Methods of Treating Cancer

Abstract

The present invention relates to a method of treating cancer in a patient.

Description

[Technical field]

[0001] The present invention relates to the use of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands, or a pharma- ceutically acceptable salt thereof, in combination with a cancer immunotherapeutic agent for the treatment of cancer. The present invention also provides a composition comprising a heterotandem bicyclic peptide complex comprising one or more pharma- ceutically acceptable CD137-binding peptide ligands, or a pharma- ceutically acceptable salt thereof. [Background technology]

[0002] Cyclic peptides are able to bind protein targets with high affinity and target specificity and are therefore an attractive class of molecules for therapeutic drug development. Indeed, several cyclic peptides have already been successfully used in the clinic, e.g., the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target and the low conformational flexibility of the cyclic structure. Typically, macrocycles are used to bind protein targets, e.g., the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å 2 ) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 (603 Å) that binds to urokinase-type plasminogen activator. 2 ; Zhao et al. (2007), J Struct Biol 160 (1), 1-10), which bind to surfaces measuring several hundred square angstroms.

[0003] Due to the cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss upon binding to the target, and therefore higher binding affinity. The lower flexibility also locks the target-specific conformation, increasing binding specificity compared to linear peptides. This effect is exemplified by potent and selective inhibitors of matrix metalloproteinase 8 (MMP-8), which lose selectivity against other MMPs when ring-opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are more pronounced in multicyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

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

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

[0006] It has been found by the present invention that a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharma- ceutically acceptable salt thereof significantly increases tumor infiltrating immune cells and immune responses. For example, the transcriptional analysis in Example 1 shows a significant increase in immune cell score and mRNA of several T cell chemotactic chemokines / cytokines by treatment with each of BCY12491 and BT7480. Thus, in one embodiment, the present invention provides a method for increasing immune responses in cancer patients, 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 pharma- ceutically acceptable salt thereof.

[0007] It has also been found by the present invention that the combination of a heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof comprising one or more CD137-binding peptide ligands and a cancer immunotherapy agent significantly improves antitumor activity compared to each single agent treatment. For example, the combination therapy of BCY12491 and the PD-1 antagonist pembrolizumab in Example 2 significantly increases antitumor activity compared to each single agent treatment. Thus, in one embodiment, the present invention provides a method of treating cancer in a patient, comprising administering to the patient a heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof comprising one or more CD137-binding peptide ligands and a cancer immunotherapy agent. [Brief description of the drawings]

[0008] [Figure 1]Figure 1 shows that BCY12491 modulates the tumor immune microenvironment and induces T cell infiltration. (A) MC38 tumor-bearing mice were treated with vehicle, 15 mg / kg EphA2 / CD137 heterotandem bicyclic peptide complex (BCY12491), enantiomer-free control heterotandem bicyclic peptide complex (BCY13626) q3d iv, or 2 mg / kg αCD137 q3d ip. Individual tumor volumes (normalized to tumor volume on treatment start day) are shown by treatment group. (B) Nanostring analysis of tumors shows the effect of BCY12491 and αCD137 on T cell (probe set: Cd3d, Cd3e, Cd3g, Cd6, Sh2d1a and Trat1), cytotoxic T cells (probe set: Ctsw, Gzma, Gzmb, Klrb1, Klrd1, Klrk1, Nkg7 and Prf1) and macrophage (probe set: Cd163, Cd68, Cd84 and Ms4a4a) content. (C) Nanostring analysis of tumors shows the effect of BCY12491 and αCD137 on checkpoint inhibitors Pdcd1 (protein PD-1), Cd274 (protein PD-L1) and Ctla4 (protein CTLA-4) transcription. (D) Representative images of histological sections from tumors treated with vehicle, 15 mg / kg BCY12491, BCY13626, or 2 mg / kg αCD137 Q3D and stained for mouse CD8. (B and C) *<0.05, ***p<0.001, one-way ANOVA with Dunnett's post-hoc test.

[0009] [Diagram 2] The effect of BT7480 on select cytokines / chemokines is shown. (A) Normalized linear count data is graphed on the left hand side for five different cytokine / chemokine mRNAs in MC38#13 tumor tissue following BT7480 treatment. (B) Overlay of cytotoxicity cell score and Ccl1, Ccl-17 and Ccl24 normalized RNA counts shows an initial increase in cytokine / chemokine transcripts followed by an increase in cytotoxicity cell score.

[0010] [Diagram 3]Figure 1 shows that BT7480 modulates the tumor immune microenvironment and induces CD8+ T cell infiltration. MC38#13 tumor-bearing mice were treated with vehicle, 5 mg / kg (0 h, 24 h) of BT7480 or non-binding heterotandem bicyclic peptide complex control BCY12797 (NB-BCY) iv or 2 mg / kg αCD137 ip. Nanostring analysis of tumors shows the effect of BT7480 and αCD137 over time on (A) macrophage (probe sets: Cd163, Cd68, Cd84 and Ms4a4a) and (B) cytotoxic cell (probe sets: Ctsw, Gzma, Gzmb, Klrb1, Klrd1, Klrk1, Nkg7 and Prf1) scores of tumor tissue. (C) Overlay of cytotoxic cell score and macrophage cell score shows an initial 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 Dunnett's post hoc test.

[0011] [Figure 4] Figure 1. BT7480 increases several immune checkpoint mRNAs. MC38#13 tumor-bearing mice were treated with vehicle, 5 mg / kg (0 h, 24 h) BT7480 or non-binding heterotandem bicyclic peptide complex control BCY12797 (NB-BCY) iv or 2 mg / kg αCD137 ip. Nanostring analysis of tumors shows the effect of BT7480 and αCD137 on the levels of several immune checkpoint mRNAs. *<0.05, **p<0.01, ***p<0.001 One-way ANOVA with Dunnett's post-hoc test.

[0012] [Diagram 5]Figure 1 shows that the BCY12491 + pembrolizumab combination from day 0 (after treatment initiation) leads to a 100% complete response rate by day 22. MC38 tumor-bearing mice were treated with vehicle, 5 mg / kg BCY12491 QW (0, 24 hours), 3 mg / kg pembrolizumab QW or their combination. The top graph shows the mean tumor volume from treatment initiation to day 28. Both monotherapy and combination treatment significantly affected tumor growth (***p<0.0001, mixed effects analysis with Dunnett's post-hoc test at D18 compared to vehicle). Furthermore, the combination treatment was more effective than either of the monotherapies (***p<0.0001, mixed effects analysis with Dunnett's post-hoc test at D20 comparing combination vs. monotherapy), leading to a complete response in all treated animals by day 22. The right hand graph shows the growth curves of individual tumors from the treatment cohorts.

[0013] [Figure 6] Figure 1 shows that BCY12491 + pembrolizumab combination leads to significant antitumor activity in different dosing sequences. MC38 tumor-bearing mice were treated with vehicle, 5mg / kg BCY12491 QW (0, 24 hours), 3mg / kg pembrolizumab QW or their combinations in three different dosing schedules: both BCY12491 and pembrolizumab treatments 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 graph shows the average tumor volume from the start of treatment to day 28. All combination treatments showed significant antitumor activity with 10 / 10 (BCY12491+pembrolizumab from D0), 9 / 10 (BCY12491+pembrolizumab from D5) and 8 / 10 (pembrolizumab from D0 and BCY12491 from D5) complete responses by day 42. ***p<0.0001, mixed effects analysis with Dunnett's post-hoc test at D18 compared to vehicle. Right hand graphs show growth curves of individual tumors from treatment cohorts.

[0014] [Figure 7]We show that the addition of BCY11864 to anti-PD-1 monotherapy significantly [p=0.004, log-rank (Mantel-Cox) test comparing anti-PD-1 arm with anti-PD-1+BCY11864 combination arm] increases survival (defined as reaching human endpoint, tumor volume >2000mm3) of CT26#7 (CT26 engineered to overexpress Nectin-4) bearing mice.

[0015] [Figure 8] We show that the addition of BT7480 to anti-PD-1 monotherapy increases the complete response rate (CR) in MC38#13 (MC38 engineered to overexpress Nectin-4) bearing mice.

[0016] [Figure 9] The addition of BT7480 to anti-CTLA-4 monotherapy significantly [p=0.0499, log-rank (Mantel-Cox) test comparing anti-CTLA-4 arm with anti-CTLA-4+BT7480 combination arm] increased survival (defined as reaching human endpoint, tumor volume >2000mm3) of MC38#13 (MC38 engineered to overexpress Nectin-4) bearing mice and increased complete response rate.

[0017] [Figure 10] BT7455 is shown to increase several immune checkpoint mRNAs. MC38 tumor-bearing mice were treated with vehicle, BT7455 at 8 mg / kg (0 h, 24 h) intravenously, or with 2 mg / kg anti-CD137 antibody or 10 mg / kg anti-PD-1 antibody intraperitoneally. Nanostring analysis of tumors shows the effect of treatment on the levels of several immune checkpoint mRNAs. Normalized Log2 counts of mRNA in MC38 tumor tissue at 24, 48, and 144 h are shown. *<0.05, **p<0.01, ***p<0.001 One-way ANOVA with Dunnett's post-hoc test comparing treatment vs. vehicle at the same time points.

[0018] [Figure 11]Shown are the effects of BT7455 (8mg / kg), anti-PD-1 and anti-CD137 (a urelumab analog) treatment on five select cytokines / chemokines over the 24, 48 and 24 hour time points. Shown are normalized Log2 counts of mRNA in MC38 tumor tissue at the 24, 48 and 144 hour time points. *p<0.05, **p<0.01, ****p<0.0001, 0.01 One-way ANOVA with Dunnett's post-hoc test.

[0019] [Figure 12] Figure 1 shows the effect of BT7455 (8mg / kg), anti-PD-1 and anti-CD137 (Urelumab analogue) treatment on cytotoxic cells. The effect of treatment on cytotoxic cells at 24, 48 and 144 hours is shown as cytotoxic cell type score as normalized Log2 (mean and standard deviation) score in MC38 tumor tissue. (*p<0.05, one-way ANOVA with Dunnett's post-hoc test comparing treatment vs vehicle).

[0020] [Figure 13] Transcriptional analysis confirmed significant modulation of several gene sets at early time points (48 hours) after initiation of treatment with BT7455 (*p<0.05, **p<0.01 one-way ANOVA with Dunnett's post-hoc test), whereas the effects of anti-PD-1 and urelumab analogue (anti-CD137) were not significant. The effect of treatment on gene sets is shown as signature scores (mean and standard deviation) in MC38 tumor tissue. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] 1. Description of certain embodiments of the present invention: It has been found by the present invention that a heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof comprising one or more CD137-binding peptide ligands significantly increases tumor-infiltrating immune cells and immune responses, and a combination of a heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof comprising one or more CD137-binding peptide ligands and a cancer immunotherapeutic agent significantly increases antitumor activity compared to each single agent treatment.See, for example, the data of treatment with BCY12491 and BT7480, respectively, in Example 1, and the data of treatment with BCY12491 alone, the PD-1 antagonist pembrolizumab alone, and the combination of BCY12491 and pembrolizumab, respectively, in Example 2. Thus, in one embodiment, a combination method or use of a heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof comprising one or more CD137-binding peptide ligands for increasing immune responses in cancer patients is provided. In another embodiment, there is provided a method or use of a combination of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands, or a pharma- ceutically acceptable salt thereof, and a cancer immunotherapeutic agent for the treatment of cancer in a patient.

[0022] 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 comprising one or more CD137-binding peptide ligands, or a pharma- ceutically acceptable salt thereof. In one embodiment, the present invention provides the use of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for increasing an immune response in a cancer patient.

[0023] In one embodiment, the present invention provides a method for treating cancer in a 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 pharma- ceutically acceptable salt thereof and a cancer immunotherapy agent.In one embodiment, the present invention provides the use of a heterotandem bicyclic peptide complex comprising one or more CD137 binding peptide ligands or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for treating cancer in a patient, wherein the medicament is used in combination with a cancer immunotherapy agent.

[0024] In some embodiments, the cancer is selected from those 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 Nectin-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.

[0025] In certain embodiments, the heterotandem bicyclic peptide complex comprising one or more CD137 binding peptide ligands is selected from the heterotandem bicyclic peptide complexes comprising one CD137 binding peptide ligand described herein. In certain embodiments, the heterotandem bicyclic peptide complex comprising one or more CD137 binding peptide ligands is selected from the heterotandem bicyclic peptide complexes comprising two or more CD137 binding peptide ligands described herein.

[0026] In some embodiments, the heterotandem bicyclic peptide complex is BCY11863 (also referred to as BT7480) or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is BCY13272 (also referred to 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.

[0027] In some embodiments, the cancer immunotherapy is selected from those described herein. In some embodiments, the cancer immunotherapy is a checkpoint inhibitor. In some embodiments, the cancer immunotherapy is a PD-1 antagonist. In some embodiments, the cancer immunotherapy is pembrolizumab. In some embodiments, the cancer immunotherapy is nivolumab.

[0028] In some embodiments, the present invention provides a method for increasing 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 a use of BT7480 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing 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 a cancer immunotherapeutic agent. In some embodiments, the present invention provides a 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 a cancer immunotherapeutic agent.

[0029] In some embodiments, the present invention provides a method for increasing 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 a use of BT7455 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing 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 a cancer immunotherapeutic agent.In some embodiments, the present invention provides a 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 a cancer immunotherapeutic agent.

[0030] 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 described herein, e.g., BT7480 or BT7455, or a pharma- ceutically 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 certain embodiments, the heterotandem bicyclic peptide complex is administered at a dose 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.

[0031] In some embodiments, the heterotandem bicyclic peptide complex is administered once, twice, three times, or four times per week. In some embodiments, the heterotandem bicyclic peptide complex is selected from those described herein, e.g., BT7480 or BT7455, or a pharma- ceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered once per day. In some embodiments, the heterotandem bicyclic peptide complex is administered once every two days. In some embodiments, the heterotandem bicyclic peptide complex is administered once every three days. In some embodiments, the heterotandem bicyclic peptide complex is administered once every four days. In some embodiments, the heterotandem bicyclic peptide complex is administered once every five days. In some embodiments, the heterotandem bicyclic peptide complex is administered once per week. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 1.5 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered once every two weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered once 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.

[0032] In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 1-4 weeks. In some embodiments, the heterotandem bicyclic peptide complex is selected from those described herein, e.g., BT7480 or BT7455, or a pharma- ceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 5-8 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 9-12 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 13-20 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 21-28 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, or 28 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 30 weeks or more.

[0033] In some embodiments, the heterotandem bicyclic peptide complex is administered to the patient by intravenous bolus injection. In some embodiments, the heterotandem bicyclic peptide complex is selected from those described herein, e.g., BT7480 or BT7455, or a pharma- ceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered to the patient by intravenous infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 5-10 minute infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 10-20 minute infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 20-40 minute infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 45 or 50 or 55 minute infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 1 hour infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 1-1.5 hour infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 1.5-2 hour infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about a 2-3 hour infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is more than a 3 hour infusion.

[0034] The cancer immunotherapeutic agent is administered in a dosing regimen according to FDA recommendations or approval. In some embodiments, the cancer immunotherapeutic agent is administered at a dose of about 1-20 mg / kg. In some embodiments, the cancer immunotherapeutic 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 cancer immunotherapeutic 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 cancer immunotherapeutic agent is administered at a dose of about 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg. In some embodiments, the cancer immunotherapy is administered at a dose of about 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg. In some embodiments, the cancer immunotherapy is administered once, twice, three times, or four times a week. In some embodiments, the cancer immunotherapy is administered once a day. In some embodiments, the cancer immunotherapy is administered once every two days. In some embodiments, the cancer immunotherapy is administered once every three days. In some embodiments, the cancer immunotherapy is administered once every four days. In some embodiments, the cancer immunotherapy is administered once every five days. In some embodiments, the cancer immunotherapy is administered once a week. In some embodiments, the cancer immunotherapy is administered once every 1.5 weeks. In some embodiments, the cancer immunotherapy is administered once every two weeks. In some embodiments, the cancer immunotherapy is administered once every 2.5 weeks. In some embodiments, the cancer immunotherapeutic agent is administered once every 3 weeks. In some embodiments, the cancer immunotherapeutic agent is administered once every 4 weeks. In some embodiments, the cancer immunotherapeutic agent is administered once a month. In some embodiments, the cancer immunotherapeutic agent is administered for a treatment period of about 1-4 weeks. In some embodiments, the cancer immunotherapeutic agent is administered for a treatment period of about 9-12 weeks, about 13-20 weeks, about 21-28 weeks, or about 29-36 weeks. In some embodiments, the cancer immunotherapeutic agent is administered for a treatment period of about 36 weeks or more.In some embodiments, the cancer immunotherapeutic agent is administered to the patient by intravenous injection. In some embodiments, the cancer immunotherapeutic agent is administered to the patient by intravenous infusion. In some embodiments, the intravenous infusion of the cancer immunotherapeutic agent is infused for about 5-10 minutes. In some embodiments, the intravenous infusion of the cancer immunotherapeutic agent is infused for about 10-20 minutes or about 20-40 minutes. In some embodiments, the intravenous infusion of the cancer immunotherapeutic agent is infused for about 30 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes. In some embodiments, the intravenous infusion of the cancer immunotherapeutic agent is infused for about 1-1.5 hours, about 1.5-2 hours or about 2-3 hours.

[0035] In some embodiments, the pharmaceutical preparation comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof is selected from the heterotandem bicyclic peptide complex formulations shown in the present example. In some embodiments, the heterotandem bicyclic peptide complex is selected from those described herein, for example, BT7480 or BT7455 or a pharma- ceutically acceptable salt thereof. In some embodiments, the pharmaceutical preparation comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof further comprises histidine. In some embodiments, the pharmaceutical preparation comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof and histidine are contained at about pH 7. In some embodiments, the pharmaceutical preparation comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof further comprises sucrose. In some embodiments, the pharmaceutical preparation comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof further comprises about 10% w / v sucrose. In some embodiments, the pharmaceutical preparation comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof further comprises water. In certain embodiments, the present invention provides a medicament comprising a heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof, histidine, sucrose, and water, wherein the medicament is provided at about pH 7.

[0036] Examples of heterotandem bicyclic peptide complexes In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) one or more CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, and at least three reactive groups separated by at least two loop sequences.

[0037] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) one or more CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0038] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) one CD137-binding peptide ligand; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, and at least three reactive groups separated by at least two loop sequences.

[0039] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) one CD137-binding peptide ligand; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0040] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) two or more CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, and at least three reactive groups separated by at least two loop sequences.

[0041] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) two or more CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0042] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) two CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, and at least three reactive groups separated by at least two loop sequences.

[0043] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) two CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0044] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) three CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, and at least three reactive groups separated by at least two loop sequences.

[0045] In one embodiment, the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof is (a) a first peptide ligand that binds to a moiety present on a cancer cell, conjugated via a linker to (b); (b) three CD137-binding peptide ligands; wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0046] First Peptide Ligand The term "cancer cell" as used herein includes any cell known to be involved in cancer. Cancer cells arise when the genes responsible for cell division are damaged. Carcinogenesis is caused by mutations and epimutations in the genetic material of normal cells that disrupt the normal balance between proliferation and cell death. This results in uncontrolled cell division and the evolution of such cells by natural selection in the body. The uncontrolled and often rapid proliferation of cells can lead to 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 distally (metastasize), and become life threatening.

[0047] In some embodiments, the cancer cells are selected from HT1080, A549, SC-OV-3, PC3, HT1376, NCI-H292, LnCap, MC38, MC38 #13, 4T1-D02, H322, HT29, T47D, and RKO tumor cells.

[0048] In one embodiment, the component present on cancer cells is Nectin-4.

[0049] Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which includes four members. Nectins are cell adhesion molecules that have important roles in various biological processes, such as polarization, spreading, differentiation and migration of epithelial, endothelial, immune and neuronal cells during development and adulthood. It is involved in several human pathological processes. It is the main receptor for poliovirus, herpes simplex virus and measles virus. Mutations in the genes encoding nectin-1 (PVRL1) or nectin-4 (PVRL4) cause ectodermal dysplasia syndromes, which are associated with other abnormalities. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than other members of the family. Nectin-4 is a tumor-associated antigen in 50%, 49% and 86% of breast, ovarian and lung cancers, respectively, the majority of which are tumors with poor prognosis. Its expression is not detectable in the corresponding normal tissues. In breast tumors, nectin-4 is expressed mainly in triple-negative and ERBB2+ carcinomas. Detection of soluble forms of Nectin-4 in the serum of patients with these cancers is associated with poor prognosis. Serum Nectin-4 levels increase during metastatic progression and decrease after treatment. These results suggest that Nectin-4 is a reliable target for cancer treatment. Accordingly, several anti-Nectin-4 antibodies have been described in the prior art. In particular, Enfortumab vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) that targets Nectin-4 and is currently undergoing clinical trials for the treatment of patients with solid tumors.

[0050] In certain embodiments, the first peptide ligand comprises a Nectin-4 binding bicyclic peptide ligand.

[0051] In one embodiment, the Nectin-4 binding bicyclic peptide ligand is selected from those disclosed in WO2019 / 243832, the contents of which are incorporated herein by reference in their entirety.

[0052] In one embodiment, the Nectin-4 binding bicyclic peptide ligand is C i P[1Nal][dD]C iiM[HArg]DWSTP[HyP]WC iii (SEQ ID NO:1; herein referred to as BCY8116); C i P[1Nal][dD]C ii M[HArg]D[dW]STP[HyP][dW]C iii (SEQ ID NO:2); C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:3); C i PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:4; herein referred to as BCY11414); C i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:14); [MerPro] i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:15; herein referred to as BCY12363); C i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO:16); [MerPro] i P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO:17; herein referred to as BCY12365); C i P[1Nal][dK]C ii M[HArg]HWSTP[HyP]WC iii (SEQ ID NO:18); C i P[1Nal][dK]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO:19); Ci P[1Nal][dE]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:20; herein designated BCY12368); C i P[1Nal][dA]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:21; herein referred to as BCY12369); C i P[1Nal][dE]C ii L[HArg]DWSTP[HyP]WC iii (SEQ ID NO:22; herein designated BCY12370); and C i P[1Nal][dE]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO:23; herein referred to as BCY12384) [In the ceremony, [MerPro] i , C i , C ii , C iii and [Cysam] iii represents the first (i), second (ii) and third (iii) reactive groups selected from cysteine, MerPro and Cysam; 1Nal represents 1-naphthylalanine; HArg represents homoarginine; HyP represents trans-4-hydroxy-L-proline; Sar 10 represents 10 sarcosine units, B-Ala represents beta-alanine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine. or a pharma- ceutically acceptable salt thereof.

[0053] In one embodiment, the Nectin-4 binding bicyclic peptide ligand is C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:1; herein referred to as BCY8116); C iP[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:3); and C i PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 4; herein referred to as BCY11414) [In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; HArg represents homoarginine; HyP represents trans-4-hydroxy-L-proline; and Sar 10 represents 10 sarcosine units, and B-Ala represents beta-alanine. or a pharma- ceutically acceptable salt thereof.

[0054] In one embodiment, the Nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification: SEQ ID NO:1 (herein referred to as BCY8116); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:1) (herein designated BCY8846); [PYA]-(SEQ ID NO:1) (herein referred to as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO:1) (herein referred to as BCY11016); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:2) (herein designated BCY11942); Ac-(SEQ ID NO:3) (herein referred to as BCY8831); SEQ ID NO:4 (herein referred to as BCY11414); [PYA]-[B-Ala]-(SEQ ID NO: 14) (herein referred to as BCY11143); Palmitin-yGlu-yGlu-(SEQ ID NO:14) (herein referred to as BCY12371); Ac-(SEQ ID NO:14) (herein referred to as BCY12024); Ac-(SEQ ID NO:16) (herein referred to as BCY12364); Ac-(SEQ ID NO:18) (referred to herein as BCY12366); and Ac-(SEQ ID NO:19) (herein referred to as BCY12367) [wherein YA represents 4-pentynoic acid, B-Ala represents beta-alanine, and Sar 10 represents 10 sarcosine units. or a pharma- ceutically acceptable salt thereof.

[0055] In one embodiment, the Nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification: SEQ ID NO:1 (herein referred to as BCY8116); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:1) (herein designated BCY8846); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:2) (herein designated BCY11942); Ac-(SEQ ID NO:3) (herein referred to as BCY8831); and SEQ ID NO:4 (herein referred to as BCY11414) [wherein YA represents 4-pentynoic acid, B-Ala represents beta-alanine, and Sar 10 represents 10 sarcosine units. or a pharma- ceutically acceptable salt thereof.

[0056] In certain embodiments, the Nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO:1 (herein referred to as BCY8116).

[0057] In one embodiment, the Nectin-4 binding bicyclic peptide ligand is C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:1; herein referred to as BCY8116); C i P[1Nal][dD]C ii M[HArg]D[dW]STP[HyP][dW]C iii (SEQ ID NO:2; herein designated BCY11415); and C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:3); C i PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 4; herein referred to as BCY11414) [In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively; 1Nal represents 1-naphthylalanine; HArg represents homoarginine; HyP represents hydroxyproline; Sar 10 represents 10 sarcosine units, and B-Ala represents beta-alanine. or a pharma- ceutically acceptable salt thereof.

[0058] In a further embodiment, the Nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification, SEQ ID NO:1 (herein referred to as BCY8116); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:1) (herein designated BCY8846); SEQ ID NO:2 (herein referred to as BCY11415); [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:2) (herein designated BCY11942); Ac-(SEQ ID NO:3) (herein referred to as BCY8831); and SEQ ID NO:4 (herein referred to as BCY11414) [wherein YA represents 4-pentynoic acid, B-Ala represents beta-alanine, and Sar10 represents 10 sarcosine units. or a pharma- ceutically acceptable salt thereof.

[0059] In certain embodiments, the component present on the cancer cell is EphA2.

[0060] Eph receptor tyrosine kinases (Ephs) belong to a large group of receptor tyrosine kinases (RTKs), kinases that phosphorylate tyrosine residues in proteins. Ephs and their membrane-bound ephrin ligands (ephrins) control cell positioning and tissue organization (Poliakov et al. (2004) Dev Cell 7, 465-80). Functional and biochemical Eph responses occur in higher-order ligand oligomerization states (Stein et al. (1998) Genes Dev 12, 667-678).

[0061] Among other patterning functions, various Ephs and ephrins have been shown to play roles in vascular development. Knockout of EphB4 and ephrin-B2 results in loss of the ability to remodel capillary beds into blood vessels (Poliakov et al., supra) and is embryonic lethal. Persistent expression of certain Eph receptors and ephrins has 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).

[0062] Unregulated reappearance of certain ephrins and their receptors in adults has also been observed to contribute to tumor invasion, metastasis, and neovascularization (Nakamoto et al. (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al., supra). Furthermore, certain Eph family members have been found to be overexpressed in tumor cells from a wide variety of human tumors (Brantley-Sieders et al., supra); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0063] EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humans is encoded by the EPHA2 gene.

[0064] EphA2 is often associated with disease progression, metastasis and poor prognosis and has been implicated in several human cancers, including 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), and gastric (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2012) J Thorac Oncol. 9, 301-308). (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)MolCancer Res. 3, 541-551; Li et al (2010) Tumor Biol. 31, 477-488).

[0065] Although the full role of EphA2 in cancer progression is yet to be defined, there is evidence that it interacts at various stages of cancer progression, including tumor cell proliferation, survival, invasion and angiogenesis. Downregulation of EphA2 expression suppresses tumor cancer cell proliferation (Binda et al (2012) Cancer Cell 22, 765-780), while EphA2 blockade inhibits VEGF-induced cell migration (Hess et al (2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40) as well as metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19, 1884-1886).

[0066] Antibody-drug conjugates to EphA2 have been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367-9374), and a similar approach has been tried in humans, although treatment-related adverse events led to treatment being discontinued (Annunziata et al (2013) Invest New drugs 31, 77-84).

[0067] In certain embodiments, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

[0068] In certain embodiments, the EphA2-binding bicyclic peptide ligand is selected from those disclosed in WO2019 / 122860, WO2019 / 122861, and WO2019 / 122863, the contents of each of which are incorporated herein by reference in their entirety.

[0069] In certain embodiments, the EphA2-binding bicyclic peptide ligand is C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii(SEQ ID NO:24); C i LWDPTPC ii ANLHL[HArg]C iii (SEQ ID NO:25); C i [HyP]LVNPLC ii L[K(PYA)]P[dD]W[HArg]C iii (SEQ ID NO:26); C i [HyP][K(PYA)]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:27); C i [HyP]LVNPLC ii [K(PYA)]HP[dD]W[HArg]C iii (SEQ ID NO:28); C i [HyP]LVNPLC ii LKP[dD]W[HArg]C iii (SEQ ID NO:29); C i [HyP]KVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:30); C i [HyP]LVNPLC ii KHP[dD]W[HArg]C iii (SEQ ID NO:31); C i [HyP]LVNPLC ii LHP[dE]W[HArg]C iii (SEQ ID NO:32); C i [HyP]LVNPLC ii LEP[dD]W[HArg]C iii (SEQ ID NO:33); C i [HyP]LVNPLC ii LHP[dD]WTC iii (SEQ ID NO:34); C i [HyP]LVNPLC ii LEP[dD]WTC iii (SEQ ID NO:35); Ci [HyP]LVNPLC ii LEP[dA]WTC iii (SEQ ID NO:36); C i [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO:37; herein referred to as BCY12860); C i [HyP][Cba]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:38); C i [HyP][Cba]VNPLC ii LEP[dD]WTC iii (SEQ ID NO:39); C i [HyP][Cba]VNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO:40); C i [HyP]LVNPLC ii L[3,3-DPA]P[dD]W[HArg]C iii (SEQ ID NO:41); C i [HyP]LVNPLC ii LHP[d1Nal]W[HArg]C iii (SEQ ID NO:42); C i [HyP]LVNPLC ii L[1Nal]P[dD]W[HArg]C iii (SEQ ID NO:43); C i [HyP]LVNPLC ii LEP[d1Nal]WTC iii (SEQ ID NO:44); C i [HyP]LVNPLC ii L[1Nal]P[dD]WTC iii (SEQ ID NO:45; herein referred to as BCY13119); C i [HyP][Cba]VNPLC ii LEP[dA]WTC iii(SEQ ID NO:46); C i [HyP][hGlu]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:47); C i [HyP]LVNPLC ii [hGlu]HP[dD]W[HArg]C iii (SEQ ID NO:48); C i [HyP]LVNPLC ii L[hGlu]P[dD]W[HArg]C iii (SEQ ID NO:49); C i [HyP]LVNPLC ii LHP[dNle]W[HArg]C iii (SEQ ID NO:50); C i [HyP]LVNPLC ii L[Nle]P[dD]W[HArg]C iii (SEQ ID NO:51); [MerPro] i [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO:154); C i [HyP]LVNPLC ii LHP[dD]W[HArg][Cysam] iii (SEQ ID NO:155); C i [HyP]LVNPLC ii L[His3Me]P[dD]W[HArg]C iii (SEQ ID NO:156); C i [HyP]LVNPLC ii L[His1Me]P[dD]W[HArg]C iii (SEQ ID NO:157); C i [HyP]LVNPLC ii L[4ThiAz]P[dD]W[HArg]C iii (SEQ ID NO:158); C i [HyP]LVNPLC iiLFP[dD]W[HArg]C iii (SEQ ID NO:159); C i [HyP]LVNPLC ii L[Thi]P[dD]W[HArg]C iii (SEQ ID NO:160); C i [HyP]LVNPLC ii L[3Thi]P[dD]W[HArg]C iii (SEQ ID NO:161); C i [HyP]LVNPLC ii LNP[dD]W[HArg]C iii (SEQ ID NO:162); C i [HyP]LVNPLC ii LQP[dD]W[HArg]C iii (SEQ ID NO: 163); and C i [HyP]LVNPLC ii L[K(PYA-(palmitoyl-Glu-LysN3)]P[dD]W[HArg]C iii (SEQ ID NO:164) [In the ceremony, [MerPro] i , C i , C ii , C iii and [Cysam] iii represents the first (i), second (ii) and third (iii) reactive groups selected from cysteine, MerPro and Cysam, HyP represents trans-4-hydroxy-L-proline, HArg represents homoarginine, PYA represents 4-pentynoic acid, 3,3-DPA represents 3,3-diphenylalanine, Cba represents β-cyclobutylalanine, 1Nal represents 1-naphthylalanine, hGlu represents homoglutamic acid, Thi represents 2-thienyl-alanine, 4ThiAz represents beta-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 3Thi represents 3-thienylalanine, palmitoyl-Glu-LysN3[PYA] represents [ka] and [K(PYA-(palmitoyl-Glu-LysN3)] is [ka] Nle stands for norleucine, MerPro stands for 3-mercaptopropionic acid, and Cysam stands for cysteamine. or a pharma- ceutically acceptable salt thereof.

[0070] In certain embodiments, the EphA2-binding bicyclic peptide ligand is C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:24) [In the formula, C i , C ii , C iii represents the first (i), second (ii) and tertiary (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof.

[0071] In certain embodiments, the EphA2-binding bicyclic peptide ligand is C i [HyP]LVNPLC ii LEP[d1Nal]WTC iii (SEQ ID NO:44) [In the formula, C i , C ii , C iii represents the first (i), second (ii) and tertiary (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine. or a pharma- ceutically acceptable salt thereof.

[0072] In certain embodiments, the EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal and / or C-terminal modification. A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY9594); [B-Ala]-[Sar 10 ]-A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY6099); [PYA]-A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY11813); Ac-A-[HArg]-D-(SEQ ID NO:24)-[K(PYA)] (herein referred to as BCY11814); Ac-A-[HArg]-D-(SEQ ID NO:24)-K (herein referred to as BCY12734); [NMeAla]-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY13121); [Ac]-(SEQ ID NO:24)-L[dH]G[dK] (herein referred to as BCY13125); [PYA]-[B-Ala]-[Sar 10 ]-VGP-(SEQ ID NO:25) (herein referred to as BCY8941); Ac-A-[HArg]-D-(SEQ ID NO:26) (herein referred to as BCY11815); Ac-A-[HArg]-D-(SEQ ID NO:27) (herein referred to as BCY11816); Ac-A-[HArg]-D-(SEQ ID NO:28) (herein referred to as BCY11817); Ac-A-[HArg]-D-(SEQ ID NO:29) (herein referred to as BCY12735); (Palmitoyl-Glu-LysN3)[PYA]A[HArg]D-(SEQ ID NO:29) (hereinafter referred to as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO:30) (herein referred to as BCY12736); Ac-A-[HArg]-D-(SEQ ID NO:31) (herein referred to as BCY12737); A-[HArg]-D-(SEQ ID NO:32) (herein referred to as BCY12738); A-[HArg]-E-(SEQ ID NO:32) (herein referred to as BCY12739); A-[HArg]-D-(SEQ ID NO:33) (herein referred to as BCY12854); A-[HArg]-D-(SEQ ID NO:34) (herein referred to as BCY12855); A-[HArg]-D-(SEQ ID NO:35) (herein referred to as BCY12856); A-[HArg]-D-(SEQ ID NO:35)-[dA] (herein referred to as BCY12857); (SEQ ID NO:35)-[dA] (referred to herein as BCY12861); [NMeAla]-[HArg]-D-(SEQ ID NO:35) (herein referred to as BCY13122); [dA]-ED-(SEQ ID NO:35) (referred to herein as BCY13126); [dA]-[dA]-D-(SEQ ID NO:35) (herein referred to as BCY13127); AD-(SEQ ID NO:35) (herein referred to as BCY13128); A-[HArg]-D-(SEQ ID NO:36) (herein referred to as BCY12858); A-[HArg]-D-(SEQ ID NO:37) (herein referred to as BCY12859); Ac-(SEQ ID NO:37)-[dK] (herein referred to as BCY13120); A-[HArg]-D-(SEQ ID NO:38) (herein referred to as BCY12862); A-[HArg]-D-(SEQ ID NO:39) (herein referred to as BCY12863); [dA]-[HArg]-D-(SEQ ID NO:39)-[dA] (herein referred to as BCY12864); (SEQ ID NO:40)-[dA] (referred to herein as BCY12865); A-[HArg]-D-(SEQ ID NO:41) (herein referred to as BCY12866); A-[HArg]-D-(SEQ ID NO:42) (herein referred to as BCY13116); A-[HArg]-D-(SEQ ID NO:43) (herein referred to as BCY13117); A-[HArg]-D-(SEQ ID NO:44) (herein referred to as BCY13118); [dA]-[HArg]-D-(SEQ ID NO:46)-[dA] (herein referred to as BCY13123); [d1Nal]-[HArg]-D-(SEQ ID NO:46)-[dA] (herein referred to as BCY13124); A-[HArg]-D-(SEQ ID NO:47) (herein referred to as BCY13130); A-[HArg]-D-(SEQ ID NO:48) (herein referred to as BCY13131); A-[HArg]-D-(SEQ ID NO:49) (herein referred to as BCY13132); A-[HArg]-D-(SEQ ID NO:50) (herein referred to as BCY13134); A-[HArg]-D-(SEQ ID NO:51) (herein referred to as BCY13135); (SEQ ID NO:154)-[dK] (referred to herein as BCY13129); A[HArg]D-(SEQ ID NO:155) (herein referred to as BCY13133); A[HArg]D-(SEQ ID NO:156) (herein referred to as BCY13917); A[HArg]D-(SEQ ID NO:157) (referred to herein as BCY13918); A[HArg]D-(SEQ ID NO:158) (herein referred to as BCY13919); A[HArg]D-(SEQ ID NO:159) (herein referred to as BCY13920); A[HArg]D-(SEQ ID NO:160) (referred to herein as BCY13922); A[HArg]D-(SEQ ID NO:161) (herein referred to as BCY13923); A[HArg]D-(SEQ ID NO:162) (referred to herein as BCY14047); A[HArg]D-(SEQ ID NO: 163) (herein referred to as BCY14048); and A[HArg]D- (SEQ ID NO: 164) (referred to herein as BCY14313) [wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, and Sar10 represents 10 sarcosine units, HArg represents homoarginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, and palmitoyl-Glu-LysN3[PYA] represents [ka] Represents the following. or a pharma- ceutically acceptable salt thereof.

[0073] In certain embodiments, the EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal and / or C-terminal modification. A-[HArg]-D-(SEQ ID NO:24) (referred to herein as BCY9594) (In the formula, HArg represents homoarginine.) or a pharma- ceutically acceptable salt thereof.

[0074] In certain embodiments, the EphA2-binding bicyclic peptide ligand optionally comprises an N-terminal and / or C-terminal modification. A-[HArg]-D-(SEQ ID NO: 44) (herein referred to as BCY13118) (In the formula, HArg represents homoarginine.) or a pharma- ceutically acceptable salt thereof.

[0075] In certain embodiments, the EphA2-binding bicyclic peptide ligand comprises the amino acid C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:24); and C i LWDPTPC ii ANLHL[HArg]C iii (SEQ ID NO:25) [In the formula, C i , C ii and C iiirepresent the first, second and third cysteine ​​residues, respectively; HyP represents hydroxyproline; dD represents aspartic acid in the D-configuration; and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof.

[0076] In certain embodiments, the EphA2-binding bicyclic peptide ligand comprises the amino acid C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:24) [In the formula, C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively; HyP represents hydroxyproline; dD represents aspartic acid in the D-configuration; and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof.

[0077] In one embodiment, the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification: A-HArg-D-(SEQ ID NO:24) (herein referred to as BCY9594); [B-Ala]-[Sar 10 ]-A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY6099); [PYA]-[B-Ala]-[Sar 10 ]-A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY6169); and [PYA]-[B-Ala]-[Sar 10 ]-VGP-(SEQ ID NO:25) (herein referred to as BCY8941) [wherein HArg represents homoarginine, PYA represents 4-pentynoic acid, and Sar 10 represents 10 sarcosine units, and B-Ala represents beta-alanine. or a pharma- ceutically acceptable salt thereof.

[0078] In one embodiment, the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification: A-HArg-D- (SEQ ID NO: 24) (herein referred to as BCY9594) (In the formula, HArg represents homoarginine.) or a pharma- ceutically acceptable salt thereof.

[0079] In one embodiment, the component present on the cancer cell is PD-L1.

[0080] Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in evading immune responses involved in chronic infections, such as chronic viral infections (including, for example, HIV, HBV, HCV, and HTLV, among others), chronic bacterial infections (including, for example, Helicobacter pylori, among others), and chronic parasitic infections (including Schistosoma mansoni). PD-L1 expression has been detected in several tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells, including endothelial cells, hepatocytes, muscle cells, and placenta.

[0081] PD-L1 expression is also associated with suppression of antitumor immune activity. Although tumors express antigens that can be recognized by host T cells, immune clearance of tumors is rare. Part of this deficiency is immune suppression by the tumor microenvironment. PD-L1 expression in many tumors is a component of this suppressive environment and acts in concert with other immunosuppressive signals. PD-L1 expression has been demonstrated in situ on a wide range of solid tumors, including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymic epithelial, and head and neck (Brown JA et al. 2003 Immunol. 170:1257-66; Dong H et al. 2002 Nat. Med. 8:793-800; Hamanishi J, et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003 Cancer Res. 63:6501-5; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). In addition, expression of the receptor for PD-L1, programmed cell death protein 1 (also known as PD-1 and CD279), is upregulated in tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al. 2003 Immunol. 171:4574-81).Most importantly, studies relating tumor PD-L1 expression to disease outcomes indicate that PD-L1 expression is strongly correlated with unfavorable prognosis in kidney, ovarian, bladder, breast, gastric and pancreatic cancers (Hamanishi J et al. 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007 Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). Furthermore, these studies suggest that higher levels of tumor PD-L1 expression may promote tumor stage progression and invasion of deeper tissue structures.

[0082] The PD-1 pathway may also have a role in hematological malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al. 2007 Blood 110:296-304). PD-L1 is expressed on some primary T-cell lymphomas, particularly anaplastic large cell T-lymphoma (Brown JA et al, 2003 Immunol. 170:1257-66). PD-1 is highly expressed on T cells in angioimmunoblastic lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al. 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, T cells associated with lymphocytic or histiocytic (L&H) cells express PD-1. Microarray analysis using readout of genes induced by PD-1 ligation suggests that tumor-associated T cells respond to PD-1 signals in situ 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 signals.

[0083] Studies in animal models indicate that tumor PD-L1 inhibits T cell activation and tumor cell lysis and, in some cases, increases tumor-specific T cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al. 2005 Cancer Res. 65:1089-96). Tumor-associated APCs can also utilize the PD-1:PD-L1 pathway to control antitumor T cell responses. PD-L1 expression on tumor-associated bone marrow DC populations is upregulated by tumor environmental factors (Curiel TJ et al. 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in tumor-draining lymph nodes of B16 melanoma express IDO, which potently activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells requires cellular contact with IDO-expressing DCs (Sharma MD et al. 2007 Clin. Invest. 117:2570-82).

[0084] In certain embodiments, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand.

[0085] In certain embodiments, the PD-L1-binding bicyclic peptide ligand is selected from those disclosed in WO2020 / 128526 and WO2020 / 128527, the contents of each of which are incorporated herein by reference in their entirety.

[0086] In one embodiment, the PD-L1 binding bicyclic peptide ligand is C i SAGWLTMC ii QKLHLC iii (SEQ ID NO:52); C i SAGWLTMC ii Q[K(PYA)]LHLC iii (SEQ ID NO:53); C i SKGWLTMC ii Q[K(Ac)]LHLC iii (SEQ ID NO:54); C i SAGWLTKCii Q[K(Ac)]LHLC iii (SEQ ID NO:55); C i SAGWLTMC ii K[K(Ac)]LHLC iii (SEQ ID NO:56); C i SAGWLTMC ii Q[K(Ac)]LKLC iii (SEQ ID NO:57); C i SAGWLTMC ii Q[HArg]LHLC iii (SEQ ID NO:58); and C i SAGWLTMC ii [HArg]QLNLC iii (SEQ ID NO:59) [In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively, PYA represents 4-pentynoic acid, and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof.

[0087] In one embodiment, the PD-L1-binding bicyclic peptide ligand optionally comprises an N-terminal and / or C-terminal modification: [PYA]-[B-Ala]-[Sar 10 ]-SDK-(SEQ ID NO:52) (herein referred to as BCY10043); Ac-D-[HArg]-(SEQ ID NO:52)-PSH (herein referred to as BCY11865); Ac-SDK-(SEQ ID NO:53) (herein referred to as BCY11013); Ac-SDK-(SEQ ID NO:53)-PSH (herein referred to as BCY10861); Ac-D-[HArg]-(SEQ ID NO:54)-PSH (herein referred to as BCY11866); Ac-D-[HArg]-(SEQ ID NO:55)-PSH (herein referred to as BCY11867); Ac-D-[HArg]-(SEQ ID NO:56)-PSH (herein referred to as BCY11868); Ac-D-[HArg]-(SEQ ID NO:57)-PSH (herein referred to as BCY11869); Ac-SD-[HArg]-(SEQ ID NO:58)-PSHK (herein referred to as BCY12479); and Ac-SD-[HArg]-(SEQ ID NO:59)-PSHK (herein referred to as BCY12477) [wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, and Sar 10 represents 10 sarcosine units, and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof.

[0088] In one embodiment, the PD-L1 binding bicyclic peptide ligand is C i [HArg]DWC ii HWTFSHGHPC iii (SEQ ID NO:82); C i SAGWLTMC ii QKLHLC iii (SEQ ID NO:52); and C i SAGWLTMC ii Q[K(PYA)]LHLC iii (SEQ ID NO:53) [In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively, HArg represents homoarginine, and PYA represents 4-pentynoic acid. or a pharma- ceutically acceptable salt thereof.

[0089] In one embodiment, the PD-L1-binding bicyclic peptide ligand comprises an N-terminal and / or C-terminal modification: [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO:82) (herein designated BCY8938); [PYA]-[B-Ala]-[Sar 10 ]-SDK-(SEQ ID NO:52) (herein referred to as BCY10043); NH2-SDK-(SEQ ID NO:52)-[Sar 10 ]-[K(PYA)] (herein referred to as BCY10044); NH2-SDK-(SEQ ID NO:53) (herein referred to as BCY10045); and Ac-SDK-(SEQ ID NO:53)-PSH (herein referred to as BCY10861) [PYA stands for 4-pentynoic acid, B-Ala stands for beta-alanine, and Sar 10 represents 10 sarcosine units. or a pharma- ceutically acceptable salt thereof.

[0090] In some embodiments, the component present on the cancer cell is prostate-specific membrane antigen (PSMA).

[0091] Prostate-specific membrane antigen (PSMA), also known as glutamic acid carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamic peptidase I (NAALADase I) and NAAG peptidase, is an enzyme that in humans is encoded by the FOLH1 (folate hydrolase 1) gene. Human GCPII contains 750 amino acids and has a mass of approximately 84 kDa.

[0092] Human PSMA is highly expressed in the prostate, roughly 100-fold more abundant than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, increasing 8-12-fold over levels in noncancerous prostate cells. Because of this high expression, PSMA is being developed as a potential therapeutic and imaging biomarker for some cancers. In human prostate cancer, the higher the expression in tumors, the faster the time to progression and the greater the percentage of patients who suffer from recurrence.

[0093] In certain embodiments, the first peptide ligand comprises a PSMA-binding bicyclic peptide ligand.

[0094] In some embodiments, the PSMA-binding bicyclic peptide ligand is selected from those disclosed in WO2019 / 243455 and WO2020 / 120980, the contents of each of which are incorporated herein by reference in their entirety.

[0095] In certain embodiments, the component present in the cancer cells is membrane type 1 metalloprotease (MT1-MMP).

[0096] In certain embodiments, the first peptide ligand comprises an MT1-MMP binding bicyclic peptide ligand.

[0097] In certain embodiments, the MT1-MMP binding bicyclic peptide ligand is selected from those disclosed in WO2016 / 067035, WO2017 / 191460, and WO2018 / 115204, the contents of each of which are incorporated herein by reference in their entirety.

[0098] CD137-binding peptide ligand CD137 is a member of the tumor necrosis factor (TNF) receptor family. It is also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, and is induced by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but the majority are CD8+, not CD4+ T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and cells of the blood vessel wall at sites of inflammation. One characterized activity of CD137 is its costimulatory activity of activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. In addition, it enhances immune activity and can eliminate tumors in mice.

[0099] CD137 is a T cell costimulatory receptor induced by 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 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)). Interaction with its ligand increases CD137TCR-induced T cell proliferation, cytokine production, functional maturation and extended CD8+ T cell survival (Nam et al, Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et d - l., Annu. Rev. Immunol, 23:23-68 (2005)).

[0100] Signaling through CD137 by CD137L or agonistic monoclonal antibodies (mAbs) to CD137 increases TCR-induced T cell proliferation, cytokine production, and functional maturation and prolongs CD8+ T cell survival. These effects are due to (1) activation of NF-κB, c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways and (2) regulation of antiapoptotic and cell cycle-related gene expression.

[0101] Experiments performed in both CD137- and CD137L-deficient mice further demonstrate CD137 costimulation in the generation of fully competent T cell responses.

[0102] IL-2- and IL-15-activated NK cells express CD137, and ligation of CD137 by agonistic mAbs stimulates NK cell proliferation and IFN-γ secretion, but not cytolytic activity.

[0103] Furthermore, CD137-stimulated NK cells promote the expansion of activated T cells in vitro.

[0104] Through its costimulatory function, agonistic mAbs against CD137 have been shown to promote the rejection of cardiac and skin allografts, eradicate established tumors, expand primary antiviral CD8+ T cell responses, and increase T cell cytolytic capacity. These studies support the view that CD137 signaling promotes T cell functions that may enhance immunity against tumors and infections.

[0105] In certain embodiments where a heterotandem bicyclic peptide complex comprises two or more CD137 binding peptide ligands, two or more of the CD137 binding peptide ligands have the same peptide sequence. In certain embodiments where a heterotandem bicyclic peptide complex comprises two or more CD137 binding peptide ligands, two or more of the CD137 binding peptide ligands have different peptide sequences. In certain embodiments where a heterotandem bicyclic peptide complex comprises two or more CD137 binding peptide ligands, two or more of the CD137 binding peptide ligands are identical. In certain embodiments where a heterotandem bicyclic peptide complex comprises two or more CD137 binding peptide ligands, two or more of the CD137 binding peptide ligands are different.

[0106] In certain embodiments where the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the CD137-binding peptide ligand is a CD137-binding bicyclic peptide ligand. In certain embodiments where the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, two or more of the CD137-binding peptide ligands are CD137-binding bicyclic peptide ligands.

[0107] In some embodiments, the CD137-binding bicyclic peptide ligand is selected from those disclosed in WO2019 / 025811. In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the CD137-binding peptide ligand is a CD137-binding bicyclic peptide ligand selected from those disclosed in WO2019 / 025811. In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, two or more of the CD137-binding bicyclic peptide ligands are independently selected from those disclosed in WO2019 / 025811. The contents of WO2019 / 025811 are incorporated herein by reference in their entirety.

[0108] In one embodiment, the CD137-binding bicyclic peptide ligand is C i IEEGQYC ii FADPY[Nle]C iii (SEQ ID NO:5); C i [tBuAla]PE[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:6); C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (SEQ ID NO:7); C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:8); C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (SEQ ID NO:9); C i [tBuAla]P[K(PYA)][D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:10); C i [tBuAla]PE[D-Lys(PYA)]PYC iiFADPY[Nle]C iii (SEQ ID NO:11); C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (SEQ ID NO:12); C i [tBuAla]PE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO:60); C i IEE[dK(PYA)]QYC ii FADPY[Nle]C iii (SEQ ID NO:61); C i [tBuAla]EE(dK)PYC ii FADPY[Nle]C iii (SEQ ID NO:62); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:63); C i [tBuAla]EE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:64); C i [tBuAla]PE[dK(PYA)]PYC ii FANPY[Nle]C iii (SEQ ID NO:65); C i [tBuAla]PE[dK(PYA)]PYC ii FAEPY[Nle]C iii (SEQ ID NO:66); C i [tBuAla]PE[dK(PYA)]PYC ii FA[Aad]PY[Nle]C iii (SEQ ID NO:67); C i [tBuAla]PE[dK(PYA)]PYC ii FAQPY[Nle]C iii (SEQ ID NO:68); C i[tBuAla]PE[dK(PYA)]PYC ii FADPY [Nle] [Cysam] iii (SEQ ID NO:69); [MerPro] i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:70; herein referred to as BCY12353); [MerPro] i [tBuAla]PE[dK(PYA)]PYC ii FADPY [Nle] [Cysam] iii (SEQ ID NO:71; herein designated BCY12354); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:72); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:73); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:74; herein referred to as BCY12372); C i [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeAla]Y[Nle]C iii (SEQ ID NO:75); C i [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeDAla]Y[Nle]C iii (SEQ ID NO:76); C i [tBuAla]P[K(PYA)][dA]PYC ii FADPY[Nle]C iii (SEQ ID NO:77); C i [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]Ciii (SEQ ID NO:78); C i [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:79); C i [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:80); and [MerPro] i [tBuAla]EE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO:81; herein designated BCY13137) [In the ceremony, [MerPro] i , C i , C ii , C iii and [Cysam] iii represent the first (i), second (ii) and third (iii) reactive groups selected from cysteine, MerPro and Cysam, Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, Aad represents alpha-L-aminoadipic acid, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine and NMeAla represents N-methyl-alanine. or a pharma- ceutically acceptable salt thereof.

[0109] In one embodiment, the CD137-binding bicyclic peptide ligand is C i IEEGQYC ii FADPY[Nle]C iii (SEQ ID NO:5); C i [tBuAla]PE[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:6); C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (SEQ ID NO:7); C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:8); C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (SEQ ID NO:9); C i [tBuAla]P[K(PYA)][D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:10); C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:11); and C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (SEQ ID NO:12) [In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively; Nle represents norleucine; tBuAla represents t-butyl-alanine; and PYA represents 4-pentynoic acid. or a pharma- ceutically acceptable salt thereof.

[0110] In one embodiment, the CD137-binding bicyclic peptide ligand is C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:11) [In the formula, C i , C ii and C iii represent the first, second, and third cysteine ​​residues, respectively; tBuAla represents t-butyl-alanine; PYA represents 4-pentynoic acid; and Nle represents norleucine. or a pharma- ceutically acceptable salt thereof.

[0111] In one embodiment, the CD137 binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification, Ac-A-(SEQ ID NO:5)-Dap (herein referred to as BCY7732); Ac-A-(SEQ ID NO:5)-Dap(PYA) (herein referred to as BCY7741); Ac-(SEQ ID NO:6)-Dap (herein referred to as BCY9172); Ac-(SEQ ID NO:6)-Dap(PYA) (herein referred to as BCY11014); Ac-A-(SEQ ID NO:7)-Dap (herein referred to as BCY8045); Ac-(SEQ ID NO:8)-A (herein referred to as BCY8919); Ac-(SEQ ID NO:9)-A (herein referred to as BCY8920); Ac-(SEQ ID NO:10)-A (herein referred to as BCY8927); Ac-(SEQ ID NO:11)-A (herein referred to as BCY8928); (SEQ ID NO:11)-A (herein designated BCY14601); Ac-A-(SEQ ID NO:12)-A (herein referred to as BCY7744); Ac-(SEQ ID NO:60)-Dap(PYA) (herein referred to as BCY11144); Ac-A-(SEQ ID NO:61)-K (herein referred to as BCY11613); Ac-(SEQ ID NO:62)-Dap(PYA) (herein referred to as BCY12023); Ac-(SEQ ID NO:63) (herein referred to as BCY12149); Ac-(SEQ ID NO:64) (herein referred to as BCY12143); Ac-(SEQ ID NO:65) (herein referred to as BCY12147); Ac-(SEQ ID NO:66) (herein referred to as BCY12145); Ac-(SEQ ID NO:67) (herein referred to as BCY12146); Ac-(SEQ ID NO:68) (herein referred to as BCY12150); Ac-(SEQ ID NO:69) (herein referred to as BCY12352); Ac-(SEQ ID NO:72)-[1,2-diaminoethane] (herein referred to as BCY12358); [palmitic acid]-[yGlu]-[yGlu]-(SEQ ID NO:73) (herein designated BCY12360); Ac-(SEQ ID NO:75) (herein referred to as BCY12381); Ac-(SEQ ID NO:76) (herein referred to as BCY12382); Ac-(SEQ ID NO:77)-K (herein referred to as BCY12357); Ac-(SEQ ID NO:78)-[dA] (herein referred to as BCY13095); [Ac]-(SEQ ID NO:78)-K (herein referred to as BCY13389); Ac-(SEQ ID NO:79)-[dA] (herein referred to as BCY13096); and Ac-(SEQ ID NO:80) (herein referred to as BCY13097) (wherein Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid.) or a pharma- ceutically acceptable salt thereof.

[0112] In one embodiment, the CD137 binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification, Ac-A-(SEQ ID NO:5)-Dap (herein referred to as BCY7732); Ac-A-(SEQ ID NO:5)-Dap(PYA) (herein referred to as BCY7741); Ac-(SEQ ID NO:6)-Dap (herein referred to as BCY9172); Ac-(SEQ ID NO:6)-Dap(PYA) (herein referred to as BCY11014); Ac-A-(SEQ ID NO:7)-Dap (herein referred to as BCY8045); Ac-(SEQ ID NO:8)-A (herein referred to as BCY8919); Ac-(SEQ ID NO:9)-A (herein referred to as BCY8920); Ac-(SEQ ID NO:10)-A (herein referred to as BCY8927); Ac-(SEQ ID NO:11)-A (herein referred to as BCY8928); and Ac-A-(SEQ ID NO:12)-A (herein referred to as BCY7744) (wherein Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid.) or a pharma- ceutically acceptable salt thereof.

[0113] In one embodiment, the CD137 binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification, Ac-(SEQ ID NO:11)-A (herein referred to as BCY8928) (In the formula, Ac represents an acetyl group.) or a pharma- ceutically acceptable salt thereof.

[0114] In certain embodiments, wherein 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 (herein referred to as BCY8928), where Ac represents an acetyl group, or a pharma- ceutically acceptable salt thereof.

[0115] In certain embodiments, wherein the heterotandem bicyclic peptide complex comprises two CD137-binding peptide ligands, both of the CD137-binding peptide ligands have the same peptide sequence, which includes Ac-(SEQ ID NO:11)-A (herein referred to as BCY8928), where Ac represents an acetyl group, or a pharma- ceutically acceptable salt thereof.

[0116] Linker Heterotandem bicyclic peptide complexes containing two or more CD137-binding peptide ligands It is recognized that the first peptide ligand may be conjugated to two or more second peptide ligands via any suitable linker. Typically, the design of the linker is such that three bicyclic peptides are present in such a way that they can bind unhindered to each target, either alone, or while simultaneously binding to both target receptors. Furthermore, the linker must allow binding to both targets simultaneously while maintaining an appropriate distance between the target cells that leads to the desired functional outcome. The nature of the linker can be adjusted to increase the length, rigidity, or solubility to optimize the desired functional outcome. The linker can also be designed to allow binding of more than one bicycle to the same target. Increasing the valency of either of the binding peptides may affect the affinity of the heterotandem to the target cell or help induce oligomerization of one or both of the target receptors.

[0117] In some embodiments, the linker is a branched linker allowing for one first peptide on one end and two or more second peptides on the other end.

[0118] In one embodiment, the branched linker is [ka] N-(acid-PEG3)-N-bis(PEG3-azide); [ka] Trimesic acid-[PEG 10 ]3; [ka] TCA-[Peg 10 ]3; [ka] Tet-[Peg 10 ]4; and [ka] BAPG-(Peg5)2 is selected from.

[0119] In one embodiment, the branched linker is [ka] N-(Acid-PEG3)-N-Bis(PEG3-Azide) It is.

[0120] Heterotandem bicyclic peptide complexes containing one CD137-binding peptide ligand It is recognized that the first peptide ligand can be conjugated to the second peptide ligand via any suitable linker. Typically, the design of the linker is such that the two bicyclic peptides are present in such a way that they can bind unhindered to each target, either alone, or while simultaneously binding to both target receptors. Furthermore, the linker must allow binding to both targets simultaneously while maintaining an appropriate distance between the target cells that leads to the desired functional outcome. The nature of the linker can be adjusted to increase the length, rigidity, or solubility to optimize the desired functional outcome. The linker can also be designed to allow binding of more than one bicycle to the same target. Increasing the valency of either of the binding peptides can affect the affinity of the heterotandem to the target cell or help induce oligomerization of one or both of the target receptors.

[0121] In some embodiments, the linker is selected from the following sequences: -PEG5- and TCA-[PEG 10 ]3.

[0122] The structures of these linkers are detailed below: [ka] -PEG5-; and [ka] TCA-[PEG 10 ]3.

[0123] In some embodiments, the linker is selected from the following sequences: -CH2-, -PEG5-, -PEG 10 -,-PEG 12 -,-PEG 23 -,-PEG 24 -,-PEG 15 -Sar5-,-PEG 10 -Sar 10 -,-PEG5-Sar 15 -, -PEG5-Sar5-, -B-Ala-Sar 20 -,-B-Ala-Sar 10 -PEG 10 -,-B-Ala-Sar5-PEG 15 - and -B-Ala-Sar5-PEG5-.

[0124] In some embodiments, the linker is selected from the following: [ka] [ka]

[0125] Heterotandem bicyclic peptide conjugates In certain embodiments, wherein the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises a nectin-4-binding bicyclic peptide ligand bound to a TATA scaffold, each of the two or more CD137-binding bicyclic peptide ligands being bound to a TATA scaffold, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table A. [Table 1]

[0126] In certain embodiments, the heterotandem bicyclic peptide complex is selected from BCY11027, BCY11863 and BCY11864. In certain embodiments, the heterotandem bicyclic peptide complex is selected from BCY11863 and BCY11864.

[0127] The heterotandem bicyclic peptide complex BCY11863 (also called BT7480) consists of the nectin-4 specific peptide BCY8116 linked to two CD137 specific peptides (both of which are BCY8928) via N-(acid-PEG3)-N-bis(PEG3-azide) linkers, as shown diagrammatically below. [ka]

[0128] CD137 is a homotrimeric protein, and the natural ligand CD137L exists as a homotrimer expressed or secreted by immune cells. The biology of CD137 is highly dependent on multimerization to induce CD137 activity in immune cells. One way to generate CD137 multimerization is cellular crosslinking of CD137-specific agonists through interaction with specific receptors present on other cells. The advantage of the heterotandem complex of the present invention is that the presence of two or more peptide ligands specific for immune cell components such as CD137 provides more effective clustering of CD137. For example, BCY11863 shows strong CD137 activation and induces robust IL-2 and IFN-γ cytokine 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 cynomolgus monkeys.

[0129] The heterotandem bicyclic peptide complex BCY11027 is a TCA-[Peg 10 It consists of the nectin-4 specific peptide BCY11015 linked to two CD137 specific peptides (both of which are BCY8928) via a .3 linker. [ka]

[0130] We found that the Nectin-4 / CD137 heterotandem BCY11027 induced target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors and induced Nectin-4-dependent changes in several immune markers (normalized to vehicle) and %CD8+ki67+T cells in patient-derived samples that correlated with Nectin-4 expression levels.

[0131] In certain embodiments, wherein the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises a nectin-4-binding bicyclic peptide ligand bound to a TATA scaffold, each of the two or more CD137-binding bicyclic peptide ligands being bound to a TATA scaffold, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table B. [Table 2]

[0132] In one embodiment, the heterotandem bicyclic peptide complex comprises two or more CD137-binding peptide ligands, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand bound to a TATA scaffold, each of the two or more CD137-binding bicyclic peptide ligands is bound to a TATA scaffold, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table C. [Table 3] [Table 4] [Table 5] [Table 6]

[0133] In certain embodiments, the heterotandem bicyclic peptide complex is selected from BCY12491, BCY12730, BCY13048, BCY13050, BCY13053, and BCY13272.

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

[0135] In one embodiment, the heterotandem bicyclic peptide conjugate is BCY12491.

[0136] The heterotandem bicyclic peptide complex BCY12491 consists of the EphA2-specific peptide BCY9594 linked to two CD137-specific peptides (both of which are BCY8928) via N-(acid-PEG3)-N-bis(PEG3-azide) linkers, as shown in the diagram below. [ka]

[0137] BCY12491 It was found that BCY12491 produced a significant anti-tumor response as well as modulation (increase) of tumor-infiltrating immune cells and immune responses.

[0138] In one embodiment, the heterotandem bicyclic peptide complex is BCY13272.

[0139] The heterotandem bicyclic peptide complex BCY13272 consists of the EphA2-specific peptide BCY13118 linked to two CD137-specific peptides (both of which are BCY8928) via N-(acid-PEG3)-N-bis(PEG3-azide) linkers, as shown below. [ka]

[0140] BCY13272 BCY13272 was found to have significant antitumor effects in a mouse MC38 tumor model.

[0141] In certain embodiments where 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 a TATA scaffold, and each of the two or more CD137-binding bicyclic peptide ligands linked to a TATA scaffold and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table D. [Table 7]

[0142] In certain embodiments where the heterotandem bicyclic peptide complex comprises one CD137 binding peptide ligand, the first peptide ligand comprises a PD-L1 binding bicyclic peptide ligand bound to a TATA scaffold, and the one CD137 binding peptide ligand is bound to a TATA scaffold, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table E. [Table 8]

[0143] In certain embodiments, the heterotandem bicyclic peptide complex is selected from BCY12375 and BCY12021.

[0144] In certain embodiments where the heterotandem bicyclic peptide complex comprises one CD137 binding peptide ligand, the first peptide ligand comprises a PD-L1 binding bicyclic peptide ligand linked to a TATA scaffold, and the one CD137 binding peptide ligand linked to a TATA scaffold, and the heterotandem bicyclic peptide complex is selected from the complexes listed in Table E-2. [Table 9]

[0145] In one embodiment, the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand bound to a TATA scaffold, and the one CD137-binding peptide ligand is bound to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table F. [Table 10] [Table 11]

[0146] In certain embodiments, the heterotandem bicyclic peptide complex is selected from BCY13035, BCY13040, BCY13253, BCY13254, BCY13340, and BCY13342.

[0147] In one embodiment, the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand bound to a TATA scaffold, and the one CD137-binding peptide ligand is bound to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table F-2. [Table 12]

[0148] In one embodiment, the heterotandem bicyclic peptide conjugate is BCY7985, wherein the CD137-specific peptide BCY7859 is PEGylated. 12 via the N-terminal PYA group of the EphA2-specific peptide BCY6169. [ka]

[0149] In one embodiment, where the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand bound to a TATA scaffold, and the one CD137-binding peptide ligand is bound to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table G. [Table 13]

[0150] In certain embodiments, the heterotandem bicyclic peptide complex is selected from BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661, and BCY12969.

[0151] In one embodiment, where the heterotandem bicyclic peptide complex comprises one CD137-binding peptide ligand, the first peptide ligand comprises a nectin-4 binding bicyclic peptide ligand bound to a TATA scaffold, and the one CD137-binding peptide ligand is bound to a TATA scaffold, and the heterotandem complex is selected from the complexes listed in Table G-2. [Table 14]

[0152] In certain embodiments, the heterotandem bicyclic peptide conjugates are selected from those disclosed in US patent application Ser. No. 17 / 062,662, the contents of which are incorporated herein by reference in their entirety.

[0153] In some embodiments, the heterotandem bicyclic peptide conjugate is selected from those disclosed in U.S. Patent Publication 20190307836, the contents of which are incorporated herein by reference in their entirety.

[0154] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as those in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques of molecular biology, genetics and biochemical methods are used, which are incorporated herein by reference (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) 4). th ed., John Wiley & Sons, Inc.).

[0155] nomenclature numbering When indicating amino acid residue positions within the compounds of the present invention, cysteine ​​residues (C i , C ii and C iii ) are omitted from the numbering since they are invariant, and therefore the numbering of amino acid residues in SEQ ID NO:1 is as follows: C i -P1-1Nal2-dD3-C ii -M4-HArg5-D6-W7-S8-T9-P 10 -HyP 11 -W 12 -C iii (Sequence number 1).

[0156] For the purposes of this invention, it is assumed that all bicyclic peptides are cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) or 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) to yield trisubstituted structures. Cyclization with TBMB and TATA results in the C i , C ii and C iii occurs in.

[0157] molecular form N- or C-terminal extensions to the bicyclic core sequence are added to the left or right of the sequence separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail is shown as follows: βAla-Sar10-A-(SEQ ID NO:X).

[0158] Reverse peptide sequence In light of the description in Nair et al (2003) J Immunol 170(3), 1362-1373, it is anticipated that the peptide sequences disclosed herein will also be useful in their retro-inverso forms. For example, the sequences may be in reverse order (i.e., N-terminus becomes C-terminus and vice versa), as well as their stereochemistry (i.e., D-amino acids become L-amino acids and vice versa). For the avoidance of doubt, the full name or one-letter or three-letter code of an amino acid is intended herein to represent an L-amino acid, unless otherwise specified. If such an amino acid is intended to represent a D-amino acid, the amino acid is preceded by a lowercase d in square brackets, e.g., [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0159] Advantages of peptide ligands Certain heterotandem bicyclic peptide complexes of the present invention have several advantageous properties that are believed to make them suitable as drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. These advantageous properties include - species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability. Heterotandem bicyclic peptide complexes should ideally demonstrate stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that heterotandem bicyclic peptide lead candidates can be developed in animal models and confidently administered to humans; - a desirable solubility profile, which is a function of charge and the ratio of hydrophilic to hydrophobic residues and intra / inter-molecular H-bonds that are important for formulation and absorption purposes; - Selectivity. Certain heterotandem bicyclic peptide complexes of the invention exhibit good selectivity over other targets; - Optimal plasma half-life in circulation. Clinical applications and treatment regimens may necessitate the development of heterotandem bicyclic peptide complexes for short exposure in acute disease management situations or heterotandem bicyclic peptide complexes with longer retention in circulation and therefore optimal for management of more chronic disease states. Other factors that lead to a desirable plasma half-life are the need for sustained exposure for maximum therapeutic efficacy versus the attendant toxicity of sustained exposure of the drug. Importantly, the selected heterotandem bicyclic peptide complexes are required to achieve the in vitro EC of the compound. 50 Data are provided herein showing antitumor efficacy when administered at a frequency that does not maintain plasma concentrations above 100 nm. This is in contrast to large recombinant biological (i.e., antibody-based) approaches of CD137 agonism or bispecific CD137 agonism (Segal et al., Clin Cancer Res., 23(8):1929-1936 (2017), Claus et al., Sci Trans Med., 11(496): eaav5989, 1-12 (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889 (2019)). Without being bound by theory, the reason for this observation is believed to be due to the fact that the heterotandem bicyclic complexes have a relatively low molecular weight (typically <15 kDa), are fully synthetic, and are tumor-targeted agonists of CD137; i.e., they have a relatively short plasma half-life, but good tumor penetration and retention. Data are provided herein that fully support these advantages. For example, antitumor efficacy with humanized CD137 in a mouse syngeneic rodent model is shown daily or every 3 days. Furthermore, intraperitoneal pharmacokinetic data show that circulating concentrations of the conjugate are greater than the in vitro EC 50The heterotandem bicyclic peptide complexes as defined herein exhibit plasma half-lives of <3 hours, which are consistently lower than the plasma levels. Furthermore, tumor pharmacokinetic data indicate that the levels of the heterotandem bicyclic complexes in tumor tissues may be higher and more persistent compared to plasma levels. It is recognized that this observation forms an important further aspect of the invention. Thus, according to a further aspect of the invention, the heterotandem bicyclic peptide complexes as defined herein are administered in vitro to determine the in vitro EC 50 In accordance with another aspect of the present invention, there is provided a method of treating cancer comprising administering to a subject a therapeutically effective amount of the conjugate to a subject at a dosing frequency that does not maintain a plasma concentration of the conjugate above 100 mg / kg. - immune memory. Coupling of cancer cell-binding bicyclic peptide ligands with immune cell-binding bicyclic peptide ligands provides the synergistic advantage of immune memory. Data are provided herein showing that selected heterotandem bicyclic peptide complexes of the present invention not only eradicate tumors, but also that none of the inoculated complete responder mice develop tumors upon re-administration of tumorigenic factors (see FIG. 5). This indicates that treatment with selected heterotandem bicyclic peptide complexes of the present invention induces immunogenic memory in complete responder mice. This is a substantial clinical advantage to prevent recurrence of the tumor once it has been initially controlled and eradicated.

[0160] Peptide Ligands A peptide ligand as used herein refers to a peptide covalently bound to a molecular scaffold. Typically, such a peptide contains two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond to the scaffold, and a sequence encompassed by the reactive groups, called a loop sequence, because the peptide forms a loop when bound to the scaffold. In this case, the peptide contains at least three reactive groups selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine, and forms at least two loops on the scaffold.

[0161] Reactive Groups The molecular scaffold of the present invention can be attached to a polypeptide through the functional or reactive groups of the polypeptide. These are typically formed from the side chains of certain amino acids found in polypeptide polymers. Such reactive groups can be cysteine ​​side chains, lysine side chains or any other suitable reactive groups such as N-terminal amine groups or penicillamine. Details of suitable reactive groups can be found in WO2009 / 098450.

[0162] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartic acid or glutamic acid, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including azide, keto-carbonyl, alkyne, vinyl or aryl halide groups. The amino and carboxyl groups at the termini of polypeptides can also serve as reactive groups to form covalent bonds to molecular scaffolds / cores.

[0163] The polypeptides of the invention contain at least three reactive groups. They may also contain four or more reactive groups. The more reactive groups that are used, the more loops that can be formed in the molecular scaffold.

[0164] In a preferred embodiment, a polypeptide having three reactive groups is produced. The reaction of said polypeptide with a molecular scaffold / molecular core having a three-part rotational symmetry produces a single product isomer. The production of a single product isomer is preferred for several reasons. The nucleic acid of the compound library only codes for the primary sequence of the polypeptide, and does not code for the isomeric state of the molecule formed by the reaction of the polypeptide with the molecular core. If only two product isomers can be formed, the assignment of the nucleic acid to the product isomer is unambiguously defined. If multiple product isomers are formed, the nucleic acid does not provide information about the nature of the product isomer isolated during the screening or selection process. The formation of a single product isomer is also advantageous if a specific member of the library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold produces a single product isomer, not a mixture of isomers.

[0165] In another embodiment, a polypeptide having four reactive groups is produced. The reaction of said polypeptide with a molecular scaffold / molecular core having tetrahedral symmetry produces two product isomers. Even if two different product isomers are encoded by one and the same nucleic acid, the isomerism of the isolated isomers can be determined by chemical synthesis of both isomers, and the two isomers can be separated and both isomers can be tested for binding to target ligands.

[0166] In some embodiments of the present invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups.The use of orthogonal reactive groups allows the orthogonal reactive groups to be directed to specific positions on the molecular core.The linking strategy involving orthogonal reactive groups can be used to limit the number of product isomers formed.In other words, by selecting one or more of at least three bonds with a separate or different reactive group from that selected for the remaining at least three bonds, a specific order of binding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold can be usefully achieved.

[0167] In other embodiments, a reactive group of a polypeptide of the invention is reacted with a molecular linker, where the linker is capable of reacting with a molecular scaffold such that in the final conjugated state the linker is interposed between the molecular scaffold and the polypeptide.

[0168] In some embodiments, the amino acids of the members of a library or set of polypeptides can be replaced with any natural or unnatural amino acid. Excluded from these replaceable amino acids are those that have a functional group for crosslinking to the molecular core of the polypeptide such that the loop sequence alone is replaceable. The replaceable polypeptide sequences have random sequences, constant sequences or sequences with random and constant amino acids. The amino acids with reactive groups are located at defined positions within the polypeptide as the position of these amino acids determines the loop size.

[0169] In one embodiment, the polypeptide having three reactive groups has the sequence (X): lY(X) m Y(X) n Y(X) o where Y represents an amino acid having a reactive group, X represents a random amino acid, m and n are numbers from 3 to 6 that define the length of the intervening polypeptide segment, which may be the same or different, and o is a number from 0 to 20 that defines the length of the adjacent polypeptide segment.

[0170] Alternatives to thiol-mediated conjugation can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively, these techniques can be used to modify the polypeptide or attach further moieties (e.g. small molecules of interest different from the molecular scaffold) after selection or isolation according to the invention - in this embodiment, if so, obviously the attachment does not have to be covalent and can include non-covalent attachment. These methods can be used instead of (or in combination with) the thiol-mediated method by producing phage displaying proteins and peptides carrying unnatural amino acids with the requisite chemically reactive groups in combination with small molecules with complementary reactive groups, or by incorporating unnatural amino acids into chemically synthesized or recombinantly synthesized polypeptides if the molecules are produced after the selection / isolation phase. Further details can be found in WO2009 / 098450 or Heinis et al., Nat Chem Biol 2009, 5 (7), 502-7.

[0171] In certain embodiments, the reactive groups are selected from cysteine, 3-mercaptopropionic acid and / or cysteamine residues.

[0172] Pharmaceutically acceptable salts It will be appreciated that salt forms are within the scope of the present invention and that a peptide ligand includes the ligand in its salt form.

[0173] The salts of the present invention can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base form of the compound with the appropriate base or acid in water or an organic solvent, or a mixture of the two.

[0174] Acid addition salts (mono or di-salts) can be formed from a wide variety of acids, both inorganic and organic. Examples of acid addition salts are acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor 10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cycla acid, and the like. , dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g. D-glucuronic acid), glutamic acid (e.g. L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g. hydrobromic acid, hydrochloric acid, iodine acid, etc.) hydric acid), isethionic acid, lactic acid (e.g. (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, The mono- or di-salts formed with acids selected from the group consisting of carboxylic acid, pamoic 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, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid and valeric acid as well as acylated amino acids and cation exchange resins.

[0175] One particular group of salts consists of the salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0176] If the compound has a functional group that is anionic or may be anionic (e.g., -COOH is -COO - Salts can be formed with organic or inorganic bases which form suitable cations. Examples of suitable inorganic cations are alkali metal ions, e.g. Li + , Na + and K. + , alkaline earth metal cations, e.g. Ca 2+ and Mg 2+ and other cations, e.g. Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are included, but are not limited to. Examples of some suitable substituted ammonium ions include methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0177] When the compounds of the present invention contain an amine function, they may be reacted with alkylating agents to form quaternary ammonium salts by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0178] modified derivative It will be appreciated that modified derivatives of the peptide ligands described herein are within the scope of the present invention. Examples of suitable modified derivatives include N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues; addition of one or more amino acid residues with alanine, replacement of one or more L-amino acid residues with one or more D-amino acid residues, addition of one or more non-naturally occurring amino acid residues with alanine ... The modifications include one or more selected from N-alkylation of an amide bond; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; replacement of the hydrogen of the alpha-carbon of one or more amino acid residues with other chemical groups, modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive agents for functionalizing the amino acid, and introduction or replacement of amino acids that introduce orthogonal reactivity suitable for functionalization, for example azide or alkyne group bearing amino acids that allow functionalization with alkyne or azide bearing moieties, respectively.

[0179] In some embodiments, modified derivatives include N-terminus and / or C-terminus modification.In further embodiments, modified derivatives include N-terminus modification using suitable amino-reactive chemistry and / or C-terminus modification using suitable carboxy-reactive chemistry.In further embodiments, said N-terminus or C-terminus modification comprises the addition of effector groups, including but not limited to cytotoxic agents, radiochelators or chromophores.

[0180] In certain embodiments, the modified derivative comprises an N-terminal modification. In further embodiments, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, an N-terminal cysteine ​​group (which group is referred to herein as C i(referred to as "bicyclic peptide") is capped with acetic anhydride or other suitable reagent during peptide synthesis to acetylate the N-terminus of the molecule. This embodiment offers the advantage of eliminating a potential recognition point for aminopeptidases and avoiding possible degradation of the bicyclic peptide.

[0181] In certain embodiments, the N-terminal modification includes the addition of a molecular spacer group that facilitates the conjugation of an effector group and retention of the bicyclic peptide's ability to target its target.

[0182] In certain embodiments, the modified derivative comprises a C-terminal modification. In further embodiments, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine ​​group (which group is referred to herein as C iii (referred to as "amyl group") is synthesized as an amide during peptide synthesis to amidate the C-terminus of the molecule. This embodiment has the advantage of eliminating a potential recognition point for carboxypeptidases, reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0183] In certain embodiments, the modified derivatives include the replacement of one or more amino acid residues with one or more non-natural amino acid residues, which may be selected from non-natural amino acids that have isosteric / isoelectronic side chains that are not recognized by degradative proteases and have no adverse effect on targeting ability.

[0184] Alternatively, unnatural amino acids can be used that have constrained amino acid side chains such that proteolytic hydrolysis of neighboring peptide bonds is conformationally and sterically hindered. In particular, these are considered to be proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib) and cycloamino acids, a simple derivative being amino-cyclopropyl carboxylic acid.

[0185] In certain embodiments, the modified derivatives include the addition of a spacer group. In certain embodiments, the modified derivatives include the addition of an N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii ) is included.

[0186] In some embodiments, the modified derivative comprises replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In some embodiments, the modified derivative comprises replacing a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0187] In some embodiments, the modified derivatives include the replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In another embodiment, the modified derivatives include the replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged amino acid residues versus hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues can affect the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. The two can combine to affect the half-life, distribution volume and exposure of the peptide drug, allowing it to be tailored according to clinical endpoints. Furthermore, the correct combination and number of charged versus hydrophobic amino acid residues can reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

[0188] In certain embodiments, the modified derivatives include the replacement of one or more L-amino acid residues with one or more D-amino acid residues, an embodiment believed to increase proteolytic stability due to steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

[0189] Modified derivatives include the removal of any amino acid residue and substitution with alanine in D-amino acids to stabilize the β-turn conformation. This embodiment offers the advantage of removing potential proteolytic attack sites.

[0190] It should be noted that each of the above modifications serves to purposefully improve the efficacy or stability of the peptide. Further efficacy improvements based on the modifications can be achieved via the following mechanisms: - incorporation of hydrophobic moieties to exploit the hydrophobic effect, leading to low off-rates, so that high affinity is achieved; - incorporation of charged groups that take advantage of long range ionic interactions leading to fast on-rates and high affinity (see e.g. Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - Incorporation of further constraints into the peptide, for example by constraining the amino acid side chains correctly so that entropy loss is minimal upon target binding, constraining the main chain torsion angles so that entropy loss is minimal upon target binding, and introducing further cyclization into the molecule for the same reasons. (For reviews, see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

[0191] Examples of modified heterotandem bicyclic peptide conjugates of the invention include those listed in Tables H and I below: [Table 15] [Table 16]

[0192] Isotope Variation The present invention includes all pharma- ceutically acceptable (radio)isotope-labeled peptide ligands of the invention in which one or more atoms have been replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, as well as peptide ligands of the invention having attached thereto a metal chelating group capable of bearing a relevant (radio)isotope (referred to as an "effector") and peptide ligands of the invention in which a functional group has been covalently replaced with a relevant (radio)isotope or an isotopically labeled functional group.

[0193] Examples of isotopes suitable for inclusion in the peptide ligands of the invention include isotopes of hydrogen, e.g. 2 H(D) and 3 H(T), an isotope of carbon, e.g. 11 C. 13 C and 14 C, an isotope of chlorine, e.g. 36 Cl, an isotope of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I and 131 I, an isotope of nitrogen, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O and 18 O, isotopes of phosphorus, e.g. 32 P, isotopes of sulfur, e.g. 35 S, isotopes of copper, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Isotopes of Y and lutetium, e.g. 177 Isotopes of Lu and bismuth, e.g. 213 Contains Bi.

[0194] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating a radioisotope, are useful for drug and / or substrate tissue distribution studies and clinical evaluation of the presence and / or absence of Nectin-4 targets in diseased tissues. The peptide ligands of the invention further have valuable diagnostic properties that can be used to detect or identify complex formation between the labeled compound and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods can use compounds used with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). The radioisotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14 C is particularly useful for this purpose in view of its ease of incorporation and ready means of detection.

[0195] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H (D), can offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferable in certain situations.

[0196] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) testing for target occupancy.

[0197] Isotopically labeled compounds of the peptide ligands of the invention may generally be prepared by conventional techniques known to those of skill in the art or by methods analogous to those described in the accompanying Examples, substituting an appropriate isotopically labeled reagent for the unlabeled reagent previously used.

[0198] Molecular scaffolds Molecular scaffolds are described, for example, in WO2009 / 098450 and the references cited therein, in particular WO2004 / 077062 and WO2006 / 078161.

[0199] As noted in the above references, the molecular scaffold can be a small molecule, such as a small organic molecule.

[0200] In some embodiments, the molecular scaffold can be a polymer, hi some embodiments, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates.

[0201] In certain embodiments, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0202] The molecular scaffold may contain chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0203] In certain embodiments, the molecular scaffold may comprise or consist of a hexahydro-1,3,5-triazine, in particular 1,3,5-triacryloylhexahydro-1,3,5-triazine ('TATA') or a derivative thereof.

[0204] The molecular scaffolds of the invention comprise chemical groups that enable functional groups of the polypeptides of the encoded libraries of the invention to form covalent bonds with the molecular scaffold, the chemical groups being selected from a wide range of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

[0205] Scaffold reactive groups that can be used to react with the thiol groups of cysteines in molecular scaffolds are alkyl halides (or alternatively halogenoalkanes or haloalkanes).

[0206] Examples include bromomethylbenzene (TBMB is an example scaffold reactive group) or iodoacetamide. Other scaffold reactive groups used for selective attachment of compounds to cysteines in proteins are maleimides, α-unsaturated carbonyl-containing compounds and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an α-unsaturated carbonyl-containing compound is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of an α-halomethylcarbonyl-containing compound is N,N',N''-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a naturally occurring amino acid with similar reactivity to cysteine ​​and can be used in the same reactions. Thus, where cysteine ​​is recited, substitution with selenocysteine ​​is typically acceptable unless the context suggests otherwise.

[0207] synthesis The peptides of the present invention can be produced by synthesis using standard techniques followed by reaction with molecular scaffolds in vitro. Standard chemistry can be used in this implementation. This allows for rapid large-scale production of soluble material for further downstream experiments or validation. Such methods can be achieved using conventional chemistry, such as that disclosed in Timmerman et al (supra).

[0208] Thus, the present invention also relates to the production of a selected polypeptide or conjugate as set forth herein, where the production comprises optional further steps as explained below, in certain embodiments, these steps are carried out on the final product polypeptide / conjugate produced by chemical synthesis.

[0209] If desired, amino acid residues in the polypeptide of interest may be substituted during preparation of the conjugate or complex.

[0210] The peptides can be extended, for example, by the incorporation of other loops, thus introducing multispecificity.

[0211] To extend peptides, they can simply be chemically extended using orthogonally protected lysines (and analogues) at the N- or C-terminus or within a loop using standard solid- or solution-phase chemistry. Activated or activatable N- or C-termini can be introduced using standard (bio)conjugation techniques. Alternatively, they can be added enzymatically using fragment condensation or native chemical ligation as described, for example, in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or subtiligase as described, for example, in (Chang et al. Proc Natl Acad Sci US A. 1994 Dec 20; 91(26):12544-8 or Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0212] Alternatively, peptides can be extended or modified by further conjugation through disulfide bonds.This has the added advantage that the first and second peptides can dissociate from each other when entering the reducing environment of cells.In this case, a molecular scaffold (e.g. TATA) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; then, additional cysteine ​​or thiol is added to the N-terminus or C-terminus of the first peptide, and this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form disulfide-bonded bicyclic peptide-peptide conjugate.

[0213] Similar techniques for the synthesis / coupling of the two bicyclic and bispecific macrocycles may be equally applicable to produce tetraspecific molecules.

[0214] Furthermore, the addition of other functional or effector groups can be achieved in the same manner using appropriate chemistry, coupling at the N-terminus or C-terminus or via side chains, In certain embodiments, the coupling is performed in a manner that does not block the activity of either entity.

[0215] 2. Compounds and Definitions: The term "pharmaceutically acceptable salts" as used herein refers to salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., within the scope of reasonable medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as by using ion exchange. Other pharma- ceutically acceptable salts include those of adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bisulfate, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfate, ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, hemisulfate, heptanoic acid, hexanoic acid, hydroiodic acid, 2-hydroxy-ethanoic acid, and 1,2-dihydro-1,2-trimethylolpropanesulfate. These include phenylsulfonic acid, lactobionic acid, lactic acid, lauric acid, lauryl sulfate, malic acid, maleic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pectinic acid, persulfate, 3-phenylpropionic acid, phosphoric acid, pivalic acid, propionic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecanoic acid, and valerate.

[0216] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include non-toxic ammonium, quaternary ammonium and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates and arylsulfonates.

[0217] Unless otherwise stated, structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also intended to include compounds which differ only by the presence of one or more isotopically enriched atoms. For example, the replacement of hydrogen with deuterium or tritium or the replacement of a carbon with a heteroatom. 13 C or 14 Compounds having this structure including C-rich carbon replacements are within the scope of this invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or as therapeutic agents according to the present invention.

[0218] As used herein, the term "about" refers to within 20% of the recited value. In some embodiments, the term "about" refers to within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the recited value.

[0219] As used herein, the term "mg / kg" refers to milligrams of drug per kilogram of the body weight of the subject being administered.

[0220] 3. Pharmaceutically acceptable compositions According to one embodiment, the present invention provides a pharmaceutical composition comprising a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier, adjuvant or vehicle. In one embodiment, the present invention provides a pharmaceutical composition for use in the treatment of cancer comprising a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands or a pharma- ceutically acceptable salt thereof, a cancer immunotherapeutic agent and a pharma- ceutically acceptable carrier, adjuvant or vehicle.

[0221] In one embodiment, the present invention provides a pharmaceutical composition comprising BT7480 or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier, adjuvant or vehicle.In one embodiment, the present invention provides a pharmaceutical composition comprising BT7480 or a pharma- ceutically acceptable salt thereof, a cancer immunotherapeutic agent and a pharma- ceutically acceptable carrier, adjuvant or vehicle for use in the treatment of cancer.

[0222] In one embodiment, the present invention provides a pharmaceutical composition comprising BT7455 or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier, adjuvant or vehicle.In one embodiment, the present invention provides a pharmaceutical composition for use in the treatment of cancer comprising BT7455 or a pharma- ceutically acceptable salt thereof, a cancer immunotherapeutic agent and a pharma- ceutically acceptable carrier, adjuvant or vehicle.

[0223] In some embodiments, the composition comprises a pharmaceutically acceptable carrier, adjuvant or vehicle.The term "pharmaceutically acceptable carrier, adjuvant or vehicle" refers to a non-toxic carrier, adjuvant or vehicle that does not impair the pharmacological activity of the compound that is formulated together.The pharmaceutically acceptable carrier, adjuvant or vehicle that can be used in the composition of the present invention includes, but is not limited to, ion exchanger, alumina, aluminum stearate, lecithin, serum protein such as human serum albumin, buffer substances such as phosphoric acid, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acid, water, salt or electrolyte such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylic acid, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol and wool fat.

[0224] The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0225] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally or intravenously. The compositions of the present invention in sterile injectable form may be aqueous or oily suspensions. These suspensions may be formulated by techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used are, inter alia, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conveniently used as solvents or suspending media.

[0226] For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides.Fatty acids such as oleic acid and its glyceride derivatives are useful for the preparation of injections, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions.These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions.Other commonly used surfactants, such as twin, span and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, can also be used for formulation purposes.

[0227] The pharmaceutically acceptable composition of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.For tablets for oral use, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate can also be typically added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are required for oral use, active ingredient is combined with emulsifying and suspending agents.If desired, certain sweeteners, flavors or coloring agents can also be added.

[0228] Alternatively, the pharma- ceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

[0229] The pharma- ceutically acceptable compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0230] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical-transdermal patches may also be used.

[0231] For topical application, the provided pharma- ceutically acceptable composition can be formulated into a suitable ointment containing the 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 petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, the provided pharma-ceutically acceptable composition can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma-ceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0232] For ophthalmic use, the provided pharma- ceutically acceptable compositions may be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, with or without a preservative such as benzalkonium chloride, or, preferably, as a solution in isotonic, pH-adjusted sterile saline. Alternatively, for ophthalmic use, the pharma-ceutically acceptable compositions may be formulated in an ointment such as petrolatum.

[0233] The pharma- ceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation.Such compositions may be prepared according to techniques well known in the art of pharmaceutical formulation, and may be formulated as saline solutions employing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0234] The pharma- ceutically acceptable composition of the present invention can also be formulated for oral administration. Such formulations can be administered with or without food. In some embodiments, the pharma- ceutically acceptable composition of the present invention is administered without food. In other embodiments, the pharma- ceutically acceptable composition of the present invention is administered with food.

[0235] The specific dosage and treatment regimen for any particular patient will depend on a wide variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, dietary habits, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the invention in the composition will also depend on the particular compound in the composition.

[0236] 4. Methods of Treating Cancer According to one embodiment, the present invention provides a method of treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of a heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof comprising one or more CD137-binding peptide ligands and a cancer immunotherapeutic agent.

[0237] In certain embodiments, the present invention provides the use of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands, or a pharma- ceutically acceptable salt thereof, in combination with a cancer immunotherapeutic agent for the treatment of cancer.

[0238] In one embodiment, 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 a cancer immunotherapeutic agent. In one embodiment, the present invention provides the use of BT7480 or a pharmaceutically acceptable salt thereof in combination with a cancer immunotherapeutic agent for the treatment of cancer.

[0239] In one embodiment, 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 a cancer immunotherapeutic agent.In one embodiment, the present invention provides the use of BT7455 or a pharmaceutically acceptable salt thereof in combination with a cancer immunotherapeutic agent for the treatment of cancer.

[0240] Examples of cancer 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 high MT1-MMP expression. For example, Adley et al. reported that MT1-MMP is expressed at high levels in ovarian clear cell carcinoma (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 February; 112(2): 319-324).

[0241] In certain embodiments, the cancer is associated with Nectin-4. In certain embodiments, the cancer is high Nectin-4 expression.

[0242] In certain embodiments, the cancer is associated with EphA2. In certain embodiments, the cancer is high EphA2 expression.

[0243] In certain embodiments, the cancer is associated with PD-L1. In certain embodiments, the cancer is high PD-L1 expression.

[0244] In some embodiments, the cancer is associated with PSMA. In some embodiments, the cancer is high PSMA expression.

[0245] In some embodiments, the cancer is bladder cancer. In some embodiments, the bladder cancer is selected from the group consisting of basal, p53-like and luminal.

[0246] In some embodiments, the cancer is endometrial cancer.In some embodiments, the endometrial cancer is selected from the group consisting of MMR-D, POLE EDM, p53 WT, p53 abnormal, type I, type II, carcinoma, carcinosarcoma, endometrioid adenocarcinoma, serous carcinoma, clear cell carcinoma, mucinous carcinoma, mixed or undifferentiated carcinoma, mixed serous and endometrioid, mixed serous and low grade endometrioid and undifferentiated.

[0247] In some embodiments, the cancer is esophageal cancer. In some embodiments, the esophageal cancer is selected from the group consisting of adenocarcinoma (EAC), squamous cell carcinoma (ESCC), chromosomal instability (CIN), Epstein-Barr virus (EBV), genomic stable (GS) and microsatellite unstable (MSI).

[0248] In some embodiments, the cancer is glioblastoma. In some embodiments, the glioblastoma is selected from the group consisting of proneural, neural, classical and mesenchymal.

[0249] 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.

[0250] In some embodiments, the cancer is multiple myeloma. In some embodiments, the multiple myeloma is selected from the group consisting of hyperdiploid, non-hyperdiploid, cyclin D translocation, MMSET translocation, MAF translocation and unclassified.

[0251] In some embodiments, the cancer is ovarian cancer, hi some embodiments, the ovarian cancer is selected from the group consisting of clear cell, endometrioid, mucinous, high-grade serous and low-grade serous ovarian cancer.

[0252] In some embodiments, the cancer is pancreatic cancer, hi some embodiments, the pancreatic cancer is selected from the group consisting of squamous, pancreatic progenitor, immunogenic, and ADEX (dysdifferentiation of endocrine and exocrine) pancreatic cancer.

[0253] 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.

[0254] 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 T790M EGFR-expressing lung adenocarcinoma.

[0255] 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.

[0256] In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is colorectal adenocarcinoma. In some embodiments, the colorectal adenocarcinoma is high pgp expressing colorectal adenocarcinoma.

[0257] In some embodiments, the cancer is gastric cancer. In some embodiments, the gastric cancer is FGFR amplified gastric cancer.

[0258] In some embodiments, the cancer is head and neck cancer. In some embodiments, the head and neck cancer is nasal septum squamous cell carcinoma.

[0259] 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).

[0260] Cancer, in certain embodiments, includes leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovial tumor, mesothelioma, Ewin's disease, and leukemia, including leukemia, leukemia, and ... cancer, 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, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0261] In some embodiments, the cancer is a glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0262] In some embodiments, the cancer is an acoustic neuroma, astrocytoma (e.g., grade I - pilocytic astrocytoma, grade II - low-grade astrocytoma, grade III - anaplastic astrocytoma, or grade IV - glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brain stem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET) tumor, or schwannoma. In some embodiments, the cancer is a type more commonly found in children than adults, such as brain stem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor tumor (PNET), or rhabdoid tumor. In some embodiments, the patient is an adult. In certain embodiments, the patient is a child or a pediatric patient.

[0263] In other embodiments, the cancer is mesothelioma, hepatobiliary (liver and bile duct), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal (stomach, colorectal and duodenal), uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, Cancer, including, but not limited to, 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, brain stem glioma, pituitary adenoma, adrenal cortical carcinoma, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0264] In some embodiments, the cancer is selected from hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; biliary hepatoma cancer; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid cancer; adrenal cortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0265] In certain 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 papillary serous carcinoma (UPSC), hepatic bile duct cancer, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenal cortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0266] In some embodiments, the cancer is a solid tumor, such as sarcoma, carcinoma or lymphoma.Solid tumors generally include an abnormal mass of tissue that typically does not include cysts or liquid areas.In some embodiments, the cancer is 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 cancer or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; biliary hepatoma cancer ; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid carcinoma; adrenal cortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0267] In certain embodiments, the cancer is selected from renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal carcinoma, colorectal cancer, colon cancer, rectal cancer, anal cancer, ovarian cancer, ovarian epithelial cancer, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatic bile duct cancer, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, anaplastic thyroid cancer, adrenal cortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, brain cancer, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0268] In certain embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatic bile duct cancer, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenal cortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0269] 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 cancer. In some embodiments, the cancer is ovarian epithelial cancer. 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 hepatic bile duct cancer. 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 anaplastic thyroid carcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. 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 neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenstrom's macroglobulinemia. In some embodiments, the cancer is medulloblastoma.

[0270] In certain embodiments, the cancer is a virus-associated cancer, including human immunodeficiency virus (HIV)-associated solid tumor, human papillomavirus (HPV)-16 positive untreatable solid tumor, and adult T-cell leukemia, a highly aggressive form of CD4+ T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and characterized by clonal integration of HTLV-I in leukemia cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); and virus-associated tumors in gastric, nasopharyngeal, cervical, vaginal, vulvar, head and neck squamous cell carcinoma, and Merkel cell carcinoma (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; See also https: / / clinicaltrials.gov / ct2 / show / NCT02426892).

[0271] In some embodiments, the cancer is melanoma cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is small cell lung cancer (SCLC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0272] In some embodiments, cancer is treated by stopping the further growth of tumor.In some embodiments, cancer is treated by reducing tumor size (e.g., volume or mass) by at least 5%, 10%, 25%, 50%, 75%, 90% or 99% compared to the size of tumor before treatment.In some embodiments, cancer is treated by reducing tumor mass in patient by at least 5%, 10%, 25%, 50%, 75%, 90% or 99% compared to the tumor mass before treatment.

[0273] The heterotandem bicyclic peptide complexes and compositions according to the method of the present invention may be administered in any amount and using any route of administration effective for treating or reducing the severity of cancer. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the disease or condition, the specific agent, its method of administration, and the like. The heterotandem bicyclic peptide complexes are preferably formulated into dosage unit forms for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically separated unit of agent appropriate for the patient being treated. However, it is understood that the total daily usage of the heterotandem bicyclic peptide complexes and compositions of the present invention will be determined by the treating physician within the scope of reasonable medical judgment. The specific effective dosage level for a particular patient or organism will depend on a wide variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used; the age, weight, general health, sex and dietary habits of the patient; the time of administration, route of administration and excretion rate of the specific compound being used; duration of treatment; drugs used in combination or simultaneously with the specific compound being used and similar factors well known in the medical arts. The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0274] The pharma- ceutically acceptable compositions of the invention may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as a powder, ointment or drops), bucally, or as an oral or nasal spray, etc., depending on the severity of the disease or disorder being treated. In certain embodiments, the heterotandem bicyclic peptide complexes of the invention may be administered orally or parenterally, one or more times daily, at dosage levels of about 0.01 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 25 mg / kg of subject body weight per day, to obtain the desired therapeutic effect.

[0275] Liquid dosage forms for oral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavors and aromatics.

[0276] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated as known in the art using suitable dispersing or wetting agents and suspending agents.Sterile injectable preparations can be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol.Acceptable vehicles and solvents that can be used are, among others, water, Ringer's solution, USP and isotonic sodium chloride solution.In addition, sterile, fixed oils are conveniently used as solvents or suspending media.For this purpose, any non-irritating fixed oils can be used, including synthetic mono- or diglycerides.In addition, fatty acids, such as oleic acid, are used in injectable preparations.

[0277] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0278] In order to prolong the effect of the compounds of the present invention, it is often desirable to slow the absorption of the compounds from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material that is poorly water soluble. The rate of absorption of the compound then depends on its rate of dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form can be accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are prepared by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the compound to polymer ratio and the nature of the specific polymer used, the compound release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues.

[0279] Compositions for rectal or vaginal administration are preferably suppositories which may be prepared by mixing the heterotandem bicyclic peptide complexes of the present invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which is solid at ambient temperature but liquid at body temperature and thus will melt in the rectum or vaginal cavity and release the active compound.

[0280] 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, pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicic acids, and sodium carbonate, e) solution retarders, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) humectants, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0281] Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules, using additives such as lactose or milk sugar and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills and granules can be coated and shelled, such as enteric coatings and other coatings well known in the pharmaceutical art. They can also contain opacifying agents if desired, and can be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules, using additives such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0282] The active compound may be in microencapsulated form with one or more additives as described above. The solid dosage forms of tablets, dragees, capsules, pills and granules may be coated and shelled, such as enteric coating, release control coating and other coatings well known in the pharmaceutical art. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose or starch. Such dosage forms may also contain additional substances other than inert diluents, such as magnesium stearate and microcrystalline cellulose, as is common practice, and other tableting aids. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents, and may be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0283] For example, dosage forms for topical or transdermal administration of the heterotandem bicyclic peptide complexes comprising one or more CD137-binding peptide ligands described herein include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharma- ceutically acceptable carrier and any needed preservatives or buffers, if necessary. Ophthalmic formulations, ear drops and eye drops are also contemplated as being within the scope of the invention. Additionally, the invention contemplates the use of transdermal patches, which have the added advantage of controlling delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0284] Co-administration of heterotandem bicyclic peptide conjugates and cancer immunotherapeutic agents For example, the heterotandem bicyclic peptide complexes described herein and the cancer immunotherapeutic agent can be administered separately as part of a multiple dose regimen.Alternatively, for example, the heterotandem bicyclic peptide complexes described herein and the cancer immunotherapeutic agent can be mixed together as a single composition in a single dosage form.In some embodiments, the heterotandem bicyclic peptide complex is BT7480 or BT7455 or a pharmaceutically acceptable salt thereof.

[0285] In some embodiments, for example, the heterotandem bicyclic peptide complex described herein is administered separately from the cancer immunotherapeutic agent. In some embodiments, for example, the heterotandem bicyclic peptide complex described herein and the cancer immunotherapeutic agent are administered simultaneously. In some embodiments, for example, the heterotandem bicyclic peptide complex described herein and the cancer immunotherapeutic agent are administered sequentially. In some embodiments, for example, the heterotandem bicyclic peptide complex described herein and the cancer immunotherapeutic agent are administered within a certain time period of each other, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 18 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours of each other. In some embodiments, for example, the heterotandem bicyclic peptide complex described herein and the cancer immunotherapeutic agent are administered more than 24 hours apart. In some embodiments, for example, a heterotandem bicyclic peptide complex described herein and a cancer immunotherapeutic agent are administered within 1, 2, 3, 4, 5, 6, or 7 days of each other. In some embodiments, for example, a heterotandem bicyclic peptide complex described herein and a cancer immunotherapeutic agent are administered more than one week apart. In some embodiments, for example, a heterotandem bicyclic peptide complex described herein and a cancer immunotherapeutic agent are administered within 1, 2, 3, 4, or 5 weeks of each other.

[0286] As used herein, the terms "combination," "combined," and related terms refer to simultaneous or sequential administration of the therapeutic agents of the present invention. For example, the heterotandem bicyclic peptide complexes described herein may be administered simultaneously or sequentially with a cancer immunotherapeutic agent, either in separate unit dosage forms or together in a single unit dosage form. Thus, in some embodiments, the present invention provides a single unit dosage form that includes, for example, a heterotandem bicyclic peptide complex described herein, a cancer immunotherapeutic agent, and optionally a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0287] The amounts of, for example, a heterotandem bicyclic peptide conjugate described herein and a cancer immunotherapeutic agent that may be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular method of administration. Preferably, the compositions of the present invention are formulated to allow for the administration of a dosage of, for example, 0.001 to 100 mg / kg body weight / day of a heterotandem bicyclic peptide conjugate described herein.

[0288] For example, a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands described herein and a cancer immunotherapeutic agent may act synergistically, and thus, the amount of, for example, a heterotandem bicyclic peptide complex described herein and a cancer immunotherapeutic agent in such a composition may be less than would be necessary in a monotherapy utilizing only that therapeutic agent.

[0289] The amount of cancer immunotherapeutic agent present in the compositions of the invention may not exceed the amount normally administered in a composition containing it as the sole active agent. Preferably, the amount of cancer immunotherapeutic agent in the compositions disclosed herein is about 50% to 100% of the amount normally present in a composition containing the agent as the sole therapeutically active agent. In certain embodiments, the cancer immunotherapeutic agent is administered at a dosage 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 term "normally administered" refers to the amount approved for an FDA approved therapeutic agent according to the FDA package insert.

[0290] 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, stents and catheters. Vascular stents are used, for example, to overcome restenosis (re-narrowing of the vessel wall after injury). However, patients who use 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 pharma- ceutically acceptable composition that includes a kinase inhibitor. Implantable devices coated with the compounds of the present invention are another embodiment of the present invention.

[0291] 5. Examples of cancer immunotherapy agents As used herein, the term "cancer immunotherapeutic agent" refers to an agent that is effective in enhancing, stimulating and / or upregulating immune response in a subject. In some embodiments, for example, administration of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands described herein and a cancer immunotherapeutic agent has a synergistic effect in the treatment of cancer.

[0292] The cancer immunotherapeutic agent can be, for example, a small molecule drug, an antibody, or a biological agent or a small molecule. Examples of biological cancer immunotherapeutic agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is humanized or human.

[0293] In certain embodiments, the cancer immunotherapeutic agent is a T cell (i) stimulatory (including costimulatory) receptor agonist or (ii) inhibitory (including costimulatory) signal antagonist, both of which result in enhanced antigen-specific T cell responses.

[0294] Some of the stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). One important family of membrane-bound ligands that bind to costimulatory 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 costimulatory or co-inhibitory receptors are the molecules of the TNF family that bind to cognate TNF receptor family members, CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RAN. K, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, Contains EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0295] In certain embodiments, the cancer immunotherapeutic agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF and other immunosuppressive cytokines) or stimulates T cell activation for stimulating an immune response.

[0296] In some embodiments, the combination of a heterotandem bicyclic peptide complex comprising one or more CD137-binding peptide ligands described herein and a cancer immunotherapeutic agent can stimulate a T cell response. In some embodiments, the heterotandem bicyclic peptide complex is BT7480 or BT7455, or a pharma- ceutical acceptable salt thereof. In certain embodiments, the cancer immunotherapeutic agent is (i) an antagonist of proteins that inhibit T cell activation, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4 (e.g., immune checkpoint inhibitors); 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.

[0297] In some embodiments, the cancer immunotherapy agent is an antagonist of the inhibitory receptor of NK cells or an agonist of the activating receptor of NK cells.In some embodiments, the cancer immunotherapy agent is an antagonist of KIR, such as lirilumab.

[0298] In certain embodiments, the cancer immunotherapeutic 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).

[0299] In certain embodiments, the cancer immunotherapeutic agent is selected from one or more of agonistic agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, agents that systemically increase the frequency of antagonist and anti-tumor T cells, agents that overcome alternative immune suppressive pathways within the tumor microenvironment (e.g., inhibitory receptor binding (e.g., PD-L1 / PD-1 interaction) blockade, Treg depletion or inhibition (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), metabolic enzyme inhibition such as IDO or restoring / preventing T cell energy or exhaustion), and agents that induce innate immune activation and / or inflammation at the tumor location.

[0300] In some embodiments, the cancer immunotherapy agent is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an antagonist CTLA-4 antibody. In some embodiments, the antagonist CTLA-4 antibody is Yervoy (ipilimumab) or tremelimumab.

[0301] In some embodiments, the cancer immunotherapy is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the cancer immunotherapy is an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is an antagonist PD-1 antibody. In some embodiments, the antagonist PD-1 antibody is Opdivo (nivolumab), Keytruda (pembrolizumab) or MEDI-0680 (AMP-514; WO2012 / 145493). In some embodiments, the cancer immunotherapy can be pidilizumab (CT-011). In some embodiments, the cancer immunotherapy is a recombinant protein consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, referred to as AMP-224.

[0302] In some embodiments, the cancer immunotherapy agent is a PD-L1 antagonist.In some embodiments, the PD-L1 antagonist is an antagonist 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).

[0303] In some embodiments, the cancer immunotherapy agent is a LAG-3 antagonist.In some embodiments, the LAG-3 antagonist is an antagonist 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).

[0304] In some embodiments, the cancer immunotherapeutic 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 urelumab or PF-05082566 (WO12 / 32433).

[0305] In some embodiments, the cancer immunotherapy agent is a GITR agonist.In some embodiments, the GITR agonist is an agonist GITR antibody.In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO006 / 105021, WO009 / 009116) or MK-4166 (WO11 / 028683).

[0306] In certain embodiments, the cancer immunotherapeutic agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In certain embodiments, the IDO antagonist is selected from epacadostat (INCB024360, Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmatinib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906 / KD108 (Phytoceutica); kynurenine degrading enzyme (Kynase, Ikena Oncology, formerly known as Kyn Therapeutics); and NLG-919 (WO09 / 73620, WO009 / 1156652, WO11 / 56652, WO12 / 142237).

[0307] In some embodiments, the cancer immunotherapeutic agent is an OX40 agonist.In some embodiments, the OX40 agonist is an agonist OX40 antibody.In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0308] In some embodiments, the cancer immunotherapy agent is an OX40L antagonist.In some embodiments, the OX40L antagonist is an antagonist OX40 antibody.In some embodiments, the OX40L antagonist is RG-7888 (WO06 / 029879).

[0309] In some embodiments, the cancer immunotherapeutic agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonist CD40 antibody. In some embodiments, the cancer immunotherapeutic agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonist CD40 antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[0310] In some embodiments, the cancer immunotherapeutic agent is a CD27 agonist.In some embodiments, the CD27 agonist is an agonist CD27 antibody.In some embodiments, the CD27 antibody is varlilumab.

[0311] In certain embodiments, the cancer immunotherapeutic agent is MGA271 (directed against B7H3) (WO11 / 109400).

[0312] In some embodiments, the cancer immunotherapeutic agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolizumab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intetumumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

[0313] In some embodiments, the cancer immunotherapeutic agent is an immune stimulatory agent. 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 sustained antitumor responses in an increasing number of tumor tissues, including certain tumor types not traditionally considered susceptible to immunotherapy. See, e.g., 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) (登録商標) , Bristol-Myers Squibb) (also known as ONO-4538, MDX1106 and BMS-936558) has shown the potential to improve overall survival in patients with RCC who have experienced disease progression during or after prior anti-angiogenic therapy.

[0314] In certain embodiments, the immunomodulatory therapy specifically induces apoptosis of tumor cells. Approved immunomodulatory therapies that may be used in the present invention include pomalidomide (Pomalyst®, Celgene); lenalidomide (Revlimid®, Celgene); ingenol mebutate (Picato®, LEO Pharma).

[0315] In some embodiments, the cancer immunotherapy agent is cancer vaccine.In some embodiments, the cancer vaccine is selected from sipuleucel-T (Provenge®, Dendreon / Valeant Pharmaceuticals), which is approved for the treatment of asymptomatic or minimally symptomatic metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (Immulidic®, BioVex / Amgen, formerly known as T-VEC), which is a genetically modified oncolytic virus therapy approved for the treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma.In certain embodiments, the cancer immunotherapeutic agent is PexaVec (PexaVec / JX-594, SillaJen / formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); Peraleorep (Reolysin®, Oncolytics), a variant of respiratory enteric orphan virus (reovirus) that does not replicate in RAS-inactivated cells in multiple 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). Biotech); ovarian cancer (NCT02028117); enadenotucirev (NG-348, PsiOxus, formerly known as ColoAd1), an adenovirus engineered to express full-length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein in metastatic or progressive epithelial tumors such as in colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); melanoma (NCT03003676); and ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF in peritoneal disease, colorectal cancer, or ovarian cancer (NCT02963831); peritoneal dissemination (NCT01443260); fallopian tube cancer, ovarian cancer (NCT The therapeutic options are selected from oncolytic virus therapies such as GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH), a vaccinia virus engineered to express beta-galactosidase (beta-gal) / beta-glucuronidase or beta-gal / human sodium iodide symporter (hNIS), respectively, which are being tested in bladder cancer (NCT02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818).

[0316] In certain embodiments, the cancer immunotherapeutic agent is JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase capable of converting the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapeutics targeting refractory RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus designated Ad5 / 3-E2F-delta24-hTNFα-IRES-hIL20; and antigen-specific CD8 + and VSV-GP (Vira Therapeutics), a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express antigens designed to generate a T cell response.

[0317] In some embodiments, the cancer immunotherapeutic agent is a T cell engineered to express a chimeric antigen receptor, or CAR. Such T cells engineered to express a chimeric antigen receptor are referred to as CAR-T cells.

[0318] CARs have been constructed that consist of a binding domain, which may be derived from a natural ligand, a single-chain variable fragment (scFv) derived from a cell surface antigen-specific monoclonal antibody fused to an endodomain, which is the functional end of the T cell receptor (TCR), capable of generating an activation signal in T lymphocytes, such as the CD3-zeta signaling domain from the TCR. Upon antigen binding, such CARs couple to endogenous signaling pathways in effector cells and generate activation signals similar to those initiated by the TCR complex.

[0319] For example, in one embodiment, the CAR-T cell is one of those described in US Patent 8,906,682 (June et al.; incorporated herein by reference in its entirety), which discloses CAR-T cells engineered to contain an extracellular domain with an antigen-binding domain (e.g., a domain that binds CD19) fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). When expressed in T cells, CARs can redirect antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Over 200 clinical trials using CAR-T are currently underway in a wide range of indications [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0320] In some embodiments, the immunostimulant is an activator of retinoic acid receptor-related orphan receptor gamma (RORγt).RORγt is a transcription factor that plays an important role in the differentiation and maintenance of type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) T cells, as well as the differentiation of IL-17 expressing natural immune cell subpopulations, such as NK cells.In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials for the treatment of solid tumors (NCT02929862).

[0321] In some embodiments, the immunostimulant is a toll-like receptor (TLR) agonist or activator.Suitable TLR activators include TLR9 agonists or activators, such as SD-101 (Dynavax).SD-101 is an immunostimulatory CpG being tested for B-cell, follicular and other lymphomas (NCT02254772).TLR8 agonists or activators that can be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals), being tested for head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT02431559).

[0322] Other cancer immunotherapeutics that can be used in the present invention include urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; varlilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirilumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; and andecaliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[0323] In certain embodiments, the immune stimulant is selected from elotuzumab, mifamurtide, agonists or activators of Toll-like receptors, and activators of RORγt.

[0324] In some embodiments, the immunostimulatory therapy is recombinant human interleukin 15 (rhIL-15). rhIL-15 has been clinically tested as a treatment for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888) and leukemia (NCT02689453). In some embodiments, the immunostimulatory agent is recombinant human interleukin 12 (rhIL-12). In some embodiments, the IL-15-based immunotherapy is heterodimeric IL-15 (hetIL-15, Novartis / Admune), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed to a soluble IL-15 binding protein IL-15 receptor alpha chain (IL15:sIL-15RA), 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 certain embodiments, the recombinant human interleukin-12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724 or NCT02542124.

[0325] In some embodiments, the cancer immunotherapeutic agent is selected from those described in Jerry L. Adams et al., "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, Vol. 14, pages 603-622, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cancer immunotherapeutic agent is selected from the examples described in Table 1 of Jerry L. Adams et al. In some embodiments, the cancer immunotherapeutic agent is a small molecule that targets an immuno-oncology target selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the cancer immunotherapeutic agent is a small molecule agent selected from those listed in Table 22 of Jerry L. Adams et al.

[0326] In some embodiments, the cancer immunotherapeutic agent is selected from small molecule cancer immunotherapeutic agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pages 319-329, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cancer immunotherapeutic agent is an agent that targets a pathway described in Peter L. Toogood.

[0327] In some embodiments, the cancer immunotherapeutic 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 cancer immunotherapeutic 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, the bispecific T cell engager (BiTE®) antibody construct activates T cells to release cytokines that induce upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates T cells, which leads to induction of bystander cell lysis. In some embodiments, the bystander cells are in a solid tumor. In some embodiments, the bystander cells to be lysed are in proximity to the BiTE®-activated T cells. In some embodiments, the bystander cells comprise tumor-associated antigen (TAA)-negative cancer cells. In some embodiments, the bystander cells comprise EGFR-negative cancer cells. In some embodiments, the cancer immunotherapeutic is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the cancer immunotherapeutic is an ex vivo expanded tumor-infiltrating T cell. In certain embodiments, the cancer immunotherapeutic agent is a bispecific antibody construct or a chimeric antigen receptor (CAR) that directly links T cells to a tumor-associated surface antigen (TAA).

[0328] Examples of checkpoint inhibitors In certain embodiments, the cancer immunotherapeutic is an immune checkpoint inhibitor described herein.

[0329] The term "checkpoint inhibitors" as used herein refers to agents useful for preventing cancer cells from evading the immune system of a patient. One of the major mechanisms of antitumor immune destruction is known as "T cell exhaustion", which is the result of chronic exposure to antigens leading to the upregulation of inhibitory receptors. These inhibitory receptors serve as immune checkpoints to prevent uncontrolled immune responses.

[0330] PD-1 and co-inhibitory receptors such as cytotoxic T-lymphocyte antigen 4 (CTLA-4, B- and T-lymphocyte attenuator (BTLA; CD272), T-cell immunoglobulin and mucin domain-3 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD223) and others are often referred to as checkpoint regulators. They serve as molecular "gatekeepers" that allow extracellular signaling to direct whether or not cell cycle progression and other intracellular signaling processes should proceed.

[0331] In one embodiment, the immune checkpoint inhibitor is an antibody against PD-1, which binds to the programmed cell death 1 receptor (PD-1) and prevents the receptor from binding to the inhibitory ligand PDL-1, thus abolishing the ability of the tumor to suppress the host anti-tumor immune response.

[0332] In some embodiments, the checkpoint inhibitor is a biological therapy 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 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 ligand, or a combination thereof. In some embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from 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 ligand, or a combination thereof. In some embodiments, the checkpoint inhibitor is an immunostimulant, a T cell growth factor, an interleukin, an antibody, a 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 a further embodiment, the vaccine is a dendritic cell (DC) vaccine.

[0333] Checkpoint inhibitors include any agent that blocks or inhibits an inhibitory 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. Exemplary checkpoint molecules that may be targets for blocking or inhibition include CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules, and inhibiting total NK, gamma delta and memory CD8 +(expressed on αβ)T cells), CD160 (also called 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 antigen-binding fragments thereof, other binding proteins, biologic therapies, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, and CGEN-15049. Exemplary immune checkpoint inhibitors include, but are not limited to, tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-Hl; 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.

[0334] In some embodiments, the immune checkpoint inhibitor is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. 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).

[0335] In certain embodiments, the checkpoint inhibitor is selected from the group consisting of lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirilumab, IPH2101, pembrolizumab (KEYTRUDA®), and tremelimumab.

[0336] In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of REGN2810 (Regeneron), an anti-PD-1 antibody being tested in patients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pidilizumab (CureTech), also known as CT-011, an antibody that binds to PD-1 that is in clinical trials for generalized large B-cell lymphoma and multiple myeloma; avelumab (BAVENCIO®, Pfizer / Merck), a fully human IgG1 anti-PD-L1 antibody in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer. KGaA), also known as MSB0010718C; or PDR001 (Novartis), an inhibitory antibody that binds to PD-1 that is in clinical trials for non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Tremelimumab (CP-675,206; AstraZeneca) is a fully human monoclonal antibody against CTLA-4 that is being tested in clinical trials for several indications, including mesothelioma, colorectal cancer, renal cancer, breast cancer, lung cancer and non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, endometrial cancer, metastatic cancer in the liver, liver cancer, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplastic thyroid cancer, urothelial carcinoma, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody being tested in Phase 1 clinical trials for advanced solid tumors (NCT02694822).

[0337] In some embodiments, the checkpoint inhibitor is an inhibitor of T cell immunoglobulin mucin-containing protein-3 (TIM-3). TIM-3 inhibitors that can be used in the present invention include TSR-022, LY3321367 and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody that is being tested in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody that is being tested in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody that is being tested in advanced malignancies (NCT02608268).

[0338] In some embodiments, the checkpoint inhibitor is an inhibitor of TIGIT, a T cell immunoreceptor with Ig and ITIM domains or a certain T cell and NK cell immunoreceptor.The TIGIT inhibitors that can be used in the present invention include BMS-986207 (Bristol-Myers Squibb), anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT03119428).

[0339] In some embodiments, the checkpoint inhibitor is an inhibitor of lymphocyte activation gene-3 (LAG-3). LAG-3 inhibitors that can be used in the present invention include BMS-986016 and REGN3767 and IMP321. BMS-986016 (Bristol-Myers Squibb), an anti-LAG-3 antibody, is being tested in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody, and is being tested in malignant tumors (NCT03005782). IMP321 (Immutep SA) is a LAG-3-Ig fusion protein, and is being tested in melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934).

[0340] Checkpoint inhibitors that can be used in the present invention include OX40 agonists. OX40 agonists in clinical trials include PF-04518600 / PF-8600 (Pfizer), an agonist anti-OX40 antibody in metastatic renal cancer (NCT03092856) and advanced cancers and neoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonist anti-OX40 antibody in a Phase 1 cancer trial (NCT02528357); and agonist anti-OX40 antibodies in advanced solid tumors (NCT02318394 and NCT02705482). MEDI0562 (Medimmune / AstraZeneca), an agonist anti-OX40 antibody in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705) (Medimmune / AstraZeneca); MEDI6469, an agonist anti-OX40 antibody (Medimmune / AstraZeneca), 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), an agonist anti-OX40 antibody in advanced cancers (NCT02737475).

[0341] Checkpoint inhibitors that can be used in the present invention include CD137 (also known as 4-1BB) agonists. CD137 agonists in clinical trials include utomilumab (PF-05082566, Pfizer), an agonist anti-CD137 antibody in generalized large B-cell lymphoma (NCT02951156) and advanced cancers and neoplasms (NCT02554812 and NCT05082566); urelumab (BMS-663513; Bristol-Myers Squibb), an agonist anti-CD137 antibody in melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981); and CTX-471 (Compass Therapeutics), an agonist anti-CD137 antibody in metastatic or locally advanced malignancies (NCT03881488).

[0342] Checkpoint inhibitors that can be used in the present invention include CD27 agonists. CD27 agonists in clinical trials include varlilumab (CDX-1127, Celldex Therapeutics), an agonistic anti-CD27 antibody in squamous cell head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0343] Checkpoint inhibitors that can be used in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists in clinical trials include TRX518 (Leap Therapeutics), an agonist anti-GITR antibody in melanoma and other malignant solid tumors (NCT01239134 and NCT02628574); GWN323 (Novartis), an agonist anti-GITR antibody in solid tumors and lymphomas (NCT02740270); INCAGN01876 (Incyte / Agenus), an agonist anti-GITR antibody in advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonist anti-GITR antibody in solid tumors (NCT02132754) and human IgG1 (Navire) in advanced solid tumors (NCT02583165). These include MEDI1873 (Medimmune / AstraZeneca), an agonistic hexameric GITR-ligand molecule with an Fc domain.

[0344] Checkpoint inhibitors that can be used in the present invention include inducible T-cell symbiont (ICOS, also known as CD278) agonists. ICOS agonists in clinical trials include MEDI-570 (Medimmune), an agonist anti-ICOS antibody in lymphoma (NCT02520791); GSK3359609 (Merck), an agonist anti-ICOS antibody in Phase 1 (NCT02723955); and JTX-2011 (Jounce Therapeutics), an agonist anti-ICOS antibody in Phase 1 (NCT02904226).

[0345] Checkpoint inhibitors that can be used in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors in clinical trials include lirilumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263) and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma) in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody binding to the 3 domain of the long cytoplasmic tail (KIR3DL2) in lymphoma (NCT02593045).

[0346] Checkpoint inhibitors that can be used in the present invention include CD47 inhibitors of the interaction between CD47 and signal regulatory protein alpha (SIRPa). CD47 / SIRPa inhibitors in clinical trials include ALX-148 (Alexo Therapeutics), an antagonist variant of (SIRPa) that binds to CD47 and blocks CD47 / SIRPa-mediated signaling, in Phase 1 (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein made by linking the N-terminal CD47-binding domain of SIRPa with the Fc domain of human IgG1, which acts by binding to human CD47 and blocking the delivery of a "don't feed" signal to macrophages, in Phase 1 clinical trials (NCT02890368 and NCT02663518). Therapeutics), CC-90002 (Celgene), an anti-CD47 antibody, in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven Inc.) in colorectal neoplasia and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[0347] Checkpoint inhibitors that can be used in the present invention include CD73 inhibitors. CD73 inhibitors in clinical trials include MEDI9447 (Medimmune), an anti-CD73 antibody in solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody in solid tumors (NCT02754141).

[0348] Checkpoint inhibitors that can be used in the present invention include agonists of the Stimulator of Interferon Genes protein (STING, also known as transmembrane protein 173 or TMEM173). Agonists of STING in clinical trials include MK-1454 (Merck), an agonist synthetic cyclic dinucleotide in lymphoma (NCT03010176); and ADU-S100 (MIW815; Aduro Biotech / Novartis), an agonist synthetic cyclic dinucleotide in Phase 1 (NCT02675439 and NCT03172936).

[0349] Checkpoint inhibitors that can be used in the present invention include CSF1R inhibitors. CSF1R inhibitors in clinical trials include pexidartinib (PLX3397, Plexxikon), a small molecule CSF1R inhibitor in colorectal cancer, pancreatic cancer, metastatic and advanced cancers (NCT02777710) and melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST) and ovarian cancer (NCT02452424); and pexidartinib (PLX3397, Plexxikon), a small molecule CSF1R inhibitor in pancreatic cancer (NCT03153410), melanoma (NCT03101254) and solid tumors (SCD03101254). These include IMC-CS4 (LY3022855; Lilly), an anti-CSF-1R antibody in tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid methylamide, Novartis), an orally available inhibitor of CSF1R in advanced solid tumors (NCT02829723).

[0350] Checkpoint inhibitors that can be used in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors in clinical trials include monalizumab (IPH2201, Innate Pharma), an anti-NKG2A antibody in head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0351] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pidilizumab. EXAMPLES

[0352] The following examples illustrate the above invention; 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 test models known per se to those skilled in the relevant field.

[0353] Example 1: Transcriptional profiling studies Profiling study with BCY12491 For transcription and immunohistochemical (IHC) analysis, 1 × 10 cells were injected subcutaneously into 6- to 8-week-old female huCD137-C57B / 6J mice (Biocytogen). 6 MC38 cells were implanted. The average tumor volume was approximately 240 mm 3When the mice reached 10 days of age, they were randomized into treatment groups and treated with vehicle (25 mM histidine, 10% sucrose, pH 7), 15 mg / kg BCY12491, 15 mg / kg BCY13626 (non-binding control) intravenously or 2 mg / kg anti-CD137 antibody urelumab intraperitoneally. Treatments were given three times Q3D, tumor growth was monitored by caliper measurements, and tumor tissue was harvested 1 hour after the last dose on day 6. A portion of the tumor tissue was used for RNA isolation for transcriptional analysis, and a portion of the tumor tissue was used for formalin-fixed paraffin-embedded (FFPE) sample preparation for IHC analysis. RNA was isolated from tumor tissue using the RNAeasy kit (Qiagen), and transcriptional analysis was performed using the nCounter Mouse PanCancer IO 360 panel (Nanostring) from 100 ng RNA / tumor. Data were analyzed using nSolver Analysis Software with the advanced analysis probe set ns_mm_io_360_v1.0 (Nanostring).CD8+ tumor infiltrating cells were stained in FFPE tissue sections using anti-mouse CD8 antibody (Abcam, # ab217344) and Ventana Discovery OmniMap anti Rabbit-HRP Kit (Ventana #760 4310).

[0354] The data are shown in Figure 1.

[0355] Findings: Transcriptional analysis shows a significant increase in immune cell scores, such as cytotoxic cell score, T cell score and macrophage cell score, in tumor tissues treated with EPhA2 heterotandem bicyclic peptide complex BCY12491 when compared to tumors from vehicle-treated mice. Anti-CD137 antibody treatment also significantly increased cytotoxic cell score and T cell score in tumor tissues, but to a lesser extent than BCY12491. No changes in immune cell scores were observed in tumor tissues from non-binding control (BCY13626)-treated animals. IHC analysis of CD8+ cells in tumor tissues showed a strong infiltration of CD8+ cells in tumors from BCY12491-treated mice when compared to tumors from vehicle- or non-binding agent BCY13626-treated mice. Some increase in CD8+ cell infiltration was also observed in tumors from anti-CD137 antibody-treated mice. These changes in immune cell scores and CD8+ cells in tumor tissues indicate that agonism of CD137 in tumor tissues by the EphA2 / CD137 heterotandem bicyclic peptide complex BCY12491 leads to a significant modulation (increase) of tumor-infiltrating immune cells and immune responses.

[0356] Testing with BT7480 For transcription and immunohistochemical (IHC) analysis, 1 × 10 cells were injected subcutaneously into 6- to 8-week-old female huCD137-C57B / 6J mice (Biocytogen). 6 MC38#13 (MC38 cells engineered to express Nectin-4) cells were implanted. The average tumor volume was approximately 255 mm 3When the tumor reached 0 h, mice were randomized into treatment groups receiving vehicle, BT7480 (BCY00011863), non-binding factor BCY control BCY00012797 (BCY12797) or αCD137 antibody (Urelumab analog). BT7480 and its non-binding control were administered intravenously at 5 mg / kg (in 25 mM histidine HCl, 10% sucrose, pH 7; vehicle) at 0 and 24 h, and Urelumab analog was administered intraperitoneally at 2 mg / kg in PBS at BIW (0 h, 72 h) dose and schedule. Tumors from BT7480-treated mice were harvested at 24 h (after 0 h dose), 48 h (24 h after the last 0 h and 24 h dose), 96 h (72 h after the last 0 h and 24 h dose), and 144 h (120 h after the last 0 h and 24 h dose). Tumors from αCD137-treated mice were harvested 144 hours after the start of treatment. Tumors from vehicle-treated mice were harvested at 0 hours, 24 hours and 144 hours (120 hours after the last 0 and 24 hours). RNA was isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcriptional analysis was performed using the nCounter Mouse PanCancer IO 360 panel (Nanostring) from 100ng RNA / tumor. Data were analyzed using nSolver Analysis Software with the advanced analysis probe set ns_mm_io_360_v1.0 (Nanostring).

[0357] The data are shown in Figures 2-4.

[0358] Findings: Transcriptional analysis showed a significant early (24 hour) increase in mRNA for several T cell chemotactic chemokines / cytokines, including Ccl1, Ccl17, and Ccl24, among others, that are secreted by myeloid cells and are thought to result in the recruitment of T cells at the site of chemokine secretion. Transcriptional analysis also confirmed a significant increase in immune cell scores, including cytotoxicity cell score and macrophage cell score, in tumor tissue with BT7480 treatment when compared to tumors from vehicle-treated mice. The macrophage cell score began to increase 24 hours after BT7480 administration, reaching a significant increase from the 24 hour vehicle readout by 48 hours. On the other hand, the cytotoxicity cell score began to increase from 48 hours after the start of treatment and increased until 144 hours, when the cytotoxicity cell score was significantly increased compared to vehicle-treated tumors at 144 hours. Overlay of the cytotoxicity cell score and normalized mRNA counts for Ccl1, Ccl17 and Ccl24 in response to BT7480 shows how increased Ccl1, Ccl17 and Ccl24 transcription precedes the increase in the cytotoxicity cell score.

[0359] Overlay of macrophage and cytotoxic cell scores in response to BT7480 shows how the increase in macrophage cell score precedes the increase in cytotoxic cell score.

[0360] Transcriptional analysis showed increased or a trend towards significant increases in mRNA for several different immune checkpoints, including CTLA-4 (Ctla4), PD-1 (Pdcd1), PD-L1 (Cd274), LAG3 (Lag3), TIM3 (Havcr2), PD-L2 (Pdcd1lg2) and TIGIT (Tigit), supporting the concept of BT7480 combination with checkpoint inhibitors.

[0361] Example 2: Efficacy study of BCY12491 and pembrolizumab combination For tumor growth analysis, 1 × 10 6- to 8-week-old female huCD137 / huPD-1-C57B / 6J mice (Biocytogen) were subcutaneously injected with 6MC38 cells were implanted. The average tumor volume was approximately 92 mm. 3 When tumor size reached 0.01, mice were randomized into treatment groups and treated with vehicle (25 mM histidine, 10% sucrose, pH 7), 5 mg / kg BCY12491 (0, 24 h) intravenously or 3 mg / kg anti-PD-1 antibody pembrolizumab or a combination of BCY12491 and pembrolizumab intraperitoneally. Combination treatments were given on three different dosing schedules: BCY12491 and pembrolizumab treatment started simultaneously on day 0, BCY12491 treatment started on day 1 and pembrolizumab treatment started on day 5, or pembrolizumab treatment started on day 0 and BCY12491 treatment started on day 5. Treatments were given four times weekly and tumor growth was monitored by caliper measurements.

[0362] The data are shown in Figures 5 and 6.

[0363] Findings: Both BCY12491 and pembrolizumab monotherapy and their combination demonstrated significant antitumor activity when compared to vehicle controls (all *** p<0.0001, mixed effects analysis with Dunnett's post-hoc test comparing treatment vs vehicle on day 18). Furthermore, the combination treatment was more effective than either of the monotherapies ( *** p<0.0001, mixed effects analysis with Dunnett's post-hoc test at D20 comparing combination vs. monotherapy), resulting in a complete response in all treated animals by day 22. In contrast, these treatment regimens and schedules resulted in 2 / 10 complete responses in the BCY12491 monotherapy treated cohort and 3 / 10 complete responses in the pembrolizumab monotherapy treated cohort. Alternating sequences of combinations of BCY12491 and pembrolizumab (BCY12491 treatment starting on day 0 and pembrolizumab treatment starting on day 5 or vice versa) also resulted in significant antitumor activity (both ***p<0.0001, mixed effects analysis with Dunnett's post-hoc test comparing treatment vs vehicle on day 18), both schedules resulted in 9 / 10 complete responses (BCY12491 treatment started on day 0 and pembrolizumab treatment started on day 5) and 8 / 10 complete responses (pembrolizumab treatment started on day 0 and BCY12491 treatment started on day 5) in treated mice by day 42.

[0364] Example 3: Efficacy study of BCY11864 and anti-PD-1 combination For tumor growth analysis, 6- to 8-week-old female Balb / c-huCD137- mice (Gempharmatech) were implanted subcutaneously with 3 × 10+e5 CT26#7 cells (CT26 cells engineered to overexpress Nectin-4). The mean tumor volume was approximately 80 mm. 3 When tumor size reached 100 mm, mice were randomized into treatment groups and treated with vehicle (25 mM histidine, 10% sucrose, pH 7), 10 mg / kg BCY11864 (0, 24 h) intravenously, or 10 mg / kg anti-PD-1 antibody (RMP1-14) or a combination of BCY11864 and anti-PD-1 antibody intraperitoneally. Treatments were given weekly and tumor growth was monitored by caliper measurements. A tumor size of >2000 mm was considered to be reached as the human endpoint. 3 Animals with tumors were sacrificed. The study was terminated 66 days after treatment initiation, at which point only two animals (both in the combination treatment arms) remained on the study (one was a complete responder and one was still undergoing tumor size regression).

[0365] The data is shown in FIG.

[0366] Findings: The addition of BCY11864 to anti-PD-1 monotherapy significantly improved survival (human endpoint, i.e., tumor volume >2000 mm) of CT26#7-bearing mice. 3 The anti-PD-1 arm and the anti-PD-1 + BCY11864 combination arm showed a significant increase in PD-1-associated cerebrovascular event (outcome measured as time to reach baseline) (p=0.004, Mantel-Cox log-rank test comparing the anti-PD-1 arm with the anti-PD-1 + BCY11864 combination arm).

[0367] Example 4. Efficacy Study of BT7480 in Combination with Anti-PD-1 and Anti-Ctla-4 For tumor growth analysis, 6-8 week-old female C57BL / 6J-huCD137- mice (Biocytogen) were implanted subcutaneously with 1×10+e6 MC38#13 cells (MC38 cells engineered to overexpress Nectin-4). The mean tumor volume was approximately 100 mm. 3 Upon reaching 18 days of age, mice were randomized into treatment groups and treated intraperitoneally with vehicle (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 BT7480 / anti-PD-1 and BT7480 / anti-Ctla-4 combinations. Treatments were given twice weekly (BIW) for 2 weeks and tumor growth was monitored by caliper measurements up to 33 days after treatment initiation. A tumor size of >2000 mm was considered to be reached as the human endpoint. 3 Tumor-bearing animals were sacrificed.

[0368] The data are shown in Figures 8 and 9.

[0369] Findings: The addition of BT7480 to anti-PD-1 monotherapy increased the complete response rate (CR) from 0 / 8 (BT7480 and anti-PD-1 monotherapy arms) to 2 / 8 in the BT7480 / anti-PD-1 combination treatment arm. The addition of BT7480 to anti-CTLA-4 monotherapy increased the complete response rate (CR) from 0 / 8 or 1 / 8 (BT7480 and anti-CTLA-4 monotherapy arms, respectively) to 4 / 8 in the BT7480 / anti-CTLA-4 combination treatment arm by day 33 after treatment initiation. Furthermore, the addition of BT7480 to anti-CTLA-4 monotherapy improved survival (human endpoint, i.e., tumor volume >2000 mm) of MC38#13-bearing mice. 3 The anti-CTLA-4 arm and the anti-CTLA-4+BT7480 combination arm showed a significant increase in CR-positive patients (outcome measured as the time to reach CR-positive outcome) (p=0.0499, Mantel-Cox log-rank test comparing the anti-CTLA-4 arm with the anti-CTLA-4+BT7480 combination arm).

[0370] Example 5. Transcriptional profiling studies with BT7455 For transcriptional profiling analysis of the effects of BT7455 on the immune tumor microenvironment, 6-8 week old female huCD137-C57B / 6J mice (Biocytogen) were implanted subcutaneously with 1x10+E6 MC38 cells. The mean tumor volume was approximately 350 mm. 3 When the tumor reached 0.5 mg / mL, mice were randomized into treatment groups receiving vehicle, BT7455, αCD137 antibody (urelumab analogue), or αPD-1 antibody. BT7455 was administered intravenously at 8 mg / kg (in 25 mM histidine HCl, 10% sucrose, pH 7; vehicle) at 0 and 24 hours, and urelumab analogue and αPD-1 antibody were administered intraperitoneally at 2 mg / kg (urelumab analogue) or 10 mg / kg (αPD-1 antibody) in PBS at 0 hour. Tumors from vehicle, BT7455, urelumab analogue, and αPD-1 antibody-treated mice were harvested at 24 hours, 48 ​​hours, and 144 hours after the start of treatment. RNA was isolated from tumor tissue using the RNAeasy kit (Qiagen), and transcriptional analysis was performed using the nCounter Mouse PanCancer IO 360 panel (Nanostring) from 100 ng RNA / tumor. Data were analyzed using nSolver Analysis Software with the advanced analysis probe set ns_mm_io_360_v1.0 (Nanostring).

[0371] The data are shown in Figures 10-13.

[0372] Findings: Transcriptional analysis showed a significant increase in mRNA for several different immune checkpoints after BT7455 treatment, including CTLA-4 (Ctla4), PD-1 (Pdcd1), PD-L1 (Cd274), LAG3 (Lag3), TIM3 (Havcr2), PD-L2 (Pdcd1lg2) and TIGIT (Tigit), supporting the concept of BT7455 combination with checkpoint inhibitors. Transcriptional analysis also showed a significant early (24-48 hour time point) increase in mRNA for several T cell chemotactic chemokines / cytokines, including Ccl1, Ccl17 and Ccl24 among others, which are secreted by myeloid cells and are thought to result in the recruitment of T cells at the site of chemokine secretion. Transcriptional analysis also showed a significant increase in immune cell scores, such as cytotoxicity cell scores, in tumor tissue with BT7455 treatment when compared to tumors from vehicle or anti-PD-1 or anti-CD137 treated mice. BT7455 treatment induced significant early (48 h) regulation of several gene sets, including those associated with cytokine and chemokine signaling, cytotoxicity, apoptosis and NK-kappa B signaling gene sets.

Claims

1. A pharmaceutical composition for treating cancer, comprising a heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof, characterized in that it is used in combination with a cancer immunotherapy agent, wherein the heterotandem bicyclic peptide complex is (a) a first peptide ligand that binds to a component present on a cancer cell, conjugated via a linker to (b); (b) one or more CD137-binding peptide ligands wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, and wherein each of the peptide ligands comprises a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

2. Heterotandem bicyclic peptide complexes (a) a first peptide ligand that binds to a component present on a cancer cell, conjugated via a linker to (b); (b) one or more CD137-binding peptide ligands 2. The pharmaceutical composition of claim 1, wherein each of the peptide ligands comprises a polypeptide comprising a molecular scaffold that forms a covalent bond with a cysteine ​​residue of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

3. Heterotandem bicyclic peptide complexes (a) a first peptide ligand that binds to a component present on a cancer cell, conjugated via a linker to (b); (b) two or more CD137-binding peptide ligands 3. The pharmaceutical composition of claim 1 or 2, comprising:

4. CD137-binding peptide ligand C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO: 11); C ｉ IEEGQYC ii FADPY[Nle]C iii (SEQ ID NO:5); C ｉ [tBuAla]PE[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:6); C ｉ IEEGQYC ii F[D-Ala]DPY[Nle]C iii (SEQ ID NO:7); C ｉ [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (SEQ ID NO:8); C ｉ [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (Allocation number 9); C ｉ [tBuAla]P[K(PYA)][D-Ala]PYC ii FADPY[Nle]C iii (Allocation number 10); (SEQ ID NO:11)-A (herein designated BCY14601); C ｉ IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (SEQ ID NO: 12); C ｉ [tBuAla]PE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO:60); C ｉ IEE[dK(PYA)]QYC ii FADPY[Nle]C iii (SEQ ID NO:61); C ｉ [tBuAla]EE(dK)PYC ii FADPY[Nle]C iii (SEQ ID NO:62); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:63); C ｉ [tBuAla]EE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:64); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FANPY[Nle]C iii (SEQ ID NO:65); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FAEPY[Nle]C iii (SEQ ID NO:66); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FA[Aad]PY[Nle]C iii (SEQ ID NO:67); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FAQPY[Nle]C iii (Allocation number 68); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle][Cysam] iii (SEQ ID NO:69); [MerPro] ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:70; herein designated BCY12353); [MerPro] ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle][Cysam] iii (SEQ ID NO:71; herein designated BCY12354); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:72); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:73); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:74; herein designated BCY12372); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeAla]Y[Nle]C iii (SEQ ID NO:75); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FAD[NMeDAla]Y[Nle]C iii (SEQ ID NO:76); C ｉ [tBuAla]P[K(PYA)][dA]PYC ii FADPY[Nle]C iii (SEQ ID NO:77); C ｉ [tBuAla]PE[dK(PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:78); C ｉ [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:79); C ｉ [tBuAla]PE[dK(Me,PYA)]PYC ii FADPY[Nle]C iii (SEQ ID NO:80); and [MerPro] ｉ [tBuAla]EE[dK]PYC ii FADPY[Nle]C iii (SEQ ID NO:81; herein referred to as BCY13137) [In the formula, [MerPro] ｉ , C ｉ , C ii , C iii and [Cysam] iii represents a first (i), second (ii) and third (iii) reactive group selected from cysteine, MerPro and Cysam, Nle represents norleucine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, Aad represents alpha-L-aminoadipic acid, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine and NMeAla represents N-methyl-alanine. The pharmaceutical composition according to any one of claims 1 to 4, comprising an amino acid sequence selected from the following, or a pharma- ceutically acceptable salt thereof:

5. CD137-binding peptide ligand C ｉ [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (Allocation number 11) [In the formula, C ｉ , C ii and C iii represent the first, second and third cysteine ​​residues, respectively; tBuAla represents t-butyl-alanine; PYA represents 4-pentynoic acid; and Nle represents norleucine. or a pharma- ceutically acceptable salt thereof.

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

7. The CD137-binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification, Ac-(SEQ ID NO:11)-A (herein referred to as BCY8928) [In the formula, Ac represents an acetyl group.] 7. The pharmaceutical composition according to claim 1, comprising:

8. A pharmaceutical composition according to any of claims 1 to 7, wherein the heterotandem bicyclic peptide complex comprises two CD137-binding peptide ligands, both of which have the same peptide sequence, and the peptide sequence comprises Ac-(sequence number 11)-A (herein referred to as BCY8928) (wherein Ac represents an acetyl group).

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

10. Nectin-4-binding bicyclic peptide ligands C ｉ P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:1; herein designated BCY8116); C ｉ P[1Nal][dK](Sar １０ -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:3); C ｉ PFGC ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:4; herein designated BCY11414); C ｉ P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 14); [MerPro] ｉ P[1Nal][dK]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:15; herein designated BCY12363); C ｉ P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO: 16); [MerPro] ｉ P[1Nal][dK]C ii M[HArg]DWSTP[HyP]W[Cysam] iii (SEQ ID NO:17; herein designated BCY12365); C ｉ P[1Nal][dK]C ii M[HArg]HWSTP[HyP]WC iii (SEQ ID NO: 18); C ｉ P[1Nal][dK]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO: 19); C ｉ P[1Nal][dE]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:20; herein designated BCY12368); C ｉ P[1Nal][dA]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:21; herein designated BCY12369); C ｉ P[1Nal][dE]C ii L[HArg]DWSTP[HyP]WC iii (SEQ ID NO:22; herein designated BCY12370); and C ｉ P[1Nal][dE]C ii M[HArg]EWSTP[HyP]WC iii (SEQ ID NO:23; herein designated BCY12384) [In the formula, [MerPro] ｉ , C ｉ , C ii , C iii and [Cysam] iii represents a first (i), second (ii) and third (iii) reactive group selected from cysteine, MerPro and Cysam; 1Nal represents 1-naphthylalanine; HArg represents homoarginine; HyP represents trans-4-hydroxy-L-proline; Sar １０ represents 10 sarcosine units, B-Ala represents beta-alanine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine. or a pharma- ceutically acceptable salt thereof.

11. the Nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification; SEQ ID NO:1 (herein designated BCY8116); [PYA]-[B-Ala]-[Sar １０ ] - (SEQ ID NO:1) (herein designated BCY8846); [PYA]-(SEQ ID NO:1) (herein referred to as BCY11015); [PYA]-[B-Ala]-(SEQ ID NO:1) (herein referred to as BCY11016); [PYA]-[B-Ala]-[Sar １０ ] - (SEQ ID NO:2) (herein designated BCY11942); Ac-(SEQ ID NO:3) (herein referred to as BCY8831); SEQ ID NO:4 (herein designated BCY11414); [PYA]-[B-Ala]-(SEQ ID NO:14) (herein referred to as BCY11143); Palmitic-yGlu-yGlu- (SEQ ID NO: 14) (herein referred to as BCY12371); Ac-(SEQ ID NO:14) (herein referred to as BCY12024); Ac-(SEQ ID NO:16) (herein referred to as BCY12364); Ac-(SEQ ID NO:18) (herein referred to as BCY12366); and Ac-(SEQ ID NO:19) (herein referred to as BCY12367) [Wherein, YA represents 4-pentynoic acid, B-Ala represents beta-alanine, and Sar １０ represents 10 sarcosine units.

11. The pharmaceutical composition of claim 9 or 10, comprising:

12. The pharmaceutical composition of any of claims 9 to 11, wherein the Nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO: 1 (herein designated BCY8116).

13. The pharmaceutical composition of any of claims 9 to 12, wherein the heterotandem bicyclic peptide complex is selected from those listed in Tables A and B, such as BCY11027, BCY11863 and BCY11864, or a pharma- ceutically acceptable salt thereof.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the heterotandem bicyclic peptide complex is BCY11863 or a pharma- ceutically acceptable salt thereof.

15. The pharmaceutical composition of claim 14, wherein the heterotandem bicyclic peptide complex is administered at 0.1 to 75 mg / kg.

16. A pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer cells have high Nectin-4 expression.

17. The pharmaceutical composition of any of claims 1 to 8, wherein the component present in the cancer cell is EphA2 and the first peptide ligand comprises an EphA2-binding bicyclic peptide ligand.

18. EphA2-binding bicyclic peptide ligands Ci[HyP]LVNPLCiiLEP[d1Nal]WTCiii (SEQ ID NO:44); C ｉ [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:24); C ｉ LWDPTPC ii ANLHL[HArg]C iii (SEQ ID NO:25); C ｉ [HyP]LVNPLC ii L[K(PYA)]P[dD]W[HArg]C iii (SEQ ID NO:26); C ｉ [HyP][K(PYA)]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:27); C ｉ [HyP]LVNPLC ii [K(PYA)]HP[dD]W[HArg]C iii (SEQ ID NO:28); C ｉ [HyP]LVNPLC ii LKP[dD]W[HArg]C iii (SEQ ID NO:29); C ｉ [HyP]KVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:30); C ｉ [HyP]LVNPLC ii KHP[dD]W[HArg]C iii (SEQ ID NO:31); C ｉ [HyP]LVNPLC ii LHP[dE]W[HArg]C iii (SEQ ID NO:32); C ｉ [HyP]LVNPLC ii LEP[dD]W[HArg]C iii (SEQ ID NO:33); C ｉ [HyP]LVNPLC ii LHP[dD]WTC iii (SEQ ID NO:34); C ｉ [HyP]LVNPLC ii LEP[dD]WTC iii (SEQ ID NO:35); C ｉ [HyP]LVNPLC ii LEP[dA]WTC iii (SEQ ID NO:36); C ｉ [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO:37; herein designated BCY12860); C ｉ [HyP][Cba]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:38); C ｉ [HyP][Cba]VNPLC ii LEP[dD]WTC iii (SEQ ID NO:39); C ｉ [HyP][Cba]VNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO:40); C ｉ [HyP]LVNPLC ii L[3,3-DPA]P[dD]W[HArg]C iii (SEQ ID NO:41); C ｉ [HyP]LVNPLC ii LHP[d1Nal]W[HArg]C iii (SEQ ID NO:42); C ｉ [HyP]LVNPLC ii L[1Nal]P[dD]W[HArg]C iii (SEQ ID NO:43); C ｉ [HyP]LVNPLC ii L[1Nal]P[dD]WTC iii (SEQ ID NO:45; herein designated BCY13119); C ｉ [HyP][Cba]VNPLC ii LEP[dA]WTC iii (SEQ ID NO:46); C ｉ [HyP][hGlu]VNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:47); C ｉ [HyP]LVNPLC ii [hGlu]HP[dD]W[HArg]C iii (SEQ ID NO:48); C ｉ [HyP]LVNPLC ii L[hGlu]P[dD]W[HArg]C iii (SEQ ID NO:49); C ｉ [HyP]LVNPLC ii LHP[dNle]W[HArg]C iii (SEQ ID NO:50); C ｉ [HyP]LVNPLC ii L[Nle]P[dD]W[HArg]C iii (SEQ ID NO:51); [MerPro] ｉ [HyP]LVNPLC ii L[3,3-DPA]P[dD]WTC iii (SEQ ID NO: 154); C ｉ [HyP]LVNPLC ii LHP[dD]W[HArg][Cysam] iii (SEQ ID NO: 155); C ｉ [HyP]LVNPLC ii L[His3Me]P[dD]W[HArg]C iii (SEQ ID NO: 156); C ｉ [HyP]LVNPLC ii L[His1Me]P[dD]W[HArg]C iii (SEQ ID NO: 157); C ｉ [HyP]LVNPLC ii L[4ThiAz]P[dD]W[HArg]C iii (SEQ ID NO: 158); C ｉ [HyP]LVNPLC ii LFP[dD]W[HArg]C iii (SEQ ID NO: 159); C ｉ [HyP]LVNPLC ii L[Thi]P[dD]W[HArg]C iii (SEQ ID NO: 160); C ｉ [HyP]LVNPLC ii L[3Thi]P[dD]W[HArg]C iii (SEQ ID NO: 161); C ｉ [HyP]LVNPLC ii LNP[dD]W[HArg]C iii (SEQ ID NO: 162); C ｉ [HyP]LVNPLC ii LQP[dD]W[HArg]C iii (SEQ ID NO: 163); and C ｉ [HyP]LVNPLC ii L[K(PYA-(Palmitoyl-Glu-LysN ３ )]P[dD]W[HArg]C iii (SEQ ID NO:164) [In the formula, [MerPro] ｉ , C ｉ , C ii , C iii and [Cysam] iii represents a first (i), second (ii) and third (iii) reactive group selected from cysteine, MerPro and Cysam; HyP represents trans-4-hydroxy-L-proline; HArg represents homoarginine; PYA represents 4-pentynoic acid; 3,3-DPA represents 3,3-diphenylalanine; Cba represents β-cyclobutylalanine; 1N al represents 1-naphthylalanine, hGlu represents homoglutamic acid, Thi represents thienyl-alanine, 4ThiAz represents beta-(4-thiazolyl)-alanine, His1Me represents N1-methyl-L-histidine, His3Me represents N3-methyl-L-histidine, 3Thi represents 3-thienylalanine, palmitoyl-Glu-LysN ３ [PYA] 【Chemistry 1】 represents [K(PYA-(palmitoyl-Glu-LysN ３ )]but 【Chemistry 2】 where Nle represents norleucine, MerPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine.

20. The pharmaceutical composition of claim 17, comprising an amino acid sequence selected from the group consisting of: (i) an EphA2-binding bicyclic peptide ligand C ｉ [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (SEQ ID NO:24) [In the formula, C ｉ , C ii , C iii represents the first (i), second (ii) and tertiary (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and HArg represents homoarginine. or a pharma- ceutically acceptable salt thereof; and / or (ii) an EphA2-binding bicyclic peptide ligand Ci [HyP]LVNPLCii LEP[d1Nal]WTCiii (SEQ ID NO: 44) (wherein C i , C ii , C iii represent the first (i), second (ii), and tertiary (iii) cysteine ​​groups, HyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine.) or a pharma- ceutically acceptable salt thereof, 19. A pharmaceutical composition according to claim 17 or 18.

20. The EphA2-binding bicyclic peptide ligand comprising an N-terminal modification, A-[HArg]-D-(SEQ ID NO:24) (herein designated BCY9594); [B-Ala]-[Sar 10 ]-A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY6099); [PYA]-A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY11813); Ac-A-[HArg]-D-(SEQ ID NO:24)-[K(PYA)] (herein referred to as BCY11814); Ac-A-[HArg]-D-(SEQ ID NO:24)-K (herein designated BCY12734); [NMeAla]-[HArg]-D-(SEQ ID NO:24) (herein designated BCY13121); [Ac]-(SEQ ID NO:24)-L[dH]G[dK] (herein referred to as BCY13125); [PYA]-[B-Ala]-[Sar 10 ]-VGP-(SEQ ID NO:25) (herein designated BCY8941); Ac-A-[HArg]-D-(SEQ ID NO:26) (herein referred to as BCY11815); Ac-A-[HArg]-D-(SEQ ID NO:27) (herein referred to as BCY11816); Ac-A-[HArg]-D-(SEQ ID NO:28) (herein referred to as BCY11817); Ac-A-[HArg]-D-(SEQ ID NO:29) (herein referred to as BCY12735); (Palmitoyl-Glu-LysN 3 )[PYA]A[HArg]D-(SEQ ID NO:29) (hereinafter referred to as BCY14327); Ac-A-[HArg]-D-(SEQ ID NO:30) (herein referred to as BCY12736); Ac-A-[HArg]-D-(SEQ ID NO:31) (herein referred to as BCY12737); A-[HArg]-D-(SEQ ID NO:32) (herein designated BCY12738); A-[HArg]-E-(SEQ ID NO:32) (herein referred to as BCY12739); A-[HArg]-D-(SEQ ID NO:33) (herein designated BCY12854); A-[HArg]-D-(SEQ ID NO:34) (herein designated BCY12855); A-[HArg]-D-(SEQ ID NO:35) (herein designated BCY12856); A-[HArg]-D-(SEQ ID NO:35)-[dA] (herein designated BCY12857); (SEQ ID NO:35)-[dA] (herein designated BCY12861); [NMeAla]-[HArg]-D-(SEQ ID NO:35) (herein designated BCY13122); [dA]-ED-(SEQ ID NO:35) (herein referred to as BCY13126); [dA]-[dA]-D-(SEQ ID NO:35) (herein designated BCY13127); AD- (SEQ ID NO: 35) (herein referred to as BCY13128); A-[HArg]-D-(SEQ ID NO:36) (herein designated BCY12858); A-[HArg]-D-(SEQ ID NO:37) (herein referred to as BCY12859); Ac-(SEQ ID NO:37)-[dK] (herein referred to as BCY13120); A-[HArg]-D-(SEQ ID NO:38) (herein designated BCY12862); A-[HArg]-D-(SEQ ID NO:39) (herein designated BCY12863); [dA]-[HArg]-D-(SEQ ID NO:39)-[dA] (herein designated BCY12864); (SEQ ID NO:40)-[dA] (herein designated BCY12865); A-[HArg]-D-(SEQ ID NO:41) (herein designated BCY12866); A-[HArg]-D-(SEQ ID NO:42) (herein designated BCY13116); A-[HArg]-D-(SEQ ID NO:43) (herein designated BCY13117); A-[HArg]-D-(SEQ ID NO:44) (herein designated BCY13118); [dA]-[HArg]-D-(SEQ ID NO:46)-[dA] (herein designated BCY13123); [d1Nal]-[HArg]-D-(SEQ ID NO:46)-[dA] (herein designated BCY13124); A-[HArg]-D-(SEQ ID NO:47) (herein designated BCY13130); A-[HArg]-D-(SEQ ID NO:48) (herein designated BCY13131); A-[HArg]-D-(SEQ ID NO:49) (herein designated BCY13132); A-[HArg]-D-(SEQ ID NO:50) (herein designated BCY13134); A-[HArg]-D-(SEQ ID NO:51) (herein designated BCY13135); (SEQ ID NO:154)-[dK] (herein referred to as BCY13129); A[HArg]D-(SEQ ID NO:155) (herein designated BCY13133); A[HArg]D-(SEQ ID NO:156) (herein designated BCY13917); A[HArg]D-(SEQ ID NO:157) (herein designated BCY13918); A[HArg]D-(SEQ ID NO:158) (herein referred to as BCY13919); A[HArg]D-(SEQ ID NO:159) (herein designated BCY13920); A[HArg]D-(SEQ ID NO:160) (herein designated BCY13922); A[HArg]D-(SEQ ID NO:161) (herein designated BCY13923); A[HArg]D-(SEQ ID NO:162) (herein designated BCY14047); A[HArg]D-(SEQ ID NO:163) (herein referred to as BCY14048); and A[HArg]D- (SEQ ID NO: 164) (referred to herein as BCY14313) [wherein YA represents 4-pentynoic acid, B-Ala represents beta-alanine, Sar 10 represents 10 sarcosine units, HArg represents homoarginine, NMeAla represents N-methyl-alanine, 1Nal represents 1-naphthylalanine, palmitoyl-Glu-LysN 3 [PYA] is 【Chemistry 3】 Represents the following.

20. The pharmaceutical composition according to any one of items 17 to 19, comprising the compound according to claim 17, or a pharma- ceutical acceptable salt thereof.

21. the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification; A-[HArg]-D-(SEQ ID NO:24) (herein referred to as BCY9594) (In the formula, HArg represents homoarginine.) 21. The pharmaceutical composition of any one of claims 17 to 20, comprising:

22. the EphA2-binding bicyclic peptide ligand comprises an N-terminal modification; A-[HArg]-D-(SEQ ID NO:44) (herein referred to as BCY13118) (In the formula, HArg represents homoarginine.) 22. The pharmaceutical composition of any one of claims 17 to 21, comprising:

23. 23. The pharmaceutical composition of any of claims 17 to 22, wherein the heterotandem bicyclic peptide complex is selected from those listed in Table C, such as BCY12491, BCY12730, BCY13048, BCY13050, BCY13053 and BCY13272, or a pharma- ceutically acceptable salt thereof.

24. The pharmaceutical composition of any of claims 1 to 8 and 17 to 23, wherein the heterotandem bicyclic peptide complex is BCY13272 or a pharma- ceutically acceptable salt thereof.

25. A pharmaceutical composition of any of claims 1 to 8 and 17 to 24, wherein the cancer cells have high EphA2 expression.

26. 26. The pharmaceutical composition of any of claims 1 to 25, wherein the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA).

27. ​​A cancer immunotherapy agent (i) a small molecule, antibody or biologic therapy; and / or (ii) an antagonist of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, or TIM-4 The pharmaceutical composition according to any one of claims 1 to 25,

28. The pharmaceutical composition of any one of claims 1 to 28, wherein the cancer immunotherapy agent is a checkpoint inhibitor.

29. A checkpoint inhibitor (i) an antibody; and / or (ii) an antagonist of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3 or TIGIT 29. The pharmaceutical composition of claim 28,

30. 30. The pharmaceutical composition of claim 28 or 29, wherein the checkpoint inhibitor is a PD-1 antagonist.

31. The pharmaceutical composition of any of claims 28 to 30, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

32. The pharmaceutical composition of claim 31, wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

33. The pharmaceutical composition of claim 28 or 29, wherein the checkpoint inhibitor is an anti-PD-L1 antibody.

34. The pharmaceutical composition of claim 33, wherein the anti-PD-L1 antibody is durvalumab or atezolizumab.

35. 30. The pharmaceutical composition of claim 28 or 29, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

36. 36. The pharmaceutical composition of claim 35, wherein the anti-CTLA-4 antibody is ipilimumab.

37. The pharmaceutical composition of any of claims 1 to 36, wherein the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof and the cancer immunotherapeutic agent are administered simultaneously or sequentially.

38. The pharmaceutical composition of any of claims 1 to 37, wherein the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof and the cancer immunotherapeutic agent are administered within 1, 2, 3, 4, 5, 6 or 7 days of each other.

39. The pharmaceutical composition of any one of claims 1 to 38, wherein the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof is administered by intravenous infusion.

40. The pharmaceutical composition of any of claims 1 to 39, wherein the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof is administered once a week or twice a week.

41. A pharmaceutical composition for the treatment of cancer comprising BT7480 or a pharma- ceutically acceptable salt thereof, wherein the pharmaceutical composition is used in combination with a checkpoint inhibitor.

42. The pharmaceutical composition of claim 41, wherein the cancer has high Nectin-4 expression.

43. 1. A pharmaceutical composition for the treatment of cancer comprising BT7455 or a pharma- ceutical acceptable salt thereof, wherein the pharmaceutical composition is used in combination with a checkpoint inhibitor.

44. 44. The pharmaceutical composition of claim 43, wherein the cancer is high EphA2 expression.

45. A pharmaceutical composition comprising: (i) further contains histidine; (ii) further comprising sucrose; and / or (iii) The use of any of claims 41 to 44, wherein (a) the medicament is a formulation comprising BT7480 or a pharma- ceutically acceptable salt thereof, histidine, sucrose and water, at a pH of 7; or (b) a formulation comprising BT7455 or a pharma- ceutically acceptable salt thereof, histidine, sucrose and water, at a pH of 7.

46. ​​(i) The cancer is a solid tumor; (ii) the cancer is a solid tumor, the solid tumor being a sarcoma, carcinoma or lymphoma; and / or (iii) the cancer is colon cancer.

46. ​​The pharmaceutical composition of any one of claims 1 to 45.

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