] In some embodiments, the branched chain linker is selected from: N-(acid-PEG3)-N-bis(PEG3-azide); benzotriic acid-[Peg10]3; TCA-[Peg10]3; Tet-[Peg10]4; and BAPG-(Peg5)2.

[0120] In some embodiments, the branched linker is: N-(acid-PEG3)-N-bis(PEG3-azide). A heterotandem bicyclic peptide complex containing a CD137-binding peptide ligand.

[0121] It will be understood that the first peptide ligand can bind to the second peptide ligand via any suitable linker. Typically, the linker is designed such that the two bicyclic peptides bind to their respective targets unimpeded, either individually or simultaneously, to both target receptors. Furthermore, the linker should allow simultaneous binding to two targets while maintaining an appropriate distance between the target cells, which will produce the desired functional outcome. The properties of the linker can be modulated to increase length, stiffness, or solubility to optimize the desired functional outcome. The linker can also be designed to allow more than one bicyclic linker to the same target. Increasing the valence of either binding peptide can increase the affinity of the heterotandem peptide for the target cells or may help induce oligomerization of one or both target receptors.

[0122] In one embodiment, the linker system is selected from the following sequences: -PEG5- and TCA-[PEG10]3.

[0123] The structural representation of these connectors is described in detail below: -PEG5-; and TCA-[PEG10]3.

[0124] In some embodiments, the linker system is selected from the following sequences: -CH2-, -PEG5-, -PEG10-, -PEG12-, -PEG23-, -PEG24-, -PEG15-Sar5-, -PEG10-Sar10-, -PEG5-Sar15-, -PEG5-Sar5-, -B-Ala-Sar20-, -B-Ala-Sar10-PEG10-, -B-Ala-Sar5-PEG15- and -B-Ala-Sar5-PEG5-.

[0125] In some embodiments, the linker system is selected from the following: heterotandem bicyclic peptide complexes.

[0126] In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises a binding protein-4 binding bicyclic peptide ligand linked to the TATA backbone, each of the two or more CD137-binding bicyclic peptide ligands being linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table A: Table A (Binding Protein-4:CD137; 1:2) Complex number BCY code for binding protein-4 Connection point connector CD137 BCY number Connection point BCY11863 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12484 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12143 dLys(PYA)4 BCY10918 BCY11015 N-end PYA Benzotriic acid-[Peg] 10 ]3 BCY8928 dLys(PYA)4 BCY10919 BCY11015 N-end PYA Benzotriic acid-[Peg] 10 ]3 BCY11014 C-end Dap (PYA) BCY11027 BCY11015 N-end PYA TCA-[Peg 10 ]3 BCY8928 dLys(PYA)4 BCY11385 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY11014 C-end Dap (PYA) BCY11864 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY7744 dLys(PYA)4 BCY12485 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12149 dLys(PYA)4 BCY12486 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12147 dLys(PYA)4 BCY12586 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12352 dLys(PYA)4 BCY12487 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12145 dLys(PYA)4 BCY12490 BCY12024 dLys3 N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys(PYA)4 BCY12587 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12353 dLys(PYA)4 BCY12588 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12354 dLys(PYA)4 BCY12589 BCY12371 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys(PYA)4 BCY12590 BCY12384 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys(PYA)4 BCY12760 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12381 dLys(PYA)4 BCY12761 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12382 dLys(PYA)4 BCY13390 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 BCY14602 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY14601 dLys(PYA)4 BCY15155 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY14601 BCY8928 dLys(PYA)4 dLys(PYA)4

[0127] In some embodiments, the heterotandem bicyclic peptide complex is selected from BCY11027, BCY11863, and BCY11864. In some embodiments, the heterotandem bicyclic peptide complex is selected from BCY11863 and BCY11864.

[0128] The heterotandem bicyclic peptide complex BCY11863 (also known as BT7480) consists of a protein-4 specific peptide BCY8116 that is linked to two CD137 specific peptides (both of which are BCY8928) via an N-(acid-PEG3)-N-bis(PEG3-azide) linker, as illustrated in the image below.

[0129] CD137 is a homotrimeric protein, and its natural ligand CD137L exists as a homotrimeric form expressed on or secreted by immune cells. The biology of CD137 is highly dependent on polymerization to induce CD137 activity in immune cells. One way to generate CD137 polymerization is through cellular cross-linking via interaction of a CD137-specific agonist with a specific receptor present on another cell. The advantage of the heterotandem complex of the present invention is that the presence of two or more peptide ligands specific to immune cell components such as CD137 provides for more efficient aggregation of CD137. For example, BCY11863 has been found to exhibit strong CD137 activation and induce stable IL-2 and IFN-γ intercytokine secretion, and BCY11863 shows an excellent PK profile with a terminal half-life of 4.1 hours in SD rats and 5.3 hours in rhesus monkeys.

[0130] The heterotandem bicyclic peptide complex BCY11027 is composed of a protein-4 specific peptide BCY11015 that is linked to two CD137 specific peptides (both of which are BCY8928) via the TCA-[Peg10]3 linker, as illustrated below:

[0131] It has been found that binding protein-4 / CD137 heterotandem BCY11027 induces target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors, and induces several immunomarkers (normalized to mediators) related to binding protein-4 expression levels and binding protein-4-dependent changes in CD8+ki67+ T cells in patient-derived samples.

[0132] In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises a binding protein-4-binding bicyclic peptide ligand linked to the TATA backbone, each of the two or more CD137-binding bicyclic peptide ligands being linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table B: Table B (Binding protein-4:CD137; 1:3) Complex number BCY code for binding protein-4 Connection point connector CD137 BCY number Connection point BCY11021 BCY11016 N-end PYA Tet-[Peg 10 ]4 BCY7744 dLys(PYA)4 BCY11022 BCY11016 N-end PYA Tet-[Peg 10 ]4 BCY8928 dLys(PYA)4

[0133] In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand linked to the TATA backbone, each of the two or more CD137-binding bicyclic peptide ligands being linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table C: Table C (EphA2:CD137; 1:2) Complex number EphA2 BCY number Connection point connector CD137 BCY number Connection point BCY12491 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12723 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12143 dLys (PYA)4 BCY12724 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12149 dLys (PYA)4 BCY12725 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12147 dLys (PYA)4 BCY12726 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12145 dLys (PYA)4 BCY12728 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12150 dLys (PYA)4 BCY12729 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12352 dLys (PYA)4 BCY12730 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12353 dLys (PYA)4 BCY12731 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12354 dLys (PYA)4 BCY12732 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12360 dLys (PYA)4 BCY12973 BCY12734 C-end Lys N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12974 BCY12735 Lys8 N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12975 BCY12736 Lys2 N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12976 BCY12737 Lys7 N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12977 BCY12738 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12978 BCY12739 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY12979 BCY9594 N-end BAPG-(Peg5)2 BCY8928 dLys (PYA)4 BCY13042 BCY12854 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13043 BCY12855 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13044 BCY12856 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13045 BCY12857 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13046 BCY12858 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13047 BCY12859 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13048 BCY12860 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13049 BCY12861 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13050 BCY12862 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13051 BCY12863 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13052 BCY12864 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13053 BCY12865 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13054 BCY12866 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13138 BCY12856 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12353 dLys (PYA)4 BCY13139 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY13137 dLys (PYA)4 BCY13140 BCY12856 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY13137 dLys (PYA)4 BCY13270 BCY13116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13271 BCY13117 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13272 BCY13118 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13273 BCY13119 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13274 BCY13120 C-end dLys N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13275 BCY13121 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13276 BCY13122 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13277 BCY13123 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13278 BCY13124 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13280 BCY13126 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13281 BCY13127 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13282 BCY13128 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13284 BCY13130 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13285 BCY13131 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13286 BCY13132 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13288 BCY13134 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13289 BCY13135 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13341 BCY12865 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12353 dLys (PYA)4 BCY13343 BCY12860 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY12353 dLys (PYA)4 BCY13279 BCY13125 C-end dLys N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13283 BCY13129 C-end dLys N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY13287 BCY13133 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14049 BCY13917 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14050 BCY13918 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14051 BCY13919 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14052 BCY13920 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14053 BCY13922 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14054 BCY13923 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14055 BCY14047 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14056 BCY14048 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14334 BCY14313 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14335 BCY14327 Lys 8 N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys (PYA)4 BCY14413 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys (PYA)4 dLys (PYA)4 BCY14414 BCY13118 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 BCY15217 BCY13118 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY14601 BCY14601 dLys(PYA)4 dLys(PYA)4 BCY15218 BCY13118 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY14601 dLys(PYA)4 dLys(PYA)4

[0134] In some embodiments, the heterotandem bicyclic peptide complex is selected from: BCY12491, BCY12730, BCY13048, BCY13050, BCY13053 and BCY13272.

[0135] In some embodiments, the heterotandem bicyclic peptide complex is selected from: BCY12491, BCY12730, BCY13048, BCY13050 and BCY13053.

[0136] In some embodiments, the heterotandem bicyclic peptide complex is BCY12491.

[0137] The heterotandem bicyclic peptide complex BCY12491 is composed of EphA2-specific peptides BCY9594, which are linked to two CD137-specific peptides (both of which are BCY8928) via an N-(acid-PEG3)-N-bis(PEG3-azide) linker, as illustrated below:

[0138] It has been found that BCY12491 induces a significant anti-tumor response and regulates (increases) tumor-infiltrating immune cells and immune responses.

[0139] In some embodiments, the heterotandem bicyclic peptide complex is BCY13272.

[0140] The heterotandem bicyclic peptide complex BCY13272 is composed of EphA2-specific peptide BCY13118, which is linked to two CD137-specific peptides (both of which are BCY8928) via an N-(acid-PEG3)-N-bis(PEG3-azide) linker, as illustrated below:

[0141] It has been found that BCY13272 induces a significant antitumor effect in a mouse MC38 tumor model.

[0142] In some embodiments, when the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand linked to the TATA backbone, each of the two or more CD137-binding bicyclic peptide ligands being linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table D: Table D (PD-L1:CD137; 1:2) Complex number PD-L1 BCY number Connection point connector CD137 BCY number Connection point BCY11780 BCY10861 Lys(PYA)9 TCA-[Peg 10 ]3 BCY8928 dLys4 BCY12662 BCY12479 C-end Lys N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys(PYA)4 BCY12722 BCY12477 C-end Lys N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 dLys(PYA)4

[0143] In some embodiments, when the heterotandem bicyclic peptide complex comprises a CD137-binding peptide ligand, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand linked to the TATA backbone, the CD137-binding peptide ligand being linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table E: Table E (PD-L1:CD137; 1:1) Complex number PD-L1 BCY number Connection point connector CD137 BCY number Connection point BCY12229 BCY11865 Lys9 Peg5 BCY8928 dLys(PYA)4 BCY12230 BCY11866 Lys2 Peg5 BCY8928 dLys(PYA)4 BCY12231 BCY11867 Lys7 Peg5 BCY8928 dLys(PYA)4 BCY12232 BCY11868 Lys8 Peg5 BCY8928 dLys(PYA)4 BCY12242 BCY11869 Lys11 Peg5 BCY8928 dLys(PYA)4 BCY12375 BCY10861 Lys(PYA)9 Peg5 BCY12023 dLys4 BCY12663 BCY12479 C-end Lys Peg5 BCY8928 dLys(PYA)4 BCY12796 BCY12477 C-end Lys Peg5 BCY8928 dLys(PYA)4 BCY12021 BCY10861 Lys(PYA)9 Peg5 BCY11144 dLys4

[0144] In some embodiments, the hetero-linked bicyclic peptide complex is selected from BCY12375 and BCY12021.

[0145] In some embodiments, where the hetero-linked bicyclic peptide complex includes a CD137-binding peptide ligand, the first peptide ligand includes a PD-L1-binding bicyclic peptide ligand linked to the TATA backbone, the CD137-binding peptide ligand being linked to the TATA backbone, and the hetero-linked bicyclic peptide complex is selected from the complexes listed in Table E-2: Table E-2 (PD-L1:CD137; 1:1) Complex number PD-L1 BCY number Connection point connector CD137 BCY serial number Connection point BCY8939 BCY8938 N-end PYA -PEG12 - BCY7732 C-end Dap BCY10580 BCY10043 N-end PYA -PEG 12 - BCY9172 C-end Dap BCY10581 BCY10044 C-end Lys(PYA) -PEG 12 - BCY9172 C-end Dap BCY10582 BCY10045 Lys(PYA)9 -PEG 12 - BCY9172 C-end Dap BCY11017 BCY10861 Lys(PYA)9 -PEG 12 - BCY8919 Lys3 BCY11018 BCY10861 Lys(PYA)9 -PEG 12 - BCY8920 dLys4 BCY11019 BCY10861 Lys(PYA)9 -PEG 12 - BCY9172 C-end Dap BCY11376 BCY10861 Lys(PYA)9 -CH2- BCY8919 Lys3 BCY11377 BCY10861 Lys(PYA)9 -CH2- BCY8920 dLys4 BCY11378 BCY10861 Lys(PYA)9 -CH2- BCY9172 C-end Dap BCY11379 BCY10861 Lys(PYA)9 -PEG5- BCY8919 Lys3 BCY11380 BCY10861 Lys(PYA)9 -PEG5- BCY8920 dLys4 BCY11381 BCY10861 Lys(PYA)9 -PEG5- BCY9172 C-end Dap

[0146] In some embodiments, where the hetero-chain bicyclic peptide complex includes a CD137-binding peptide ligand, the first peptide ligand includes an EphA2-binding bicyclic peptide ligand linked to the TATA backbone, the CD137-binding peptide ligand being linked to the TATA backbone, and the hetero-chain complex is selected from the complexes listed in Table F: Table F (EphA2:CD137; 1:1) Complex number EphA2 BCY code Connection point connector CD137 BCY serial number Connection point BCY12233 BCY11813 N-end PYA Peg5 BCY8920 dLys4 BCY12234 BCY11814 C-end Lys(PYA) Peg5 BCY8920 dLys4 BCY12235 BCY11815 Lys(PYA) 8 Peg5 BCY8920 dLys4 BCY12236 BCY11816 Lys(PYA)2 Peg5 BCY8920 dLys4 BCY12237 BCY11817 Lys(PYA)7 Peg5 BCY8920 dLys4 BCY12711 BCY9594 N-end Peg5 BCY12143 dLys (PYA)4 BCY12712 BCY9594 N-end Peg5 BCY12149 dLys (PYA)4 BCY12713 BCY9594 N-end Peg5 BCY12147 dLys (PYA)4 BCY12714 BCY9594 N-end Peg5 BCY12145 dLys (PYA)4 BCY12715 BCY9594 N-end Peg5 BCY12146 dLys (PYA)4 BCY12717 BCY9594 N-terminus Peg5 BCY12352 dLys (PYA)4 BCY12718 BCY9594 N-terminus Peg5 BCY12353 dLys (PYA)4 BCY12719 BCY9594 N-terminus Peg5 BCY12354 dLys (PYA)4 BCY12720 BCY9594 N-terminus [[ID=4​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ BCY12965 BCY12738 N-end Peg5 BCY8928 dLys (PYA)4 BCY12966 BCY12739 N-end Peg5 BCY8928 dLys (PYA)4 BCY13029 BCY12854 N-end Peg5 BCY8928 dLys (PYA)4 BCY13030 BCY12855 N-end Peg5 BCY8928 dLys (PYA)4 BCY13031 BCY12856 N-end Peg5 BCY8928 dLys (PYA)4 BCY13032 BCY12857 N-end Peg5 BCY8928 dLys (PYA)4 BCY13033 BCY12858 N-end Peg5 BCY8928 dLys (PYA)4 BCY13034 BCY12859 N-end Peg5 BCY8928 dLys (PYA)4 BCY13035 BCY12860 N-end Peg5 BCY8928 dLys (PYA)4 BCY13036 BCY12861 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13037 BCY12862 N-terminus Peg5 BCY8928 dLys (PYA)4 BCY13038 BCY12863<000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​dLys (PYA)4 BCY13143 BCY12856 N-terminal Peg5 BCY13137 dLys (PYA)4 BCY13250 BCY13116 N-terminal Peg5 BCY8928 dLys (PYA)4 BCY13251 BCY13117 N-terminal Peg5 BCY8928 dLys (PYA)4 BCY13252 BCY13118 N-terminal Peg5 BCY8928 dLys (PYA)4 BCY13253 BCY13119 N-terminal Peg5 BCY8928 dLys (PYA)4 BCY13254 BCY13120 C-terminal dLys Peg5 BCY8928 dLys (PYA)4 BCY13255 BCY13121<00,01329>N-terminal Peg5 BCY8928 dLys (PYA)4 BCY13256 BCY13122 N-terminal Peg5 BCY8928 dLys (PYA)4 BCY13257 BCY13123 N-end Peg5 BCY8928 dLys (PYA)4 BCY13258 BCY13124 N-end Peg5 BCY8928 dLys (PYA)4 BCY13260 BCY13126 N-end Peg5 BCY8928 dLys (PYA)4 BCY13261 BCY13127 N-end Peg5 BCY8928 dLys (PYA)4 BCY13262 BCY13128 N-end Peg5 BCY8928 dLys (PYA)4 BCY13264 BCY13130 N-end Peg5 BCY8928 dLys (PYA)4 BCY13265 BCY13131 N-end Peg5 BCY8928 dLys (PYA)4 BCY13266 BCY13132 N-end Peg5 BCY8928 dLys (PYA)4 BCY13268 BCY13134 N-end Peg5 BCY8928 dLys (PYA)4 BCY13269 BCY13135 N-end Peg5 BCY8928 dLys (PYA)4 BCY13340 BCY12865 N-end Peg5 BCY12353 dLys (PYA)4 BCY13342 BCY12860 N-end Peg5 BCY12353 dLys (PYA)4

[0147] In some embodiments, the heterotandem bicyclic peptide complex is selected from: BCY13035, BCY13040, BCY13253, BCY13254, BCY13340 and BCY13342.

[0148] In some embodiments, when the heterotandem bicyclic peptide complex comprises a CD137-binding peptide ligand, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand linked to the TATA backbone, the CD137-binding peptide ligand being linked to the TATA backbone, and the heterotandem complex is selected from the complexes listed in Table F-2: Table F-2 (EphA2:CD137; 1:1) Complex number EphA2 BCY number Connection point connector CD137 BCY number Connection point BCY9173 BCY6169 N-end PYA -PEG 12 - BCY9172 C-end Dap BCY7985 BCY6169 N-end PYA -PEG12 - BCY7732 C-end Dap BCY8942 BCY6169 N-end PYA -PEG 12 - BCY8045 C-end Dap BCY8943 BCY8941 N-end PYA -PEG 12 - BCY7732 C-end Dap BCY9647 BCY6099 N-end -PEG 10 - BCY7741 C-end Dap (PYA) BCY9648 BCY6099 N-end -PEG 23 - BCY7741 C-end Dap (PYA) BCY9655 BCY6099 N-end -PEG 15 -Sar5- BCY7741 C-end Dap (PYA) BCY9656 BCY6099 N-end -PEG 10 -Sar 10 - BCY7741 C-end Dap (PYA) BCY9657 BCY6099 N-end -PEG5-Sar 15 - BCY7741 C-end Dap (PYA) BCY9658 BCY6099 N-end -PEG5-Sar5- BCY7741 C-end Dap (PYA) BCY9659 BCY6099 N-end -PEG5- BCY7741 C-end Dap (PYA) BCY9758 BCY6099 N-end -PEG 24 - BCY7732 C-end Dap BCY10568 BCY6169 N-end PYA -PEG 12 - BCY8919 Lys3 BCY10570 BCY6169 N-end PYA -PEG 12 - BCY8920 dLys4 BCY10574 BCY9594 N-end -PEG5- BCY8927 Lys (PYA)3 BCY10575 BCY9594 N-end -PEG5- BCY8928 dLys (PYA)4 BCY10576 BCY9594 N-end -PEG5- BCY11014 C-end Dap (PYA) BCY10577 BCY6169 N-end -CH2- BCY9172 C-end Dap

[0149] In some embodiments, the heterotandem bicyclic peptide complex is BCY7985, wherein the CD137-specific peptide BCY7859 is linked to the N-terminal PYA group of the EphA2-specific peptide BCY6169 via PEG12.

[0150] In some embodiments, when the heterotandem bicyclic peptide complex includes a CD137-binding peptide ligand, the first peptide ligand includes a binding protein-4-binding bicyclic peptide ligand linked to the TATA backbone, the CD137-binding peptide ligand being linked to the TATA backbone, and the heterotandem complex is selected from the complexes listed in Table G: Table G (Binding protein-4:CD137; 1:1) Complex number BCY code for binding protein-4 Connection point connector CD137 BCY number Connection point BCY11616 BCY8116 N-end Peg5 BCY7744 dLys(PYA)4 BCY12238 BCY12024 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12377 BCY8116 N-end Peg5 BCY12143 dLys(PYA)4 BCY12379 BCY8116 N-end Peg5 BCY12149 dLys(PYA)4 BCY12572 BCY8116 N-terminal Peg5 BCY12352 dLys(PYA)4 BCY12573 BCY8116 N-terminal Peg5 BCY12353 dLys(PYA)4 BCY12574 BCY8116 N-terminal Peg5 BCY12354 dLys(PYA)4 BCY12575 BCY8116 N-terminal Peg5 BCY12360 dLys(PYA)4 BCY12576 BCY12363 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12577 BCY12364 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12578 BCY12365 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12579 BCY12366 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12580 BCY12367 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12581 BCY12368 N-end Peg5 BCY8928 dLys(PYA)4 BCY12582 BCY12369 N-end Peg5 BCY8928 dLys(PYA)4 BCY12583 BCY12370 N-end Peg5 BCY8928 dLys(PYA)4 BCY12584 BCY12371 dLys3 Peg5 BCY8928 dLys(PYA)4 BCY12585 BCY12384 N-end Peg5 BCY8928 dLys(PYA)4 BCY12709 BCY8116 N-end Peg5 BCY12381 dLys(PYA)4 BCY12710 BCY8116 N-end Peg5 BCY12382 dLys(PYA)4 BCY11468 BCY11016 N-end PYA TCA-[Peg10]3 BCY8928 dLys(PYA)4 BCY11618 BCY11143 N-end PYA Peg5 BCY8920 dLys4 BCY11776 BCY8116 N-end Peg5 BCY11144 C-end Dap (PYA) BCY11860 BCY11143 N-end PYA Peg5 BCY8920 dLys4 BCY12020 BCY11016 N-end PYA Peg5 BCY11144 C-end Dap (PYA) BCY12661 BCY11015 N-end PYA Peg5 BCY12023 dLys4 BCY12969 BCY8116 N-end Peg5 BCY12358 dLys(PYA)4

[0151] In some embodiments, the hetero-linked bicyclic peptide complex is selected from: BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661 and BCY12969.

[0152] In some embodiments, where the hetero-coupling bicyclic peptide complex includes a CD137-binding peptide ligand, the first peptide ligand includes a binding protein-4 binding bicyclic peptide ligand linked to the TATA backbone, the CD137-binding peptide ligand being linked to the TATA backbone, and the hetero-coupling complex is selected from the complexes listed in Table G-2: Table G-2 (Binding protein-4:CD137; 1:1) Complex number BCY code for binding protein-4 Connection point connector CD137 BCY number Connection point BCY8854 BCY8846 N-end PYA -PEG 12 - BCY7732 C-end Dap BCY9350 BCY11942 N-end PYA -PEG 12 - BCY7732 C-end Dap BCY9351 BCY8846 N-end PYA -PEG 12 - BCY8045 C-end Dap BCY9399 BCY8116 N-end -PEG 10 - BCY7741 C-end Dap (PYA) BCY9400 BCY8116 N-end -PEG 23 - BCY7741 C-end Dap (PYA) BCY9401 BCY8116 N-end -B-Ala-Sar 20 - BCY7741 C-end Dap (PYA) BCY9403 BCY8116 N-end -B-Ala-Sar 10 -PEG 10 - BCY7741 C-end Dap (PYA) BCY9405 BCY8116 N-end -B-Ala-Sar5-PEG 15 - BCY7741 C-end Dap (PYA) BCY9406 BCY8116 N-end -B-Ala-Sar5-PEG5- BCY7741 C-end Dap (PYA) BCY9407 BCY8116 N-end -PEG 15 -Sar5- BCY7741 C-end Dap (PYA) BCY9408 BCY8116 N-end -PEG 10 -Sar 10 - BCY7741 C-end Dap (PYA) BCY9409 BCY8116 N-end -PEG5-Sar 15 - BCY7741 C-end Dap (PYA) BCY9410 BCY8116 N-end -PEG5-Sar5- BCY7741 C-end Dap (PYA) BCY9411 BCY8116 N-end -PEG5- BCY7741 C-end Dap (PYA) BCY9759 BCY8116 N-end -PEG 24 - BCY7732 C-end Dap BCY10000 BCY8846 N-end PYA -PEG 12 - BCY9172 C-end Dap BCY10567 BCY8846 N-end PYA -PEG 12 - BCY8919 Lys3 BCY10569 BCY8846 N-end PYA -PEG 12 - BCY8920 dLys4 BCY10571 BCY8116 N-end -PEG5- BCY8927 Lys(PYA)3 BCY10572 BCY8116 N-end -PEG5- BCY8928 dLys (PYA)4 BCY10573 BCY8116 N-end -PEG5- BCY11014 C-end Dap (PYA) BCY10578 BCY8846 N-end PYA -CH2- BCY9172 C-end Dap BCY10917 BCY8831 dLys(Sar 10)-(B-Ala))4 -PEG 12 - BCY11014 C-end Dap (PYA) BCY11020 BCY8831 dLys(Sar 10 )-(B-Ala))4 -PEG5- BCY11014 C-end Dap (PYA) BCY11373 BCY8116 N-end -CH2- BCY8927 Lys(PYA)3 BCY11374 BCY8116 N-end -CH2- BCY8928 dLys (PYA)4 BCY11375 BCY8116 N-end -CH2- BCY11014 C-end Dap (PYA) BCY11616 BCY8116 N-end -PEG5- BCY7744 dLys (PYA)4 BCY11617 BCY8116 N-end -PEG5- BCY11506 Lys(PYA)4 BCY11857 BCY11414 N-end -PEG5- BCY7744 dLys (PYA)4 BCY11858 BCY11414 N-end -PEG5- BCY8928 dLys (PYA)4 BCY11859 BCY11415 N-end -PEG5- BCY8928 dLys (PYA)4

[0153] In some embodiments, the heterotandem bicyclic peptide complex is selected from the heterotandem bicyclic peptide complexes disclosed in U.S. Patent Application 17 / 062,662, the contents of which are incorporated herein by reference in their entirety.

[0154] In some embodiments, the heterotandem bicyclic peptide complex is selected from those heterotandem bicyclic peptide complexes disclosed in U.S. Patent Publication 20190307836, the contents of which are incorporated herein by reference in their entirety.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (such as peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry). Standard techniques used in molecular biology, genetics, and biochemistry (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th ed., John Wiley & Sons, Inc.), are incorporated herein by reference. Nomenclature Numbering

[0156] When referring to the positions of amino acid residues in the compounds of the present invention, cysteine ​​residues (Ci, Cii and Ciii) are omitted from the numbering because they are unchanging. Therefore, the amino acid residues in SEQ ID NO: 1 are numbered as follows: Ci-P1-1Nal2-dD3-Cii-M4-HArg5-D6-W7-S8-T9-P10-HyP11-W12-Ciii (SEQ ID NO: 1).

[0157] For the purposes of this specification, it is assumed that all bicyclic peptides are cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) or 1,1',1''-(1,3,5-tris(triyl)propane-1,3,5-triyl)triprop-2-en-1-one (TATA) to produce trisubstituted structures. Cyclization with TBMB and TATA occurs at Ci, Cii, and Ciiii. Molecular Type

[0158] The N-terminal or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence, separated by a hyphen. For example, the N-terminal βAla-Sar10-Ala tail will be represented as: βAla-Sar10-A-(SEQ ID NO: X). Reverse peptide sequence

[0159] Given the findings of Nair et al. (2003) J Immunol 170(3), 1362-1373, it is assumed that the peptide sequences disclosed herein will also be found to have their reverse-reverse properties. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa) and its stereochemistry is also reversed (i.e., the D-amino acid becomes the L-amino acid, and vice versa). To avoid doubt, unless otherwise stated, references to amino acids in this document, whether by their full name or by their single-letter or three-letter code, are intended to be L-amino acids. If an amino acid is intended to be represented as a D-amino acid, it will begin with a lowercase d in square brackets, such as [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc. Advantages of peptide ligands

[0160] Certain heterotandem bicyclic peptide complexes of the present invention possess a number of advantageous properties that enable them to be considered suitable drug molecules for injection, inhalation, nasal, ocular, oral, or topical administration. These advantageous properties include: - Species cross-reactivity. This is a typical requirement for preclinical pharmacokinetic and pharmacodynamic assessments; - Protease stability. Ideally, the heterotandem bicyclic peptide complex should exhibit stability against plasma proteases, epithelial ("membrane-anchored") proteases, pepsin and intestinal proteases, pulmonary surface proteases, intracellular proteases, and the like. Protease stability should be maintained across different species to enable the development of heterotandem bicyclic peptide lead candidates in animal models and safe administration to humans; - Required solubility profile. This is a function of the ratio of charged residues and hydrophilic to hydrophobic residues, as well as intramolecular / intermolecular H bonds, which is crucial for formulation and absorption purposes; - Selectivity. Certain heterotandem bicyclic peptide complexes of the present invention exhibit superior selectivity compared to other targets; - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop heterotandem bicyclic peptide complexes for short-term exposure in acute disease management settings, or heterotandem bicyclic peptide complexes with enhanced retention in circulation and therefore optimal for managing more chronic disease states. Other factors driving the desired plasma half-life include the requirement for sustained exposure to maximize therapeutic efficacy and the toxicology associated with sustained drug exposure. Crucially, this article presents data showing that the selected heterotandem bicyclic peptide complexes exhibited antitumor efficacy when administered at frequencies not maintaining plasma concentrations above the compound's in vitro EC50. This contrasts with larger recombinant biological (i.e., antibody-based) approaches that utilize CD137 agonists or bispecific CD137 agonists (Segal et al., Clin Cancer Res., 23(8):1929-1936 (2017), Claus et al., Sci Trans Med., 11(496): eaav5989, 1-12 (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889 (2019)). Unbound by theory, this observation is attributed to the fact that heterotandem bicyclic complexes have relatively low molecular weights (typically <15 kDa), are fully synthetic, and are tumor-targeting agonists of CD137. Consequently, heterotandem bicyclic complexes exhibit relatively short plasma half-lives but good tumor penetrance and retention. Data in this paper adequately support these advantages. For example, it showed antitumor efficacy in genotype rodent models of mice with humanized CD137, either daily or every 3 days.Furthermore, intraperitoneal pharmacokinetic data show a plasma half-life of <3 hours, which predicts that the circulating concentration of the complex will continue to decline below the in vitro EC50 between doses. In addition, tumor pharmacokinetic data show that the concentration of the heterotandem bicyclic complex in tumor tissue may be higher and more persistent than in plasma. It will be understood that this observation forms another important aspect of the present invention. Therefore, according to another aspect of the present invention, a method for treating cancer is provided, comprising administering a heterotandem bicyclic peptide complex as defined herein at a dosing frequency that does not maintain a plasma concentration of the complex above the in vitro EC50. - Immune Memory. The synergistic advantage of coupling cancer cells to bicyclic peptide ligands with immune cells to provide immune memory is presented herein. Data presented herein show that the selected heterotandem bicyclic peptide complex of the present invention not only eradicates tumors but also, upon re-administration of the oncogenic agent, none of the fully responsive mice inoculated with the complex developed tumors (see Figure 5). This indicates that treatment with the selected heterotandem bicyclic peptide complex of the present invention induces immunogenic memory in fully responsive mice. This has significant clinical advantages in preventing recurrence of the tumor after it has been initially controlled and eradicated. Peptide ligands.

[0161] As mentioned herein, a peptide coordination system refers to a peptide covalently bound to a molecular backbone. Typically, such peptides contain two or more reactive groups (i.e., cysteine ​​residues) capable of forming covalent bonds with the backbone, and a sequence between these reactive groups that is referred to as a cyclic sequence because it forms a ring when the peptide is bound to the backbone. In the present case, the peptide contains at least three reactive groups selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine and forms at least two rings on the backbone. Reactive groups

[0162] The molecular backbone of the present invention can be attached to the polypeptide via functional groups or reactive groups on the polypeptide. These are typically formed by side chains of specific amino acids found in polypeptide polymers. Such reactive groups can be cysteine ​​side chains, lysine side chains, or N-terminal amino groups, or any other suitable reactive groups, such as penicillamine. Details of suitable reactive groups can be found in WO 2009 / 098450.

[0163] Examples of reactive groups in natural amino acids include the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartic acid or glutamic acid, the carboxyl group of arginine, the phenolic group of tyrosine, or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups, including azides, ketone-carbonyl groups, alkynes, vinyl groups, or aryl halogen groups. The amino and carboxyl groups at the ends of peptides can also act as reactive groups to form covalent bonds with the molecular skeleton / molecular core.

[0164] The polypeptides of the present invention contain at least three reactive groups. These polypeptides may also contain four or more reactive groups. The more reactive groups used, the more rings can be formed in the molecular backbone.

[0165] In a preferred embodiment, a polypeptide having three reactive groups is generated. The reaction of these polypeptides with a molecular backbone / molecular core having three times the rotational symmetry produces a single product isomer. The generation of a single product isomer is advantageous for several reasons. The nucleic acids in the compound library encode only the first-order sequence of the polypeptide, not the isomeric state of the molecule formed when the polypeptide reacts with the molecular core. If only one product isomer can be formed, the allocation of nucleic acids to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acids cannot provide information about the properties of the product isomers isolated in a screening or selection process. The formation of a single product isomer is also advantageous when synthesizing specific library members of the invention. In this case, the chemical reaction of the polypeptide with the molecular backbone produces a single product isomer rather than a mixture of isomers.

[0166] In another embodiment, a polypeptide having four reactive groups is generated. The polypeptide reacts with a molecular backbone / core having tetrahedral symmetry to produce two product isomers. Even if the two different product isomers are encoded by the same nucleic acid, the isomeric properties of the isolated isomers can be determined by chemically synthesizing the two isomers, isolating the two isomers, and testing the binding of the two isomers to the target ligand.

[0167] In one embodiment of the invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows these orthogonal reactive groups to be directed to specific sites on the molecular core. Linkage strategies involving orthogonal reactive groups can be used to limit the number of product isomers formed. In other words, by selecting reactive groups that are different from or different from the reactive groups selected for one or more of at least three bonds compared to the remaining reactive groups selected for at least three bonds, a specific order in which specific reactive groups of the polypeptide are bound to or directed to specific positions on the molecular backbone can be effectively achieved.

[0168] In another embodiment, the reactive groups of the polypeptide of the present invention are reacted with molecular linkers, wherein the linkers are capable of reacting with the molecular backbone such that the linkers are inserted between the molecular backbone and the polypeptide in a final bound state.

[0169] In some embodiments, the amino acids of library members or polypeptide sets may be replaced by any natural or non-natural amino acids. These exchangeable amino acids exclude those carrying functional groups for crosslinking the polypeptide to the molecular core, such that only the ring sequence is exchangeable. The exchangeable polypeptide sequence has a random sequence, a constant sequence, or a sequence with both random and constant amino acids. Because the position of these amino acids determines the ring size, amino acids with reactive groups are located at defined positions within the polypeptide.

[0170] In one embodiment, the polypeptide having three reactive groups has the sequence (X)lY(X)mY(X)nY(X)o, where Y represents an amino acid having a reactive group, X represents a random amino acid, m and n are numbers between 3 and 6 that define the length of the inserted polypeptide segment (they may be the same or different), and l and o are numbers between 0 and 20 that define the length of the side-attached polypeptide segment.

[0171] Thiol-mediated binding alternatives can be used to link the molecular backbone to peptides via covalent interactions. Alternatively, these techniques can be used to modify or link other parts (such as small molecules of interest different from the molecular backbone) to peptides after other parts have been selected or isolated according to the invention, in which case the subsequent apparent link does not need to be covalent and can encompass non-covalent links. These methods can be used instead of thiol-mediated methods (or combinations thereof) by generating phages that exhibit non-natural amino acids carrying essential chemically reactive groups and small molecules carrying complementary reactive groups, or by incorporating non-natural amino acids into chemically or recombinantly synthesized peptides during molecule formation after the selection / isolation stage. Further details can be found in WO 2009 / 098450 or Heinis et al., Nat Chem Biol 2009, 5(7), 502-7.

[0172] In some embodiments, the reactive group is selected from cysteine, 3-mercaptopropionic acid, and / or cysteine ​​residues. Pharmaceutically acceptable salts.

[0173] It will be understood that salt forms are within the scope of the present invention, and references to peptide ligands include salt forms of such ligands.

[0174] The salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. Generally, such salts can be prepared by reacting the free acidic or basic form of these compounds with a suitable acid or base in water, in an organic solvent, or in a mixture of both.

[0175] Acid addition salts (single or disalts) can be formed from a wide variety of acids (both inorganic and organic). Examples of acid addition salts include mono- or di-salts formed from acids selected from the group consisting of: acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetaminophen, butyric acid, (+)camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, decanoic acid, hexanoic acid, caprylic acid, cinnamic acid, citric acid, cyclohexylaminesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactosic acid, gentian acid, glucoheponic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-semi-oxyglutarate. Glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), hydroxyethyl sulfonic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, dihydroxynaphthoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanate, p-toluenesulfonic acid, undecenoic acid and valeric acid, as well as acetylated amino acids and cation exchange resins.

[0176] A specific group of salts consists of salts formed from the following: acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, hydroxyethyl sulfonic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid, ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid, and lactobionic acid. One specific salt is a hydrochloride salt. Another specific salt is an acetate salt.

[0177] If the compound is an anion or has a functional group that can be an anion (e.g., -COOH can be -COO-), the salt can be formed from an organic or inorganic base that produces a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+, and K+; alkaline earth metal cations such as Ca2+ and Mg2+; and other cations such as Al3+ or Zn+. Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+). Some examples of suitable substituted ammonium ions are derived from the following ammonium ions: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, benzylamine, phenylbenzylamine, choline, meglumine, and thiazoline, as well as amino acids such as lysine and arginine. One example of a common quaternary ammonium ion is N(CH3)4+.

[0178] When the compounds of the present invention contain amine functional groups, these compounds can be formed into quaternary ammonium salts by methods well known to those skilled in the art, such as by reacting with an alkylating agent. Such quaternary ammonium compounds are within the scope of the present invention. Modified derivatives

[0179] It will be understood that modified derivatives of peptide ligands as defined herein are within the scope of this invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (such as substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; substitution of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; substitution of one or more amino acid residues with alanine; substitution of one or more L-amino acid residues with one or more D-amino acid residues. Substitution; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; substitution of one or more peptide bonds; modification of peptide backbone length; substitution of hydrogen on the α-carbon of one or more amino acid residues with another chemical group; modification of amino acids (such as cysteine, lysine, glutamic acid / aspartic acid, and tyrosine) with suitable amine, thiol, carboxylic acid, and phenol reactive reagents to functionalize such amino acids; and introduction or substitution of amino acids, wherein the introduction of such amino acids is suitable for orthogonal reactivity of functionalization, such as amino acids carrying azide or alkynyl groups, which allow functionalization with the alkynyl or azide-carrying portions, respectively.

[0180] In some embodiments, the modified derivative comprises N-terminal and / or C-terminal modifications. In another embodiment, the modified derivative comprises N-terminal modification using a suitable amino-reactive chemical reagent and / or C-terminal modification using a suitable carboxyl-reactive chemical reagent. In yet another embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to cytotoxic agents, radiochelates, or chromophores.

[0181] In some embodiments, the modified derivative comprises an N-terminal modification. In another embodiment, the N-terminal modification comprises an N-terminal acetylation group. In this embodiment, the N-terminal cysteine ​​group (referred to herein as the Ci group) is capped with acetic anhydride or other suitable reagent during peptide synthesis to produce an N-terminal acetylated molecule. This embodiment provides the advantage of removing a potential recognition site for aminopeptidases and avoiding the possibility of bicyclic peptide degradation.

[0182] In some embodiments, the N-terminal modification includes the addition of a molecular spacer group, which helps to bind the effector group and retain the efficacy of the bicyclic peptide for its target.

[0183] In some embodiments, the modified derivative comprises a C-terminal modification. In another embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine ​​group (referred to herein as the Ciii group) is synthesized into an amide during peptide synthesis, thereby producing a C-terminal amide-modified molecule. This embodiment provides the advantage of removing a potential recognition site for carboxypeptidase and reducing the likelihood of hydrolytic degradation of the bicyclic peptide protein.

[0184] In some embodiments, the modified derivative comprises one or more amino acid residues replaced by one or more non-natural amino acid residues. In this embodiment, non-natural amino acids having isomeric / isoelectron side chains that are neither recognized by degrading proteases nor have any adverse effect on target efficacy may be selected.

[0185] Alternatively, non-natural amino acids with restricted amino acid side chains may be used, thereby hindering the conformational and steric lysis of adjacent peptide bonds. Specifically, such non-natural amino acids include proline analogs, large side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid Aib), and cyclic amino acids, with simple derivatives being amino-cyclopropylcarboxylic acid.

[0186] In some embodiments, the modified derivative comprises the addition of a spacer group. In some embodiments, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine ​​(Ci) and / or the C-terminal cysteine ​​(Ciii).

[0187] In some embodiments, the modified derivative comprises one or more oxidation-sensitive amino acid residues replaced by one or more antioxidant amino acid residues. In some embodiments, the modified derivative comprises tryptophan residues replaced by naphthylalanine or alanine residues. This embodiment provides the advantage of improved pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0188] In some embodiments, the modified derivative comprises one or more charged amino acid residues replaced by one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises one or more hydrophobic amino acid residues replaced by one or more charged amino acid residues. The proper balance of charged and hydrophobic amino acid residues is an important characteristic of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (specifically, arginine) can affect the interaction between the peptide and the phospholipid membrane on the cell surface. The combination of both can affect the half-life, distribution volume, and exposure of the peptide drug, and can be adjusted according to clinical endpoints. Furthermore, the proper combination and number of charged and hydrophobic amino acid residues can reduce stimulation at the injection site (where the peptide drug has already been administered subcutaneously).

[0189] In some embodiments, the modified derivative comprises one or more L-amino acid residues replaced by one or more D-amino acid residues. It is believed that this embodiment increases protein hydrolytic stability by steric hindrance and by the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0190] When the D-amino acid stabilizes the β-turn conformation, the modified derivative comprises the removal of any amino acid residue and substitution with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

[0191] It should be noted that each of the above modifications is intended to intentionally improve the potency or stability of the peptide. Other potency improvements based on modifications can be achieved through the following mechanisms: - incorporation of hydrophobic moieties that utilize hydrophobic interactions and produce a lower dissociation rate, resulting in higher affinity; - incorporation of charged groups that utilize long-distance ion interactions, resulting in a faster association rate and higher affinity (see, for example, Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - incorporation of additional constraints into the peptide by, for example, properly restricting the side chains of amino acids to minimize entropy loss at target binding, restricting the torsion angle of the main chain to minimize entropy loss at target binding, and introducing additional cyclization into the molecule for the same reason. (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0192] Examples of the modified heterotandem bicyclic peptide complexes of the present invention include those listed in Tables H and I below: Table H: (EphA2:CD137; 1:2) Complex number EphA2 BCY number Connection point connector CD137 BCY number Connection point Modifier BCY14415 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys (PYA)4 dLys (PYA)4 Peg12-Biotin BCY14416 BCY9594 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys (PYA)4 dLys (PYA)4 Alexa Fluor® 488 BCY14417 BCY13118 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 Peg12-Biotin BCY14418 BCY13118 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 Alexa Fluor® 488 Table I: (Binding protein-4:CD137; 1:2) Complex number BCY code for binding protein-4 Connection point connector CD137 BCY number Connection point Modifier BCY13582 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928, BCY13389 dLys(PYA)4 dLys(PYA)4 Biotin-Peg12 BCY13583 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928, BCY13389 dLys(PYA)4 dLys(PYA)4 Alexa Fluor 488 BCY13628 BCY8116 N-end N-(acid-PEG3)-N-bis(PEG3-azide) BCY8928, BCY13389 dLys(PYA)4 dLys(PYA)4 Cyanine 5 Isotope variants

[0193] This invention includes all pharmaceutically acceptable (radioactive) isotopically labeled peptide ligands of the present invention, wherein one or more atoms are replaced by atomic substitutions having the same number of atoms but atomic masses or mass numbers different from those usually found in nature; and peptide ligands of the present invention wherein a metal chelating group (referred to as an "effecton") is attached, such metal chelating group being capable of accommodating the relevant (radioactive) isotope; and peptide ligands of the present invention wherein certain functional groups are covalently substituted with the relevant (radioactive) isotope or isotopically labeled functional groups.

[0194] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include the following isotopes: hydrogen, such as 2H (D) and 3H (T); carbon, such as 11C, 13C and 14C; chlorine, such as 36Cl; fluorine, such as 18F; iodine, such as 123I, 125I and 131I; nitrogen, such as 13N and 15N; oxygen, such as 15O, 17O and 18O; phosphorus, such as 32P; sulfur, such as 35S; copper, such as 64Cu; gallium, such as 67Ga or 68Ga; yttrium, such as 90Y; and ilium, such as 177Lu; and bismuth, such as 213Bi.

[0195] Certain isotopically labeled peptide ligands of the present invention (e.g., those with radioactive isotopes) are suitable for drug and / or receptor tissue distribution studies and for clinically assessing the presence of binding protein-4 in diseased tissues. The peptide ligands of the present invention can further possess valuable diagnostic properties because they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may use compounds labeled with labeling reagents such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, amylopectin, and luciferase). Radioactive isotopes tritium (i.e., 3H(T)) and carbon-14 (i.e., 14C) are particularly suitable for this purpose due to their ease of incorporation and readily available detection methods.

[0196] Substitution with a heavier isotope such as deuterium (i.e., 2H (D)) can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dose requirement, and may therefore be preferred in some cases.

[0197] Substitution with positron emission isotopes (such as 11C, 18F, 15O and 13N) can be used in positron emission tomography (PET) studies to examine target occupancy.

[0198] The isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by means of conventional techniques known to those skilled in the art, or by means of a process similar to that described in the accompanying examples, using a suitable isotopically labeled reagent instead of the previously used unlabeled reagent. Molecular backbone

[0199] The molecular skeleton is described, for example, in WO 2009 / 098450 and the references cited therein (especially WO 2004 / 077062 and WO 2006 / 078161).

[0200] As noted in the aforementioned literature, the molecular skeleton can be a small molecule, such as a small organic molecule.

[0201] In one embodiment, the molecular backbone may be a macromolecule. In one embodiment, the molecular backbone is a macromolecule composed of amino acids, nucleotides, or carbohydrates.

[0202] In one embodiment, the molecular backbone includes reactive groups capable of reacting with the functional groups of the polypeptide to form covalent bonds.

[0203] The molecular skeleton may contain chemical groups that form bonds with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, formic acid, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleic amides, alkyl halides and acetylated halides.

[0204] In one embodiment, the molecular skeleton may comprise or may consist of hexahydro-1,3,5-tris(TATA), especially 1,3,5-tripropenylhexahydro-1,3,5-tris(TATA) or a derivative thereof.

[0205] The molecular skeleton of the present invention contains chemical groups that allow the functional groups of the polypeptides encoded by the present invention to form covalent bonds with the molecular skeleton. These chemical groups are selected from a wide range of functional groups, including amines, thiols, alcohols, ketones, aldehydes, nitriles, formic acid, esters, alkenes, alkynes, anhydrides, succinimides, maleamides, azides, alkyl halides, and acyl halides.

[0206] The skeletal reactive group that reacts with the thiol group of cysteine ​​can be an alkyl halide (or also called a haloalkane or haloalkane) on the molecular skeleton.

[0207] Examples include bromomethylbenzene (a skeleton reactive group exemplified by TBMB) or iodoacetamide. Other skeleton reactive groups used to selectively couple the compound to cysteine ​​in proteins are maleamides, compounds containing α-unsaturated carbonyl groups, and compounds containing α-halomethyl carbonyl groups. Examples of maleimides that can be used as the molecular skeleton in this invention include: tris(2-maleiminoethyl)amine, tris(2-maleiminoethyl)benzene, and tris(maleimino)benzene. An example of a compound containing an α-unsaturated carbonyl group is 1,1',1''-(1,3,5-tris(2-triyl)prop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). One example of a compound containing an α-halomethyl carbonyl group is N,N',N''-(phenyl-1,3,5-triyl)-(2-bromoacetamide). Selenocysteine ​​is also a natural amino acid with similar reactivity to cysteine ​​and can be used in the same reactions. Therefore, unless the context otherwise suggests, substituted selenocysteine ​​is generally acceptable whenever cysteine ​​is mentioned. Synthesis

[0208] The peptides of this invention can be synthesized using standard techniques and then reacted with the molecular backbone in vitro. Standard chemical methods can be used when performing this operation. This enables the rapid, large-scale preparation of soluble materials for further downstream experiments or validation. Such methods can be implemented using conventional chemical methods such as those disclosed in Timmerman et al. (see above).

[0209] Therefore, the present invention also relates to the manufacture of selected polypeptides or conjugates as described herein, wherein the manufacture comprises other steps, as may be selected as explained below. In one embodiment, these steps are performed on a final product polypeptide / conjugate obtained by chemical synthesis.

[0210] Depending on the circumstances, the amino acid residues in the polypeptide of interest may be substituted during the manufacture of the conjugate or complex.

[0211] Peptides can also be extended to incorporate, for example, another ring and thus introduce multiple specificities.

[0212] is an extended peptide that can only be chemically extended using standard solid-phase or solution-phase chemical methods with orthogonally protected lysine (and analogues) at its N-terminus or C-terminus or within the ring. Standard (biological) binding techniques can be used to introduce an activated or activatable N-terminus or C-terminus. Alternatively, addition can be carried out by fragment condensation or native chemical conjugation, as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779); or by enzymes, such as subtiligase, as described in (Chang et al. Proc Natl Acad Sci US A. 20 Dec 1994; 91(26):12544-8 or Hikari et al. Bioorganic & Medicinal Chemistry Letters Vol. 18, No. 22, 15 November 2008, pp. 6000-6003).

[0213] Alternatively, the peptide can be extended or modified by further binding via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other in a reducing environment within the cell. In this case, a molecular backbone (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; another cysteine ​​or thiol can then be attached to the N-terminus or C-terminus of the first peptide, such that this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide, thereby forming a disulfide-linked bicyclic peptide-peptide conjugate.

[0214] Similar techniques are also applicable to the synthesis / coupling of two bicyclic and bispecific macrocyclic compounds, which may result in the formation of tetraspecific molecules.

[0215] In addition, the addition of other functional groups or effector groups can be achieved in the same manner, using appropriate chemical methods, at the N-terminus or C-terminus, or via side-chain coupling. In one embodiment, coupling is carried out in a manner that does not inhibit the activity of any entity. 2. Compounds and definitions:

[0216] As used herein, the term "medically acceptable salt" means salts that, to the extent of reasonable medical judgment, are suitable for contact with human and lower animal tissues without adverse toxicity, irritation, or anaphylactic reactions or the like, and for which the benefit / risk ratio is proportionate. Medically acceptable salts are well known in the art. For example, SM Berge et al. describe medically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Medically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by the amino group with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods used in this technique (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, p-pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and their analogues.

[0217] Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N+(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and similar salts. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using relative ions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, low-carbon alkyl sulfonate, and aryl sulfonate.

[0218] Unless otherwise stated, the structures described herein are intended to include all isomers (e.g., mirror-image isomers, non-mirror-image isomers, and geometrical isomers (or configurational isomers) of such structures; for example, R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E configurational isomers. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of mirror-image, non-mirror-image, and geometrical isomers (or configurational isomers), are within the scope of the present invention. Unless otherwise stated, all tautomers of the compounds of the present invention are within the scope of the present invention. Furthermore, unless otherwise stated, the structures described herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures of the present invention, including those with hydrogen replaced by deuterium or tritium or carbon replaced by 13C- or 14C-enriched carbon, are within the scope of the present invention. Such compounds are suitable as, for example, analytical tools, probes in bioanalysis, or therapeutic agents according to the present invention.

[0219] As used herein, the term “about” means within 20% of a given value. In some embodiments, the term “about” means within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of a given value.

[0220] As used herein, the term "mg / kg" refers to the number of milligrams of drug per kilogram of body weight of an individual taking the drug. 3. Pharmaceutically acceptable compositions

[0221] According to some embodiments, the present invention provides a pharmaceutical composition comprising a heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof, the heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands; and a pharmaceutically acceptable carrier, adjuvant, or mediator. In some embodiments, the present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising a heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof, the heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands; an immuno-oncology agent; and a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0222] In some embodiments, the present invention provides a pharmaceutical composition comprising BT7480 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or mediator. In some embodiments, the present invention provides a pharmaceutical composition for treating cancer comprising BT7480 or a pharmaceutically acceptable salt thereof, an immuno-oncology agent, and a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0223] In some embodiments, the present invention provides a pharmaceutical composition comprising BT7455 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or mediator. In some embodiments, the present invention provides a pharmaceutical composition for treating cancer comprising BT7455 or a pharmaceutically acceptable salt thereof, an immuno-oncology agent, and a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0224] In some embodiments, the composition comprises a pharmaceutically acceptable carrier, adjuvant, or catalyst. The term "pharmaceutically acceptable carrier, adjuvant, or catalyst" means a non-toxic carrier, adjuvant, or catalyst that does not impair the pharmacological activity of the compound formulated therewith. Pharmaceutically acceptable carriers, adjuvants, or catalysts that can be used in the compositions of the present invention include, but are not limited to, ion exchangers; alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances, such as phosphates; glycine; sorbic acid; potassium sorbate; a mixture of partial glycerides of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate; disodium hydrogen phosphate; potassium hydrogen phosphate; sodium chloride; zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; cellulose-based substances; polyethylene glycol; sodium carboxymethyl cellulose; polyacrylates; waxes; polyethylene-polyoxypropylene-block polymers; polyethylene glycol; and lanolin.

[0225] As used herein, the term “patient” means animal, preferably mammal, and most preferably human.

[0226] The compositions of the present invention can be administered orally, non-enterically, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implantable reservoir. As used herein, the term "non-enteric" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasheathic, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions of the present invention can be an aqueous or oily suspension. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in this art. The sterile injectable formulation can also be a sterile injectable solution or suspension in a non-toxic, non-enteric acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable mediators and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally used as solvents or suspension media.

[0227] For this purpose, any mild, non-volatile oil, including synthetic monoglycerides or diglycerides, may be used. Fatty acids (such as oleic acid and its glyceride derivatives) are suitable for the preparation of injectable formulations, as are naturally pharmaceutically acceptable oils (such as olive oil or castor oil, especially in their polyoxyethylene form). Such oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms (including emulsions and suspensions). Other commonly used surfactants (such as Tween, Span, and other emulsifiers) or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0228] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, suitable diluents include lactose and dried corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. Sweeteners, flavoring agents, or coloring agents may also be added as needed.

[0229] Alternatively, the pharmaceutically acceptable compositions of the present invention can be administered rectally as suppositories. These suppositories can be prepared by mixing the pharmaceutical agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0230] The pharmaceutically acceptable compositions of the present invention can also be administered topically, especially when the therapeutic target includes areas or organs easily accessible by topical application, including diseases of the eyes, skin, or lower intestine. Suitable topical formulations can be readily prepared for use in each of these areas or organs.

[0231] For local application in the lower intestine, it may be administered as a rectal suppository formulation (see above) or as a suitable enema formulation. A local percutaneous patch may also be used.

[0232] For topical application, the provided pharmaceutically acceptable compositions may be formulated in a suitable ointment form containing an active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white paraffin, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified waxes, and water. Alternatively, the provided pharmaceutically acceptable compositions may be formulated in a suitable emulsion or cream form containing an active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl wax, cetearyl alcohol, 2-octyldodecyl alcohol, benzyl alcohol, and water.

[0233] For ocular use, the provided pharmaceutically acceptable composition may be formulated as a micronized suspension, with or without a preservative (such as benzylalkonium chloride), in isotonic pH-adjusted sterile saline, or preferably as a solution in isotonic pH-adjusted sterile saline. Alternatively, for ocular use, the pharmaceutically acceptable composition may be formulated as an ointment (such as paraffin).

[0234] The pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceutical formulation and can be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, fluorocarbons and / or other known solubilizers or dispersants.

[0235] The pharmaceutically acceptable compositions of the present invention can also be formulated for oral administration. Such formulations may or may not be administered with food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are administered without food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0236] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health condition, sex, diet, administration time, excretion rate, drug combination, the judgment of the treating physician, and the severity of the specific disease being treated. The amount of the compound of the present invention in the composition also depends on the specific compound in the composition. 4. Method for treating cancer

[0237] In some embodiments, the present invention provides a method of treating a patient’s cancer, comprising administering to the patient a therapeutically effective amount of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof and an immuno-oncology agent.

[0238] In some embodiments, the present invention provides the use of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharmaceutically acceptable salt thereof, and an immuno-oncology agent for the treatment of cancer.

[0239] In some embodiments, the present invention provides a method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of BT7480 or a pharmaceutically acceptable salt thereof and an immuno-oncology agent. In some embodiments, the present invention provides the use of BT7480 or a pharmaceutically acceptable salt thereof and an immuno-oncology agent for treating cancer.

[0240] In some embodiments, the present invention provides a method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of BT7455 or a pharmaceutically acceptable salt thereof and an immuno-oncology agent. In some embodiments, the present invention provides the use of BT7455 or a pharmaceutically acceptable salt thereof and an immuno-oncology agent for treating cancer. Exemplary Cancer

[0241] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is associated with MT1-MMP. In some embodiments, the cancer is characterized by high MT1-MMP expression. For example, Adley et al. have reported high expression of MT1-MMP in clear cell carcinoma of the ovary (Adley et al. "Expression of Membrane Type 1 Matrix Metalloproteinase (MMP-14) in Epithelial Ovarian Cancer: High Level Expression in Clear Cell Carcinoma" Gynecol Oncol. 2009 Feb; 112(2): 319-324).

[0242] In some embodiments, cancer is associated with binding protein-4. In some embodiments, cancer is characterized by high binding protein-4 activity.

[0243] In some embodiments, cancer is associated with EphA2. In some embodiments, cancer is characterized by high EphA2 levels.

[0244] In some embodiments, cancer is associated with PD-L1. In some embodiments, cancer exhibits high PD-L1 expression.

[0245] In some embodiments, cancer is associated with PSMA. In some embodiments, cancer is characterized by high PSMA expression.

[0246] In some embodiments, the cancer is bladder cancer. In some embodiments, the bladder cancer is selected from the group consisting of: the base, p53-like structure, and the lumen.

[0247] In some embodiments, the cancer is endometrial cancer. In some embodiments, endometrial cancer is selected from the group consisting of: MMR-D, POLE EDM, p53 WT, p53 abnormality, type I, type II, carcinoma, carcinosarcoma, endometrioid adenocarcinoma, serous carcinoma, clear cell carcinoma, mucinous carcinoma, mixed or undifferentiated carcinoma, mixed serous and endometrioid carcinoma, mixed serous and low-grade endometrioid carcinoma and undifferentiated carcinoma.

[0248] In some embodiments, the cancer is esophageal cancer. In some embodiments, esophageal cancer is selected from the group consisting of: adenocarcinoma (EAC), squamous cell carcinoma (ESCC), chromosomal instability (CIN), Ebola virus (EBV), genomic stability (GS), and microsatellite instability (MSI).

[0249] In some embodiments, the cancer is glioblastoma. In some embodiments, glioblastoma is selected from the group consisting of: protoneurocytes, neuromorphs, typical cells, and mesenchymal cells.

[0250] In some embodiments, the cancer is mesothelioma. In some embodiments, the mesothelioma is selected from the group consisting of: pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, epithelioid mesothelioma, sarcomatoid mesothelioma, biphasic mesothelioma, and malignant mesothelioma.

[0251] In some embodiments, the cancer is multiple myeloma. In some embodiments, multiple myeloma is selected from the group consisting of: hyperdiploid, non-hyperdiploid, cyclin D translocation, MMSET translocation, MAF translocation, and unspecified type.

[0252] In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is selected from the group consisting of: clear cell, endometrioid, mucinous, high-grade serous, and low-grade serous ovarian cancers.

[0253] In some embodiments, the cancer is pancreatic cancer. In some embodiments, the pancreatic cancer is selected from the group consisting of squamous, pancreatic precursor cell, immunogenic, and ADEX (abnormally differentiated endocrine-exocrine) pancreatic cancer.

[0254] In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is selected from the group consisting of AZGP1 (subtype I), MUC1 (subtype II), and MUC1 (subtype III) prostate cancer.

[0255] In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is met-amplified squamous NSCLC, squamous cell NSCLC with wild-type EGFR, or lung adenocarcinoma expressing T790M EGFR.

[0256] In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the breast cancer is basaloid triple-negative breast cancer.

[0257] In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is colorectal adenocarcinoma. In some embodiments, the colorectal adenocarcinoma is a colorectal adenocarcinoma with high pgp expression.

[0258] In some embodiments, the cancer is gastric cancer. In some embodiments, the gastric cancer is FGFR-amplified gastric cancer.

[0259] In some embodiments, the cancer is head and neck cancer. In some embodiments, the head and neck cancer is squamous cell carcinoma of the nasal septum.

[0260] In some embodiments, the cancer is a sarcoma. In some embodiments, the sarcoma is a fibrosarcoma. In some embodiments, the fibrosarcoma is an N-ras mutant / IDH1 mutant soft tissue sarcoma (STS).

[0261] In one embodiment, cancer includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia. Macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphoendothelial sarcoma, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor). Tumors, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as neuroblastoma), neurotubercula, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0262] In some embodiments, the cancer is glioma, astrocytoma, glioblastoma multiforme (GBM, also known as neuroblastoma), neuroblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0263] In some embodiments, the cancer is acoustic neuroma, astrocytoma (e.g., grade I - pilocytic astrocytoma, grade II - low-grade astrocytoma, grade III - degenerative astrocytoma, or grade IV - glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymal ependymoma, neuroblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal (PNET) tumor, or schwannoma. In some embodiments, the cancer is a type more common in children than in adults, such as brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), neuroblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdomyosarcoma. In some embodiments, the patient is an adult. In some embodiments, the patient is a child or a pediatric patient.

[0264] In another embodiment, the cancers include, but are not limited to, mesothelioma, hepatobiliary (liver and bile duct) cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, gastrointestinal (stomach, colon, rectum and duodenum) cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, and parathyroid cancer. Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, non-Hodgkin's lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, multiple myeloma, bile duct carcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the aforementioned cancers.

[0265] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial carcinoma or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; bile duct hepatocellular carcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing sarcoma; degenerative thyroid cancer; adrenocortical adenoma; pancreatic cancer; pancreatic duct cancer or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; head and neck squamous cell carcinoma (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibroma-1-related malignant peripheral nerve sheath tumor (MPNST); Waldenström's macroglobulinemia; or neurotubular cell tumor.

[0266] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine serous papillary carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, degenerative thyroid cancer, adrenocortical adenoma, pancreatic cancer, pancreatic duct cancer, pancreatic adenocarcinoma, glioma, neurofibroma-1-related malignant peripheral nerve sheath tumor (MPNST), Waldenström's macroglobulinemia, or neurotubular cell tumor.

[0267] In some embodiments, the cancer is a solid tumor, such as sarcoma, carcinoma, or lymphoma. Solid tumors generally comprise abnormal tissue masses that typically do not include cysts or fluid-filled areas. In some embodiments, the cancer is selected from renal cell carcinoma or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma or liver cancer; melanoma; breast cancer; colorectal carcinoma or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; Hepatocellular carcinoma of the bile duct; soft tissue and synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; degenerative thyroid carcinoma; adrenocortical carcinoma; pancreatic cancer; pancreatic duct carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland carcinoma; glioma or brain cancer; neurofibroma-1-related malignant peripheral nerve sheath tumor (MPNST); Waldenström's macroglobulinemia; or neurotubular cell tumor.

[0268] In some embodiments, the cancer is selected from renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal cancer, colon cancer, rectal cancer, anal cancer, ovarian cancer, ovarian epithelial cancer, ovarian tumor, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, degenerative thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic duct cancer, pancreatic adenocarcinoma, glioma, brain cancer, neurofibroma-1-related malignant peripheral nerve sheath tumor (MPNST), Waldenström's macroglobulinemia, or neurotubular cell tumor.

[0269] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), cholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, degenerative thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic duct cancer, pancreatic adenocarcinoma, glioma, neurofibroma-1-related malignant peripheral nerve sheath tumor (MPNST), Waldenström's macroglobulinemia, or neurotubular cell tumor.

[0270] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer or ovarian tumor. In some embodiments, the cancer is ovarian epithelial carcinoma. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is degenerative thyroid cancer. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic duct cancer. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is neurofibroma-1 related MPNST. In some embodiments, the cancer is Waldenström's macroglobulinemia. In some embodiments, the cancer is neuroblastoma.

[0271] In some embodiments, the cancer is a virus-related cancer, including human immunodeficiency virus (HIV)-related solid tumors, human papillomavirus (HPV)-16-positive incurable solid tumors, and adult T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and a highly invasive form of CD4+ T-cell leukemia characterized by pure lineage integration of HTLV-I into leukemia cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); as well as virus-related tumors in gastric cancer, nasopharyngeal carcinoma, cervical cancer, vaginal cancer, vulvar cancer, head and neck squamous cell carcinoma, and Merkel cell carcinoma. (See https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; also see https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892)

[0272] In some embodiments, the cancer is melanoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0273] In some embodiments, cancer is treated by inhibiting further growth of the tumor. In some embodiments, cancer is treated by reducing the size (e.g., volume or mass) of the tumor relative to its size before treatment by at least 5%, 10%, 25%, 50%, 75%, 90%, or 99%. In some embodiments, cancer is treated by reducing the number of tumors in the patient relative to the number of tumors before treatment by at least 5%, 10%, 25%, 50%, 75%, 90%, or 99%.

[0274] The heterotandem bicyclic peptide complexes and compositions of the present invention can be administered in any amount and via any route of administration effective for treating or reducing the severity of cancer. The precise amount required will vary from individual to individual, depending on the individual's species, age and general condition, the severity of the disease or symptom, the specific drug, the mode of administration, and similar factors. For ease of administration and uniformity of dosing, the heterotandem bicyclic peptide complexes are preferably formulated in dose units. As used herein, "dose unit form" refers to a physically discrete unit of the drug suitable for the patient to be treated. However, it will be understood that the total daily dosage of the heterotandem bicyclic peptide complexes and compositions of the present invention will be determined by the attending physician within the bounds of reasonable medical judgment. The specific effective dose for any particular patient or organism will depend on a number of factors, including the condition to be treated and its severity; the activity of the specific compound used; the specific combination used; the patient's age, weight, general health condition, sex, and diet; the time of administration, route of administration, and rate of excretion of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in medical technology. As used herein, the term "patient" means animal, preferably mammal, and most preferably human.

[0275] The pharmaceutically acceptable compositions of the present invention may be administered to humans and other animals orally, rectally, non-enterally, intracerebrospinally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, or drops), buccally (e.g., by oral or nasal spray), or similarly, depending on the severity of the disease or condition being treated. In some embodiments, the heterotandem bicyclic peptide complex of the present invention may be administered orally or non-enterally once or more times daily at a dose of about 0.01 mg / kg to about 100 mg / kg or about 1 mg / kg to about 25 mg / kg body weight / day to achieve the desired therapeutic effect.

[0276] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in this art, such as water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol; and mixtures thereof. In addition to inert diluents, the oral composition may also include adjuvants, such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers.

[0277] Injectable formulations can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. Sterile injectable formulations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic, non-enteric-acceptable diluents or solvents, for example, in a solution in 1,3-butanediol. Acceptable mediators and solvents include water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids such as oleic acid can be used in the preparation of injectable formulations.

[0278] The injectable formulation may be sterilized, for example, by filtration through a bacterial trapping filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0279] To prolong the effect of the compounds of the present invention, it is generally necessary to slow the absorption of compounds administered subcutaneously or intramuscularly. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of the compound depends on its dissolution rate, which in turn depends on the crystal size and crystal form. Alternatively, delayed absorption of compounds administered non-enterovenously can be achieved by dissolving or suspending the compound in an oil-based medium. Injectable storage forms are manufactured by forming microcapsule matrices of the compound in a biodegradable polymer such as polylactide-polyglycolic acid. The rate of compound release can be controlled depending on the ratio of compound to polymer and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoester) and poly(anhydride). Reservoir-type injectable formulations can also be prepared by encapsulating the compound in tissue-compatible liposomes or microemulsions.

[0280] The composition is preferably prepared by mixing the heterotandem bicyclic peptide complex of the present invention with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol or suppository wax) for transrectal or transvaginal administration.

[0281] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and / or the following: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants, such as glycerin; d) disintegrants, such as agar-agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blockers, such as paraffin; f) absorption enhancers, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

[0282] Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or toffee and high molecular weight polyethylene glycol and its analogues. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in pharmaceutical compounding techniques. They may contain emulsifiers and may also have compositions that release the active ingredient, either only or preferentially, in a portion of the intestine, as appropriate, in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar solid compositions can also be used as fillers in soft-filled and hard-filled gelatin capsules using excipients such as lactose or toffee and high molecular weight polyethylene glycol and its analogues.

[0283] The active compound may also be microencapsulated together with one or more excipients as noted above. Solid dosage forms of tablets, sugar-coated pills, capsules, pellets, and granules may be prepared with coatings and shells, such as enteric coatings, release-controlled coatings, and other coatings well known in pharmaceutical formulation techniques. In such solid dosage forms, the active compound may be blended with at least one inert diluent (such as sucrose, lactose, or starch). As is commonly practiced, such dosage forms may also contain additional substances besides inert diluents, such as tablet lubricants and other tablet-making aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage form may also contain buffers. They may contain emulsifiers and may also have compositions that release the active ingredient, as appropriate, only or preferentially in a portion of the intestine. Examples of encapsulation compositions that may be used include polymers and waxes.

[0284] Dosage forms for topical or transdermal administration (e.g.,) of heterotandem bicyclic peptide complexes comprising one or more CD137-binding peptide ligands as described herein include ointments, pastes, creams, emulsions, gels, powders, solutions, sprays, inhalers, or patches. The active ingredient is infused under sterile conditions with a pharmaceutically acceptable carrier and, if desired, any desired preservatives or buffers. Ophthalmic formulations, ear drops, and eye drops are also covered within the scope of this invention. Additionally, this invention covers the use of transdermal patches, which have the added advantage of providing controlled delivery of compounds to the body. Such dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium. Absorption enhancers can also be used to increase the transdermal amount of the compound. The rate can be controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel. Co-administration of heterotandem bicyclic peptide complexes and immuno-oncology agents.

[0285] (For example) the heterotandem bicyclic peptide complex and immuno-oncology agent as described herein may be administered separately as part of a multiple-dose regimen. Alternatively, (for example) the heterotandem bicyclic peptide complex and immuno-oncology agent as described herein may be mixed together in a single composition as a single-dose formulation. In some embodiments, the heterotandem bicyclic peptide complex is BT7480 or BT7455 or a pharmaceutically acceptable salt thereof.

[0286] In some embodiments, for example, the hetero tandem bicyclic peptide complex as described herein is administered separately from the immuno-oncology agent. In some embodiments, for example, the hetero tandem bicyclic peptide complex as described herein and the immuno-oncology agent are administered simultaneously. In some embodiments, for example, the hetero tandem bicyclic peptide complex as described herein and the immuno-oncology agent are administered sequentially. In some embodiments, for example, the hetero tandem bicyclic peptide complex as described herein and the immuno-oncology agent are administered at intervals of time, such as within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, for example, the hetero tandem bicyclic peptide complex as described herein and the immuno-oncology agent are administered at intervals greater than 24 hours. In some embodiments, (for example) the hetero tandem bicyclic peptide complex and immuno-oncology agent as described herein are administered at intervals of 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, (for example) the hetero tandem bicyclic peptide complex and immuno-oncology agent as described herein are administered at intervals greater than one week. In some embodiments, (for example) the hetero tandem bicyclic peptide complex and immuno-oncology agent as described herein are administered at intervals of 1, 2, 3, 4, or 5 weeks.

[0287] As used herein, the terms "combination" and related terms refer to the simultaneous or sequential administration of a therapeutic agent according to the invention. For example, a heterotandem bicyclic peptide complex as described herein may be administered simultaneously or sequentially with an immuno-oncology agent in a single unit dose, or together in a single unit dose. Therefore, in some embodiments, the invention provides a single unit dose comprising, for example, a heterotandem bicyclic peptide complex as described herein, an immuno-oncology agent, and, where appropriate, a pharmaceutically acceptable carrier, adjuvant, or mediator.

[0288] The amount of heterotandem bicyclic peptide complexes and immuno-oncology agents that can be combined with carrier materials to produce a single dose form (e.g., as described herein) will vary depending on the host being treated and the specific administration method. Preferably, the compositions of the present invention should be formulated such that a dose (e.g., as described herein) of the heterotandem bicyclic peptide complex can be administered in the range of 0.001-100 mg / kg body weight / day.

[0289] (For example) heterotandem bicyclic peptide complexes comprising one or more CD137-binding peptide ligands as described herein and immuno-oncology agents can act synergistically. Therefore, the amount of (for example) heterotandem bicyclic peptide complexes and immuno-oncology agents in such compositions may be less than that required in monotherapy using only the therapeutic agent.

[0290] The amount of the immuno-oncology agent present in the compositions of the present invention may not exceed the amount normally administered in a composition comprising it as the sole active agent. Preferably, the amount of the immuno-oncology agent in the compositions disclosed in the present invention will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the sole therapeutic active agent. In some embodiments, the immuno-oncology agent is administered at a dose of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount normally administered as a monotherapy. As used herein, the phrase "normally administered" means the amount of an FDA-approved therapeutic agent approved for administration according to the FDA label insert.

[0291] The pharmaceutical compositions of the present invention can also be incorporated into compositions for coating implantable medical devices such as prostheses, artificial valves, vascular grafts, endovascular stents, and catheters. Vascular stents have been used, for example, to overcome restenosis (restenosis of the blood vessel wall after injury). However, patients using stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition containing a kinase inhibitor. Implantable devices coated with the compounds of the present invention are another embodiment of the present invention. 5. Exemplary Immuno-oncology Agents

[0292] As used herein, the term "immuno-oncology agent" refers to an agent that effectively enhances, stimulates, and / or upregulates an individual's immune response. In some embodiments, administration of an immuno-oncology agent and (for example) a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands as described herein has a synergistic effect in the treatment of cancer.

[0293] Immuno-oncology agents may be, for example, small molecule drugs, antibodies, or biomolecules or small molecules. Examples of biological immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies, and interleukins. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a humanized or human antibody.

[0294] In some embodiments, the immuno-oncology agent is (i) a agonist that stimulates (including co-stimulates) receptors or (ii) an antagonist that inhibits (including co-inhibits) signals on T cells, both of which induce an amplified antigen-specific T cell response.

[0295] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to members of the homologous TNF receptor family, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, and CD137. (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT βR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0296] In some embodiments, the immuno-oncology agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF and other immunosuppressive cytokines) or a cytokine that stimulates T cell activation to stimulate an immune response.

[0297] In some embodiments, (for example) a combination of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands as described herein with an immuno-oncology agent can stimulate a T-cell response. In some embodiments, the heterotandem bicyclic peptide complex is BT7480 or BT7455 or a pharmaceutically acceptable salt thereof. In some embodiments, the immuno-oncology agent is: (i) an antagonist of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galactoglobulin 9, CEACAM-1, BTLA, CD69, galactoglobulin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; or (ii) an agonist of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0298] In some embodiments, the immuno-oncology agent is an antagonist of an inhibitory receptor on NK cells or an agonist of an activating receptor on NK cells. In some embodiments, the immuno-oncology agent is an antagonist of KIR, such as lirilumab.

[0299] In some embodiments, the immuno-oncology agent is an agent that inhibits or depletes macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249; WO13169264; WO14 / 036357).

[0300] In some embodiments, the immuno-oncology agent is selected from: agonists that connect to positive co-stimulatory receptors; agents that attenuate signaling by inhibiting receptors, antagonists and one or more agents that systematically increase the frequency of anti-tumor T cells; agents that overcome unique immunosuppressive pathways within the tumor microenvironment (e.g., blocking receptor binding (e.g., PD-L1 / PD-1 interaction), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by depletion of ex vivo anti-CD25 beads), inhibiting metabolic enzymes such as IDO or reversing / preventing T cell energy or depletion); and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0301] In some embodiments, the immuno-oncology agent is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an antagonistic CTLA-4 antibody. In some embodiments, the antagonistic CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab.

[0302] In some embodiments, the immuno-oncology agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the immuno-oncology agent is an antibody or its antigen-binding portion that specifically binds to the planned death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is an antagonistic PD-1 antibody. In some embodiments, the antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 145493). In some embodiments, the immuno-oncology agent may be pidilizumab (CT-011). In some embodiments, the immuno-oncology agent is a recombinant protein, referred to as AMP-224, consisting of the extracellular domain (B7-DC) of PD-L2 fused to the Fc portion of IgG1.

[0303] In some embodiments, the immuno-oncology agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonistic PD-L1 antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG7446; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174).

[0304] In some embodiments, the immuno-oncology agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonistic LAG-3 antibody. In some embodiments, the LAG3 antibody is BMS-986016 (WO10 / 19570, WO14 / 08218) or IMP-731 or IMP-321 (WO08 / 132601, WO009 / 44273).

[0305] In some embodiments, the immuno-oncology agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is an agonist CD137 antibody. In some embodiments, the CD137 antibody is urerutumab or PF-05082566 (WO12 / 32433).

[0306] In some embodiments, the immuno-oncology agent is a GITR activator. In some embodiments, the GITR activator is an activating GITR antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO006 / 105021, WO009 / 009116) or MK-4166 (WO11 / 028683).

[0307] In some embodiments, the immuno-oncology agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some embodiments, the IDO antagonist is selected from epacadostat (INCB024360, Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmanitib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906 / KD108 (Phytoceutica); kynase (Ikena Oncology, formerly known as Kyn Therapeutics); and NLG-919 (WO09 / 73620, WO009 / 1156652, WO11 / 56652, WO12 / 142237).

[0308] In some embodiments, the immuno-oncology agent is an OX40 activator. In some embodiments, the OX40 activator is an activating OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0309] In some embodiments, the immuno-oncology agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40 antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO06 / 029879).

[0310] In some embodiments, the immuno-oncology agent is a CD40 agonist. In some embodiments, the CD40 agonist is a agonist CD40 antibody. In some embodiments, the immuno-oncology agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonist CD40 antibody. In some embodiments, the CD40 antibody is lucarumumab or dacetuzumab.

[0311] In some embodiments, the immuno-oncology agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonist CD27 antibody. In some embodiments, the CD27 antibody is varlilumab.

[0312] In some embodiments, the immuno-oncology agent is MGA271 (targeting B7H3) (WO11 / 109400).

[0313] In some embodiments, the immuno-oncology agents are abagovomab, adalimumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolimusab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, icardostat, epratuzumab, indomod, and inotuzumab. (ozogamicin), intelumumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, olatatumab, pembrolizumab, pilizumab, rituximab, ticilimumab, samalizumab, or trimetumab.

[0314] In some embodiments, the immuno-oncology agent is an immunostimulant. For example, antibodies that block the PD-1 and PD-L1 inhibitory axis can release activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses and increase the number of tumor tissue structures, including some tumor types not known to be sensitive to immunotherapy. See, for example, Okazaki, T. et al. (2013) Nat. Immunol. 14, 1212-1218; Zou et al. (2016) Sci. Transl. Med. 8. The anti-PD-1 antibody nivolumab (OPDIVO®, Bristol-Myers Squibb, also known as ONO-4538, MDX1106, and BMS-936558) has been shown to improve overall survival in RCC patients who have experienced disease progression during or after anti-angiogenic therapy.

[0315] In some embodiments, the immunomodulatory therapeutic agent specifically induces apoptosis in tumor cells. Approved immunomodulatory therapeutic agents that can be used in this invention include pomalidomide (POMALYST®, Celgene); lenalidomide (REVLIMID®, Celgene); and ingenol mebutate (PICATO®, LEO Pharma).

[0316] In some embodiments, the immuno-oncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from sipuleucel-T (PROVENGE®, Dendreon / Valeant Pharmaceuticals), which has been approved for the treatment of asymptomatic or mildly symptomatic metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (IMLYGIC®, BioVex / Amgen, formerly known as T-VEC), a genetically modified oncolytic virus therapy approved for the treatment of unresectable cutaneous, subcutaneous, and nodular lesions of melanoma.In some embodiments, immuno-oncology agents are selected from oncolytic virus therapies, such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly known as Jennerex Biotherapeutics), an engineered thymidine kinase-(TK-)-deficient vaccinia virus expressing GM-CSF, used for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep (REOLYSIN®, Oncolytics) Biotech, a variant of the respiratory enteric-coated orphan virus (reovirus) that cannot replicate in non-RAS-activated cells, is used in numerous cancers, including colorectal cancer (NCT01622543), prostate cancer (NCT01619813), head and neck squamous cell carcinoma (NCT01166542), pancreatic adenocarcinoma (NCT00998322), and non-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev (NG-348, PsiOxus (formerly known as ColoAd1), an engineered adenovirus expressing a full-length CD80 and antibody fragment specific to the T-cell receptor CD3 protein, is used for ovarian cancer (NCT02028117), metastatic or advanced epithelial tumors such as colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly known as Oncos), an engineered adenovirus expressing GM-CSF, is used for melanoma (NCT03003676) and peritoneal diseases, colorectal cancer, or ovarian cancer (NCT02963831); GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux) GmbH, engineered vaccinia virus expressing either β-galactosidase (β-gal) / β-glucuronidase or β-gal / human sodium iodide transporter (hNIS), is being investigated for use in peritoneal cancer (NCT01443260), fallopian tube cancer, and ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF, is being investigated for use in bladder cancer (NCT02365818).

[0317] In some embodiments, the immuno-oncology agents are selected from JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK-deficient and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which can convert the prodrug 5-fluorocytosine into the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapeutic agents targeting refractory RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus called Ad5 / 3-E2F-δ24-hTNFα-IRES-hIL20; and VSV-GP (ViraTherapeutics) is a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningovirus (LCMV), which can be further engineered to express antigens designed to enhance antigen-specific CD8+ T cell responses.

[0318] In some embodiments, the immuno-oncology agent is a T cell engineered to represent a chimeric antigen receptor or CAR. T cells engineered to represent this chimeric antigen receptor are called CAR-T cells.

[0319] A CAR has been constructed, which consists of the following: a binding domain, which may be derived from a natural ligand; and a single-chain variable fragment (scFv), derived from a monoclonal antibody specific for cell surface antigens, fused with an intracellular domain that is the functional terminus of the T cell receptor (TCR), such as the CD3-ζ signaling domain from the TCR capable of generating activation signals in T lymphocytes. Upon antigen binding, this type of CAR links to the endogenous signaling pathway in effector cells and generates an activation signal similar to that induced by the TCR complex.

[0320] For example, in some embodiments, the CAR-T cells are one of those cells described in U.S. Patent 8,906,682 (June et al.; incorporated herein by reference in its entirety), which discloses CAR-T cells engineered to include an extracellular domain having an antigen-binding domain (such as a domain binding to CD19) fused to an intracellular signaling domain of a T-cell antigen receptor complex ζ chain (such as CD3ζ). When expressed in T cells, the CAR is able to redirect antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Currently, more than 200 clinical trials using CAR-T in a wide range of indications are underway. [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0321] In some embodiments, the immunostimulant is an activator of retinoic acid receptor-associated orphan receptor γ (RORγt). RORγt is a transcription factor that plays a key role in the differentiation and maintenance of the type 17 effector subset of CD4+ (Th17) and CD8+ (Tc17) T cells and in the differentiation of innate immune cell subsets expressing IL-17 (such as NK cells). In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in a clinical trial for the treatment of solid tumors (NCT02929862).

[0322] In some embodiments, the immunostimulant is a agonist or activator of a TLR-like receptor (TLR). Suitable activators of TLRs include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG and is being investigated for use in B-cell lymphoma, follicular lymphoma, and other lymphomas (NCT02254772). A agonist or activator of TLR8 that can be used in this invention includes motolimod (VTX-2337, VentiRx Pharmaceuticals), which is being investigated for use in head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT02431559).

[0323] Other immuno-oncology agents that can be used in this invention include urilumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; valimumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lireliumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; andecaliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[0324] In some embodiments, the immunostimulant is selected from elotuzumab, mifamurtide, agonists or activators of dorsal-like receptors and activators of RORγt.

[0325] In some embodiments, the immunostimulatory agent is recombinant human interleukin-15 (rhIL-15). rhIL-15 has been tested in clinical trials as a therapy for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888) and leukemia (NCT02689453). In some embodiments, the immunostimulator is recombinant human interleukin-12 (rhIL-12). In some embodiments, the IL-15-based immunotherapy agent is heterodimeric IL-15 (hetIL-15, Novartis / Admune), a synthetic fusion complex (IL15:sIL-15RA) consisting of an endogenous IL-15 complexed with the α chain of the soluble IL-15-binding protein IL-15 receptor, which has been tested in Phase 1 clinical trials for melanoma, renal cell carcinoma, non-small cell lung cancer, and head and neck squamous cell carcinoma (NCT02452268). In some embodiments, recombinant human interleukin-12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724, or NCT02542124.

[0326] In some embodiments, the immuno-oncology agents are selected from those described in Jerry L. Adams et al., "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, Vol. 14, pp. 603-622, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agents are selected from the examples described in Table 1 of Jerry L. Adams et al. In some embodiments, the immuno-oncology agents are small molecules targeting immuno-oncology targets selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the immuno-oncology agents are small molecule agents selected from those listed in Table 2 of Jerry L. Adams et al.

[0327] In some embodiments, the immuno-oncology agent is selected from the small molecule immuno-oncology agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents", Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pp. 319-329, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agent is an agent that targets the pathway described in Peter L. Toogood.

[0328] In some embodiments, the immuno-oncology agent is selected from those described in Sandra L. Ross et al., "Bispecific T cell engager (BITE®) antibody constructs can mediate bystander tumor cell killing", PLoS ONE 12(8): e0183390, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agent is a bispecific T cell engager (BITE®) antibody construct. In some embodiments, the bispecific T cell engager (BITE®) antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager (BITE®) antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager (BITE®) antibody construct activates T cells. In some embodiments, a bispecific T-cell conjugate (BITE®) antibody construct activates T cells, releasing cytokines that induce upregulation of intercellular adhesion molecule-1 (ICAM-1) and FAS on neighboring cells. In some embodiments, the bispecific T-cell conjugate (BITE®) antibody construct activates T cells, thereby inducing the lysis of neighboring cells. In some embodiments, the neighboring cells are in a solid tumor. In some embodiments, the lysed neighboring cells are adjacent to the BITE®-activated T cells. In some embodiments, the neighboring cells comprise tumor-associated antigen (TAA)-negative cancer cells. In some embodiments, the neighboring cells comprise EGFR-negative cancer cells. In some embodiments, the immuno-oncology agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the immuno-oncology agent is ex vivo expanded tumor-infiltrating T cells. In some embodiments, the immuno-oncology agent is a bispecific antibody construct or chimeric antigen receptor (CAR) that directly links T cells to tumor-associated surface antigens (TAAs). Exemplary checkpoint inhibitors

[0329] In some embodiments, the immuno-oncology agent is an immune checkpoint inhibitor as described herein.

[0330] As used herein, the term "checkpoint inhibitor" refers to agents that prevent cancer cells from evading a patient's immune system. One of the main mechanisms of antitumor immune destruction is called "T-cell exhaustion," which is caused by prolonged exposure to antigens that have induced upregulation of inhibitory receptors. These inhibitory receptors act as immune checkpoints to prevent uncontrolled immune responses.

[0331] PD-1 and its co-inhibitory receptors (such as cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuation factor (BTLA; CD272), T cell immunoglobulin and mucin domain-3 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD223) and others) are commonly referred to as checkpoint regulators. They act as "gatekeepers" of molecules that allow extracellular information to indicate whether cell cycle progression and other intracellular communication processes should continue.

[0332] In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1. PD-1 binds to the planned cell death 1 receptor (PD-1) to prevent the receptor from binding to the inhibitory ligand PDL-1, thereby suppressing the tumor's ability to suppress the host's anti-tumor immune response.

[0333] In some embodiments, the checkpoint inhibitor is a biological therapeutic agent or a small molecule. In some embodiments, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. In some embodiments, the checkpoint inhibitor inhibits checkpoint proteins selected from the following: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or a combination thereof. In some embodiments, the checkpoint inhibitor interacts with ligands selected from the following checkpoint proteins: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof. In some embodiments, the checkpoint inhibitor is an immunostimulant, T-cell growth factor, interleukin, antibody, vaccine, or a combination thereof. In some embodiments, the interleukin is IL-7 or IL-15. In some embodiments, the interleukin is glycosylated IL-7. In one extramorphic sample, the vaccine is a dendritic cell (DC) vaccine.

[0334] Checkpoint inhibitors include any agent that blocks or inhibits a suppressive pathway of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Illustrated checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 molecule family and expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies or their antigen-binding fragments, other binding proteins, biological therapeutics, or small molecules that bind to and block or inhibit the activity of one or more of the following: CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Illustrative immune checkpoint inhibitors include, but are not limited to, trametumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody), and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0335] In some embodiments, the immune checkpoint inhibitor is selected from PD-1 antagonists, PD-L1 antagonists, and CTLA-4 antagonists. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (OPDIVO®), ipilimumab (YERVOY®), and pembrolizumab (KEYTRUDA®). In some embodiments, the checkpoint inhibitor is selected from nivolumab (anti-PD-1 antibody, OPDIVO®, Bristol-Myers Squibb); pembrolizumab (anti-PD-1 antibody, KEYTRUDA®, Merck); ipilimumab (anti-CTLA-4 antibody, YERVOY®, Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody, IMFINZI®, AstraZeneca); and atezolizumab (anti-PD-L1 antibody, TECENTRIQ®, Genentech).

[0336] In some embodiments, the checkpoint inhibitor is selected from the group consisting of: larizumab (MK-3475), nivolumab (BMS-936558), pilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lireliumab, IPH2101, pembrolizumab (KEYTRUDA®), and trametumab.

[0337] In some embodiments, the immune checkpoint inhibitor is REGN2810 (Regeneron), an anti-PD-1 antibody tested in patients with basal cell carcinoma (NCT03132636), NSCLC (NCT03088540), squamous cell carcinoma of the skin (NCT02760498), lymphoma (NCT02651662), and melanoma (NCT03002376); pilithumab (CureTech), also known as CT-011, an antibody that binds to PD-1 in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avelumab (BAVENCIO®, Pfizer / Merck). KGaA (also known as MSB0010718C) is a fully human IgG1 anti-PD-L1 antibody used in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, kidney cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer; or PDR001 (Novartis) is a PD-1-binding inhibitory antibody used in clinical trials for non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Trimetazaprine (CP-675,206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4 that has been investigated in clinical trials for a wide range of indications, including: mesothelioma, colorectal cancer, renal cell carcinoma, breast cancer, lung and non-small cell lung cancer, pancreatic duct adenocarcinoma, pancreatic cancer, germ cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, endometrial cancer, metastatic liver cancer, hepatocellular carcinoma, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic degenerative thyroid cancer, urothelial carcinoma, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody that has been investigated in a Phase 1 clinical trial for advanced solid tumors (NCT02694822).

[0338] In some embodiments, the checkpoint inhibitor is an inhibitor of T-cell immunoglobulin mucin (TIM-3), which contains protein-3. TIM-3 inhibitors that can be used in this invention include TSR-022, LY3321367, and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody studied in advanced malignant disease (NCT02608268).

[0339] In some embodiments, the checkpoint inhibitor is an inhibitor of a T-cell immune receptor having both Ig and ITIM domains or of TIGIT (an immune receptor on certain T cells and NK cells). TIGIT inhibitors that can be used in this invention include BMS-986207 (Bristol-Myers Squibb), an anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and an anti-TIGIT monoclonal antibody (NCT03119428).

[0340] In some embodiments, the checkpoint inhibitor is an inhibitor of lymphocyte activation gene-3 (LAG-3). LAG-3 inhibitors that can be used in this invention include BMS-986016, REGN3767, and IMP321. BMS-986016 (Bristol-Myers Squibb) (an anti-LAG-3 antibody) has been studied in glioblastoma and glioma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody and has been studied in malignant diseases (NCT03005782). IMP321 (Immutep SA) is a LAG-3-Ig fusion protein that has been studied in melanoma (NCT02676869), adenocarcinoma (NCT02614833), and metastatic breast cancer (NCT00349934).

[0341] Checkpoint inhibitors that can be used in this invention include OX40 agonists. OX40 agonists studied in clinical trials include: PF-04518600 / PF-8600 (Pfizer), an agonist anti-OX40 antibody used in metastatic renal cell carcinoma (NCT03092856) and advanced cancer and tumors (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonist anti-OX40 antibody used in a phase 1 cancer trial (NCT02528357); MEDI0562 Medimmune / AstraZeneca, a activating anti-OX40 antibody for use in advanced solid tumors (NCT02318394 and NCT02705482); MEDI6469, a activating anti-OX40 antibody (Medimmune / AstraZeneca) for use in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705); and BMS-986178 (Bristol-Myers Squibb), a activating anti-OX40 antibody for use in advanced cancer (NCT02737475).

[0342] Checkpoint inhibitors that can be used in this invention include CD137 (also known as 4-1BB) agonists. CD137 agonists investigated in clinical trials include utomilumab (PF-05082566, Pfizer), an agonist anti-CD137 antibody used in diffuse large B-cell lymphoma (NCT02951156) and advanced cancers and tumors (NCT02554812 and NCT05082566); utomilumab (BMS-663513, Bristol-Myers Squibb), an agonist anti-CD137 antibody used in melanoma and skin cancer (NCT02652455) and glioblastoma and glioma (NCT02658981); and CTX-471 (Compass Therapeutics), an agonist anti-CD137 antibody used in metastatic or locally advanced malignant disease (NCT03881488).

[0343] Checkpoint inhibitors that can be used in this invention include CD27 agonists. CD27 agonists studied in clinical trials include: valimumab (CDX-1127, Celldex Therapeutics), an agonist anti-CD27 antibody used in squamous cell head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma and glioblastoma (NCT02335918), lymphoma (NCT01460134) and glioma and astrocytoma (NCT02924038).

[0344] Checkpoint inhibitors that can be used in this invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists investigated in clinical trials include: TRX518 (Leap Therapeutics), an agonist anti-GITR antibody for malignant melanoma and other malignant solid tumors (NCT01239134 and NCT02628574); GWN323 (Novartis), an agonist anti-GITR antibody for solid tumors and lymphomas (NCT 02740270); INCAGN01876 (Incyte / Agenus), an agonist anti-GITR antibody for advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonist anti-GITR antibody for solid tumors (NCT02132754); and MEDI1873 (Medimmune / AstraZeneca), an antibody for advanced solid tumors (NCT02583165) containing human IgG1. Fc domain-promoting hexameric GITR-ligand molecules.

[0345] Checkpoint inhibitors that can be used in this invention include inducible T-cell costimulators (ICOS, also known as CD278) agonists. ICOS agonists studied in clinical trials include: MEDI-570 (Medimmune), an agonist anti-ICOS antibody used in lymphoma (NCT02520791); GSK3359609 (Merck), an agonist anti-ICOS antibody used in phase 1 (NCT02723955); and JTX-2011 (Jounce Therapeutics), an agonist anti-ICOS antibody used in phase 1 (NCT02904226).

[0346] Checkpoint inhibitors that can be used in this invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors being studied in clinical trials include: lireuromab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody used in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma) used in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody (KIR3DL2) that binds to three domains of the long cytoplasmic tail used in lymphoma (NCT02593045).

[0347] Checkpoint inhibitors that can be used in this invention include CD47 inhibitors of the interaction between CD47 and signal regulatory protein α (SIRPa). CD47 / SIRPa inhibitors being studied in clinical trials include: ALX-148 (Alexo Therapeutics), an antagonistic variant of SIRPa that binds to CD47 and prevents CD47 / SIRPa-mediated signaling in a phase 1 trial (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein formed by linking the N-terminal CD47-binding domain of SIRPa to the Fc domain of human IgG1 in phase 1 clinical trials (NCT02890368 and NCT02663518), which works by binding to human CD47 and preventing it from delivering its "do not eat" signal to macrophages; CC-90002 (Celgene), an anti-CD47 antibody used in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc., which is used for colorectal cysts and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[0348] Checkpoint inhibitors that can be used in this invention include CD73 inhibitors. CD73 inhibitors studied in clinical trials include MEDI9447 (Medimmune), an anti-CD73 antibody for solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody for solid tumors (NCT02754141).

[0349] Checkpoint inhibitors that can be used in this invention include agonists of interferon gene-stimulating protein (STING, also known as transmembrane protein 173 or TMEM173). STING agonists studied in clinical trials include: MK-1454 (Merck), an agonist synthetic cyclic dinucleotide used in lymphoma (NCT03010176); and ADU-S100 (MIW815, Aduro Biotech / Novartis), an agonist synthetic cyclic dinucleotide used in phase 1 trials (NCT02675439 and NCT03172936).

[0350] The checkpoint inhibitors that can be used in this invention include CSF1R inhibitors. CSF1R inhibitors studied in clinical trials include: pexidartinib (PLX3397, Plexxikon), a small molecule CSF1R inhibitor used in colorectal cancer, pancreatic cancer, metastatic and advanced cancer (NCT02777710), as well as melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST), and ovarian cancer (NCT02452424); and IMC-CS4 (LY3022855, Lilly), an anti-CSF-1R antibody used in pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT02718911); and BLZ945. (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid methyl amide, Novartis), an orally effective inhibitor of CSF1R for use in advanced solid tumors (NCT02829723).

[0351] Checkpoint inhibitors that can be used in this invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors studied in clinical trials include monazumab (IPH2201, Innate Pharma), an anti-NKG2A antibody used in head and neck sarcoma (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0352] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avirumab, durvalumab, atezolizumab, or pilizumab. Examples

[0353] The following examples illustrate the invention described above; however, they are not intended to limit the scope of the invention in any way. The beneficial effects of the pharmaceutical compounds, combinations, and compositions of the present invention can also be determined by other testing models known to those skilled in the art. Example 1: Transcription mapping analysis study using BCY12491 mapping analysis study

[0354] For transcriptional and immunohistochemical (IHC) analysis, 1 × 10⁶ MC38 cells were subcutaneously implanted into 6-8 week old female huCD137-C57B / 6J (Biocytogen) mice. When the mean tumor volume reached approximately 240 mm³, mice were randomly assigned to treatment groups and treated intravenously with a mordant (25 mM histidine, 10% sucrose, pH 7), 15 mg / kg BCY12491, or 15 mg / kg BCY13626 (non-binding control), or intraperitoneally with 2 mg / kg anti-CD137 antibody urerutumab. Treatment was administered at three doses Q3D, and tumor growth was monitored by caliper measurement. Tumor tissue was collected on day 6 one hour after the last dose. A portion of tumor tissue was used for RNA isolation and transcriptional analysis, while another portion was used in a formalin-fixed paraffin-embedded (FFPE) sample preparation for IHC analysis. RNA lines were isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcribed using the nCounter Mouse PanCancer IO 360 panel (Nanostring) at a concentration of 100 ng RNA / tumor. Data were analyzed using nSolver analysis software with the advanced analytical probe set ns_mm_io_360_v1.0 (Nanostring). CD8+ tumor-infiltrating cells were stained in FFPE tissue sections using an anti-mouse CD8 antibody (Abcam, #ab217344) and the Ventana Discovery OmniMap anti-rabbit-HRP kit (Ventana #760 4310).

[0355] The data is shown in Figure 1.

[0356] Results: Transcriptional analysis revealed that, compared with tumors from mice treated with the EPhA2 heterotandem bicyclic peptide complex BCY12491, immune cell scores such as cytotoxic cell score, T cell score, and macrophage cell score were significantly increased in tumor tissues. Anti-CD137 antibody treatment also significantly increased cytotoxic cell and T cell scores in tumor tissues, but to a lesser extent than BCY12491. No changes in immune cell scores were observed in tumor tissues from animals treated with the unbound control (BCY13626). IHC analysis of CD8+ cells in tumor tissues confirmed strong CD8+ cell infiltration in tumors from mice treated with BCY12491 compared with tumors from mice treated with either the mediated or unbound BCY13626. Some increase in CD8+ cell infiltration was also observed in tumors from mice treated with the anti-CD137 antibody. These changes in immune cell scores and CD8+ cell counts in tumor tissue indicate that the EphA2 / CD137 heterotandem bicyclic peptide complex BCY12491 promotes the activity of CD137 in tumor tissue, leading to a significant regulation (increase) of tumor-infiltrating immune cells and immune responses. Studies using BT7480...

[0357] For transcriptional and immunohistochemical (IHC) analysis, 1 × 10⁶ MC38#13 cells (engineered to express protein-binding protein-4) were subcutaneously implanted into 6-8 week old female huCD137-C57B / 6J (Biocytogen) mice. When the mean tumor volume reached approximately 255 mm³, the mice were randomly assigned to treatment groups to receive a mediator, BT7480 (BCY00011863), a non-binding agent BCY control BCY00012797 (BCY12797), or an αCD137 antibody (urerulumab analogue). BT7480 and its unbound control were administered intravenously at 0 h and 24 h at 5 mg / kg (in 25 mM histidine HCl, 10% sucrose, pH 7; catalyst), and a urorigumab analogue was administered intraperitoneally at 2 mg / kg during PBS BIW (0 h, 72 h) doses and schedules. Tumors from mice treated with BT7480 were collected at 24 h (after 0 h administration), 48 h (24 h after the last 0 h and 24 h administration), 96 h (72 h after the last 0 h and 24 h administration), and 144 h (120 h after the last 0 h and 24 h administration). Tumors from mice treated with αCD137 were collected 144 h after the start of treatment. Tumors from mice treated with the catalyst were collected 24 h and 144 h after 0 h administration (120 h after the last 0 h and 24 h administration). RNA lines were isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcriptional analysis was performed using the nCounter Mouse PanCancer IO 360 panel (Nanostring) with 100 ng RNA / tumor. Data were analyzed using nSolver analysis software with advanced analytical probes ns_mm_io_360_v1.0 (Nanostring).

[0358] The data is shown in Figures 2 to 4.

[0359] Results: Transcriptional analysis revealed a significant early (24-hour) increase in the mRNA of several T-cell chemokines / cytokines (such as Ccl1, Ccl17, and Ccl24, which are thought to be secreted by bone marrow cells, thereby causing T-cell recruitment to chemokine secretion sites). Transcriptional analysis also revealed a significant increase in immune cell scores, such as cytotoxic cell scores and macrophage cell scores, in tumor tissues after BT7480 treatment compared to tumors from mice treated with the chemokine. Macrophage cell scores began to increase 24 hours after BT7480 administration and increased significantly from the 24-hour chemokine reading to 48 hours. On the other hand, when cytotoxic cell scores were significantly increased compared to tumors treated with the chemokine at 144 hours, cytotoxic cell scores began to increase 48 hours after treatment initiation and continued to increase until 144 hours. The summation of cytotoxic cell scores in response to BT7480 and normalized mRNA counts of Ccl1, Ccl17, and Ccl24 will confirm how the increase in Ccl1, Ccl17, and Ccl24 transcription precedes the increase in cytotoxic cell scores.

[0360] The superposition of macrophage and cytotoxic cell scores in response to BT7480 will confirm how the increase in macrophage cell score precedes the increase in cytotoxic cell score.

[0361] Transcriptional analysis revealed a trend of increased or significantly increased mRNA levels at several different immune checkpoints supporting the concept of combining BT7480 with a checkpoint inhibitor. These immune checkpoints include CTLA-4 (Ctla4), PD-1 (Pdcd1), PD-L1 (Cd274), LAG3 (Lag3), TIM3 (Havcr2), PD-L2 (Pdcd1lg2), and TIGIT (Tigit). Example 2: Efficacy study using the combination of BCY12491 and pembrolizumab.

[0362] For tumor growth analysis, 1×10⁶ MC38 cells were subcutaneously implanted into 6-8 week old female huCD137 / huPD-1-C57B / 6J mice (Biocytogen). When the average tumor volume reached approximately 92 mm³, the mice were randomly assigned to treatment groups and treated intravenously with a mordant (25 mM histidine, 10% sucrose, pH 7) and 5 mg / kg BCY12491 (0, 24 h), or intraperitoneally with 3 mg / kg anti-PD-1 antibody pembrolizumab or a combination of BCY12491 and pembrolizumab. Combination therapy was administered in three different dosing schedules: simultaneous BCY12491 and pembrolizumab treatment starting on day 0; BCY12491 treatment starting on day 1 and pembrolizumab treatment starting on day 5; or pembrolizumab treatment starting on day 0 and BCY12491 treatment starting on day 5. Treatment was administered four times weekly, and tumor growth was monitored using a caliper.

[0363] The data is shown in Figures 5 and 6.

[0364] Results: When compared with the carcass control, BCY12491 and pembrolizumab monotherapy and their combination therapy demonstrated significant antitumor activity (all p < 0.0001, mixed-effects analysis and post-Dunnet test, which compared treatment with carcass at D18). Furthermore, the combination therapy was more effective than either monotherapy alone (p < 0.0001, mixed-effects analysis and post-Dunnet test, which compared combination with monotherapy at D20), resulting in complete responses in all treated animals up to day 22. In contrast, these treatment regimens and schedules resulted in 2 / 10 complete responses in the BCY12491 monotherapy group and 3 / 10 complete responses in the pembrolizumab monotherapy group. Alternating sequences of BCY12491 and pembrolizumab (BCY12491 treatment starting on day 0 followed by pembrolizumab treatment starting on day 5, or vice versa) also produced significant antitumor activity (***p < 0.0001 for both, mixed-effects analysis and post-Dunnet test, which compared treatment with the mordant at D18). Both sequences resulted in 9 / 10 complete responses (BCY12491 treatment starting on day 0 followed by pembrolizumab treatment starting on day 5) and 8 / 10 complete responses (pembrolizumab treatment starting on day 0 followed by BCY12491 treatment starting on day 5) in treated mice up to day 42. Example 3: Efficacy study of BCY11864 and anti-PD-1 combination.

[0365] For tumor growth analysis, 3 × 10⁺e⁵ CT26#7 cells (engineered to overexpress protein-binding protein-4) were subcutaneously implanted into 6-8 week old female Balb / c-huCD137 mice (Gempharmatech). When the average tumor volume reached approximately 80 mm³, mice were randomly assigned to treatment groups and treated intravenously with a mordant (25 mM histidine, 10% sucrose, pH 7) and 10 mg / kg BCY11864 (0, 24 h), or intraperitoneally with 10 mg / kg anti-PD-1 antibody (RMP1-14) or a combination of BCY11864 and anti-PD-1 antibody. Treatment was administered weekly, and tumor growth was monitored by caliper measurement. Animals with tumors >2000 mm³ were euthanized when they reached the humanitarian endpoint. The study was terminated on day 66 after the start of treatment, at which point only two animals (both in the combined treatment team) remained in the study (one a complete responder and the other whose tumor size was still regressing).

[0366] The data is shown in Figure 7.

[0367] Results: Adding BCY11864 to anti-PD-1 monotherapy significantly (p=0.004, Mantel-Cox log-rank test, which compares anti-PD-1 with the anti-PD-1+BCY11864 combination group) increased the survival time of mice carrying CT26#7 (results were measured as the time to reach the humanitarian endpoint (i.e., tumor volume > 2000 mm3)). Example 4. Efficacy study of BT7480 in combination with anti-PD-1 and anti-Ctla-4

[0368] For tumor growth analysis, 1×10+e6 MC38#13 cells (engineered to overexpress protein-binding protein-4) were subcutaneously implanted into 6-8 week old female C57Bl / 6J-huCD137 mice (Biocytogen). When the mean tumor volume reached approximately 100 mm3, mice were randomly assigned to treatment groups and treated intraperitoneally with a mordant (25 mM histidine, 10% sucrose, pH 7), 1 mg / kg BT7480, 5 mg / kg anti-PD-1 (RMP 1-14), 5 mg / kg anti-Ctla-4 (9H10), or a combination of BT7480 / anti-PD-1 and BT7480 / anti-Ctla-4. Treatment was administered twice weekly (BIW) for 2 weeks, and tumor growth was monitored by caliper measurement until day 33 after the start of treatment. Animals with tumors >2000 mm3 were euthanized when they reached the human end of their lives.

[0369] The data is shown in Figures 8 and 9.

[0370] Results: Adding BT7480 to anti-PD-1 monotherapy increased the complete response rate (CR) from 0 / 8 (in the BT7480 and anti-PD-1 monotherapy group) to 2 / 8 in the BT7480 / anti-PD-1 combination therapy group. By day 33 of treatment, adding BT7480 to anti-Ctla-4 monotherapy increased the complete response rate (CR) from 0 / 8 or 1 / 8 (in the BT7480 and anti-Ctla-4 monotherapy groups, respectively) to 4 / 8 in the BT7480 / anti-Ctla-4 combination therapy group. Furthermore, adding BT7480 to anti-CTLA-4 monotherapy significantly (p=0.0499, Mantel-Cox log-rank test, which compares anti-CTLA-4 with the anti-CTLA-4 + BT7480 combination) increased the survival time of mice carrying MC38#13 (the result was measured as the time to reach the humane endpoint (i.e., tumor volume > 2000 mm3)). Example 5. Transcriptional profiling analysis using BT7455.

[0371] Transcriptional profiling analysis of the effects of BT7455 on the immunotumor microenvironment was performed by subcutaneously implanting 1×10+E6 MC38 cells into 6-8 week old female huCD137-C57B / 6J (Biocytogen) mice. When the mean tumor volume reached approximately 350 mm3, the mice were randomly assigned to treatment groups to receive a mediator, BT7455, αCD137 antibody (uregumab analogue), or αPD-1 antibody. BT7455 was administered intravenously at 0 h and 24 h at 8 mg / kg (in 25 mM histidine HCl, 10% sucrose, pH 7; mediator), and at 0 h, uregumab analogue and αPD-1 antibody were administered intraperitoneally in PBS at 2 mg / kg (uregumab analogue) or 10 mg / kg (αPD-1 antibody). Tumors were collected from mice treated with the mediated agent, BT7455, urrelumab analogue, and αPD-1 antibody at 24h, 48h, and 144h after treatment initiation. RNA lines were isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcribed using the nCounter Mouse PanCancer IO 360 panel (Nanostring) at 100 ng RNA / tumor. Data were analyzed using nSolver analysis software with the advanced analytical probe set ns_mm_io_360_v1.0 (Nanostring).

[0372] The data is shown in Figures 10 to 13.

[0373] Results: Transcriptional analysis revealed a significant increase in mRNAs of several different immune checkpoints supporting the concept of combining BT7455 with checkpoint inhibitors after BT7455 treatment. These immune checkpoints included CTLA-4 (Ctla4), PD-1 (Pdcd1), PD-L1 (Cd274), LAG3 (Lag3), TIM3 (Havcr2), PD-L2 (Pdcd1lg2), and TIGIT (Tigit). Transcriptional analysis also revealed a significant early (24 to 48-hour) increase in mRNAs of several T-cell chemokines / cytokines (such as Ccl1, Ccl17, and Ccl24, which are thought to be secreted by bone marrow cells, thereby causing T-cell recruitment at chemokine secretion sites). Transcriptional analysis also revealed that, compared with tumors from mice treated with mediated agents or anti-PD-1 or anti-CD137, BT7455 treatment significantly increased immune cell scores (such as cytotoxic cell scores) in tumor tissues. BT7455 treatment induced significant early (48-hour) regulation of several gene sets, including those associated with cytokine and chemokine signaling, cytotoxicity, apoptosis, and NK-κB signaling gene sets. [Simplified Explanation of the Diagram]

[0008] Figure 1 illustrates how BCY12491 modulates the tumor immune microenvironment and drives T cell infiltration. (A) Mice carrying MC38 tumors were treated with a mediator, 15 mg / kg EphA2 / CD137 heterotandem bicyclic peptide complex (BCY12491), mirror-image isomer unbound control heterotandem bicyclic peptide complex (BCY13626) q3d iv, or 2 mg / kg αCD137 q3d ip. Individual tumor volumes (normalized to tumor volume on the day treatment began) are shown and grouped according to treatment. (B) Tumor nanostring analysis demonstrates the effects of BCY12491 and αCD137 on the levels of T cells (probe set: Cd3d, Cd3e, Cd3g, Cd6, Sh2d1a, and Trat1), cytotoxic cells (probe set: Ctsw, Gzma, Gzmb, Klrb1, Klrd1, Klrk1, Nkg7, and Prf1), and macrophages (probe set: Cd163, Cd68, Cd84, and Ms4a4a). (C) Tumor nanostring analysis demonstrates the effects of BCY12491 and αCD137 on the transcription of checkpoint inhibitors Pdcd1 (protein PD-1), Cd274 (protein PD-L1), and Ctla4 (protein CTLA-4). (D) Presents representative images of tissue sections from mouse tumors treated with mordant, 15 mg / kg BCY12491, BCY13626, or 2 mg / kg αCD137 Q3D and stained with CD8. (B and C) *<0.05, ***p<0.001, one-way ANOVA and Dunnett's post test.

[0009] Figure 2 illustrates the effect of BT7480 on selected intercytokines / chemokines. (A) The curves are on the left-hand side, showing the normalized linear counts of five different intercytokine / chemokine mRNAs in MC38#13 tumor tissue after BT7480 treatment. (B) The overlay plot of cytotoxicity scores and normalized RNA counts of Ccl1, Ccl-17, and Ccl24 shows an early increase in these intercytokine / chemokine transcripts, followed by an increase in cytotoxicity scores.

[0010] Figure 3 illustrates how BT7480 modulates the tumor immune microenvironment and drives CD8+ T cell infiltration. Mice carrying MC38#13 tumors were treated with a mediator, 5 mg / kg (0h, 24h) of BT7480, or the unbound heterotandem bicyclic peptide complex control BCY12797 (NB-BCY) iv or 2 mg / kg αCD137 ip. Nanofiber analysis of tumors shows the effects of BT7480 and αCD137 over time on the scores of (A) macrophages (probe set: Cd163, Cd68, Cd84, and Ms4a4a) and (B) cytotoxic cells (probe set: Ctsw, Gzma, Gzmb, Klrb1, Klrd1, Klrk1, Nkg7, and Prf1) in tumor tissue. (C) Overlapping plot of cytotoxic cell score and macrophage cell score shows an early increase in macrophage cell score, followed by an increase in cytotoxic cell score. (A and B) *<0.05, **p<0.01, one-way ANOVA and post-Dunnett test.

[0011] Figure 4 illustrates the increase in several immune checkpoint mRNAs induced by BT7480. Mice carrying MC38#13 tumors were treated with a mediator, 5 mg / kg (0h, 24h) of BT7480 or the unbound heterotandem bicyclic peptide complex control BCY12797 (NB-BCY) iv, or 2 mg / kg αCD137 ip. Tumor nanostring analysis shows the effects of BT7480 and αCD137 on the levels of several immune checkpoint mRNAs. *<0.05, **p<0.01, ***p<0.001, one-way ANOVA and post-Dunnett assay.

[0012] Figure 5 depicts the 100% complete response rate achieved by the BCY12491 + pembrolizumab combination up to day 22, starting from day 0 (after treatment initiation). Mice carrying MC38 tumors were treated with a mediator, 5 mg / kg BCY12491 QW (0, 24h), 3 mg / kg pembrolizumab QW, or a combination thereof. The top plot shows the mean tumor volume from treatment initiation to day 28. Both monotherapy and combination therapy significantly affected tumor growth (***p < 0.0001, mixed-effects analysis and post-Dunnet test, compared with mediator at D18). Furthermore, combination therapy was more effective than either monotherapy (***p < 0.0001, mixed-effects analysis and post-Dunnet test, compared with monotherapy at D20), resulting in a complete response in all treated animals up to day 22. The right-hand curves show the growth curves of individual tumors from the treatment population.

[0013] Figure 6 illustrates the significant antitumor activity produced by the BCY12491 + pembrolizumab combination under different dose sequences. Mice carrying MC38 tumors were treated with the following three different dosing schedules: 5 mg / kg BCY12491 QW (0, 24h), 3 mg / kg pembrolizumab QW, or combinations thereof: BCY12491 and pembrolizumab treatment both started on day 0; BCY12491 treatment started on day 0 followed by pembrolizumab treatment on day 5; or pembrolizumab treatment started on day 0 followed by BCY12491 treatment on day 5. The top plot shows the mean tumor volume from the start of treatment to day 28. All combination therapies demonstrated significant antitumor activity, with complete responses observed in 10 / 10 (BCY12491 + pembrolizumab from D0), 9 / 10 (BCY12491 + pembrolizumab from D5 from D0), and 8 / 10 (pembrolizumab from D0 and BCY12491 from D5) by day 42. ***p < 0.0001, mixed-effects analysis and post-Dunnet test, compared with mediators at day 18. The right-hand curve shows the growth curves of individual tumors from the treatment population.

[0014] Figure 7 depicts the addition of BCY11864 to anti-PD-1 monotherapy significantly [p=0.004, log-rank (Mantel-Cox) test, which compares anti-PD-1 and anti-PD-1+BCY11864 combination group] increased the survival of mice carrying CT26#7 (CT26 engineered to overexpress binding protein-4 (Nectin-4)) (defined as reaching the humanitarian endpoint, tumor volume >2000 mm3).

[0015] Figure 8 illustrates the increase in complete response (CR) rate in mice carrying MC38#13 (MC38 engineered to overexpress binding protein-4) by adding BT7480 to anti-PD-1 monotherapy.

[0016] Figure 9 illustrates that adding BT7480 to anti-CTLA-4 monotherapy significantly [p=0.0499, log-rank (Mantel-Cox) test, compared with anti-CTLA-4 and anti-CTLA-4+BT7480 combination group] increased the survival (defined as reaching the human endpoint, tumor volume >2000 mm3) and increased the complete response rate in mice carrying MC38#13 (MC38 engineered to overexpress binding protein-4).

[0017] Figure 10 illustrates the increase in several immune checkpoint mRNAs induced by BT7455. Mice carrying MC38 tumors were treated intravenously with the carboxin, 8 mg / kg (0h, 24h) of BT7455, or intraperitoneally with 2 mg / kg of anti-CD137 antibody or 10 mg / kg of anti-PD-1 antibody. Nanofiber analysis of the tumors shows the effect of treatment on the levels of several immune checkpoint mRNAs. Normalized Log2 counts of mRNAs in MC38 tumor tissues at 24h, 48h, and 144h are shown. *<0.05, **p<0.01, ***p<0.001, one-way ANOVA and post-Dunnett's assays compare the treatment with the carboxin at the same time points.

[0018] Figure 11 depicts the effects of BT7455 (8 mg / kg), anti-PD-1, and anti-CD137 (urelumab analogue) treatment across 24, 48, and 24-hour time points on five selected intercytokines / chemokines. Normalized Log2 counts of mRNA in MC38 tumor tissues at 24, 48, and 144-hour time points are shown. *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA and post-Dunnett assay.

[0019] Figure 12 illustrates the effects of BT7455 (8 mg / kg), anti-PD-1, and anti-CD137 (uregula) treatment on cytotoxic cells. The effects of treatment on cytotoxic cells at 24, 48, and 144 hours are shown as cytotoxic cell type scores as standardized Log2 (mean with standard deviation) scores in MC38 tumor tissue. (*p < 0.05, one-way ANOVA and post-Dunnet test, which compare the treatment agent with the carboxyl agent).

[0020] Figure 13 depicts the significant regulation of several gene sets by BT7455 at an early time point (48h) after treatment initiation, as revealed by transcriptional analysis (*p<0.05, **p<0.01, one-way ANOVA and post-Dunnett test), while the effects of anti-PD-1 and urrelumab analogs (anti-CD137) were not significant. The effects of treatment on gene sets are shown as the signature score (mean with standard deviation) in MC38 tumor tissue.

Claims

1. A method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of a heterotonandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof and an immuno-oncology agent, wherein the heterotonandem bicyclic peptide complex comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) one or more CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two ring sequences, and a molecular backbone, the molecular backbone covalently bonded to the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

2. The method of claim 1, wherein the heterotandem bicyclic peptide complex comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) one or more CD137-binding peptide ligands via a linker; wherein each of the peptide ligands comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two ring sequences, and a molecular backbone covalently bonded to the cysteine ​​residues of the polypeptide such that at least two polypeptide rings are formed on the molecular backbone.

3. The method of claim 1 or 2, wherein the heterotandem bicyclic peptide complex comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) two or more CD137-binding peptide ligands via a linker.

4. The method of claim 1 or 2, wherein the heterotandem bicyclic peptide complex comprises: (a) a first peptide ligand that binds to a component present on a cancer cell; and (b) two or more CD137-binding peptide ligands via a linker.

5. The method of any one of claims 1 to 4, wherein the CD137-binding peptide ligand comprises an amino acid sequence selected from the following: CiIEEGQYCiiFADPY[Nle]Ciii (SEQ ID NO: 5); Ci[tBuAla]PE[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 6); CiIEEGQYCiiF[D-Ala]DPY[Nle]Ciii (SEQ ID NO: 7); Ci[tBuAla]PK[D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 8); Ci[tBuAla]PE[D-Lys]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 9); Ci[tBuAla]P[K(PYA)][D-Ala]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 10); Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 11); (SEQ ID NO: 11)-A (referred to herein as BCY14601); CiIEE[D-Lys(PYA)]QYCiiFADPY(Nle)Ciii(SEQ ID NO: 12); Ci[tBuAla]PE[dK]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 60); CiIEE[dK(PYA)]QYCiiFADPY[Nle]Ciii(SEQ ID NO: 61); Ci[tBuAla]EE(dK)PYCiiFADPY[Nle]Ciii(SEQ ID NO: 62); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 63); Ci[tBuAla]EE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 64); Ci[tBuAla]PE[dK(PYA)]PYCiiFANPY[Nle]Ciii(SEQ ID NO: 65); 66); Ci[tBuAla]PE[dK(PYA)]PYCiiFA[Aad]PY[Nle]Ciii (SEQ ID NO: 67);Ci[tBuAla]PE[dK(PYA)]PYCiiFAQPY[Nle]Ciii(SEQ ID NO: 68); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle][Cysam]iii(SEQ ID NO: 69); [MerPro]i[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 70; name in text BCY12353); [MerPro]i[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle][Cysam]iii(SEQ ID NO: 71; name in text BCY12354); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 72); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 73); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 74; name in text BCY12372); Ci[tBuAla]PE[dK(PYA)]PYCiiFAD[NMeAla]Y[Nle]Ciii(SEQ ID NO: 75); Ci[tBuAla]PE[dK(PYA)]PYCiiFAD[NMeDAla]Y[Nle]Ciii(SEQ ID NO: 76); Ci[tBuAla]P[K(PYA)][dA]PYCiiFADPY[Nle]Ciii(SEQ ID NO: 77); Ci[tBuAla]PE[dK(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 78); Ci[tBuAla]PE[dK(Me,PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 79); Ci[tBuAla]PE[dK(Me,PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 80); and [MerPro]i[tBuAla]EE[dK]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 81; referred to in the text as BCY13137);Wherein [MerPro]i, Ci, Cii, Ciii, and [Cysam]iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam; Nle represents leucine; tBuAla represents tert-butyl-alanine; PYA represents 4-pentynic acid; Aad represents α-L-aminoadipic acid; MerPro represents 3-mercaptopropionic acid; Cysam represents cysteine; and NMeAla represents N-methyl-alanine, or a pharmaceutically acceptable salt thereof.

6. The method of any one of claims 1 to 5, wherein the CD137-binding peptide ligand comprises the following amino acid sequence: Ci[tBuAla]PE[D-Lys(PYA)]PYCiiFADPY[Nle]Ciii (SEQ ID NO: 11); wherein Ci, Cii, and Ciii represent the first, second, and third cysteine ​​residues, respectively, tBuAla represents tert-butyl-alanine, PYA represents 4-pentyneic acid, and Nle represents leucine, or a pharmaceutically acceptable salt thereof.

7. The method of any one of claims 1 to 6, wherein the CD137-binding bicyclic peptide ligand comprises N-terminal and C-terminal modifications and comprises: Ac-A-(SEQ ID NO: 5)-Dap (hereinafter referred to as BCY7732); Ac-A-(SEQ ID NO: 5)-Dap(PYA) (hereinafter referred to as BCY7741); Ac-(SEQ ID NO: 6)-Dap (hereinafter referred to as BCY9172); Ac-(SEQ ID NO: 6)-Dap(PYA) (hereinafter referred to as BCY11014); Ac-A-(SEQ ID NO: 7)-Dap (hereinafter referred to as BCY8045); Ac-(SEQ ID NO: 8)-A (hereinafter referred to as BCY8919); Ac-(SEQ ID NO: 9)-A (hereinafter referred to as BCY8920); Ac-(SEQ ID NO: 10)-A (Referred to herein as BCY8927); Ac-(SEQ ID NO: 11)-A (Referred to herein as BCY8928); Ac-A-(SEQ ID NO: 12)-A (Referred to herein as BCY7744); Ac-(SEQ ID NO: 60)-Dap(PYA) (Referred to herein as BCY11144); Ac-A-(SEQ ID NO: 61)-K (Referred to herein as BCY11613); Ac-(SEQ ID NO: 62)-Dap(PYA) (Referred to herein as BCY12023); Ac-(SEQ ID NO: 63) (Referred to herein as BCY12149); Ac-(SEQ ID NO: 64) (Referred to herein as BCY12143); Ac-(SEQ ID NO: 65) (Referred to herein as BCY12147); Ac-(SEQ ID NO: 66) (referred to as BCY12145 in this article); Ac-(SEQ ID NO: 67) (referred to as BCY12146 in this article); Ac-(SEQ ID NO: 68) (referred to as BCY12150 in this article); Ac-(SEQ ID NO: 69) (referred to as BCY12352 in this article); Ac-(SEQ ID NO: 72)-[1,2-diaminoethane] (referred to as BCY12358 in this article); [palmitic acid]-[yGlu]-[yGlu]-(SEQ ID NO: 73) (referred to as BCY12360 in this article); Ac-(SEQ ID NO: 75) (referred to as BCY12381 in this article); Ac-(SEQ ID NO: 76) (referred to as BCY12382 in this article);Ac-(SEQ ID NO: 77)-K (referred to herein as BCY12357); Ac-(SEQ ID NO: 78)-[dA] (referred to herein as BCY13095); [Ac]-(SEQ ID NO: 78)-K (referred to herein as BCY13389); Ac-(SEQ ID NO: 79)-[dA] (referred to herein as BCY13096); and Ac-(SEQ ID NO: 80) (referred to herein as BCY13097); wherein Ac represents acetyl, Dap represents diaminopropionic acid, and PYA represents 4-pentyneic acid, or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1 to 7, wherein the CD137-binding bicyclic peptide ligand comprises N-terminal and C-terminal modifications and comprises: Ac-(SEQ ID NO: 11)-A (referred to herein as BCY8928); wherein Ac represents acetyl or a pharmaceutically acceptable salt thereof.

9. The method of any one of claims 1 to 8, wherein the component present on the cancer cell is Nectin-4, and the first peptide ligand comprises Nectin-4 binding a bicyclic peptide ligand.

10. The method of claim 9, wherein the binding protein-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: CiP[1Nal][dD]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 1; referred to herein as BCY8116); CiP[1Nal][dK](Sar10-(B-Ala))CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 3); CiPFGCiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 4; referred herein as BCY11414); CiP[1Nal][dK]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 14); [MerPro]iP[1Nal][dK]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 15; referred to herein as BCY12363); CiP[1Nal][dK]CiiM[HArg]DWSTP[HyP]W[Cysam]iii (SEQ ID NO: 16); [MerPro]iP[1Nal][dK]CiiM[HArg]DWSTP[HyP]W[Cysam]iii (SEQ ID NO: 17; referred herein as BCY12365); CiP[1Nal][dK]CiiM[HArg]HWSTP[HyP]WCiii (SEQ ID NO: 18); CiP[1Nal][dK]CiiM[HArg]EWSTP[HyP]WCiii (SEQ ID NO: 19); CiP[1Nal][dE]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 20; referred to herein as BCY12368); CiP[1Nal][dA]CiiM[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 21; referred to herein as BCY12369); CiP[1Nal][dE]CiiL[HArg]DWSTP[HyP]WCiii (SEQ ID NO: 22; referred to herein as BCY12370); and CiP[1Nal][dE]CiiM[HArg]EWSTP[HyP]WCiii (SEQ ID NO: 23; referred to herein as BCY12384);Wherein [MerPro]i, Ci, Cii, Ciii, and [Cysam]iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam; 1Nal represents 1-naphthylalanine; HArg represents high-arginine; HyP represents trans-4-hydroxy-L-proline; Sar10 represents 10 sarcosine units; B-Ala represents β-alanine; MerPro represents 3-mercaptopropionic acid; and Cysam represents cysteine, or a pharmaceutically acceptable salt thereof.

11. The method of claim 9 or 10, wherein the binding protein-4 binding bicyclic peptide ligand optionally includes an N-terminal modification and comprises: SEQ ID NO: 1 (hereinafter referred to as BCY8116); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846); [PYA]-(SEQ ID NO: 1) (hereinafter referred to as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO: 1) (hereinafter referred to as BCY11016); [PYA]-[B-Ala]-[Sar10]-(SEQ ID NO: 2) (hereinafter referred to as BCY11942); Ac-(SEQ ID NO: 3) (hereinafter referred to as BCY8831); SEQ ID NO: 4 (hereinafter referred to as BCY11414); [PYA]-[B-Ala]-(SEQ ID NO: 14) (referred to herein as BCY11143); palmitic acid-yGlu-yGlu-(SEQ ID NO: 14) (referred to herein as BCY12371); Ac-(SEQ ID NO: 14) (referred to herein as BCY12024); Ac-(SEQ ID NO: 16) (referred to herein as BCY12364); Ac-(SEQ ID NO: 18) (referred to herein as BCY12366); and Ac-(SEQ ID NO: 19) (referred to herein as BCY12367); wherein PYA represents 4-pentynic acid, B-Ala represents β-alanine, and Sar10 represents 10 sarcosine units, or a pharmaceutically acceptable salt thereof.

12. The method of any one of claims 9 to 11, wherein the binding protein-4 binding bicyclic peptide ligand comprises SEQ ID NO: 1 (referred to herein as BCY8116).

13. The method of any one of claims 9 to 12, wherein the heterotandem bicyclic peptide complex is selected from the heterotandem bicyclic peptide complexes listed in Tables A and B, such as BCY11027, BCY11863 and BCY11864 or their pharmaceutically acceptable salts.

14. The method of any one of claims 1 to 8, wherein the component present on the cancer cell is EphA2, and the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

15. The method of claim 14, wherein the EphA2-binding bicyclic peptide ligand comprises an amino acid sequence selected from the following: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 24); CiLWDPTPCiiANLHL[HArg]Ciii (SEQ ID NO: 25); Ci[HyP]LVNPLCiiL[K(PYA)]P[dD]W[HArg]Ciii (SEQ ID NO: 26); Ci[HyP][K(PYA)]VNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 27); Ci[HyP]LVNPLCii[K(PYA)]HP[dD]W[HArg]Ciii (SEQ ID NO: 28); Ci[HyP]LVNPLCiiLKP[dD]W[HArg]Ciii (SEQ ID NO: 29). NO: 29); Ci[HyP]KVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 30); Ci[HyP]LVNPLCiiKHP[dD]W[HArg]Ciii (SEQ ID NO: 31); Ci[HyP]LVNPLCiiLEP[dD]W[HArg]Ciii(SEQ ID NO: 33); Ci[HyP]LVNPLCiiLHP[dD]WTCiii(SEQ ID NO: 34); Ci[HyP]LVNPLCiiLEP[dD]WTCiii(SEQ ID NO: 35); Ci[HyP]LVNPLCiiLEP[dA]WTCiii(SEQ ID NO: 36); Ci[HyP]LVNPLCiiL[3,3-DPA]P[dD]WTCiii (SEQ ID NO: 37; referred to herein as BCY12860); Ci[HyP][Cba]VNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 38); Ci[HyP][Cba]VNPLCiiLEP[dD]WTCiii (SEQ ID NO: 39); Ci[HyP][Cba]VNPLCiiL[3,3-DPA]P[dD]WTCiii (SEQ ID NO: 40); Ci[HyP]LVNPLCiiL[3,3-DPA]P[dD]W[HArg]Ciii (SEQ ID NO: 41);Ci[HyP]LVNPLCiiLHP[d1Nal]W[HArg]Ciii (SEQ ID NO: 42); Ci[HyP]LVNPLCiiL[1Nal]P[dD]W[HArg]Ciii (SEQ ID NO: 43); Ci[HyP]LVNPLCiiLEP[d1Nal]WTCiii (SEQ ID NO: 44); Ci[HyP]LVNPLCiiL[1Nal]P[dD]WTCiii (SEQ ID NO: 45; herein referred to as BCY13119); Ci[HyP][Cba]VNPLCiiLEP[dA]WTCiii (SEQ ID NO: 46); Ci[HyP][hGlu]VNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 47); Ci[HyP]LVNPLCii[hGlu]HP[dD]W[HArg]Ciii (SEQ ID NO: 48); Ci[HyP]LVNPLCiiL[hGlu]P[dD]W[HArg]Ciii (SEQ ID NO: 49); Ci[HyP]LVNPLCiiLHP[dNle]W[HArg]Ciii (SEQ ID NO: 50); Ci[HyP]LVNPLCiiL[Nle]P[dD]W[HArg]Ciii (SEQ ID NO: 51); [MerPro]i[HyP]LVNPLCiiL[3,3-DPA]P[dD]WTCiii (SEQ ID NO: 154); Ci[HyP]LVNPLCiiLHP[dD]W[HArg][Cysam]iii (SEQ ID NO: 155); Ci[HyP]LVNPLCiiL[His3Me]P[dD]W[HArg]Ciii (SEQ ID NO: 156); Ci[HyP]LVNPLCiiL[His1Me]P[dD]W[HArg]Ciii (SEQ ID NO: 157); Ci[HyP]LVNPLCiiL[4ThiAz]P[dD]W[HArg]Ciii (SEQ ID NO: 158); Ci[HyP]LVNPLCiiLFP[dD]W[HArg]Ciii (SEQ ID NO: 159); Ci[HyP]LVNPLCiiL[Thi]P[dD]W[HArg]Ciii (SEQ ID NO: 160); Ci[HyP]LVNPLCiiL[3Thi]P[dD]W[HArg]Ciii (SEQ ID NO: 161);Ci[HyP]LVNPLCiiLNP[dD]W[HArg]Ciii(SEQ ID NO: 162); Ci[HyP]LVNPLCiiLQP[dD]W[HArg]Ciii(SEQ ID NO: 163); and Ci[HyP]LVNPLCiiL[K(PYA-(palmitoyl-Glu-LysN3))]P[dD]W[HArg]Ciii(SEQ ID NO: 164); Where [MerPro]i, Ci, Cii, Ciii, and [Cysam]iii represent the first (i), second (ii), and third (iii) reactive groups selected from cysteine, MerPro, and Cysam; HyP represents trans-4-hydroxy-L-proline; HArg represents homoarginine; PYA represents 4-pentynic acid; 3,3-DPA represents 3,3-diphenylalanine; Cba represents β-cyclobutylalanine; 1Nal represents 1-naphthylalanine; hGlu represents homoglutamic acid; Thi represents... The symbols represent: thienyl-alanine, 4ThiAz represents β-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histamine, His3Me represents N3-methyl-L-histamine, 3Thi represents 3-thienylalanine, palmityl-Glu-LysN3[PYA] represents: [K(PYA-(palmityl-Glu-LysN3))] represents: Nle represents leucine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteine, or a pharmaceutically acceptable salt thereof.

16. The method of claim 14 or 15, wherein the EphA2-binding bicyclic peptide ligand comprises the following amino acid sequence: Ci[HyP]LVNPLCiiLHP[dD]W[HArg]Ciii (SEQ ID NO: 24); wherein Ci, Cii, and Ciii represent first (i), second (ii), and third (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and HArg represents arginine, or a pharmaceutically acceptable salt thereof.

17. The method of any one of claims 14 to 16, wherein the EphA2-binding bicyclic peptide ligand comprises the following amino acid sequence: Ci[HyP]LVNPLCiiLEP[d1Nal]WTCiii (SEQ ID NO: 44); wherein Ci, Cii, and Ciii represent first (i), second (ii), and third (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and d1Nal represents 1-naphthylalanine, or a pharmaceutically acceptable salt thereof.

18. The method of any one of claims 14 to 17, wherein the EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises: A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY9594); [B-Ala]-[Sar10]-A-[HArg]-D-(SEQ ID NO: 24) (hereinafter referred to as BCY6099); [PYA]-A-[HArg]-D-(SEQ NO: 24) (hereinafter referred to as BCY11813); Ac-A-[HArg]-D-(SEQ ID NO: 24)-[K(PYA)] (hereinafter referred to as BCY11814); Ac-A-[HArg]-D-(SEQ ID NO: 24)-K (hereinafter referred to as BCY12734); [NMeAla]-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY13121); [Ac]-(SEQ ID NO: 24)-L[dH]G[dK] (referred to herein as BCY13125); [PYA]-[B-Ala]-[Sar10]-VGP-(SEQ ID NO: 25) (referred to herein as BCY8941); Ac-A-[HArg]-D-(SEQ ID NO: 26) (referred to herein as BCY11815); Ac-A-[HArg]-D-(SEQ ID NO: 27) (referred to herein as BCY11816); Ac-A-[HArg]-D-(SEQ ID NO: 28) (referred to herein as BCY11817); Ac-A-[HArg]-D-(SEQ ID NO: 29) (Hereinafter referred to as BCY12735); (palmitinyl-Glu-LysN3)[PYA]A[HArg]D-(SEQ ID NO: 29) (Hereinafter referred to as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO: 30) (Hereinafter referred to as BCY12736); Ac-A-[HArg]-D-(SEQ ID NO: 31) (Hereinafter referred to as BCY12737); A-[HArg]-D-(SEQ ID NO: 32) (Hereinafter referred to as BCY12738); A-[HArg]-E-(SEQ ID NO: 32) (Hereinafter referred to as BCY12739); A-[HArg]-D-(SEQ ID NO: 33) (Hereinafter referred to as BCY12854); A-[HArg]-D-(SEQ ID NO: 34) (Refered as BCY12855 in this article);A-[HArg]-D-(SEQ ID NO: 35) (referred to herein as BCY12856); A-[HArg]-D-(SEQ ID NO: 35)-[dA] (referred to herein as BCY12857); (SEQ ID NO: 35)-[dA] (referred to herein as BCY12861); [NMeAla]-[HArg]-D-(SEQ ID NO: 35) (referred to herein as BCY13122); [dA]-ED-(SEQ ID NO: 35) (referred to herein as BCY13126); [dA]-[dA]-D-(SEQ ID NO: 35) (referred to herein as BCY13127); AD-(SEQ ID NO: 35) (referred to herein as BCY13128); A-[HArg]-D-(SEQ ID NO: 36) (referred to herein as BCY12858); A-[HArg]-D-(SEQ ID NO: 37) (referred to herein as BCY12859); Ac-(SEQ ID NO: 37)-[dK] (referred to herein as BCY13120); A-[HArg]-D-(SEQ ID NO: 38) (referred to herein as BCY12862); A-[HArg]-D-(SEQ ID NO: 39) (referred to herein as BCY12863); [dA]-[HArg]-D-(SEQ ID NO: 39)-[dA] (referred to herein as BCY12864); (SEQ ID NO: 40)-[dA] (referred to herein as BCY12865); A-[HArg]-D-(SEQ ID NO: 41) (referred to herein as BCY12866); A-[HArg]-D-(SEQ ID NO: 42) (referred to herein as BCY13116); A-[HArg]-D-(SEQ ID NO: 43) (referred to herein as BCY13117); A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); [dA]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13123); [d1Nal]-[HArg]-D-(SEQ ID NO: 46)-[dA] (referred to herein as BCY13124); A-[HArg]-D-(SEQ ID NO: 47) (referred to herein as BCY13130); A-[HArg]-D-(SEQ ID NO: 48) (referred to herein as BCY13131);A-[HArg]-D-(SEQ ID NO: 49) (referred to herein as BCY13132); A-[HArg]-D-(SEQ ID NO: 50) (referred to herein as BCY13134); A-[HArg]-D-(SEQ ID NO: 51) (referred to herein as BCY13135); (SEQ ID NO: 154)-[dK] (referred to herein as BCY13129); A[HArg]D-(SEQ ID NO: 155) (referred to herein as BCY13133); A[HArg]D-(SEQ ID NO: 156) (referred to herein as BCY13917); A[HArg]D-(SEQ ID NO: 157) (referred to herein as BCY13918); A[HArg]D-(SEQ ID NO: 158) (referred to herein as BCY13919); A[HArg]D-(SEQ ID NO: 159) (referred to herein as BCY13920); A[HArg]D-(SEQ ID NO: 160) (referred to herein as BCY13922); A[HArg]D-(SEQ ID NO: 161) (referred to herein as BCY13923); A[HArg]D-(SEQ ID NO: 162) (referred to herein as BCY14047); A[HArg]D-(SEQ ID NO: 163) (referred to herein as BCY14048); and A[HArg]D-(SEQ ID NO: 164) (referred to herein as BCY14313); Where PYA represents 4-pentynic acid, B-Ala represents β-alanine, Sar10 represents 10 sarcosine units, HArg represents high-arginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, and palmityl-Glu-LysN3[PYA] represents: , or a pharmaceutically acceptable salt thereof.

19. The method of any one of claims 14 to 18, wherein the EphA2-binding bicyclic peptide ligand may include an N-terminal modification and comprises: A-[HArg]-D-(SEQ ID NO: 24) (referred to herein as BCY9594); wherein HArg represents arginine, or a pharmaceutically acceptable salt thereof.

20. The method of any one of claims 14 to 19, wherein the EphA2-binding bicyclic peptide ligand may include an N-terminal modification and comprises: A-[HArg]-D-(SEQ ID NO: 44) (referred to herein as BCY13118); wherein HArg represents arginine, or a pharmaceutically acceptable salt thereof.

21. The method of any one of claims 14 to 20, wherein the heterotandem bicyclic peptide complex is selected from the heterotandem bicyclic peptide complexes listed in Table C, such as BCY12491, BCY12730, BCY13048, BCY13050, BCY13053 and BCY13272 or a pharmaceutically acceptable salt thereof.

22. The method of any one of claims 1 to 21, wherein the molecular skeleton is 1,1',1''-(1,3,5-trisyl-1,3,5-triyl)triprop-2-en-1-one (TATA).

23. The method of any one of claims 1 to 22, wherein the immuno-oncology agent is a checkpoint inhibitor.

24. The method of claim 23, wherein the checkpoint inhibitor is a PD-1 antagonist.

25. The method of any one of claims 1 to 24, wherein the heterotandem bicyclic peptide complex or its pharmaceutically acceptable salt and the immuno-oncology agent are administered simultaneously or sequentially.

26. The method of any one of claims 1 to 25, wherein the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof and the immuno-oncology agent are administered at intervals of 1, 2, 3, 4, 5, 6 or 7 days.

27. A method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of BT7480 or a medically acceptable salt thereof and an immuno-oncology agent.

28. The method of claim 27, wherein the immuno-oncology agent is a checkpoint inhibitor.

29. The method of claim 28, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

30. The method of claim 29, wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

31. The method of claim 28, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

32. The method of claim 31, wherein the anti-PD-L1 antibody is durvalumab or atezolizumab.

33. The method of claim 28, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

34. The method of claim 33, wherein the anti-CTLA-4 antibody is ipilimumab.

35. The method of any of claims 27 to 34, wherein BT7480 or its pharmaceutically acceptable salt and the immuno-oncology agent are administered simultaneously or sequentially.

36. The method of any of claims 27 to 35, wherein BT7480 or a pharmaceutically acceptable salt thereof and the immuno-oncology agent are administered at intervals of 1, 2, 3, 4, 5, 6 or 7 days.

37. The method of any of claims 27 to 36, wherein BT7480 or its medically acceptable salt is administered by intravenous infusion.

38. The method of any of claims 27 to 37, wherein BT7480 or its medically acceptable salt is administered once a week.

39. The method of any of claims 27 to 37, wherein BT7480 or its medically acceptable salt is administered twice a week.

40. The method of any one of claims 27 to 39, wherein BT7480 or a pharmaceutically acceptable salt thereof is administered at a dose of about 0.1 to 75 mg / kg.

41. Use of BT7480 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer, wherein the medicament is used in combination with a checkpoint inhibitor.

42. The use as claimed in claim 41, wherein the drug further comprises histidine.

43. As claimed in claim 41 or 42, wherein the drug further comprises sucrose.

44. The use of any of claims 41 to 43, wherein the drug is a formulation having a pH of about 7, the formulation comprising BT7480 or a pharmaceutically acceptable salt thereof, histidine, sucrose and water.

45. The method of any one of claims 27 to 40 or the use of any one of claims 41 to 44, wherein the cancer is characterized by high binding protein-4.

46. ​​The method of any of claims 27 to 40 or the use of any of claims 41 to 44, wherein the cancer is a solid tumor.

47. The method or use as described in claim 46, wherein the solid tumor is a sarcoma, carcinoma, or lymphoma.

48. A method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of BT7455 or a medically acceptable salt thereof and an immuno-oncology agent.

49. The method of claim 48, wherein the immuno-oncology agent is a checkpoint inhibitor.

50. The method of claim 49, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

51. The method of claim 50, wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

52. The method of claim 49, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

53. The method of claim 52, wherein the anti-PD-L1 antibody is divaluromab or atezolizumab.

54. The method of claim 49, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

55. The method of claim 54, wherein the anti-CTLA-4 antibody is ipilimumab.

56. The method of any of claims 48 to 55, wherein BT7455 or a pharmaceutically acceptable salt thereof and the immuno-oncology agent are administered simultaneously or sequentially.

57. The method of any of claims 48 to 56, wherein BT7455 or a pharmaceutically acceptable salt thereof and the immuno-oncology agent are administered at intervals of 1, 2, 3, 4, 5, 6 or 7 days.

58. The method of any of claims 48 to 57, wherein BT7455 or a medically acceptable salt thereof is administered by intravenous infusion.

59. The method of any of claims 48 to 58, wherein BT7455 or its medically acceptable salt is administered once a week.

60. The method of any of claims 48 to 58, wherein BT7455 or its medically acceptable salt is administered twice a week.

61. Use of BT7455 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer, wherein the medicament is used in combination with a checkpoint inhibitor.

62. As claimed in claim 61, wherein the drug further comprises histidine.

63. As claimed in claim 61 or 62, wherein the drug further comprises sucrose.

64. The use of any of claims 61 to 63, wherein the drug is a formulation having a pH of about 7, the formulation comprising BT7455 or a pharmaceutically acceptable salt thereof, histidine, sucrose and water.

65. The method of any of claims 48 to 60 or the use of any of claims 61 to 64, wherein the cancer is characterized by high EphA2 expression.