CN116963757A

CN116963757A - Methods of treating cancer

Info

Publication number: CN116963757A
Application number: CN202280019734.7A
Authority: CN
Inventors: N·基恩; G·E·穆德; J·拉登兰塔; K·赫罗夫; S·巴图拉; P·E·布兰迪什; P·乌帕德亚亚; K·麦克唐奈尔
Original assignee: BicycleTx Ltd
Current assignee: BicycleTx Ltd
Priority date: 2021-01-11
Filing date: 2022-01-11
Publication date: 2023-10-27

Abstract

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

Description

Methods of treating cancer

Technical Field

The present invention relates to the use of a heteroconcatemeric (heteroelectrotandem) bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, in combination with an immunooncology agent for the treatment of cancer. The invention also provides a pharmaceutically acceptable composition comprising a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof.

Background

Cyclic peptides are capable of binding protein targets with high affinity and target specificity and thus are attractive molecular classes for developing therapeutic agents. Indeed, some cyclic peptides have been successfully used in clinic, such as, for example, the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporin, or the anticancer drug octreotide (Driggers et al (2008), nat Rev Drug Discov (7), 608-24). Good binding properties result from the relatively large interaction surface formed between the peptide and the target and reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to hundreds of square angstroms of surface, such as, for example, the cyclopeptide CXCR4 antagonist CVX15 # Wu et al (2007), science 330, 1066-71), a cyclopeptide having Arg-Gly-Asp motif binding to integrin αVb3>(Xiong et al (2002), science 296 (5565), 151-5) or a ring binding to urokinase-type plasminogen activatorPeptide inhibitor upain-1 (>Zhao et al (2007), J Struct Biol 160 (1), 1-10).

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

The different groups have previously left polypeptides with cysteine residues to the synthetic molecular structure (Kemp and McNamara (1985), J.Org.chem.; timmerman et al (2005), chemBiochem). Meloen and colleagues have used tris (bromomethyl) benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops to a synthetic backbone for structural simulation of protein surfaces (Timmerman et al (2005), chemBioChem). Methods for producing candidate drug compounds are disclosed in WO2004/077062 and WO2006/078161, wherein the compounds are produced via linking a cysteine-containing polypeptide to a molecular backbone such as, for example, tris (bromomethyl) benzene.

Phage display-based combinatorial approaches have been developed to generate and screen large double-loop peptide libraries for targets of interest (Heinis et al (2009), nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, it contains three cysteine residues and two six random amino acid regions (Cys- (Xaa) ₆ -Cys-(Xaa) ₆ -Cys) is displayed on phage and cyclized by covalently linking the cysteine side chain to a small molecule (tris- (bromomethyl) benzene).

Disclosure of Invention

It has now been found that a heterotandem bicyclic peptide complex comprising one or more CD137 binding peptide ligands, or a pharmaceutically acceptable salt thereof, causes a significant increase in tumor infiltrating immune cells and immune responses. See, e.g., the transcriptional analysis in example 1, which shows a significant increase in immune cell scores and mRNA for some T cell chemotactic chemokines/cytokines following treatment of each of BCY12491 and BT 7480. Accordingly, in one aspect, 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 CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof.

It has also been found that a combination of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, and an immunooncology agent significantly improves antitumor activity compared to each of the single agent treatments. See, for example, that combination therapy of BCY12491 in example 2 with the PD-1 antagonist pampareizumab (Pembrolizumab) results in more pronounced antitumor activity than treatment with each single agent. Accordingly, in one aspect, the present invention provides a method for treating cancer in a patient comprising administering to the patient a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, and an immunooncology agent.

Drawings

Figure 1 depicts BCY12491 modulates tumor immune microenvironment and drives T cell infiltration. (A) Mice bearing MC38 tumors were treated with vehicle, 15mg/kg EphA2/CD137 heterotandem bicyclic peptide complex (BCY 12491), enantiomer-non-binding control heterotandem bicyclic peptide complex (BCY 13626) q3d intravenous (i.v.) or 2mg/kg αcd137 q3d intraperitoneal (i.p.). Individual tumor volumes (normalized to the tumor volume on the day of treatment initiation) are shown as groups by treatment. (B) Nanostring analysis of tumors showed the effect of BCY12491 and αCD137 on the T cell (probe set: cd3d, cd3e, cd3g, cd6, sh2d1a and Trat 1), cytotoxic cell (probe set: ctsw, gzma, gzmb, klrb1, klrd1, klrk1, nkg and Prf 1) and macrophage (probe set: cd163, cd68, cd84 and Ms4a4 a) content. (C) Nanostring analysis of tumors showed the effect of BCY12491 and αcd137 on transcription of checkpoint inhibitors Pdcd1 (protein PD-1), CD274 (protein PD-L1) and Ctla4 (protein Ctla-4). (D) Representative images from tissue sections of tumors treated with vehicle, 15mg/kg BCY12491, BCY13626 or 2mg/kg αcd 137Q 3D and stained for mouse CD8 are shown. (B and C) < 0.05, < p < 0.001, one-way ANOVA with Dunnett's post test.

Figure 2 depicts the effect of BT7480 on selected cytokines/chemokines. (A) On the left hand side of the graph, normalized linear count data for 5 different cytokine/chemokine mrnas in the MC38#13 tumor tissue after BT7480 treatment is shown. (B) The overlay of cytotoxicity cell scores and the normalized RNA counts of Ccl1, ccl-17 and Ccl24 shows early increases in these cytokine/chemokine transcripts followed by increases in cytotoxicity cell scores.

Figure 3 depicts BT7480 modulating tumor immune microenvironment and driving cd8+ T cell infiltration. Mice bearing the MC38#13 tumor were treated intravenously or intraperitoneally with vehicle, 5mg/kg (0 h, 24 h) BT7480 or unbound heterotandem bicyclic peptide complex control BCY12797 (NB-BCY) or 2mg/kg αCD 137. Nanostring analysis of tumors showed the effect of BT7480 and αcd137 on (a) macrophages (probe sets: CD163, CD68, CD84 and Ms4a4 a) and (B) cytotoxic cells (probe sets: ctsw, gzma, gzmb, klrb1, klrd1, klrk1, nkg and Prf 1) scores in tumor tissue over time. (C) The overlapping plots of cytotoxicity and macrophage cell scores demonstrate an early increase in macrophage cell score followed by an increase in cytotoxicity cell score. (a and B) < 0.05, < p < 0.01, one-way ANOVA with Dunnett post test.

Figure 4 depicts that BT7480 results in an increase in some immune checkpoint mrnas. Mice bearing the MC38#13 tumor were treated intravenously or intraperitoneally with vehicle, 5mg/kg (0 h, 24 h) BT7480 or unbound heterotandem bicyclic peptide complex control BCY12797 (NB-BCY) or 2mg/kg αCD 137. Nanostring analysis of tumors showed the effect of BT7480 and αcd137 on the levels of some immune checkpoint mRNA. * < 0.05, < p < 0.01, < p < 0.001, one-way ANOVA with Dunnett post test.

Fig. 5 depicts that BCY12491 +palbociclib combined from day 0 (after treatment initiation) produced 100% complete response by day 22. Mice bearing MC38 tumors were treated with vehicle, 5mg/kg BCY12491 QW (0, 24 h), 3mg/kg Paborrelizumab QW, or combinations thereof. The top panel shows the average tumor volume from the start of treatment to day 28. Both monotherapy and combination therapy significantly affected tumor growth (< 0.0001, mixed effect analysis compared to Dunnett post test, comparison at D18 with vehicle). Furthermore, the combination treatment was more effective than either of the monotherapy (p < 0.0001), mixed effect analysis and Dunnett post test, comparing the combination to the monotherapy at D20), resulting in a complete response in all treated animals by day 22. The right curve shows the growth curve of individual tumors from the treated population.

Figure 6 depicts BCY12491 +palbociclib mab combination produced significant anti-tumor activity with different order of administration. Mice bearing MC38 tumors were treated with vehicle, 5mg/kg BCY12491 QW (0, 24 h), 3mg/kg pamglizumab QW or combinations thereof with three different dosing schedules: BCY12491 and palbociclizumab treatment all began on day 0; BCY12491 treatment started on day 0 followed by palbociclizumab treatment started on day 5; or palbociclib treatment started on day 0 followed by BCY12491 treatment started on day 5. The top panel shows the average tumor volume from the start of treatment to day 28. All combination treatments showed significant anti-tumor activity with complete responses of 10/10 (BCY 12491 +palbocizumab from D0), 9/10 (BCY 12491 +palbocizumab from D5 from D0) and 8/10 (palboc Li Zhushan antibody from D0 and BCY12491 from D5) by day 42. * P < 0.0001, mixed effect analysis and Dunnett post test, comparison with vehicle at D18. The right hand curve shows the growth curve of individual tumors from the treated population.

Fig. 7 depicts the addition of BCY11864 to anti-PD-1 monotherapy significance [ p=0.004, log rank (Mantel-Cox) test, comparing anti-PD-1 and anti-PD-1+bcy11864 combination groups ]Increased portabilitySurvival (defined as reaching the humane endpoint, tumor volume > 2000 mm) of mice with CT26#7 (CT 26 engineered to overexpress Purpurin-4 (Nectin-4)) ³ )。

Figure 8 depicts that addition of BT7480 to anti-PD-1 monotherapy increases the rate of Complete Response (CR) in mice carrying MC38#13 (MC 38 engineered to overexpress petin-4).

Figure 9 depicts the addition of BT7480 to anti-CTLA-4 monotherapy significantly [ p=0.0499, log rank (Mantel-Cox) test, comparing anti-CTLA-4 and anti-CTLA-4+bt7480 combination groups]Increasing survival (defined as reaching the humane endpoint, tumor volume > 2000 mm) of mice carrying MC38#13 (MC 38 engineered to overexpress handle protein-4) ³ ) And increases the full response rate.

Figure 10 depicts BT7455 causing an increase in some immune checkpoint mrnas. Mice bearing MC38 tumors were treated intravenously with vehicle, 8mg/kg (0 h, 24 h) BT7455 or intraperitoneally with 2mg/kg anti-CD 137 antibody or 10mg/kg anti-PD-1 antibody. Nanostring analysis of tumors showed the effect of treatment on the levels of some immune checkpoint mRNA. mRNA from MC38 tumor tissue was shown to be normalized to Log2 counts at 24, 48 and 144 hour time points. * < 0.05, < p < 0.01, < p < 0.001, one-way ANOVA with Dunnett post test, comparing the therapeutic agent to vehicle at the same time point.

Figure 11 depicts the effect of BT7455 (8 mg/kg), anti-PD-1 and anti-CD 137 (Wu Ruilu mab (urelumab) analogs) treatment on 5 selected cytokines/chemokines across 24 hours, 48 hours and 24 hour time points. mRNA from MC38 tumor tissue was shown to be normalized to Log2 counts at 24, 48 and 144 hour time points. * p < 0.05, p < 0.01, p < 0.0001,0.01 one-way ANOVA with Dunnett post test.

FIG. 12 depicts the effect of BT7455 (8 mg/kg), anti-PD-1 and anti-CD 137 (Wu Ruilu mab analogs) treatment on cytotoxic cells. The effect of treatment on cytotoxic cells at 24 hours, 48 hours and 144 hours time points was shown as a cytotoxic cell type score, which was a normalized Log2 (mean and standard deviation) score in MC38 tumor tissue. P < 0.05, one-way ANOVA with Dunnett post test, comparing therapeutic agent to vehicle).

Figure 13 depicts transcriptional analysis demonstrating significant modulation of some gene sets at an early time point (48 h) after initiation of treatment by BT7455 (×p < 0.05, ×p < 0.01, one-way ANOVA with post Dunnett test), whereas the effects of anti-PD-1 and Wu Ruilu mab analogs (anti-CD 137) were not significant. The effect of treatment on the gene set was shown as a signature score (mean and standard deviation) in MC38 tumor tissue.

Detailed Description

1. Description of certain embodiments of the invention:

it has been found that a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, causes a significant increase in tumor-infiltrating immune cells and immune responses compared to each of the single agent treatments, and that a combination of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, and an immunooncology agent significantly improves antitumor activity. See, e.g., data for treatment with each of BCY12491 and BT7480 in example 1, and data for treatment with BCY12491 alone, the PD-1 antagonist pamphlet Li Zhushan antibody alone, and a combination of BCY12491 and pamphlet in example 2. Thus, in one aspect, provided herein is a method or use of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, for increasing an immune response in a cancer patient. In another aspect, provided herein is a method or use of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, in combination with an immunooncology agent for treating cancer in a patient.

In some embodiments, the 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 CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof. In some embodiments, the invention provides a use of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for increasing an immune response in a cancer patient.

In some embodiments, the 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 CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, and an immunooncology agent. In some embodiments, the invention provides a use of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating cancer in a patient, wherein the medicament is used in combination with an immunooncology agent.

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 MT 1-MMP. In some embodiments, the cancer is associated with calpain-4. In some embodiments, the cancer is associated with EphA 2. In some embodiments, the cancer is associated with PD-L1. In some embodiments, the cancer is associated with PSMA.

In some embodiments, as described herein, the heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands is selected from the group consisting of heterotandem bicyclic peptide complexes comprising one CD 137-binding peptide ligand. In some embodiments, as described herein, the heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands is selected from the group consisting of heterotandem bicyclic peptide complexes comprising two or more CD 137-binding peptide ligands.

In some embodiments, the heterotandem bicyclic peptide complex is BCY11863 (also known as BT 7480) or a pharmaceutically acceptable salt thereof. In some embodiments, the heterotandem bicyclic peptide complex is BCY13272 (also known as BT 7455) 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.

In some embodiments, the immunooncology agent is selected from the group consisting of immunooncology agents as described herein. In some embodiments, the immunooncology agent is a checkpoint inhibitor. In some embodiments, the immunooncology agent is a PD-1 antagonist. In some embodiments, the immunooncology agent is pamphlet Li Zhushan antibody. In some embodiments, the immunooncology agent is nivolumab (nivolumab).

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

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

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

In some embodiments, the heterotandem bicyclic peptide complex is administered at a frequency of 1, 2, 3, or 4 times a week. In some embodiments, the heterotandem bicyclic peptide complexes are selected from those described herein, e.g., BT7480 or BT7455, or pharmaceutically acceptable salts thereof. In some embodiments, the heterotandem bicyclic peptide complex is administered once daily. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 2 days. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 3 days. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 4 days. In some embodiments, the heterotandem bicyclic peptide complex is administered once every 5 days. In some embodiments, the heterotandem bicyclic peptide complex is administered at a frequency of once a 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 2 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 at a frequency of once every 3 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered at a frequency of once every 4 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered at a frequency of once a month.

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 complexes are selected from those described herein, e.g., BT7480 or BT7455, or pharmaceutically acceptable salts 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, 8, 12, 16, 20, 24, or 28 weeks. In some embodiments, the heterotandem bicyclic peptide complex is administered for a treatment period of about 30 weeks or more.

In some embodiments, the heterotandem bicyclic peptide complex is administered to the patient via intravenous bolus injection (bolus injection). In some embodiments, the heterotandem bicyclic peptide complexes are selected from those described herein, e.g., BT7480 or BT7455, or pharmaceutically acceptable salts thereof. In some embodiments, the heteroconcatemeric bicyclic peptide complex is administered to the patient via intravenous infusion. In some embodiments, intravenous infusion of the heterotandem bicyclic peptide complex is about 5-10 minutes infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about 10-20 minutes of infusion. In some embodiments, intravenous infusion of the heterotandem bicyclic peptide complex is about 20-40 minutes infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about 45 or 50 or 55 minutes infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is about 1 hour infusion. In some embodiments, intravenous infusion of the heterotandem bicyclic peptide complex is about 1-1.5 hours of infusion. In some embodiments, intravenous infusion of the heterotandem bicyclic peptide complex is about 1.5-2 hours of infusion. In some embodiments, intravenous infusion of the heterotandem bicyclic peptide complex is about 2-3 hours of infusion. In some embodiments, the intravenous infusion of the heterotandem bicyclic peptide complex is over 3 hours of infusion.

The immunooncology agent is administered according to a dosage regimen recommended or approved by the FDA. In some embodiments, the immunooncology agent is administered at a dose of about 1-20 mg/kg. In some embodiments, the immunooncology agent is administered at a dose of about 1-5mg/kg, about 6-10mg/kg, about 11-15mg/kg, or about 16-20 mg/kg. In some embodiments, the immunooncology agent is administered at a dose of about 1-10mg/kg, about 5-15mg/kg, or about 10-20 mg/kg. In some embodiments, the immunooncology agent is administered at a dose of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg. In some embodiments, the immunooncology agent is administered at a dose of about 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg. In some embodiments, the immunooncology agent is administered at a frequency of 1, 2, 3, or 4 times a week. In some embodiments, the immunooncology agent is administered once a day. In some embodiments, the immunooncology agent is administered once every 2 days. In some embodiments, the immunooncology agent is administered once every 3 days. In some embodiments, the immunooncology agent is administered once every 4 days. In some embodiments, the immunooncology agent is administered once every 5 days. In some embodiments, the immunooncology agent is administered at a frequency of once a week. In some embodiments, the immunooncology agent is administered at a frequency of once every 1.5 weeks. In some embodiments, the immunooncology agent is administered at a frequency of once every 2 weeks. In some embodiments, the immunooncology agent is administered at a frequency of once every 2.5 weeks. In some embodiments, the immunooncology agent is administered at a frequency of once every 3 weeks. In some embodiments, the immunooncology agent is administered at a frequency of once every 4 weeks. In some embodiments, the immunooncology agent is administered at a frequency of once a month. In some embodiments, the immunooncology agent is administered for a treatment period of about 1-4 weeks. In some embodiments, the immunooncology 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 immunooncology agent is administered for a treatment period of about 36 weeks or more. In some embodiments, the immunooncology agent is administered to the patient via intravenous injection. In some embodiments, the immunooncology agent is administered to the patient via intravenous infusion. In some embodiments, the intravenous infusion of the immunooncology agent is about 5-10 minutes infusion. In some embodiments, the intravenous infusion of the immunooncology agent is about 10-20 minutes or about 20-40 minutes. In some embodiments, the intravenous infusion of the immunooncology agent is about 30, 40, 45, 50, 55, or 60 minutes infusion. In some embodiments, the intravenous infusion of the immunooncology agent is about 1-1.5 hours, about 1.5-2 hours, or about 2-3 hours.

In some embodiments, the drug comprising the heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof is selected from the heterotandem bicyclic peptide complex formulation as shown in the examples of the invention. In some embodiments, the heterotandem bicyclic peptide complexes are selected from those described herein, e.g., BT7480 or BT7455, or pharmaceutically acceptable salts thereof. In some embodiments, the drug comprising the heteroconcatemeric bicyclic peptide complex or pharmaceutically acceptable salt thereof further comprises histidine. In some embodiments, the drug comprising the heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof and histidine is at about pH7. In some embodiments, the drug comprising the heteroconcatemeric bicyclic peptide complex or pharmaceutically acceptable salt thereof further comprises sucrose. In some embodiments, the medicament comprising the heteroconcatemeric bicyclic peptide complex or pharmaceutically acceptable salt thereof further comprises about 10% w/v sucrose. In some embodiments, the medicament comprising the heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof further comprises water. In some embodiments, the invention provides a medicament comprising a heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof, histidine, sucrose, and water, wherein the medicament is at about pH7.

Exemplary heteroconcatemeric bicyclic peptide complexes

In some embodiments, the heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof comprises:

(a) A first peptide ligand that binds a component present on a cancer cell; conjugated to via a linker

(b) One or more CD 137-binding peptide ligands;

wherein each of the peptide ligands comprises a polypeptide comprising: at least three reactive groups separated by at least two ring sequences, and a molecular scaffold forming a covalent bond with the reactive groups of the polypeptide such that at least two polypeptide rings are formed on the molecular scaffold.

(b) One or more CD 137-binding peptide ligands;

wherein each of the peptide ligands comprises a polypeptide comprising: at least three cysteine residues separated by at least two loop sequences, and a molecular scaffold forming a covalent bond with a cysteine residue of a polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

(b) A CD137 binding peptide ligand;

b) Two or more CD 137-binding peptide ligands;

(b) Two or more CD 137-binding peptide ligands;

(b) Two CD137 binding peptide ligands;

(b) Three CD137 binding peptide ligands;

First peptide ligand

References herein to the term "cancer cell" include any cell known to be involved in cancer. Cancer cells form when genes responsible for regulating cell division are damaged. Carcinogenesis is caused by mutations in the genetic material and surface mutations of normal cells, which interfere with the normal balance between proliferation and cell death. This causes uncontrolled cell division and the evolution of those cells via natural selection in vivo. Uncontrolled and often rapid cell proliferation can cause benign or malignant tumors (cancers). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors can invade other organs, spread to remote locations (metastasis) and become life threatening.

In some embodiments, the cancer cell is selected from the group consisting of HT1080, A549, SC-OV-3, PC3, HT1376, NCI-H292, lnCap, MC38#13, 4T1-D02, H322, HT29, T47D, and RKO tumor cells.

In some embodiments, the component present on the cancer cell is handle protein-4 (Nectin-4).

The handle protein-4 is a surface molecule belonging to the handle protein family, which contains 4 members. The stalk proteins are cell adhesion molecules that play an important role in various biological processes such as polarity, proliferation, differentiation and migration of epithelial cells, endothelial cells, immune cells and neuronal cells during development and adult life. It is involved in some pathological processes in humans. It is the primary receptor for polio, herpes simplex and measles viruses. Mutations in the genes encoding for either handle protein-1 (PVRL 1) or handle protein-4 (PVRL 4) cause ectodermal dysplasia syndromes associated with other abnormalities. The petiolin-4 is expressed during fetal development. In adult tissues, their expression is more restricted than that of other family members. Handle protein-4 is a tumor associated antigen in 50%, 49% and 86% of breast, ovarian and lung cancers, respectively, mainly on tumors with poor prognosis. Its expression was not detected in the corresponding normal tissue. In breast tumors, handle protein-4 is expressed mainly in triple negative and erbb2+ cancers. In the serum of patients with such cancers, detection of soluble forms of handle protein-4 correlates with poor prognosis. Serum sesamin-4 levels increased during metastatic progression and decreased after treatment. These results indicate that petin-4 can be a reliable target for the treatment of cancer. Thus, some anti-stalk protein-4 antibodies have been described in the prior art. In particular, enrolment Shan Kangwei statin (Enfortumab Vedotin, ASG-22 ME) is an antibody-drug conjugate (ADC) targeting ansa-4 and is currently being clinically investigated for treating patients suffering from solid tumors.

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

In some embodiments, the handle protein-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.

In some embodiments, the ansa-4 binding bicyclic peptide ligand comprises an amino acid sequence selected from the group consisting of:

C _i P[1Nal][dD]C _ii M[HArg]DWSTP[HyP]WC _iii (SEQ ID NO:1; referred to herein as BCY 8116);

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; referred to herein as BCY 11414);

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; referred to herein as BCY 12363);

C _i P[1Nal][dK]C _ii M[HArg]DWSTP[HyP]W[Cysam] _iii (SEQ ID NO:16)；

[MerPro] _i P[1Nal][dK]C _ii M[HArg]DWSTP[HyP]W[Cysam] _iii (SEQ ID NO:17; referred to herein as BCY 12365);

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; referred to herein as BCY 12368);

C _i P[1Nal][dA]C _ii M[HArg]DWSTP[HyP]WC _iii (SEQ ID NO:21; referred to herein as BCY 12369);

C _i P[1Nal][dE]C _ii L[HArg]DWSTP[HyP]WC _iii (SEQ ID NO:22; referred to herein as BCY 12370); and

C _i P[1Nal][dE]C _ii M[HArg]EWSTP[HyP]WC _iii (SEQ ID NO:23; referred to herein as BCY 12384);

wherein [ MerPro] _i 、C _i 、C _ii 、C _iii And [ Cysam ]] _iii Represents a first (i), a second (ii) and a 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.

C _i P[1Nal][dK](Sar ₁₀ -(B-Ala))C _ii M[HArg]DWSTP[HyP]WC _iii (SEQ ID NO: 3); and

wherein C is _i 、C _ii And C _iii Respectively representing a first, a second and a third cysteine residue, 1Nal representing 1-naphthylalanine, HArg representing homoarginine, hyP representing trans-4-hydroxy-L-proline, sar ₁₀ Represents 10 sarcosine units, and B-Ala represents beta-alanine.

In some embodiments, the ansa-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

SEQ ID NO. 1 (referred to herein as BCY 8116);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 1) (referred to herein as BCY 8846);

[ PYA ] - (SEQ ID NO: 1) (referred to herein as BCY 11015);

[ PYA ] - [ B-Ala ] - (SEQ ID NO: 1) (referred to herein as BCY 11016);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 2) (referred to herein as BCY 11942);

ac- (SEQ ID NO: 3) (referred to herein as BCY 8831);

SEQ ID NO. 4 (referred to herein as BCY 11414);

[ PYA ] - [ B-Ala ] - (SEQ ID NO: 14) (referred to herein as BCY 11143);

palmitic acid-yGlu-yGlu- (SEQ ID NO: 14) (referred to herein as BCY 12371);

Ac- (SEQ ID NO: 14) (referred to herein as BCY 12024);

ac- (SEQ ID NO: 16) (referred to herein as BCY 12364);

ac- (SEQ ID NO: 18) (referred to herein as BCY 12366); and

ac- (SEQ ID NO: 19) (referred to herein as BCY 12367);

wherein the method comprises the steps ofPYA represents 4-pentynoic acid, B-Ala represents beta-alanine, sar ₁₀ 10 sarcosine units are indicated.

SEQ ID NO. 1 (referred to herein as BCY 8116);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 1) (referred to herein as BCY 8846);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 2) (referred to herein as BCY 11942);

ac- (SEQ ID NO: 3) (referred to herein as BCY 8831); and

SEQ ID NO. 4 (referred to herein as BCY 11414);

wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, sar ₁₀ 10 sarcosine units are indicated.

In some embodiments, the ansa-4 binding bicyclic peptide ligand comprises SEQ ID NO:1 (referred to herein as BCY 8116).

C _i P[1Nal][dD]C _ii M[HArg]DWSTP[HyP]WC _iii (SEQ ID NO:1; hereinafter referred to as BCY 8116);

C _i P[1Nal][dD]C _ii M[HArg]D[dW]STP[HyP][dW]C _iii (SEQ ID NO:2; hereinafter BCY 11415); and

C _i PFGC _ii M[HArg]DWSTP[HyP]WC _iii (SEQ ID NO:4; hereinafter BCY 11414);

wherein C is _i 、C _ii And C _iii Respectively representing a first, a second and a third cysteine residue, 1Nal representing 1-naphthylalanine, HArg representing homoarginine, hyP representing hydroxyproline, sar ₁₀ Represents 10 sarcosine units, and B-Ala represents beta-alaninAnd (3) acid.

In another embodiment, the ansa-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

SEQ ID NO. 1 (referred to herein as BCY 8116);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8846);

SEQ ID NO. 2 (referred to herein as BCY 11415);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 2) (hereinafter referred to as BCY 11942);

ac- (SEQ ID NO: 3) (referred to herein as BCY 8831); and

SEQ ID NO. 4 (referred to herein as BCY 11414);

In some embodiments, the component present on the cancer cell is EphA2.

Eph receptor tyrosine kinases (ephs) belong to a large group of Receptor Tyrosine Kinases (RTKs), i.e. kinases that phosphorylate proteins at tyrosine residues. Ephs and their membrane-bound ephrin (ephrin) ligands (ephrin) control Cell localization and tissue organization (poliaov et al (2004) Dev Cell 7,465-80). Functional and biochemical Eph reactions occur in a higher ligand oligomeric state (Stein et al (1998) Genes Dev 12, 667-678).

Among other modeling functions, various ephs and ephrins have been shown to play a role in vascular development. Knockout of EphB4 and ephrin-B2 results in a lack of ability to remodel microvascular beds into blood vessels (poliaov et al, supra) and embryonic lethality. Persistent expression of some Eph receptors and ephrin has also been observed in newly formed adult microvasculature (Brantley-Sieders et al (2004) Curr Pharm Des 10, 3431-42; adams (2003) J Anat 202,105-12).

Deregulation of some hepatins 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, it has been found that some Eph family members are overexpressed on tumor cells from a variety of human tumors (Brantley-Sieders et al, supra); marme (2002) Ann Hematol 81 journal 2, S66; booth et al (2002) Nat Med 8,1360-1).

EPH receptor A2 (ephrin type a receptor 2) is a protein encoded by the EPHA2 gene in humans.

EphA2 is upregulated in a variety of cancers in humans, typically associated with disease progression, metastasis and poor prognosis, such as breast Cancer (Zelinski et al (2001) Cancer res.61,2301-2306; zhuang et al (2010) Cancer res.70,299-308; brandley-Sieders et al (2011) PLoS One6, e 24426), lung Cancer (brandan et al (2009) Cancer Prev Res (philia) 2,1039-1049; king et al (2003) Clin Cancer res.9,613-618; guo et al (2013) J thermo Oncol.8, 301-308), stomach Cancer (Nakamura et al (2005) Cancer sci.96,42-47; yn et al (2009) Dig Sci 54, 2410-2417), packet (Mudali et al (2006) support Clin Exp Metastasis, 357, projection (47) and advanced (47) Cancer, projection et al (47) and human brain Cancer (47) projection et al (2001) gastric Cancer, projection et al (2001) 35, 35-357-618; gum et al (35) and human Tumor (47) projection et al, projection 5, projection vascular Tumor (35, 35) and human projection) projection (47).

Despite evidence of interactions at multiple stages of cancer progression including tumor cell growth, survival, invasion and angiogenesis, the full role of EphA2 in cancer progression has not been defined. Down-regulation of EphA2 expression inhibits tumor Cancer Cell transmission (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), budding and angiogenesis (Cheng et al (2002) Mol Cancer res.1,2-11; lin et al (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB j.19, 1884-1886).

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 similar approaches have been tried in humans, although treatment had to be discontinued due to treatment-related adverse events (Annunziata et al (2013) Invest New drugs 31,77-84).

In some embodiments, the first peptide ligand comprises an EphA 2-binding bicyclic peptide ligand.

In some embodiments, the EphA 2-binding bicyclic peptide ligand is selected from those EphA 2-binding bicyclic peptide ligands disclosed in WO2019/122860, WO2019/122861, and WO2019/122863, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the EphA 2-binding bicyclic peptide ligand comprises an amino acid sequence selected from the group consisting of:

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; referred to herein as BCY 12860);

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; referred to herein as BCY 13119);

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)；

Wherein [ MerPro] _i 、C _i 、C _ii 、C _iii And [ Cysam ]] _iii 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-DPA represents 3, 3-diphenylalanine, cba represents β -cyclobutylalanine, 1Nal represents 1-naphthylalanine, hGlu represents homoglutamic acid, thi represents 2-thienyl-alanine, 4 thiz represents β - (4-thiazolyl) -alanine, his1Me represents N1-methyl-L-histidine, his3Me represents N3-methyl-L-histidine, 3Thi represents 3-thienyl alanine, palmitoyl-Glu-LysN ₃ [PYA]The representation is:

(palmitoyl-Glu-LysN 3) [ PYA ],

[ K (PYA- (palmitoyl-Glu-LysN) ₃ )]The representation is:

[ K (PYA (palmitoyl-Glu-LysN) ₃ ))]，

Nle represents norleucine, merPro represents 3-mercaptopropionic acid, and Cysam represents cysteamine.

In some embodiments, the EphA 2-binding bicyclic peptide ligand comprises the amino acid sequence of:

C _i [HyP]LVNPLC _ii LHP[dD]W[HArg]C _iii (SEQ ID NO:24)；

Wherein C is _i 、C _ii And C _iii Represents a first (i), a second (ii) and a third (iii) cysteine group, hyP represents trans-4-hydroxy-L-proline, and HArg represents homoarginine.

C _i [HyP]LVNPLC _ii LEP[d1Nal]WTC _iii (SEQ ID NO:44)；

wherein C is _i 、C _ii And C _iii Represents the first (i), second (ii) and third (iii) cysteine groups, hyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine.

In some embodiments, the EphA 2-binding bicyclic peptide ligand optionally comprises an N-terminal and/or C-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

a- [ HArg ] -D- (SEQ ID NO: 24) (referred to herein as BCY 9594);

[B-Ala]-[Sar ₁₀ ]-A-[HArg]-D- (SEQ ID NO: 24) (referred to herein as BCY 6099);

[ PYA ] -A- [ HArg ] -D- (SEQ NO: 24) (referred to herein as BCY 11813);

Ac-A- [ HArg ] -D- (SEQ ID NO: 24) - [ K (PYA) ] (referred to herein as BCY 11814;

Ac-A- [ HArg ] -D- (SEQ ID NO: 24) -K (referred to herein as BCY 12734);

[ NMeAla ] - [ HArg ] -D- (SEQ ID NO: 24) (referred to herein as BCY 13121);

[ Ac ] - (SEQ ID NO: 24) -L [ dH ] G [ dK ] (referred to herein as BCY 13125);

[PYA]-[B-Ala]-[Sar ₁₀ ]VGP- (SEQ ID NO: 25) (referred to herein as BCY 8941);

Ac-A- [ HArg ] -D- (SEQ ID NO: 26) (referred to herein as BCY 11815);

Ac-A- [ HArg ] -D- (SEQ ID NO: 27) (referred to herein as BCY 11816);

Ac-A- [ HArg ] -D- (SEQ ID NO: 28) (referred to herein as BCY 11817);

Ac-A- [ HArg ] -D- (SEQ ID NO: 29) (referred to herein as BCY 12735);

(palmitoyl-Glu-LysN) ₃ )[PYA]A[HArg]D- (SEQ ID NO: 29) (hereinafter referred to as BCY 14327);

Ac-A- [ HArg ] -D- (SEQ ID NO: 30) (referred to herein as BCY 12736);

Ac-A- [ HArg ] -D- (SEQ ID NO: 31) (referred to herein as BCY 12737);

a- [ HArg ] -D- (SEQ ID NO: 32) (referred to herein as BCY 12738);

a- [ HArg ] -E- (SEQ ID NO: 32) (referred to herein as BCY 12739);

a- [ HArg ] -D- (SEQ ID NO: 33) (referred to herein as BCY 12854);

a- [ HArg ] -D- (SEQ ID NO: 34) (referred to herein as BCY 12855);

a- [ HArg ] -D- (SEQ ID NO: 35) (referred to herein as BCY 12856);

a- [ HArg ] -D- (SEQ ID NO: 35) - [ dA ] (referred to herein as BCY 12857);

(SEQ ID NO: 35) - [ dA ] (referred to herein as BCY 12861);

[ NMeAla ] - [ HArg ] -D- (SEQ ID NO: 35) (referred to herein as BCY 13122);

[ dA ] -ED- (SEQ ID NO: 35) (referred to herein as BCY 13126);

[ dA ] - [ dA ] -D- (SEQ ID NO: 35) (referred to herein as BCY 13127);

AD- (SEQ ID NO: 35) (referred to herein as BCY 13128);

a- [ HArg ] -D- (SEQ ID NO: 36) (referred to herein as BCY 12858);

a- [ HArg ] -D- (SEQ ID NO: 37) (referred to herein as BCY 12859);

Ac- (SEQ ID NO: 37) - [ dK ] (referred to herein as BCY 13120);

a- [ HArg ] -D- (SEQ ID NO: 38) (referred to herein as BCY 12862);

a- [ HArg ] -D- (SEQ ID NO: 39) (referred to herein as BCY 12863);

[ dA ] - [ HArg ] -D- (SEQ ID NO: 39) - [ dA ] (referred to herein as BCY 12864);

(SEQ ID NO: 40) - [ dA ] (referred to herein as BCY 12865);

a- [ HArg ] -D- (SEQ ID NO: 41) (referred to herein as BCY 12866);

a- [ HArg ] -D- (SEQ ID NO: 42) (referred to herein as BCY 13116);

a- [ HArg ] -D- (SEQ ID NO: 43) (referred to herein as BCY 13117);

a- [ HArg ] -D- (SEQ ID NO: 44) (referred to herein as BCY 13118);

[ dA ] - [ HArg ] -D- (SEQ ID NO: 46) - [ dA ] (referred to herein as BCY 13123);

[ D1Nal ] - [ HArg ] -D- (SEQ ID NO: 46) - [ dA ] (referred to herein as BCY 13124);

a- [ HArg ] -D- (SEQ ID NO: 47) (referred to herein as BCY 13130);

a- [ HArg ] -D- (SEQ ID NO: 48) (referred to herein as BCY 13131);

a- [ HArg ] -D- (SEQ ID NO: 49) (referred to herein as BCY 13132);

a- [ HArg ] -D- (SEQ ID NO: 50) (referred to herein as BCY 13134);

a- [ HArg ] -D- (SEQ ID NO: 51) (referred to herein as BCY 13135);

(SEQ ID NO: 154) - [ dK ] (referred to herein as BCY 13129);

a [ HArg ] D- (SEQ ID NO: 155) (referred to herein as BCY 13133);

a [ HArg ] D- (SEQ ID NO: 156) (referred to herein as BCY 13917);

A [ HArg ] D- (SEQ ID NO: 157) (referred to herein as BCY 13918);

a [ HArg ] D- (SEQ ID NO: 158) (referred to herein as BCY 13919);

a [ HArg ] D- (SEQ ID NO: 159) (referred to herein as BCY 13920);

a [ HArg ] D- (SEQ ID NO: 160) (referred to herein as BCY 13922);

a [ HArg ] D- (SEQ ID NO: 161) (referred to herein as BCY 13923);

a [ HArg ] D- (SEQ ID NO: 162) (referred to herein as BCY 14047);

a [ HArg ] D- (SEQ ID NO: 163) (referred to herein as BCY 14048); and

a [ HArg ] D- (SEQ ID NO: 164) (referred to herein as BCY 14313);

wherein PYA 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]The representation is:

(palmitoyl-Glu-LysN) ₃ )[PYA]。

a- [ HArg ] -D- (SEQ ID NO: 24) (referred to herein as BCY 9594);

wherein HArg represents homoarginine.

A- [ HArg ] -D- (SEQ ID NO: 44) (referred to herein as BCY 13118);

wherein HArg represents homoarginine.

In some embodiments, the EphA 2-binding bicyclic peptide ligand comprises an amino acid sequence or a pharmaceutically acceptable salt thereof:

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)；

wherein C is _i 、C _ii And C _iii Respectively the first, second and third cysteine residues, hyP is hydroxyproline, dD is aspartic acid in the D configuration, and HArg is homoarginine.

C _i [HyP]LVNPLC _ii LHP[dD]W[HArg]C _iii (SEQ ID NO:24)；

In some embodiments, the EphA 2-binding bicyclic peptide ligand comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

A-HArg-D- (SEQ ID NO: 24) (hereinafter referred to as BCY 9594);

[B-Ala]-[Sar ₁₀ ]-A-[HArg]-D- (SEQ ID NO: 24) (hereinafter referred to as BCY 6099);

[PYA]-[B-Ala]-[Sar ₁₀ ]-A-[HArg]d- (SEQ ID NO: 24) (hereinafter referred to as BCY 6169); and

[PYA]-[B-Ala]-[Sar ₁₀ ]VGP- (SEQ ID NO: 25) (hereinafter referred to as BCY 8941);

wherein HArg represents homoarginine, PYA represents 4-pentynoic acid, sar ₁₀ Represents 10 sarcosine units, and B-Ala represents beta-alanine.

A-HArg-D- (SEQ ID NO: 24) (hereinafter referred to as BCY 9594);

wherein HArg represents homoarginine.

In some embodiments, the component present on the cancer cell is PD-L1.

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

PD-L1 expression is also involved in the inhibition of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immune clearance of tumors is rare. This failure is due in part to immunosuppression of the tumor microenvironment. PD-L1 expression on many tumors is a component of this inhibitory environment and acts synergistically with other immunosuppressive signals. PD-L1 expression has been demonstrated in situ on a wide variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymus epithelium, head and neck tumors (Brown JA et al 170:1257-66; dong H et al 2002Nat.Med.8:793-800; hamanishi J, et al 2007Proc.Natl.Acad.Sci.USA 104:3360-65; strome SE et al 2003Cancer Res.63:6501-5; inman BA et al 2007Cancer 109:1499-505; konishi J et al 2004Clin.Cancer Res.10:5094-100; nakanishi J et al 2007Cancer Immunol.Immunother.56:1173-82; nomi T et al 2007Clin.Cancer Res.13:2151-57; thompson RH et al 2004Proc.Natl.Acad.Sci.USA 101:17174-79; wu C et al 2006Acta Histochem.108:19-24). In addition, the expression of PD-L1 receptor apoptosis protein 1 (also known as PD-1 and CD 279) is up-regulated on tumor infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al 2003immunol. 171:4574-81). Most importantly, studies correlating PD-L1 expression on tumors with disease outcome showed that PD-L1 expression was largely correlated with adverse prognosis in renal, ovarian, bladder, breast, gastric and pancreatic cancers (Hamanishi J et al 2007Proc.Natl.Acad.Sci.USA 104:3360-65; inman BA et al 2007Cancer 109:1499-505; konishi J et al 2004Clin.Cancer Res.10:5094-100; nakanishi J et al 2007Cancer Immunol.Immunother.56:1173-82; nomi T et al 2007Clin.Cancer Res.13:2151-57; thompson RH et al 2004Proc.Natl.Acad.Sci.USA 101:17174-79; wu C et al 2006Acta Histochem.108:19-24). In addition, these studies indicate that higher PD-L1 expression levels on tumors can promote progression of tumor stages and invasion into deeper tissue structures.

The PD-1 pathway may also play a role in hematological malignancies. PD-L1 is expressed on multiple myeloma cells but not on normal plasma cells (Liu J et al 2007Blood 110:296-304). PD-L1 is expressed on some primary T cell lymphomas, particularly on heterogeneous large cell T lymphomas (anaplastic large cell T lymphomas) (Brown JA et al, 2003Immunol. 170:1257-66). PD-1 is highly expressed on T cells of angioimmunoblastic lymphoma and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al 2006Am.J. surg. Pathol.30:802-10). In nodular lymphocytic-dominated hodgkin's lymphoma, T cells associated with lymphocytic or histiocytological (L & H) cells express PD-1. Microarray analysis using reads of the genes induced by PD-1 ligation showed that tumor-associated T cells responded to in situ PD-1 signaling in Hodgkin's lymphoma (Chemnitz JM et al 2007Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD 4T cells in HTLV-1 mediated adult T cell leukemias and lymphomas (Shimachi T et al 2007Int.J.Cancer 121:2585-90). These tumor cells have low responsiveness to TCR signaling.

Studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and lysis of tumor cells, and in some cases causes increased tumor-specific T cell death (Dong H et al 522 Nat. Med.8:793-800; hirano F et al 2005Cancer Res.65:1089-96). Tumor-associated APCs can also utilize the PD-1:pd-L1 pathway to control anti-tumor T cell responses. PD-L1 expression on tumor-associated bone marrow DC populations is upregulated via tumor environmental factors (Curiel TJ et al 2003Nat. Med. 9:562-67). The plasmacytoid Dendritic Cells (DCs) in the tumor draining lymph nodes of B16 melanoma express IDO, which strongly activates the inhibitory activity of regulatory T cells. The inhibitory activity of IDO-treated regulatory T cells requires that the cells be contacted with IDO-expressing DCs (Sharma MD et al 2007clin. Invest. 117:2570-82).

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

In some 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.

In some embodiments, the PD-L1-binding bicyclic peptide ligand comprises an amino acid sequence selected from the group consisting of:

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 SAGWLTKC _ii 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)；

wherein C is _i 、C _ii And C _iii Respectively represent a first (i), a second (ii) and a third (iii) cysteine group, PYA represents 4-pentynoic acid and HArg represents homoarginine.

In some embodiments, the PD-L1-binding bicyclic peptide ligand optionally comprises an N-terminal and/or C-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

[PYA]-[B-Ala]-[Sar ₁₀ ]-SDK- (SEQ ID NO: 52) (referred to herein as BCY 10043);

Ac-D- [ HArg ] - (SEQ ID NO: 52) -PSH (referred to herein as BCY 11865);

Ac-SDK- (SEQ ID NO: 53) (referred to herein as BCY 11013);

Ac-SDK- (SEQ ID NO: 53) -PSH (referred to herein as BCY 10861);

Ac-D- [ HArg ] - (SEQ ID NO: 54) -PSH (referred to herein as BCY 11866);

Ac-D- [ HArg ] - (SEQ ID NO: 55) -PSH (referred to herein as BCY 11867);

Ac-D- [ HArg ] - (SEQ ID NO: 56) -PSH (referred to herein as BCY 11868);

Ac-D- [ HArg ] - (SEQ ID NO: 57) -PSH (referred to herein as BCY 11869);

Ac-SD- [ HArg ] - (SEQ ID NO: 58) -PSHK (referred to herein as BCY 12479); and

Ac-SD- [ HArg ] - (SEQ ID NO: 59) -PSHK (referred to herein as BCY 12477);

wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, sar ₁₀ Representing 10 sarcosine units, HArg represents homoarginine.

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)；

wherein C is _i 、C _ii And C _iii Respectively the first, second and third cysteine residues, HArg stands for homoarginine and PYA stands for 4-pentynoic acid.

In some embodiments, the PD-L1-binding bicyclic peptide ligand comprises an N-terminal and/or C-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 82) (hereinafter referred to as BCY 8938);

[PYA]-[B-Ala]-[Sar ₁₀ ]-SDK- (SEQ ID NO: 52) (hereinafter BCY 10043);

NH ₂ -SDK-(SEQ ID NO:52)-[Sar ₁₀ ]-[K(PYA)](hereinafter referred to as BCY 10044);

NH ₂ -SDK- (SEQ ID NO: 53) (hereinafter BCY 10045); and

Ac-SDK- (SEQ ID NO: 53) -PSH (hereinafter referred to as BCY 10861);

In some embodiments, the component present on the cancer cell is Prostate Specific Membrane Antigen (PSMA).

Prostate Specific Membrane Antigen (PSMA) (also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (naaladase I) and NAAG peptidase) are enzymes encoded by the FOLH1 (folate hydroxylase 1) gene in humans. Human GCPII contains 750 amino acids and is approximately 84kDa heavy.

Human PSMA is highly expressed in the prostate, approximately one hundred times higher than in most other tissues. In some prostate cancers, PSMA is a second most upregulated gene product, wherein levels in non-cancerous prostate cells are increased 8-fold to 12-fold. Because of this high expression, PSMA was developed as a potential biomarker for therapy and imaging of some cancers. In human prostate cancer, higher expressing tumors are associated with faster time to progression and a greater percentage of patients suffering from recurrence.

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

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.

In some embodiments, the component present on the cancer cell is a membrane 1-type metalloprotease (MT 1-MMP).

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

In some embodiments, the MT 1-MMP-binding bicyclic peptide ligand is selected from those MT 1-MMP-binding bicyclic peptide ligands disclosed in WO2016/067035, WO2017/191460, and WO2018/115204, the contents of each of which are incorporated herein by reference in their entirety.

CD137 binding peptide ligands

CD137 is a member of the Tumor Necrosis Factor (TNF) receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF 9), 4-1BB, and are induced by lymphocyte activation (ILA). CD137 may be expressed by activated T cells, but to a greater extent on cd8+ T cells than on cd4+ T cells. In addition, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and vascular wall cells at sites of inflammation. One characteristic activity of CD137 is its costimulatory activity on activated T cells. Crosslinking of CD137 enhances T cell proliferation, IL-2 secretion, survival and cytolytic activity. In addition, it can enhance immune activity to eliminate tumors in mice.

CD137 is a T cell co-stimulatory receptor induced upon TCR activation (Nam et al, curr. Cancer Drug Targets,5:357-363 (2005); waits et al, annu. Rev, immunol.,23:23-68 (2005)). In addition to their 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 to appear on antigen presenting cells including B cells, monocytes/macrophages and dendritic cells (Watts et al, annu. Rev. Immunol,23:23-68 (2005)). CD137, upon interaction with its ligand, causes increased TCR-induced T cell proliferation, cytokine production, functional maturation and prolonged CD8+ T cell survival (Nam et al Cancer Drug Targets,5:357-363 (2005), watts et al Annu.Rev.Immunol,23:23-68 (2005)).

Signaling by CD137 via CD137L or an agonistic monoclonal antibody (mAb) to CD137 results in increased TCR-induced T cell proliferation, cytokine production and functional maturation, and prolonged cd8+ T cell survival. These effects are caused by: (1) Activation of NF-. Kappa. B, c-Jun NH 2-terminal kinase/stress activated protein kinase (JNK/SAPK) and p38 Mitogen Activated Protein Kinase (MAPK) signaling pathways, and (2) control of anti-apoptotic and cell cycle-related gene expression.

Experiments performed in both CD 137-deficient mice and CD 137L-deficient mice have additionally shown the importance of CD137 co-stimulation in generating fully competent T cell responses.

IL-2 and IL-15 activated NK cells express CD137, and the attachment of an agonist mAb to CD137 stimulates NK cell proliferation and IFN-gamma secretion, but not its cytolytic activity.

In addition, CD 137-stimulated NK cells promote expansion of activated T cells in vitro.

Based on its co-stimulatory function, an agonistic mAb against CD137 has been shown to promote heart and skin allograft rejection, eradicate established tumors, expand primary antiviral cd8+ T cell responses, and increase T cell lytic potential. These studies support the notion that CD137 signaling promotes T cell function (which may enhance immunity to tumors and infections).

In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, two or more of the CD 137-binding peptide ligands have the same peptide sequence. In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, two or more of the CD 137-binding peptide ligands have different peptide sequences. In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, two or more of the CD 137-binding peptide ligands are the same. In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, two or more of the CD 137-binding peptide ligands are different.

In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the CD 137-binding peptide ligand is a CD 137-binding bicyclic peptide ligand. In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, two or more of the CD 137-binding peptide ligands are CD 137-binding bicyclic peptide ligands.

In some embodiments, the CD 137-binding bicyclic peptide ligand is selected from those disclosed in WO 2019/025811. In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the CD 137-binding peptide ligand is a CD 137-binding bicyclic peptide ligand selected from those disclosed in WO 2019/025811. In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, two or more of the CD 137-binding bicyclic peptide ligands are independently selected from those CD 137-binding bicyclic peptide ligands disclosed in WO 2019/025811. The content of WO2019/025811 is incorporated herein by reference in its entirety.

In some embodiments, the CD 137-binding bicyclic peptide ligand comprises the following amino acid sequence or a pharmaceutically acceptable salt thereof:

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)；

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; referred to herein as BCY 12353);

[MerPro] _i [tBuAla]PE[dK(PYA)]PYC _ii FADPY[Nle][Cysam] _iii (SEQ ID NO:71; referred to herein as BCY 12354);

C _i [tBuAla]PE[dK(PYA)]PYC _ii FADPY[Nle]C _iii (SEQ ID NO:72)；

C _i [tBuAla]PE[dK(PYA)]PYC _ii FADPY[Nle]C _iii (SEQ ID NO:73)；

C _i [tBuAla]PE[dK(PYA)]PYC _ii FADPY[Nle]C _iii (SEQ ID NO:74; referred to herein as BCY 12372);

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; referred to herein as BCY 13137);

wherein [ MerPro] _i 、C _i 、C _ii 、C _iii And [ Cysam ]] _iii Represents a first (i), a second (ii) and a third (iii) reactive group selected from cysteine, merPro and Cysam, nle represents norleucine, tbu ala represents tert-butyl-alanine, PYA represents 4-pentynoic acid, aad represents α -L-aminoadipic acid, merPro represents 3-mercaptopropionic acid, cysam represents cysteamine, NMeAla represents N-methyl-alanine.

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)；

wherein C is _i 、C _ii And C _iii Respectively the first, second and third cysteine residues, nle norleucine, tbuoala tert-butyl-alanine, PYA 4-pentynoic acid.

C _i [tBuAla]PE[D-Lys(PYA)]PYC _ii FADPY[Nle]C _iii (SEQ ID NO:11)；

Wherein C is _i 、C _ii And C _iii Respectively, the first, second and third cysteine residues, tBuAla for tert-butyl-alanine, PYA for 4-valeric acid, nle for norleucine.

In some embodiments, the CD 137-binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

Ac-A- (SEQ ID NO: 5) -Dap (referred to herein as BCY 7732);

Ac-A- (SEQ ID NO: 5) -Dap (PYA) (referred to herein as BCY 7741);

ac- (SEQ ID NO: 6) -Dap (referred to herein as BCY 9172);

ac- (SEQ ID NO: 6) -Dap (PYA) (referred to herein as BCY 11014);

Ac-A- (SEQ ID NO: 7) -Dap (referred to herein as BCY 8045);

ac- (SEQ ID NO: 8) -A (referred to herein as BCY 8919);

ac- (SEQ ID NO: 9) -A (referred to herein as BCY 8920);

ac- (SEQ ID NO: 10) -A (referred to herein as BCY 8927);

ac- (SEQ ID NO: 11) -A (referred to herein as BCY 8928);

(SEQ ID NO: 11) -A (referred to herein as BCY 14601);

Ac-A- (SEQ ID NO: 12) -A (referred to herein as BCY 7744);

ac- (SEQ ID NO: 60) -Dap (PYA) (referred to herein as BCY 11144);

Ac-A- (SEQ ID NO: 61) -K (referred to herein as BCY 11613);

ac- (SEQ ID NO: 62) -Dap (PYA) (referred to herein as BCY 12023);

ac- (SEQ ID NO: 63) (referred to herein as BCY 12149);

Ac- (SEQ ID NO: 64) (referred to herein as BCY 12143);

ac- (SEQ ID NO: 65) (referred to herein as BCY 12147);

ac- (SEQ ID NO: 66) (referred to herein as BCY 12145);

ac- (SEQ ID NO: 67) (referred to herein as BCY 12146);

ac- (SEQ ID NO: 68) (referred to herein as BCY 12150);

ac- (SEQ ID NO: 69) (referred to herein as BCY 12352);

ac- (SEQ ID NO: 72) - [1, 2-diaminoethane ] (referred to herein as BCY 12358);

[ palmitic acid ] - [ yGlu ] - [ yGlu ] - (SEQ ID NO: 73) (referred to herein as BCY 12360);

ac- (SEQ ID NO: 75) (referred to herein as BCY 12381);

ac- (SEQ ID NO: 76) (referred to herein as BCY 12382);

ac- (SEQ ID NO: 77) -K (referred to herein as BCY 12357);

ac- (SEQ ID NO: 78) - [ dA ] (referred to herein as BCY 13095);

[ Ac ] - (SEQ ID NO: 78) -K (referred to herein as BCY 13389);

ac- (SEQ ID NO: 79) - [ dA ] (referred to herein as BCY 13096); and

ac- (SEQ ID NO: 80) (referred to herein as BCY 13097);

wherein Ac represents acetyl, dap represents diaminopropionic acid, and PYA represents 4-pentynoic acid.

Ac-A- (SEQ ID NO: 5) -Dap (referred to herein as BCY 7732);

Ac-A- (SEQ ID NO: 5) -Dap (PYA) (referred to herein as BCY 7741);

Ac- (SEQ ID NO: 6) -Dap (referred to herein as BCY 9172);

ac- (SEQ ID NO: 6) -Dap (PYA) (referred to herein as BCY 11014);

Ac-A- (SEQ ID NO: 7) -Dap (referred to herein as BCY 8045);

ac- (SEQ ID NO: 8) -A (referred to herein as BCY 8919);

ac- (SEQ ID NO: 9) -A (referred to herein as BCY 8920);

ac- (SEQ ID NO: 10) -A (referred to herein as BCY 8927);

ac- (SEQ ID NO: 11) -A (referred to herein as BCY 8928);

Ac-A- (SEQ ID NO: 12) -A (referred to herein as BCY 7744);

ac- (SEQ ID NO: 11) -A (referred to herein as BCY 8928);

wherein Ac represents acetyl.

In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, each of the two or more CD 137-binding peptide ligands has the same peptide sequence and the peptide sequence comprises Ac- (SEQ ID NO: 11) -a (referred to herein as BCY 8928), or a pharmaceutically acceptable salt thereof, wherein Ac represents acetyl.

In some embodiments, where the heterotandem bicyclic peptide complex comprises two CD 137-binding peptide ligands, both of the two CD 137-binding peptide ligands have the same peptide sequence comprising Ac- (SEQ ID NO: 11) -a (referred to herein as BCY 8928), or a pharmaceutically acceptable salt thereof, wherein Ac represents acetyl.

Joint

Heterotandem bicyclic peptide complexes comprising two or more CD 137-binding peptide ligands

It will be appreciated that a first peptide ligand may be conjugated to two or more second peptide ligands via any suitable linker. Typically, the design of the linker will be such that the three bicyclic peptides are presented in a manner that enables them to bind their respective targets unhindered when bound to two target receptors individually or simultaneously. In addition, the linker should permit simultaneous binding of two targets while maintaining the appropriate distance between the target cells, which will produce the desired functional result. The characteristics of the joint may be adjusted to increase length, stiffness, or solubility to optimize the desired functional result. The linker may also be designed to permit more than one bicyclic ring to be attached to the same target. Increasing the valence of either binding peptide can be used to increase the affinity of the heteroconcatamer for the target cell or can help induce oligomerization of one or both of the target receptors.

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

In some embodiments, the branching linker is selected from:

n- (acid-PEG) ₃ ) -N-bis (PEG) ₃ -azide);

trimesic acid- [ Peg ₁₀ ] ₃ ；

TCA-[Peg ₁₀ ] ₃ ；

Tet-[Peg ₁₀ ] ₄ The method comprises the steps of carrying out a first treatment on the surface of the And

BAPG-(Peg ₅ ) ₂ 。

in some embodiments, the branching linker is:

n- (acid-PEG) ₃ ) -N-bis (PEG) ₃ -azide).

Heterotandem bicyclic peptide complexes comprising a CD137 binding peptide ligand

It will be appreciated that the first peptide ligand may be conjugated to the second peptide ligand via any suitable linker. Typically, the design of the linker will be such that the two bicyclic peptides are presented in a manner that enables them to bind their respective targets unhindered when bound to both target receptors alone or simultaneously. In addition, the linker should permit simultaneous binding of two targets while maintaining the appropriate distance between the target cells, which will produce the desired functional result. The characteristics of the joint may be adjusted to increase length, stiffness, or solubility to optimize the desired functional result. The linker may also be designed to permit more than one bicyclic ring to be attached to the same target. Increasing the valence of either binding peptide can be used to increase the affinity of the heteroconcatamer for the target cell or can help induce oligomerization of one or both of the target receptors.

In one embodiment, the linker is selected from the following sequences: PEG 5-and TCA[PEG ₁₀ ] ₃ 。

The structural schematic of these joints is shown in detail below:

-PEG5-; and

TCA-[PEG ₁₀ ] ₃ 。

In some embodiments, the linker is selected from the following sequences: -CH ₂ -、-PEG ₅ -、-PEG ₁₀ -、-PEG ₁₂ -、-PEG ₂₃ -、-PEG ₂₄ -、-PEG ₁₅ -Sar ₅ -、-PEG ₁₀ -Sar ₁₀ -、-PEG ₅ -Sar ₁₅ -、-PEG ₅ -Sar ₅ -、-B-Ala-Sar ₂₀ -、-B-Ala-Sar ₁₀ -PEG ₁₀ -、-B-Ala-Sar ₅ -PEG ₁₅ -and-B-Ala-Sar ₅ -PEG ₅ -。

In some embodiments, the linker is selected from the following:

/>

heteroconcatemeric bicyclic peptide complexes

In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, the first peptide ligand comprises a taglin-4-binding bicyclic peptide ligand linked to a TATA backbone, each of the two or more CD 137-binding bicyclic peptide ligands is linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in table a:

table A (handle protein-4:CD137; 1:2)

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In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY11027, BCY11863 and BCY11864. In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY11863 and BCY11864.

The heterotandem bicyclic peptide complex BCY11863 (also known as BT 7480) consists of: via N- (acid-PEG) ₃ ) -N-bis (PEG) ₃ An azide linker was linked to the two CD 137-specific peptides (both BCY 8928) the handle protein-4-specific peptide BCY8116, which is illustrated as:

CD137 is a homotrimeric protein and the natural ligand CD137L exists as a homotrimer expressed on immune cells or secreted. The biology of CD137 is highly dependent on multimerization to induce CD137 activity in immune cells. One way to generate CD137 multimerization is to crosslink the cells via interaction with specific receptors present on another cell via a CD 137-specific agonist. The heterotandem compound of the present invention has the advantages that: the presence of two or more peptide ligands specific for immune cell components such as CD137 provides for more efficient CD137 clustering. For example, BCY11863 has been found to exhibit strong CD137 activation and induce robust IL-2 and IFN- γ cytokine secretion, and BCY11863 exhibits an excellent PK profile with a terminal half-life of 4.1 hours in SD rats and 5.3 hours in cynomolgus monkeys (cyno).

Hybrid tandem bicyclic ringPeptide complex BCY11027 consists of: via TCA- [ Peg ₁₀ ] ₃ The adaptor was linked to the two CD 137-specific peptides (both BCY 8928) the ansa-4-specific peptide BCY11015, which is illustrated as:

it has been found that the aestinin-4/CD 137 heterotandem BCY11027 induces target-dependent cytokine release in ex vivo cultures of primary patient-derived lung tumors, and induces some immune markers (normalized to vehicle) and cd8+ki67+t cell% aestinin-4 dependent changes in patient-derived samples that are correlated with the expression level of aestinin-4.

In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, the first peptide ligand comprises a taglin-4-binding bicyclic peptide ligand linked to a TATA backbone, each of the two or more CD 137-binding bicyclic peptide ligands is linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in table B:

table B (handle protein-4:CD137; 1:3)

In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, the first peptide ligand comprises an EphA 2-binding bicyclic peptide ligand linked to a TATA backbone, each of the two or more CD 137-binding bicyclic peptide ligands is linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in table C:

Table C (EphA 2: CD137; 1:2)

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In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY12491, BCY12730, BCY13048, BCY13050, BCY13053, and BCY13272.

In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY12491, BCY12730, BCY13048, BCY13050, and BCY13053.

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

The heterotandem bicyclic peptide complex BCY12491 consists of: via N- (acid-PEG) ₃ ) -N-bis (PEG) ₃ An azide linker was linked to the EphA 2-specific peptide BCY9594 of two CD 137-specific peptides (both BCY 8928), which is illustrated as:

BCY12491 has been found to elicit significant anti-tumor responses and modulation (increase) of tumor-infiltrating immune cells and immune responses.

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

The heterotandem bicyclic peptide complex BCY13272 consists of: via N- (acid-PEG) ₃ ) -N-bis (PEG) ₃ An azide linker was linked to the EphA 2-specific peptide BCY13118 of two CD 137-specific peptides (both BCY 8928), which is illustrated as:

BCY13272 has been found to elicit significant anti-tumor effects in MC38 tumor models in mice.

In some embodiments, where the heterotandem bicyclic peptide complex comprises two or more CD 137-binding peptide ligands, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand linked to a TATA backbone, each of the two or more CD 137-binding bicyclic peptide ligands is linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in table D:

Table D (PD-L1: CD137; 1:2)

In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand linked to a TATA backbone, the one CD 137-binding peptide ligand is linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in table E:

table E (PD-L1: CD137; 1:1)

In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY12375 and BCY12021.

In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the first peptide ligand comprises a PD-L1-binding bicyclic peptide ligand linked to a TATA backbone, the one CD 137-binding peptide ligand is linked to the TATA backbone, and the heterotandem bicyclic peptide complex is selected from the complexes listed in table E-2:

table E-2 (PD-L1: CD137; 1:1)

In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the first peptide ligand comprises an EphA 2-binding bicyclic peptide ligand linked to a TATA backbone, the one CD 137-binding peptide ligand is linked to the TATA backbone, and the heterotandem complex is selected from the complexes listed in table F:

Table F (EphA 2: CD137; 1:1)

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In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY13035, BCY13040, BCY13253, BCY13254, BCY13340, and BCY13342.

In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the first peptide ligand comprises an EphA 2-binding bicyclic peptide ligand linked to a TATA backbone, the one CD 137-binding peptide ligand is linked to the TATA backbone, and the heterotandem complex is selected from the complexes listed in table F-2:

table F-2 (EphA 2: CD137; 1:1)

In some embodiments, the heterotandem bicyclic peptide complex is BCY7985, wherein the CD 137-specific peptide BCY7859 is via PEG ₁₂ An N-terminal PYA group linked to EphA 2-specific peptide BCY 6169:

in some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the first peptide ligand comprises a taglin-4-binding bicyclic peptide ligand linked to a TATA backbone, the one CD 137-binding peptide ligand is linked to the TATA backbone, and the heterotandem complex is selected from the complexes listed in table G:

table G (handle protein-4:CD137; 1:1)

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In some embodiments, the heterotandem bicyclic peptide complex is selected from the group consisting of: BCY11468, BCY11618, BCY11776, BCY11860, BCY12020, BCY12661, and BCY12969.

In some embodiments, where the heterotandem bicyclic peptide complex comprises one CD 137-binding peptide ligand, the first peptide ligand comprises a taglin-4-binding bicyclic peptide ligand linked to a TATA backbone, the one CD 137-binding peptide ligand is linked to the TATA backbone, and the heterotandem complex is selected from the complexes listed in table G-2:

table G-2 (handle protein-4:CD137; 1:1)

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In some embodiments, the heterotandem bicyclic peptide complexes are selected from those disclosed in U.S. patent application 17/062,662, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the heterotandem bicyclic peptide complexes are selected from those disclosed in U.S. patent publication 20190307836, the disclosure of which is incorporated herein by reference in its entirety.

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

Naming the name

Numbering device

When referring to the amino acid residue position within the compounds of the invention, the cysteine residue (C _i 、C _ii And C _iii ) Omitted from numbering, because they are unchanged, the numbering of the amino acid residues within SEQ ID NO:1 is shown below:

C _i -P ₁ -1Nal ₂ -dD ₃ -C _ii -M ₄ -HArg ₅ -D ₆ -W ₇ -S ₈ -T ₉ -P ₁₀ -HyP ₁₁ -W ₁₂ -C _iii (SEQ ID NO:1)。

for the purposes of this specification, assume that all bicyclic ringsPeptides were cyclized with TBMB (1, 3, 5-tris (bromomethyl) benzene) or 1,1',1"- (1, 3, 5-triazin-1, 3, 5-triyl) trip-2-en-1-one (TATA) and resulted in trisubstituted structures. Cyclization with TBMB and TATA occurs at C _i 、C _ii And C _iii And (3) upper part.

Molecular forms

The N-terminal or C-terminal extension of the bicyclic core sequence is added to the left or right of the sequence, separated by a hyphen. For example, the N-terminal βAla-Sar10-Ala tail will be expressed as:

βAla-Sar10-A-(SEQ ID NO:X)。

reverse peptide sequences

In view of the disclosure in Nair et al (2003) J Immunol 170 (3), 1362-1373, it is contemplated that the peptide sequences disclosed herein will also find utility in their reverse-inverted form. For example, the sequence is reversed (i.e., the N-terminal is changed to the C-terminal and vice versa) and the stereochemistry is likewise reversed (i.e., the D-amino acid is changed to the L-amino acid and vice versa). For the avoidance of doubt, reference to an amino acid in its full name or in its single letter code or three letter code is intended herein to mean an L-amino acid unless otherwise stated. If the amino acid is intended to be denoted as a D-amino acid, the amino acid will start in brackets with the lowercase letter D, e.g., [ dA ], [ dD ], [ dE ], [ dK ], [ D1Nal ], [ dNote ], etc.

Advantages of peptide ligands

Certain heterotandem bicyclic peptide complexes of the invention have a number of advantageous properties that enable them to be viewed as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic assessments;

protease stability. The heterotandem bicyclic peptide complex should ideally exhibit stability to plasma proteases, epithelial ("membrane anchored") proteases, gastric and intestinal proteases, pulmonary surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species so that the heteroconcatemeric bicyclic peptide primary candidates can be developed in animal models and administered to humans with confidence;

-a desired solubility profile. This is a function of the ratio of charged and hydrophilic to hydrophobic residues and the intramolecular/intermolecular H-bonds, which are critical for formulation and absorption purposes;

-selectivity. Certain heterotandem bicyclic peptide complexes of the invention show good selectivity over other targets;

optimum plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop a heterotandem bicyclic peptide complex for short term exposure to acute disease management environments, or to develop a heterotandem bicyclic peptide complex with enhanced retention in the circulation and thus optimal for management of more chronic disease states. Other factors driving the required plasma half-life are the requirement for sustained exposure to maximum therapeutic efficiency versus the toxicology attendant to sustained exposure of the agent.

Importantly, data are presented herein wherein the selected heterotandem bicyclic peptide complex is in vitro EC at levels that are not maintained above that of the compound ₅₀ Shows anti-tumour efficacy when administered at frequent doses of plasma concentrations. This is in contrast to the larger recombinant biological (i.e., antibody-based) methods 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, it is believed that the reason for this observation is due to the fact that: the heterotandem bicyclic complex has a relatively low molecular weight (typically < 15 kDa), is fully synthetic and is a tumor-targeted agonist of CD 137. Thus, the heterotandem bicyclic complexes have a relatively short plasma half-life, but good tumor penetrance (tumor pendance) and retention. Data is described herein that fully supports these advantages. For example, anti-tumor efficacy in a genotype rodent model in mice with humanized CD137 is shown daily or every 3 days. In addition, intraperitoneal medicineKinetic data show that plasma half-life < 3 hours, which predicts that circulating concentrations of complex will continue to drop below in vitro EC between dosing ₅₀ . Furthermore, tumor pharmacokinetic data show that the level of heterotandem bicyclic complexes in tumor tissue may be higher and more durable than plasma levels.

It will be appreciated that this observation forms another important aspect of the present invention. Thus, according to another aspect of the present invention, there is provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an EC of a compound of the present invention ₅₀ The frequency of administration of the plasma concentration of the complex of a heterotandem bicyclic peptide complex as defined herein.

-immunological memory. Coupling cancer cell-binding bicyclic peptide ligands to immune cell-binding bicyclic peptide ligands provides the synergistic advantage of immune memory. The data presented herein show that the selected heterotandem bicyclic peptide complexes of the invention not only eradicate the tumor, but that none of the vaccinated fully-responding mice developed the tumor upon re-administration of the tumorigenic agent (see fig. 5). This indicates that treatment with the selected heterotandem bicyclic peptide complexes of the invention has induced immunogenic memory in fully responsive mice. This has significant clinical advantages, preventing recurrence of the tumor after it has begun to be controlled and eradicated.

Peptide ligands

As referred to herein, peptide ligand refers to a peptide covalently bound to the backbone of a molecule. Typically, such peptides comprise two or more reactive groups (i.e., cysteine residues) capable of forming a covalent bond with the backbone and a sequence of pairs between the reactive groups, which is referred to as a loop sequence because it forms a loop when the peptide is bound to the backbone. In the present case, the peptide comprises at least three reactive groups selected from cysteine, 3-mercaptopropionic acid and/or cysteamine and forms at least two rings on the backbone.

Reactive group

The molecular scaffold of the present invention may be bound to a polypeptide via a functional group or reactive group on the polypeptide. These are typically formed from side chains of specific amino acids present in the polypeptide polymer. Such reactive groups may be cysteine side chains, lysine side chains, or N-terminal amine groups, or any other suitable reactive group, such as penicillamine. Details of suitable reactive groups can be found in WO2009/098450.

Examples of reactive groups of natural amino acids are thiol groups of cysteines, amino groups of lysines, carboxyl groups of aspartic acids or glutamic acids, guanidine groups of arginine, phenol groups of tyrosine or hydroxyl groups of serine. Unnatural amino acids can provide a wide range of reactive groups including azide, ketone-carbonyl, alkyne, vinyl, or aryl halide groups. Amino and carboxyl groups at the ends of the polypeptide may also act as reactive groups to form covalent bonds with the molecular backbone/molecular core.

The polypeptides of the invention contain at least three reactive groups. The polypeptide may also contain four or more reactive groups. The more reactive groups used, the more rings that can be formed in the molecular backbone.

In a preferred embodiment, a polypeptide having three reactive groups is produced. The reaction of the polypeptide with a molecular backbone/molecular core with triple rotational symmetry yields a single product isomer. The production of a single product isomer is advantageous for several reasons. The nucleic acid of the library of compounds encodes only the primary sequence of the polypeptide, not the isomeric state of the molecule formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of nucleic acid to the product isomer is well defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomers that are separated in the screening or selection procedure. The formation of a single product isomer is also advantageous if a particular library member of the invention can be synthesized. In this case, the chemical reaction of the polypeptide with the molecular backbone results in a single product isomer rather than a mixture of multiple isomers.

In another embodiment, a polypeptide having four reactive groups is produced. The reaction of the polypeptide with a molecular backbone/molecular core with tetrahedral symmetry yields two product isomers. Even if the two different product isomers are encoded by one and the same nucleic acid, the isomerism properties of the separated isomers can be determined by chemically synthesizing the two isomers, separating the two isomers and testing the binding of the two isomers to the target ligand.

In one embodiment of the invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows directing the orthogonal reactive groups to specific sites of the molecular core. Ligation strategies involving orthogonal reactive groups can be used to limit the number of product isomers formed. In other words, by selecting reactive groups for one or more of the at least three bonds that are different or different from reactive groups selected for the remainder of the at least three bonds, a particular order of binding or directing particular reactive groups of the polypeptide to particular positions on the molecular scaffold can be effectively achieved.

In another embodiment, the reactive group of the polypeptide of the invention is reacted with a molecular linker, wherein the linker is capable of reacting with the molecular scaffold such that the linker will be inserted between the molecular scaffold and the polypeptide in a final bonded state.

In some embodiments, the amino acids of a library member or collection of polypeptides may be substituted with any natural or unnatural amino acid. Amino acids bearing functional groups for cross-linking the polypeptide to the molecular core are excluded from these exchangeable amino acids, so that only the loop sequence is exchangeable. The exchangeable polypeptide sequences have random sequences, constant sequences or sequences with random and constant amino acids. Since the position of amino acids with reactive groups determines the ring size, these amino acids are located in defined positions within the polypeptide.

In one embodiment, the polypeptide having three reactive groups has the sequence (X) _l Y(X) _m Y(X) _n Y(X) _o Wherein Y represents an amino acid having a reactive group, X represents a random amino acid, m and n are numbers (which may be the same or different) between 3 and 6 that are the length of the inserted polypeptide segment, and l and o are numbers between 0 and 20 that are the length of the flanking polypeptide segment.

Alternatives to thiol-mediated binding can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively, these techniques may be used to modify or attach other moieties (such as small molecules of interest other than the molecular scaffold) to the polypeptide after they have been selected or isolated according to the invention, in this embodiment, then clearly the attachment need not be covalent and may encompass non-covalent attachment. These methods can be used instead of (or in combination with) thiol-mediated methods by generating phage displaying proteins and peptides carrying unnatural amino acids with requisite chemically reactive groups, as well as small molecules carrying complementary reactive groups, or by incorporating unnatural amino acids into chemical or recombinant synthetic polypeptides when the molecules are formed after the selection/isolation stage. Further details can be found in WO2009/098450 or Heinis et al, nat Chem Biol 2009,5 (7), 502-7.

In some embodiments, the reactive group is selected from cysteine, 3-mercaptopropionic acid, and/or cysteamine residues.

Pharmaceutically acceptable salts

It will be appreciated that salt forms are within the scope of the invention, and that reference to peptide ligands includes salt forms of the ligands.

The salts of the invention may be synthesized from parent compounds containing basic or acidic moieties via conventional chemical methods such as those described in Pharmaceutical Salts:Properties, selection, and Use, P.Heinrich Stahl (Editor), camille G.Wermuth (ed.), ISBN:3-90639-026-8, hardcover, page 388, august 2002. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent or in a mixture of both.

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 include mono-or di-salts formed from acids selected from the group consisting of: acetic acid, 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, cyclohexanaminosulfonic acid, 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), alpha-oxoglutarate, glycolic acid, hippuric acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+) -L-lactic acid, (+ -) -DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-) -L-malic acid, malonic acid, (+ -) -DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+) -L-tartaric acid, thiocyanate, p-toluenesulfonic acid, undecylenic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of 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 (methanesulfonic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is acetate.

If the compound is anionic or has a functional group which may be anionic (e.g., -COOH may be-COO) ^- ) The salt may be formed from an organic or inorganic base that produces the appropriate cation. Examples of suitable inorganic cations include, but are not limited to: alkali metal ions such as Li ⁺ 、Na ⁺ And K ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Alkaline earth metal cations, such as Ca ²⁺ And Mg (magnesium) ²⁺ The method comprises the steps of carrying out a first treatment on the surface of the And other cations such as Al ³⁺ Or Zn ⁺ . Examples of suitable organic cations include, but are not limited to: ammonium ion (i.e. NH) ₄ ⁺ ) And substituted ammonium ions (e.g., NH ₃ R ⁺ 、NH ₂ R ₂ ⁺ 、NHR ₃ ⁺ 、NR ₄ ⁺ ). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. One example of a common quaternary ammonium ion is N (CH ₃ ) ₄ ⁺ 。

Where the compounds of the present invention contain amine functionality, these compounds may be formed into quaternary ammonium salts according to methods well known to the skilled artisan, for example, via reaction with alkylating agents. Such quaternary ammonium compounds are within the scope of the present invention.

Modified derivatives

It will be appreciated that modified derivatives of peptide ligands as defined herein are within the scope of the invention. Examples of such suitable modified derivatives include one or more modifications selected from the group consisting of: n-terminal and/or C-terminal modification; one or more amino acid residues are substituted with one or more non-natural amino acid residues (such as one or more polar amino acid residues with one or more isosteres or isoelectric amino acid substitutions; one or more non-polar amino acid residues with other non-natural isosteres or isoelectric amino acid substitutions); adding a spacer group; substitution of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; substitution of one or more amino acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; n-alkylation of one or more amide bonds within the bicyclic peptide ligand; one or more peptide bonds are replaced with alternative bonds; modifying the length of a peptide main chain; the hydrogen on the alpha carbon of one or more amino acid residues is substituted with another chemical group; amino acids (such as cysteine, lysine, glutamic acid/aspartic acid and tyrosine) are modified with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids; and introducing or replacing an amino acid that introduces orthogonal reactivity suitable for functionalization, e.g., an azide or alkyne-bearing amino acid, that allows functionalization with alkyne or azide-bearing moieties, respectively.

In some embodiments, the modified derivative comprises an N-terminal and/or C-terminal modification. In another embodiment, wherein the modified derivative comprises an N-terminal modification with a suitable amino-reactive chemical and/or a C-terminal modification with a suitable carboxy-reactive chemical. In another embodiment, the N-terminal or C-terminal modification comprises the addition of effector groups including, but not limited to, cytotoxic agents, radiochelators, or chromophores.

In some embodiments, the modified derivative comprises an N-terminal modification. In another embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (referred to herein as C _i The groups of (c) are capped with acetic anhydride or other suitable reagent during peptide synthesis, thereby producing an N-terminally acetylated molecule. This embodiment provides the advantage of removing the potential recognition point of aminopeptidases and avoids the possibility of degradation of bicyclic peptides.

In some embodiments, the N-terminal modification comprises the addition of a molecular spacer group that aids in the conjugation of the effector group to its target and retains the potency of the bicyclic peptide to its target.

In some embodiments, the modified derivative comprises a C-terminal modification. In another embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group (referred to herein as C _iii The groups of (2) are synthesized as amides during peptide synthesis, resulting in C-terminally amidated molecules. This embodiment provides the advantage of removing the potential recognition point of the carboxypeptidase and reduces the likelihood of proteolytic degradation of the bicyclic peptide.

In some embodiments, the modified derivative comprises the substitution of one or more amino acid residues with one or more unnatural amino acid residues. In this embodiment, unnatural amino acids can be selected that have isostere/isoelectric side chains that are neither recognized by the degrading protease nor have any adverse effect on target potency.

Alternatively, unnatural amino acids with constrained amino acid side chains can be used, such that proteolysis of adjacent peptide bonds is conformationally and sterically hindered. In particular, this relates to proline analogues, bulky side chains, C alpha disubstituted derivatives (e.g. aminoisobutyric acid Aib) and cyclic amino acids as simple derivatives of amino-cyclopropylcarboxylic acids.

In some embodiments, the modified derivative comprises an added spacer group. In some embodiments, the modified derivative comprises a cysteine (C) _i ) And/or C-terminal cysteine (C) _iii ) A spacer group is added.

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

In some embodiments, the modified derivative comprises substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. Proper balance of charged amino acid residues and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the extent of plasma protein binding and thus the concentration of free available moieties in plasma, whereas charged amino acid residues (specifically arginine) can affect the interaction of peptides with phospholipid membranes on the cell surface. The two in combination can affect the half-life, distribution volume and exposure of the peptide drug and can be adjusted according to clinical endpoints. In addition, the correct combination and number of charged amino acid residues and hydrophobic amino acid residues may reduce irritation at the injection site (where the peptide drug has been administered subcutaneously).

In some embodiments, the modified derivative comprises substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by the propensity of the D-amino acid to stabilize the beta-turn conformation (Tugyi et al (2005) PNAS,102 (2), 413-418).

In stabilizing the beta turn conformation of the D-amino acid, the modified derivative comprises removal of any amino acid residue and substitution with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

It should be noted that each of the above modifications serves to intentionally improve the potency or stability of the peptide. Other efficacy improvements based on modification can be achieved via the following mechanisms:

-incorporating a hydrophobic moiety that exploits the hydrophobic effect and that gives a lower dissociation rate, so that a higher affinity is obtained;

incorporation of charged groups that utilize long-range ionic interactions, resulting in faster association rates and higher affinities (see, e.g., schreiber et al, rapid, electrostatically assisted association of proteins (1996), nature struct. Biol.3, 427-31); and

-incorporating additional restrictions into the peptide via, for example: the side chains of the amino acids are correctly constrained to minimize entropy loss upon target binding, the torsion angle of the backbone is constrained to minimize entropy loss upon target binding, and additional cyclization is introduced in the molecule for the same reason. (for reviews see Gentilucci et al, curr.pharmaceutical Design, (2010), 16,3185-203, and Nestor et al, curr.medicinal Chem (2009), 16, 4399-418).

Examples of modified heterotandem bicyclic peptide complexes of the invention include those complexes listed in tables H and I below:

table H: (EphA 2: CD137; 1:2)

Table I: (handle protein-4:CD137; 1:2)

Isotopic variation

The present invention includes all pharmaceutically acceptable (radioisotope) labelled peptide ligands of the invention in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from that commonly found in nature; and peptide ligands of the invention, having attached thereto a metal chelating group (referred to as an "effector") capable of accommodating the relevant (radioactive) isotope; and peptide ligands of the invention, wherein certain functional groups are covalently replaced with related (radioactive) isotopes or isotopically labeled functional groups.

Examples of isotopes suitable for inclusion in the peptide ligands of the invention include: isotopes of hydrogen, such as ² H (D) and ³ h (T); isotopes of carbon, such as ¹¹ C、 ¹³ C and C ¹⁴ C, performing operation; isotopes of chlorine, such as ³⁶ Cl; isotopes of fluorine, such as ¹⁸ F, performing the process; isotopes of iodine, such as ¹²³ I、 ¹²⁵ I and ¹³¹ i, a step of I; isotopes of nitrogen, such as ¹³ N and ¹⁵ n; isotopes of oxygen, such as ¹⁵ O、 ¹⁷ O and ¹⁸ O; isotopes of phosphorus, such as ³² P is as follows; isotopes of sulfur, such as ³⁵ S, S; isotopes of copper, such as ⁶⁴ Cu; isotopes of gallium, such as ⁶⁷ Ga or ⁶⁸ Ga; isotopes of yttrium, such as ⁹⁰ Y; isotopes of the fraction, e.g ¹⁷⁷ Lu; and isotopes of bismuth, such as ²¹³ Bi。

Certain isotopically labeled peptide ligands of the invention (e.g., those incorporating a radioisotope) are useful in drug and/or substrate tissue distribution studies and to assess clinically the presence or absence of a handle protein-4 target on diseased tissue. The peptide ligands of the invention may further have valuable diagnostic properties, as they may be used to detect or identify the formation of complexes between a labeled compound and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methodCompounds labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance (e.g., luminol), a luminol derivative, luciferin, jellyfish, and luciferase), and the like can be used. Radioisotope tritium (i.e ³ H (T)) and carbon-14 (i.e.) ¹⁴ C) Which is particularly suitable for this purpose because of its ease of incorporation and ready detection means.

Such as deuterium (i.e ² Heavier isotopic substitution of H (D)) may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some circumstances.

Positron-emitting isotopes (such as ¹¹ C、 ¹⁸ F、 ¹⁵ O and ¹³ n) substitution may be applicable in Positron Emission Tomography (PET) studies to examine target occupancy.

Isotopically-labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those recited in the accompanying examples, using an appropriate isotopically-labeled reagent in place of the previously employed unlabeled reagent.

Molecular skeleton

Molecular backbones are described, for example, in WO2009/098450 and the references cited therein (in particular WO2004/077062 and WO 2006/078161).

As indicated in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

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

In one embodiment, the molecular scaffold comprises a reactive group capable of reacting with a functional group of the polypeptide to form a covalent bond.

The molecular backbone may comprise chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, formic acid, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist of: hexahydro-1, 3, 5-triazines, in particular 1,3, 5-triacryloylhexahydro-1, 3, 5-triazines ("TATA") or derivatives thereof.

The molecular scaffold of the invention contains chemical groups that allow the functional groups of the polypeptides of the invention encoding the repertoire to form covalent bonds with the molecular scaffold. The chemical groups are selected from a wide range of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, formic acid, esters, alkenes, alkynes, anhydrides, maleimides, azides, alkyl halides and acyl halides.

Backbone reactive groups that react with thiol groups of cysteines may be used on the molecular backbone as alkyl halides (or also known as haloalkanes or haloalkanes).

Examples include bromomethylbenzene (a backbone reactive group exemplified by TBMB) or iodoacetamide. Other backbone reactive groups for selectively coupling compounds to cysteines in proteins are maleimides, alpha-unsaturated carbonyl containing compounds, and alpha-halomethyl carbonyl containing compounds. Examples of maleimides that can be used as the molecular skeleton in the present invention include: tris- (2-maleimidoethyl) amine, tris- (2-maleimidoethyl) benzene, tris- (maleimido) benzene. Examples of ab-unsaturated carbonyl-containing compounds are 1,1',1"- (1, 3, 5-triazin-1, 3, 5-yl) trip-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' - (benzene-1, 3, 5-triyl) tris (2-bromoacetamide). Selenocysteine is also a natural amino acid that has similar reactivity to cysteine and can be used for the same reaction. Thus, the substituent selenocysteine is generally acceptable whenever cysteine is mentioned, unless the context suggests otherwise.

Synthesis

The peptides of the invention can be prepared synthetically via standard techniques and subsequently reacted with the molecular scaffold in vitro. When doing this, standard chemical methods may be used. This enables the rapid large-scale preparation of soluble materials for use in other downstream experiments or validation. Such methods may be implemented using conventional chemical methods such as those disclosed in Timmerman et al (supra).

Thus, the present invention also relates to the preparation of a selected polypeptide or conjugate as described herein, wherein the preparation comprises optionally further steps as explained below. In one embodiment, these steps are performed on the final product polypeptide/conjugate produced via chemical synthesis.

Optionally, the amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

The peptide may also be extended to incorporate, for example, another loop and thus introduce multiple specificities.

To extend the peptide, it can only be extended chemically at its N-or C-terminus or within the ring using orthogonally protected lysines (and the like) using standard solid-or solution-phase chemistry. Standard (bio) conjugation techniques can be used to introduce an activated or activatable N-or C-terminus. Alternatively, the addition may be via fragment condensation or natural chemical ligation, e.g., as described in (Dawson et al 1994.Synthesis of Proteins by Native Chemical Ligation.Science 266:776-779); or via enzymes, for example using subtilase (Chang et al Proc Natl Acad Sci U S A1994 Dec 20;91 (26): 12544-8, or Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18,Issue 22,15November 2008,Pages 6000-6003).

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

Similar techniques are equally applicable to the synthesis/coupling of two bicyclic and bispecific macrocyclic compounds, thereby making it possible to form a tetra-specific molecule.

Furthermore, the addition of other functional or effector groups can be achieved in the same manner using suitable chemical methods at the N-terminus or C-terminus or via side chain coupling. In one embodiment, the coupling is performed in such a way that it does not block the activity of either entity.

2. Compounds and definitions:

as used herein, the term "pharmaceutically acceptable salts" refers to those salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without the undesirable toxicity, irritation, allergic response, and the like commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in J.pharmaceutical Sciences,1977,66,1-19 by S.M. Bere et al, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the 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 with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or via the use of other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorinates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfate, ethanesulfonates, formates, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoates, caprates, hydroiodinates, 2-hydroxy-ethanesulfonates, lactonates, lactates, laurates, lauryl sulfate, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectinates (pecetates), persulfates, 3-phenylpropionates, phosphates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and the like.

Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N ⁺ (C _1-4 Alkyl group ₄ And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Other pharmaceutically acceptable salts include nontoxic ammonium salts, quaternary ammonium salts, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate, as appropriate.

Unless stated otherwise, 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 configuration, Z and E double bond isomers, and Z and E conformational isomers of each asymmetric center. Thus, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the invention 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. In addition, unless stated otherwise, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, having a composition comprising hydrogen replaced by deuterium or tritium or carbon replaced by ¹³ C-or ¹⁴ C-enriched carbon-substituted compounds of the present structure are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or therapeutic agents according to the invention.

As used herein, the term "about" means within 20% of a given 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 a given value.

As used herein, the term "mg/kg" refers to milligrams of drug per kilogram of body weight of a subject taking the drug.

3. Pharmaceutically acceptable compositions

According to some embodiments, the present invention provides a pharmaceutical composition comprising: a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier, adjuvant or vehicle. In some embodiments, the invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising: a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof; an immunooncology agent; and a pharmaceutically acceptable carrier, adjuvant or vehicle.

In some embodiments, the invention provides a pharmaceutical composition comprising BT7480 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In some embodiments, the invention provides a pharmaceutical composition for treating cancer comprising BT7480 or a pharmaceutically acceptable salt thereof, an immunooncology agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

In some embodiments, the invention provides a pharmaceutical composition comprising BT7455 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. In some embodiments, the invention provides a pharmaceutical composition for treating cancer comprising BT7455 or a pharmaceutically acceptable salt thereof, an immunooncology agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

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 destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the invention include, but are not limited to: an ion exchanger; alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffer substances such as phosphates; glycine; sorbic acid; potassium sorbate; a partial glyceride mixture of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate; disodium hydrogen phosphate; potassium hydrogen phosphate; sodium chloride; zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; a cellulose-based material; polyethylene glycol; sodium carboxymethyl cellulose; a polyacrylate; a wax; polyethylene-polyoxypropylene-block polymers; polyethylene glycol; and lanolin.

As used herein, the term "patient" means an animal, preferably a mammal, and most preferably a human.

The compositions of the invention may be administered orally, parenterally, via 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 composition is administered orally, intraperitoneally, or intravenously. The sterile injectable form of the compositions of the invention may be an aqueous or oleaginous suspension. These suspensions may be formulated according to 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 as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants (such as Tween, span and other emulsifiers) or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

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

Alternatively, the pharmaceutically acceptable compositions of the present invention may be administered in the form of suppositories for rectal administration. These suppositories can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutically acceptable compositions of the present invention may also be administered topically, especially when the therapeutic goal includes an area or organ readily accessible via topical administration, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are readily prepared for use in each of these areas or organs.

Topical administration for the lower intestinal tract may be effected in rectal suppository formulations (see above) or in the form of suitable enema formulations. Topical transdermal patches may also be used.

For topical application, the provided pharmaceutically acceptable compositions may be formulated in the form of a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical application of the compounds of the invention include, but are not limited to, mineral oil, liquid paraffin, white paraffin, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying waxes and water. Alternatively, the provided pharmaceutically acceptable compositions may be formulated in a suitable lotion or cream form containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetostearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the provided pharmaceutically acceptable compositions may be formulated with or without a preservative (such as benzalkonium chloride), as a micronized suspension in isotonic, pH adjusted sterile saline, or preferably as a solution in isotonic, pH adjusted sterile saline. Alternatively, for ocular use, the pharmaceutically acceptable composition may be formulated in the form of an ointment (such as paraffin).

The pharmaceutically acceptable compositions of the present invention may also be administered via nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional dissolving or dispersing agents.

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

It will also be appreciated that the particular dosage and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the particular compound employed, the age, weight, general health, sex, diet, 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 the compound of the present invention in the composition also depends on the particular compound in the composition.

4. Methods for treating cancer

According to some embodiments, 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 comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, and an immunooncology agent.

In some embodiments, the invention provides a use of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, or a pharmaceutically acceptable salt thereof, in combination with an immunooncology agent for treating cancer.

In some embodiments, the invention provides a method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of BT7480 or a pharmaceutically acceptable salt thereof and an immunooncology agent. In some embodiments, the invention provides the use of BT7480, or a pharmaceutically acceptable salt thereof, in combination with an immunooncology agent for the treatment of cancer.

In some embodiments, the invention provides a method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of BT7455 or a pharmaceutically acceptable salt thereof and an immunooncology agent. In some embodiments, the invention provides the use of BT7455, or a pharmaceutically acceptable salt thereof, in combination with an immunooncology agent for treating cancer.

Exemplary cancers

In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is associated with MT 1-MMP. In some embodiments, the cancer is a cancer of high MT1-MMP expression. For example, adley et al have reported that MT1-MMP has high expression levels in ovarian clear cell carcinomas (Adley et al, "Expression of Membrane Type 1Matrix Metalloproteinase (MMP-14) in Epithelial Ovarian Cancer: high Level Expression in Clear Cell Carcinoma" Gynecol Oncol.2009 February;112 (2): 319-324).

In some embodiments, the cancer is associated with calpain-4. In some embodiments, the cancer is a high-stalk protein-4 expressing cancer.

In some embodiments, the cancer is associated with EphA 2. In some embodiments, the cancer is a cancer with high EphA2 expression.

In some embodiments, the cancer is associated with PD-L1. In some embodiments, the cancer is a cancer with high PD-L1 expression.

In some embodiments, the cancer is associated with PSMA. In some embodiments, the cancer is a cancer with high PSMA expression.

In some embodiments, the cancer is bladder cancer. In some embodiments, the bladder cancer is selected from the group consisting of: basal, p 53-like and luminal cancers.

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 abnormality, type I, type II, carcinoma, carcinomatosis, endometrioid adenocarcinoma, serous carcinoma, clear cell carcinoma, mucinous carcinoma, mixed or undifferentiated carcinoma, mixed serous and endometrioid carcinoma, mixed serous and lower endometrioid carcinoma and undifferentiated carcinoma.

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), chromosome Instability (CIN), epstein-barr virus (EBV), genome Stability (GS), and microsatellite instability (MSI).

In some embodiments, the cancer is neuroglioblastoma. In some embodiments, the neuroglioblastoma is selected from the group consisting of: anterior nerves, typical and mesenchymal cells.

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, sarcoidoid mesothelioma, biphasic mesothelioma and malignant mesothelioma.

In some embodiments, the cancer is multiple myeloma. In some embodiments, the multiple myeloma is selected from the group consisting of: superdiploid, non-superdiploid, cyclin D translocation, MMSET translocation, MAF translocation, and categorical.

In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is selected from the group consisting of: clear cells, endometrium-like, mucinous, advanced serous and hyposerous ovarian cancer.

In some embodiments, the cancer is pancreatic cancer. In some embodiments, the pancreatic cancer is selected from the group consisting of: squamous, pancreatic progenitor cells, immunogenic and ADEX (abnormal differentiated endocrine exocrine) pancreatic cancer.

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.

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

In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is a triple negative breast cancer. In some embodiments, the breast cancer is basal-like triple negative breast cancer.

In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is colorectal adenocarcinoma. In some embodiments, the colorectal adenocarcinoma is a colorectal adenocarcinoma that highly expresses pgp.

In some embodiments, the cancer is gastric cancer. In some embodiments, the gastric cancer is FGFR amplified gastric cancer.

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

In some embodiments, the cancer is a sarcoma. In some embodiments, the sarcoma is fibrosarcoma. In some embodiments, the fibrosarcoma is an N-ras mutation/IDH 1 mutation Soft Tissue Sarcoma (STS).

In one embodiment, the cancer includes, but is not limited to: leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myelogenous 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, lymphomas (e.g., hodgkin's or non-hodgkin's disease), waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia), multiple myelomas, heavy chain diseases, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphoendotheliosarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchi carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilm's tumor (Wilm's tumor), cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, glioblastoma multiforme (GBM also known as glioblastoma), medulloblastoma, craniopharyoma, ependymoma, pineal tumor, angioblastoma, auditory glioma, oligodendroglioma, neurosarcoma, neurofibrosarcoma, neurosarcoma Meningioma, melanoma, neuroblastoma, and retinoblastoma).

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

In some embodiments, the cancer is an acoustic neuroma, astrocytoma (e.g., grade I-wool cell astrocytoma, grade II-low astrocytoma, grade III-anaplastic astrocytoma, or grade IV-neuroglioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngeal tube tumor, brain stem glioma, ependymoma, mixed glioma, optic glioma, ependymal ependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendritic glioma, pituitary tumor, primitive Neuroectodermal (PNET) tumor, or schwannoma. In some embodiments, the cancer is of a type more common in children than in adults, such as brain stem glioma, craniopharyngeal tube tumor, ependymoma, juvenile hair cell astrocytoma (JPA), medulloblastoma, optic glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some embodiments, the patient is an adult. In some embodiments, the patient is a pediatric or pediatric patient.

In another embodiment, the cancer does not include, but is not limited to: mesothelioma, hepatobiliary (liver and bile duct) cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal region cancer, gastric cancer, gastrointestinal (gastric, colorectal and duodenal) cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulval cancer, hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myelogenous leukemia, lymphocytic lymphoma, bladder cancer, renal or ductal carcinoma, renal cell carcinoma, renal pelvis cancer, non-hodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenal cortex cancer, gall bladder cancer, multiple myeloma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

In some embodiments, the cancer is selected from: hepatocellular carcinoma, ovarian epithelial carcinoma, or fallopian tube carcinoma; papillary serous cystic adenocarcinoma or Uterine Papillary Serous Carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary tract cancer; soft tissue and synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; ewing's sarcoma; anaplastic thyroid cancer; adrenal cortex adenoma; pancreatic cancer; pancreatic duct cancer or pancreatic adenocarcinoma; gastrointestinal/Gastric (GIST) cancer; lymphomas; squamous Cell Carcinoma of Head and Neck (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1 related Malignant Peripheral Nerve Sheath Tumor (MPNST); waldenstrom macroglobulinemia; or medulloblastoma.

In some embodiments, the cancer is selected from: hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous cyst adenocarcinoma, uterine Papillary Serous Carcinoma (UPSC), hepatobiliary carcinoma, soft tissue and synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenocortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibroma-1-associated Malignant Peripheral Nerve Sheath Tumor (MPNST), waldenstrom macroglobulinemia, or medulloblastoma.

In some embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. Solid tumors typically contain abnormal tissue masses that do not typically include cysts or fluid areas. In some embodiments, the cancer is selected from: renal cell carcinoma or renal carcinoma; hepatocellular carcinoma (HCC) or hepatoblastoma or liver cancer; melanoma; breast cancer; colorectal 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 cystic adenocarcinoma or Uterine Papillary Serous Carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatobiliary tract cancer; soft tissue and synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; ewing's sarcoma; anaplastic thyroid cancer; adrenal cortex cancer; pancreatic cancer; pancreatic duct cancer or pancreatic adenocarcinoma; gastrointestinal/Gastric (GIST) cancer; lymphomas; squamous Cell Carcinoma of Head and Neck (SCCHN); salivary gland cancer; glioma or brain cancer; neurofibromatosis-1 related Malignant Peripheral Nerve Sheath Tumor (MPNST); waldenstrom macroglobulinemia; or medulloblastoma.

In some embodiments, the cancer is selected from: renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal cancer, colon cancer, rectal cancer, anal carcinoma, ovarian cancer, ovarian epithelial cancer, ovarian cancer, fallopian tube cancer, papillary serous cyst adenocarcinoma, uterine Papillary Serous Carcinoma (UPSC), hepatobiliary tract cancer, soft tissue and synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic duct cancer, pancreatic adenocarcinoma, glioma, brain cancer, neurofibroma-1-associated Malignant Peripheral Nerve Sheath Tumor (MPNST), waldenstrom macroglobulinemia, or medulloblastoma.

In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer (ovarian cancer), ovarian epithelial cancer, ovarian cancer (ovarian carcinoma), fallopian tube cancer, papillary serous cyst adenocarcinoma, uterine Papillary Serous Carcinoma (UPSC), hepatobiliary tract cancer, soft tissue and synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenocortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibroma-1-associated Malignant Peripheral Nerve Sheath Tumor (MPNST), waldenstrom macroglobulinemia, or medulloblastoma.

In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is a 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 cyst adenocarcinoma. In some embodiments, the cancer is Uterine Papillary Serous Carcinoma (UPSC). In some embodiments, the cancer is hepatobiliary cancer. In some embodiments, the cancer is soft tissue and synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is anaplastic thyroid cancer. In some embodiments, the cancer is adrenocortical cancer. In some embodiments, the cancer is pancreatic cancer or pancreatic duct cancer. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is a Malignant Peripheral Nerve Sheath Tumor (MPNST). In some embodiments, the cancer is neurofibromatosis-1 related MPNST. In some embodiments, the cancer is Waldenstrom macroglobulinemia. In some embodiments, the cancer is a medulloblastoma.

In some embodiments, the cancer is a virus-associated cancer, including Human Immunodeficiency Virus (HIV) -associated solid tumors, human Papillomavirus (HPV) -16 positive incurable solid tumors, and adult T cell leukemia, which is caused by human T cell leukemia virus type I (HTLV-I) and is a highly invasive form of cd4+ T cell leukemia characterized by clonal integration of HTLV-I in leukemia cells (see https:// clinicaltrias.gov/ct 2/show/study/NCT 02631746); and virus-associated tumors in gastric cancer, nasopharyngeal cancer, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, and merkel cell carcinoma. (see https:// clinicaltrias.gov/ct 2/show/student/NCT 02488759; see also https:// clinicaltrias.gov/ct 2/show/student/NCT 0240886;

https://clinicaltrials.gov/ct2/show/NCT02426892)。

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 lung cancer is Small Cell Lung Cancer (SCLC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

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

The heterotandem bicyclic peptide complexes and compositions according to the methods of the invention can be administered using any amount and any route of administration effective to treat or reduce the severity of cancer. The precise 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 particular agent, its mode of administration, and the like. For ease of administration and dose uniformity, the heterotandem bicyclic peptide complexes are preferably formulated in dosage unit form. The expression "dosage unit form" as used herein refers to physically discrete units of medicament suitable for the patient to be treated. However, it will be appreciated that the total daily amount of the heterotandem bicyclic peptide complexes and compositions of the invention will be determined by the attending physician within the scope of sound medical judgment. The particular effective dosage level for any particular patient or organism depends on a variety of factors including the condition to be treated and the severity of the condition; the activity of the particular compound employed; the specific composition employed; age, weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion of the particular compound being employed; duration of treatment; a medicament for use in combination or simultaneously with the particular compound employed; and similar factors well known in the medical arts. As used herein, the term "patient" means an animal, preferably a mammal, and most preferably a human.

The pharmaceutically acceptable compositions of the invention may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., via powders, ointments or drops), bucally (e.g., by oral or nasal spray), or the like, depending on the severity of the disease or condition being treated. In certain embodiments, the heterotandem bicyclic peptide complexes of the invention may be administered orally or parenterally at a dosage level of about 0.01mg/kg to about 100mg/kg or about 1mg/kg to about 25mg/kg subject body weight/day one or more times a day to achieve the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents; solubilizing agents and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; and mixtures thereof. In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable formulations, for example sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be employed are water, ringer's solution, u.s.p. And isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

To prolong the effect of the compounds of the invention, it is often desirable to slow the absorption of the compounds from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials with poor water solubility. The absorption rate of a compound depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound is achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming a matrix of microcapsules of a compound in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions which are compatible with body tissues

Compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the heterotandem bicyclic peptide complexes of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert, pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and/or the following: a) Fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid; b) Binders such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) Humectants, such as glycerin; d) Disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) Solution retarders such as paraffin; f) Absorption enhancers such as quaternary ammonium compounds; g) Wetting agents such as cetyl alcohol and glycerol monostearate; h) Absorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be used as fillers in soft-and hard-filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation arts. It may optionally contain an opacifying agent and may also have a composition which releases the active ingredient in a certain part of the intestinal tract, optionally in a delayed manner, only or preferentially. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers in soft-and hard-filled gelatin capsules using excipients such as lactose or milk sugar, high molecular weight polyethylene glycols and the like.

The active compound may also be in microencapsulated form together with one or more excipients as indicated above. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings, controlled release coatings and other coatings well known in the pharmaceutical formulation arts. In such solid dosage forms, the active compound may be admixed with at least one inert diluent, such as sucrose, lactose or starch. Such dosage forms may also contain, as is commonly practiced, additional substances other than inert diluents, for example tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. It may optionally contain an opacifying agent and may also have a composition which releases the active ingredient in a certain part of the intestinal tract, optionally in a delayed manner, only or preferentially. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, e.g., as described herein, include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any required preservatives or buffers as may be required. Ophthalmic formulations, ear drops and eye drops are also included within the scope of the present invention. In addition, the present invention encompasses the use of transdermal patches, which have the additional advantage of providing controlled delivery of compounds to the body. Such dosage forms may be prepared by dissolving or partitioning the compound in an appropriate medium. Absorption enhancers may also be used to increase the transdermal amount of the compound. The rate may be controlled by providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

Co-administration of heteroconcatemeric bicyclic peptide complexes and immunooncology agents

For example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein may be administered separately as part of a multiple dose regimen. Alternatively, for example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein may be mixed together in a single composition as a single dosage form. In some embodiments, the heterotandem bicyclic peptide complex is BT7480 or BT7455 or a pharmaceutically acceptable salt thereof.

In some embodiments, for example, a heterotandem bicyclic peptide complex as described herein is administered separately from an immunooncology agent. In some embodiments, for example, a heterotandem bicyclic peptide complex as described herein and an immunooncology agent are administered simultaneously. In some embodiments, for example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein are administered sequentially. In some embodiments, for example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein are administered within a period of time of each other, such as within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of each other. In some embodiments, for example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein are administered in greater than 24 hour intervals. In some embodiments, for example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein 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 and an immunooncology agent as described herein are administered over a period of greater than one week. In some embodiments, for example, a heterotandem bicyclic peptide complex and an immunooncology agent as described herein are administered within 1, 2, 3, 4, or 5 weeks of each other.

As used herein, the terms "combination," "combined," and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the invention. For example, a heterotandem bicyclic peptide complex as described herein can be administered simultaneously or sequentially with an immunooncology agent in separate unit dosage forms, or together in a single unit dosage form. Thus, in some embodiments, the invention provides a single unit dosage form comprising, for example, a heterotandem bicyclic peptide complex as described herein, an immunooncology agent, and optionally a pharmaceutically acceptable carrier, adjuvant, or vehicle.

The amount of the heterotandem bicyclic peptide complexes and the immunooncology agents that can be combined with the carrier material to produce a single dosage form, e.g., as described herein, will vary depending on the host treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated so that a dose of between 0.001-100mg/kg body weight/day, for example, of a heterotandem bicyclic peptide complex as described herein, can be administered.

For example, a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands and an immunooncology agent as described herein may act synergistically. Thus, the amount of a heterotandem bicyclic peptide complex and an immunooncology agent, for example, as described herein, in such compositions can be less than that required in monotherapy using only the therapeutic agent.

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

The pharmaceutical compositions of the present invention may also be incorporated into compositions for coating implantable medical devices such as prostheses, prosthetic valves, vascular grafts, stents and catheters. Vascular stents have been used, for example, to overcome restenosis (restenosis of the vessel wall following injury). However, patients using stents or other implantable devices are at risk of clot formation or platelet activation. These unwanted effects can be prevented or alleviated by pre-coating the device with a pharmaceutically acceptable composition comprising a kinase inhibitor. Implantable devices coated with a compound of the invention are another embodiment of the invention.

5. Exemplary Immunotorhinocerology agents

As used herein, the term "immunooncology agent" refers to an agent effective to enhance, stimulate, and/or up-regulate an immune response in a subject. In some embodiments, administration of an immunooncology agent with a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands, e.g., as described herein, has a synergistic effect in treating cancer.

The immunooncology agent may be, for example, a small molecule drug, an antibody or a biological molecule or a small molecule. Examples of biological immunooncology 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 a humanized or human antibody.

In some embodiments, the immunooncology agent is (i) an agonist that stimulates (including co-stimulates) receptors or (ii) an antagonist that inhibits (including co-inhibits) signals on T cells, both of which elicit an expanding antigen-specific T cell response.

Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the TNF family of molecules that bind to members of the cognate TNF receptor family, including CD40 and CD40L, OX-40, OX-40L, CD, CD27L, CD, CD30L, 4-1BBL, CD137 (4-1 BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT beta R, LIGHT, dcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin alpha/TNF beta, TNFR2, TNFR, lymphotoxin alpha 1 beta 2, FAS, FASL, RELT, DR, TROY, NGFR.

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

In some embodiments, a combination of a heterotandem bicyclic peptide complex comprising one or more CD 137-binding peptide ligands as described herein, and an immunooncology agent, for example, can stimulate a T cell response. In some embodiments, the heterotandem bicyclic peptide complex is BT7480 or BT7455 or a pharmaceutically acceptable salt thereof. In some embodiments, the immunooncology agent is: (i) Antagonists (e.g., immune checkpoint inhibitors) of proteins that inhibit T cell activation such as CTLA-4, PD-1, 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; or (ii) agonists of T cell activation stimulating proteins such as B7-1, B7-2, CD28, 4-1BB (CD 137), 4-1BBL, ICOS, ICOS-L, OX, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

In some embodiments, the immunooncology agent is an antagonist of an inhibitory receptor on NK cells or an agonist of an activating receptor on NK cells. In some embodiments, the immunooncology agent is an antagonist of KIR, such as Li Ruilu mab (lirilumab).

In some embodiments, the immunooncology 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 (WO 11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716, WO 13/132044) or FPA-008 (WO 11/140249; WO13169264; WO 14/036357).

In some embodiments, the immunooncology agent is selected from the group consisting of: agonists that bind to positive co-stimulatory receptors; a blocking agent that attenuates signaling via an agent that inhibits the receptor, an antagonist, and one or more agents that systematically increase the frequency of anti-tumor T cells; agents that overcome unique immunosuppressive pathways within the tumor microenvironment (e.g., block inhibition of receptor engagement (e.g., PD-L1/PD-1 interaction), deplete or inhibit Treg (e.g., use of anti-CD 25 monoclonal antibodies (e.g., daclizumab) or depletion via ex vivo anti-CD 25 beads), inhibit metabolic enzymes such as IDO, or reverse/prevent T cell energy or depletion); and agents that trigger innate immune activation and/or inflammation at the tumor site.

In some embodiments, the immunooncology agent is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an antagonistic CTLA-4 antibody. In some embodiments, the antagonistic CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab (tremelimumab).

In some embodiments, the immunooncology agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered via infusion. In some embodiments, the immunooncology agent 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 antagonistic PD-1 antibody. In some embodiments, the antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (palbociclizumab) or MEDI-0680 (AMP-514; WO 2012/145493). In some embodiments, the immunooncology agent may be pidotizumab (CT-011). In some embodiments, the immunooncology agent 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.

In some embodiments, the immunooncology agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonistic PD-L1 antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG 7446; WO 2010/077634), dewaruzumab (durvalumab, MEDI 4736), BMS-936559 (WO 2007/005874) and MSB0010718C (WO 2013/79174).

In some embodiments, the immunooncology agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonistic LAG-3 antibody. In some embodiments, the LAG3 antibody is BMS-986016 (WO 10/19570, WO 14/08218) or IMP-731 or IMP-321 (WO 08/132601, WO 009/44273).

In some embodiments, the immunooncology agent is a CD137 (4-1 BB) agonist. In some embodiments, the CD137 (4-1 BB) agonist is an agonistic CD137 antibody. In some embodiments, the CD137 antibody is Zuccinimide or PF-05082566 (WO 12/32433).

In some embodiments, the immunooncology agent is a GITR agonist. In some embodiments, the GITR agonist is an agonistic GITR antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO 006/105021, WO 009/009116) or MK-4166 (WO 11/028683).

In some embodiments, the immunooncology agent is an indoleamine (2, 3) -dioxygenase (IDO) antagonist. In some embodiments, the IDO antagonist is selected from Ai Kaduo stat (INCB 024360, incyte); indoximod (Indoximod, NLG-8189,NewLink Genetics Corporation); cabozatinib (capmannitib, INC280, novartis); GDC-0919 (Genntech/Roche); PF-06840003 (Pfizer); BMS F001287 (Bristol-Myers Squibb); phy906/KD108 (Phytoceutics); an enzyme that breaks down kynurenine (Kynase, ikena oncology, previously known as kyn therapeutics); and NLG-919 (WO 09/73620, WO009/1156652, WO11/56652, WO 12/142237).

In some embodiments, the immunooncology agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonistic OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

In some embodiments, the immunooncology agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40 antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO 06/029879).

In some embodiments, the immunooncology agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonistic CD40 antibody. In some embodiments, the immunooncology agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonistic CD40 antibody. In some embodiments, the CD40 antibody is Lu Kamu mab (lucatumumab) or dactyluzumab (daceatuzumab).

In some embodiments, the immunooncology agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonistic CD27 antibody. In some embodiments, the CD27 antibody is varluab (vardilumab).

In some embodiments, the immunooncology agent is MGA271 (against B7H 3) (WO 11/109400).

In some embodiments, the immunooncology agent is aba Fu Shan antibody (abago), adalimumab (adavalumab), atozuab (afutuzumab), alemtuzumab (alemtuzumab), ting An Moshan antibody (anatumomab mafenatox), apolizumab (apolizumab), atuzumab (atezolimab), abauzumab (avelumab), buermo mab (blinatumomab), BMS-936559, katuzumab (cataxomab), devaluzumab (durvalumab), ai Kaduo setuzumab (epatuzumab), indomadder, oxuzumab (inotuzumab ozogamicin), itumomab (saluzumab), ideuzumab, itumomab (isoximab), lanuzumab (labuzumab), meunab 38, melitumomab, oxuzumab (atuzumab) or oxytuzumab (atuzumab), oxypuzumab (atuzumab) or oxytuzumab (atuzumab), oxypuzumab (atuzumab) or oxytuzumab (atuzumab) and/.

In some embodiments, the immunooncology agent is an immunostimulant. For example, antibodies blocking the PD-1 and PD-L1 inhibition axes may release activated tumor-reactive T cells, and have been shown in clinical trials to induce durable anti-tumor responses, increasing the number of tumor tissue structures, including some tumor types that have not conventionally been considered sensitive to immunotherapy. See, e.g., okazaki, t.et al (2013) nat. Immunol.14,1212-1218; zou et al (2016) Sci.Transl.Med.8. anti-PD-1 antibody Nawuzumab Bristol-Myers Squibb, also known as ONO-4538, MDX1106, and BMS-936558) has shown the potential to improve overall survival in RCC patients who have undergone disease progression during or after prior anti-angiogenic therapy.

In some embodiments, the immunomodulatory therapeutic specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutic agents useful in the invention include pomalidomide (pomalidomide,celgene); lenalidomide (lenalidomide,)>Celgene); ingenol mebutate (jogenol mebutate,)>LEO Pharma)。

In some embodiments, the immunooncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from the group consisting of sipuleucel-T #Dendreon/Valeant Pharmaceuticals), which has been approved for the treatment of asymptomatic conditionsMetastatic castration-resistant (hormone refractory) prostate cancer with mild symptoms or symptoms; and talimogene laherparepvec ()>BioVex/Amgen, previously known as T-VEC), a genetically modified oncolytic virus therapy approved for the treatment of unresectable skin, subcutaneous and nodular lesions in melanoma. In some embodiments, the immunooncology agent is selected from oncolytic viral therapies such as pexastimogene devacirepvec (PexaVec/JX-594, sillajen/previously Jennerex Biotherapeutics), a thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF, for use in hepatocellular carcinoma (NCT 02562755) and melanoma (NCT 00429312); pilariprap (pelaroep,) >Oncolytics Biotech), which is a variant of respiratory enteric orphan virus (reovirus) that is non-replicable in non-RAS activated cells, is used in a number of cancers, including colorectal cancer (NCT 01622543), prostate cancer (NCT 01619813), head and neck squamous cell carcinoma (NCT 01166542), pancreatic adenocarcinoma (NCT 00998322), and non-small cell lung cancer (NSCLC) (NCT 00861627); enanaxiri (Enadenitucirev, NG-348, psioxus, previously known as ColoAd 1), an adenovirus engineered to express full length CD80 and antibody fragments specific for the T cell receptor CD3 protein, for ovarian cancer (NCT 02028117), metastatic or advanced epithelial tumors, such as colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland carcinoma (NCT 02636036); ONCOS-102 (Targovix/previously referred to as Oncos), an adenovirus engineered to express GM-CSF for melanoma (NCT 03003676) and peritoneal disease, colorectal or ovarian cancer (NCT 02963831); GL-ONC1 (GLV-1 h68/GLV-1h153,Genelux GmbH), a vaccinia virus engineered to express β -galactosidase (β -gal)/β -glucuronidase or β -gal/human sodium iodide symporter (hNIS), respectively, is being investigated for peritoneal cancer (NCT 01443260), fallopian tube cancer, ovarian cancer (NCT 02759588); or (b) CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF, for use in bladder cancer (NCT 02365818).

In some embodiments, the immunooncology agent is selected from the group consisting of: JX-929 (SillaJen/previously 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 (targaax/previously called Oncos), which are peptide-based immunotherapeutic agents targeting refractory RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus known as Ad 5/3-E2F-delta 24-hTNFα -IRES-hIL 20; and VSV-GP (ViraTherapeutics), a Vesicular Stomatitis Virus (VSV) engineered to express Glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express CD8 designed to promote antigen specificity ⁺ Antigen of T cell response.

In some embodiments, the immunooncology agent is a T cell engineered to express a chimeric antigen receptor or CAR. T cells engineered to express this chimeric antigen receptor are referred to as CAR-T cells.

A CAR has been constructed consisting of: a binding domain, which may be derived from a natural ligand; a single chain variable fragment (scFv) derived from a monoclonal antibody specific for a cell surface antigen fused to an intracellular domain that is a functional end of a T Cell Receptor (TCR), such as the CD 3-zeta signaling domain from the TCR capable of generating an activation signal in T lymphocytes. Upon antigen binding, such CARs connect to endogenous signaling pathways in effector cells and produce activation signals similar to those elicited by TCR complexes.

For example, in some embodiments, the CAR-T cell is one of the cells described in U.S. patent 8,906,682 (June et al; incorporated herein by reference in its entirety), which discloses a CAR-T cell engineered to comprise an extracellular domain having an antigen binding domain, such as a domain that binds to CD19, fused to an intracellular signaling domain of a T cell antigen receptor complex zeta chain, such as cd3ζ. When expressed in T cells, CARs are able to redirect antigen recognition based on antigen binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Currently more than 200 clinical trials employing CAR-T in a wide range of indications are underway. [ https:// clinicaltrias.gov/ct 2/resultstm=chimeric+anti+acceptors & pg=1 ].

In some embodiments, the immunostimulant is an activator of retinoic acid receptor-related orphan receptor gamma (rorγt). Roryt is a transcription factor that plays a key role in the differentiation and maintenance of type 17 effector subsets of cd4+ (Th 17) and cd8+ (Tc 17) T cells, and in the differentiation of a subset of innate immune cells expressing IL-17, such as NK cells. In some embodiments, the activator of roryt is LYC-55716 (lycra), which is currently evaluated in clinical trials for the treatment of solid tumors (NCT 02929862).

In some embodiments, the immunostimulant is an agonist or activator of a toll-like receptor (TLR). Suitable activators of TLR include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG that is being studied for use in B cell lymphomas, follicular lymphomas and other lymphomas (NCT 02254772). Agonists or activators of TLR8 useful in the present invention include motolimod (VTX-2337,VentiRx Pharmaceuticals), which is being studied for use in squamous cell carcinoma of the head and neck (NCT 02124850) and ovarian cancer (NCT 02431559).

Other immunological oncology agents useful in the present invention include: wu Ruilu monoclonal antibody (BMS-663513, bristol-Myers Squibb), which is an anti-CD 137 monoclonal antibody; varromab (CDX-1127,Celldex Therapeutics), an anti-CD 27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), which is an anti-OX 40 monoclonal antibody; li Ruilu monoclonal antibody (IPH 2102/BMS-986015,Innate Pharma,Bristol-Myers Squibb), which is an anti-KIR monoclonal antibody; mo Nali bead mab (IPH 2201, innate Pharma, astrazeneca), which is an anti-NKG 2A monoclonal antibody; andeliximab (GS-5745,Gilead Sciences), an anti-MMP 9 antibody; MK-4166 (Merck & Co.) is an anti-GITR monoclonal antibody.

In some embodiments, the immunostimulant is selected from the group consisting of eltuzumab (eltuzumab), mifamurtide (mifamurtide), an agonist or activator of toll-like receptors, and an activator of roryt.

In some embodiments, the immunostimulatory therapeutic agent is recombinant human interleukin 15 (rhIL-15). rhIL-15 has been tested clinically as a therapy for melanoma and renal cell carcinoma (NCT 01021059 and NCT 01369888) and leukemia (NCT 02689453). In some embodiments, the immunostimulant is recombinant human interleukin 12 (rhIL-12). In some embodiments, the IL-15-based immunotherapeutic agent is heterodimeric IL-15 (hetIL-15, novartis/Admune), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed with the alpha chain of the soluble IL-15 binding protein IL-15 receptor (IL 15: sIL-15 RA), which has been tested in phase 1 clinical trials against melanoma, renal cell carcinoma, non-small cell lung carcinoma and head and neck squamous cell carcinoma (NCT 02452268). In some embodiments, recombinant human interleukin 12 (rhIL-12) is NM-IL-12 (needles, inc.), NCT02544724, or NCT02542124.

In some embodiments, the immunooncology agent is selected from the group consisting of the immunooncology agents 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 immunooncology agent is selected from the examples described in table 1 of Jerry l.adams et al. In some embodiments, the immunooncology agent is a small molecule targeting an immunooncology target selected from those listed in table 2 of Jerry l.adams et al. In some embodiments, the immunooncology agent is a small molecule agent selected from those listed in table 2 of Jerry l.adams et al.

In some embodiments, the immunooncology agent is selected from Peter l.tooled, "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 immunooncology agent is an agent that targets a pathway as described in Peter l.

In some embodiments, the immunooncology agent is selected from Sandra L.Ross et al, "Bispecific T cell engagerantibody 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 immunooncology agent is a bispecific T cell engagerAntibody constructs. In some embodiments, bispecific T cell engager +.>The antibody construct is a CD19/CD3 bispecific antibody construct. In some embodiments, bispecific T cell engager +.>The antibody construct is an EGFR/CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager The antibody construct activates T cells. In some embodiments, bispecific T cell engager +.>The antibody construct activates T cells, which release cytokines that induce up-regulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on the neighboring cells. In some embodiments, bispecific T cell engager +.>The antibody construct activates T cells, which results in induced bystander cell lysis. In some embodiments, the paralytic cells are in a solid tumor. In some embodiments, the lysed bypass cells are close to +.>Activated T cells. In some embodiments, the paralytic cells comprise tumor-associated antigen (TAA) negative cancer cells. In some embodiments, the paralytic cells comprise EGFR-negative cancer cells. In some embodiments, the immunooncology agent is an antibody that blocks the PD-L1/PD1 axis and/or CTLA 4. In some embodiments, the immunooncology agent is an ex vivo expanded tumor-infiltrating T cell. In some embodiments, the immunooncology agent is a bispecific antibody construct or Chimeric Antigen Receptor (CAR) that directly links T cells to a tumor-associated surface antigen (TAA).

Exemplary checkpoint inhibitors

In some embodiments, the immunooncology agent is an immune checkpoint inhibitor as described herein.

The term "checkpoint inhibitor" as used herein relates to an agent suitable for preventing cancer cells from circumventing the immune system of a patient. One of the main mechanisms of anti-tumor immune destruction is called "T cell depletion", which is caused by prolonged exposure to antigens that have caused up-regulation of inhibitory receptors. These inhibitory receptors act as immune checkpoints in order to prevent uncontrolled immune responses.

PD-1 and co-inhibitory receptors such as cytotoxic T lymphocyte antigen 4 (CTLA-4), B and T lymphocyte attenuation factors (BTLA; CD 272), T cell immunoglobulin and mucin domain-3 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD 223) and others are commonly referred to as checkpoint modulators. It acts as a molecular "gatekeeper" that allows extracellular information to indicate whether cell cycle processes and other intracellular signaling processes should continue.

In some embodiments, the immune checkpoint inhibitor is an antibody to PD-1. PD-1 binds to the programmed cell death 1 receptor (PD-1) to prevent binding of the receptor to the inhibitory ligand PDL-1, thereby inhibiting the ability of the tumor to suppress the host's anti-tumor immune response.

In some embodiments, the checkpoint inhibitor is a biologic therapeutic 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 a checkpoint protein selected from the group consisting of: 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 combinations thereof. In some embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of: 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 combinations 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 an additional aspect, the vaccine is a Dendritic Cell (DC) vaccine.

Checkpoint inhibitors include any agent that blocks or inhibits the 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. Illustrative checkpoint molecules that can be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed in all NK, γδ and memory CD 8) ⁺ On (. Alpha.beta.) T cells), CD160 (also known as BY 55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands. Ligands of the B7 family include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 andB7-H7. Checkpoint inhibitors include antibodies or antigen binding fragments thereof, other binding proteins, biotherapeutic agents or small molecules that bind to and block or inhibit the activity of one or more of the following: CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049. Illustrative immune checkpoint inhibitors include, but are not limited to, trimeumab (CTLA-4 blocking antibody), anti-OX 40, PD-L1 monoclonal antibody (anti-B7-Hl; MEDI 4736), MK-3475 (PD-1 blocking), nivolumab (anti-PD 1 antibody), CT-011 (anti-PD 1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL 1 antibody), BMS-936559 (anti-PDL 1 antibody), MPLDL3280A (anti-PDL 1 antibody), MSB0010718C (anti-PDL 1 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.

In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of 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: nawu monoclonal antibodyIpimab->And palbociclib monoclonal antibody->In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (anti-PD-1 antibody,/-)>Bristol-Myers Squibb); palbociclib monoclonal antibody (anti-PD-1 antibody,>merck); ipilimumab (anti-CTLA-4 antibody,>Bristol-Myers Sqa uibb); dewaruzumab (anti-PD-L1 antibody,>AstraZeneca); and alemtuzumab (anti-PD-L1 antibody,/->Genentech)。

In some embodiments, the checkpoint inhibitor is selected from the group consisting of: raney monoclonal antibody (MK-3475), nawuzumab (BMS-936558), pituzumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipimab, li Ruilu monoclonal antibody, IPH2101, pabolizumabAnd trimeumab.

In some embodiments, the immune checkpoint inhibitor is: REGN2810 (Regeneron), an anti-PD-1 antibody tested in patients with basal cell carcinoma (NCT 03132636), NSCLC (NCT 03088540), cutaneous squamous cell carcinoma (NCT 02760498), lymphoma (NCT 02651662) and melanoma (NCT 03002376); pittuzumab (Curetech), also known as CT-011, an antibody that binds to PD-1 for clinical trials against diffuse large B-cell lymphoma and multiple myeloma; avermectin Pfizer/Merck KGaA), also known as MSB 0010718C), which is a fully human IgG1 anti-PD-L1 antibody for clinical trials against non-small cell lung cancer, merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer and gastric cancer; or PDR001 (Novartis), which is an inhibitory antibody that binds to PD-1, for clinical trials against non-small cell lung cancer, melanoma, triple negative breast cancer and advanced or metastatic solid tumors. Tramadol (CP-675, 206; astrazeneca) is a fully human monoclonal antibody against CTLA-4 that has been studied in clinical trials against a large number of indications, which are covered by the indicationThe method comprises the following steps: 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 cancer, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma and melanoma. AGEN-1884 (Agenus) is an anti-CTLA 4 antibody studied in a phase 1 clinical trial against advanced solid tumors (NCT 02694822).

In some embodiments, the checkpoint inhibitor is an inhibitor of protein 3 (TIM-3) comprising T cell immunoglobulin mucin. TIM-3 inhibitors useful in the present invention include TSR-022, LY3321367, and MBG453.TSR-022 (Tesaro) is an anti-TIM-3 antibody studied in solid tumors (NCT 02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody studied in solid tumors (NCT 03099109). MBG453 (Novartis) is an anti-TIM-3 antibody studied in advanced malignant disease (NCT 02608268).

In some embodiments, the checkpoint inhibitor is an inhibitor of a T cell immune receptor or TIGIT (an immune receptor on certain T cells and NK cells) having Ig and ITIM domains. TIGIT inhibitors useful in the present invention include: BMS-986207 (Bristol-Myers Squibb), an anti-TIGIT monoclonal antibody (NCT 02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibodies (NCT 03119428).

In some embodiments, the checkpoint inhibitor is an inhibitor of lymphocyte activation gene-3 (LAG-3). LAG-3 inhibitors useful in the present invention include BMS-986016 and REGN3767 and IMP321.BMS-986016 (Bristol-Myers Squibb, an anti-LAG-3 antibody) was studied in neuroglioblastoma and glioma (NCT 02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody and was studied in malignancy (NCT 03005782). IMP321 (Immutep S.A.) is a LAG-3-Ig fusion protein, which was studied in melanoma (NCT 02676869), adenocarcinoma (NCT 02614833) and metastatic breast cancer (NCT 00349934).

Checkpoint inhibitors useful in the present invention include OX40 agonists. OX40 agonists studied in clinical trials include: PF-04518600/PF-8600 (Pfizer), an agonistic anti-OX 40 antibody for use in metastatic renal cancer (NCT 03092856) and advanced cancers and tumors (NCT 02554812; NCT 05082566); GSK3174998 (Merck), an agonistic anti-OX 40 antibody for use in a phase 1 cancer assay (NCT 02528357); MEDI0562 (mediimune/AstraZeneca), an agonistic anti-OX 40 antibody used in advanced solid tumors (NCT 02318394 and NCT 02705482); MEDI6469, an agonistic anti-OX 40 antibody (Medimmune/AstraZeneca) for use in patients with colorectal cancer (NCT 02559024), breast cancer (NCT 01862900), head and neck cancer (NCT 02274155) and metastatic prostate cancer (NCT 01303705); and BMS-986178 (Bristol-Myers Squibb), which is an agonistic anti-OX 40 antibody used in advanced cancer (NCT 02737475).

Checkpoint inhibitors useful in the present invention include CD137 (also known as 4-1 BB) agonists. CD137 agonists studied in clinical trials include: wu Tuolu mab (utomiumab, PF-05082566, pfizer), an agonistic anti-CD 137 antibody for use in diffuse large B cell lymphomas (NCT 02951156) and advanced cancers and tumors (NCT 02554812 and NCT 05082566); wu Ruilu mab (BMS-663513, bristol-Myers Squibb), an agonistic anti-CD 137 antibody for use in melanoma and skin cancer (NCT 02652455) and neuroglioblastoma and glioma (NCT 02658981); and CTX-471 (Compass Therapeutics), which is an agonistic anti-CD 137 antibody for use in metastatic or locally advanced malignant disease (NCT 03881488).

Checkpoint inhibitors useful in the present invention include CD27 agonists. CD27 agonists studied in clinical trials include: varromab (CDX-1127,Celldex Therapeutics), an agonistic anti-CD 27 antibody for use in squamous cell head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma and neuroglioblastoma (NCT 02335918), lymphoma (NCT 01460134) and glioma and astrocytoma (NCT 02924038).

Checkpoint inhibitors useful in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists studied in clinical trials include: TRX518 (Leap Therapeutics), an agonistic anti-GITR antibody for use in malignant melanoma and other malignant solid tumors (NCT 01239134 and NCT 02628574); GWN323 (Novartis), an agonistic anti-GITR antibody used in solid tumors and lymphomas (NCT 02740270); INCAGN01876 (Incyte/agalus), an agonistic anti-GITR antibody for use in advanced cancers (NCT 02697591 and NCT 03126110); MK-4166 (Merck), an agonistic anti-GITR antibody for use in solid tumors (NCT 02132754); and MEDI1873 (mediimune/AstraZeneca), an agonistic hexamer GITR-ligand molecule with a human IgG1 Fc domain for use in advanced solid tumors (NCT 02583165).

Checkpoint inhibitors useful in the present invention include inducible T cell costimulatory (ICOS, also known as CD 278) agonists. ICOS agonists studied in clinical trials include: MEDI-570 (mediimune), an agonistic anti-ICOS antibody for use in lymphoma (NCT 02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody used in phase 1 (NCT 02723955); JTX-2011 (Jounce Therapeutics), an agonistic anti-ICOS antibody used in phase 1 (NCT 02904226).

Checkpoint inhibitors useful in the present invention include killing IgG-like receptor (KIR) inhibitors. KIR inhibitors studied in clinical trials include: li Ruilu mab (IPH 2102/BMS-986015,Innate Pharma/Bristol-Myers Squibb), an anti-KIR antibody for use in leukemia (NCT 01687387, NCT02399917, NCT02481297, NCT 02599649), multiple myeloma (NCT 02252263) and lymphoma (NCT 01592370); IPH2101 (1-7F9,Innate Pharma) for myeloma (NCT 01222286 and NCT 01217203); and IPH4102 (InnatePharma), an anti-KIR antibody (KIR 3DL 2) that binds to three domains of the long cytoplasmic tail for use in lymphomas (NCT 02593045).

Checkpoint inhibitors useful in the present invention include CD47 inhibitors of the interaction between CD47 and signal regulator protein α (SIRPa). CD47/SIRPa inhibitors studied in clinical trials include: ALX-148 (Alexo Therapeutics), an antagonistic variant of (SIRPa) that binds to CD47 and prevents CD47/SIRPa mediated signaling for use in phase 1 (NCT 03013218); TTI-621 (SIRPa-Fc, trillium Therapeutics), a soluble recombinant fusion protein formed by linking the N-terminal CD47 binding domain of SIRPa with the Fc domain of human IgG1, acting by binding to human CD47 and preventing it from delivering its "don't eat" signal to macrophages, is in phase 1 clinical trials (NCT 02890368 and NCT 02663518); CC-90002 (Celgene), an anti-CD 47 antibody for use in leukemia (NCT 02641002); and Hu5F9-G4 (Forty Seven, inc.) for colorectal and solid tumors (NCT 02953782), acute myelogenous leukemia (NCT 02678338), and lymphomas (NCT 02953509).

Checkpoint inhibitors useful in the present invention include CD73 inhibitors. CD73 inhibitors studied in clinical trials include MEDI9447 (mediimune), an anti-CD 73 antibody used in solid tumors (NCT 02503774); and BMS-986179 (Bristol-Myers Squibb), which is an anti-CD 73 antibody used in solid tumors (NCT 02754141).

Checkpoint inhibitors useful in the present invention include agonists of the interferon gene stimulatory protein (STING, also known as transmembrane protein 173 or TMEM 173). Agonists of STING studied in clinical trials include: MK-1454 (Merck), an agonistic synthetic cyclic dinucleotide used in lymphomas (NCT 03010176); and ADU-S100 (MIW 815, aduro Biotech/Novartis), an agonistic synthetic cyclic dinucleotide used in phase 1 (NCT 02675439 and NCT 03172936).

Checkpoint inhibitors useful in the present invention include CSF1R inhibitors. CSF1R inhibitors studied in clinical trials included: piroxicinib (PLX 3397, plexxikon), a small molecule inhibitor of CSF1R for use in colorectal cancer, pancreatic cancer, metastatic and advanced cancer (NCT 02777710), melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST) and ovarian cancer (NCT 02452424); and IMC-CS4 (LY 3022855, lilly), an anti-CSF-1R antibody for use in pancreatic cancer (NCT 03153410), melanoma (NCT 03101254) and solid tumors (NCT 02718911); 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 (NCT 02829723).

Checkpoint inhibitors useful in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors studied in clinical trials included Mo Nali bead mab (IPH 2201, innate Pharma), an anti-NKG 2A antibody used in head and neck tumors (NCT 02643550) and chronic lymphocytic leukemia (NCT 02557516).

In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of na Wu Shankang, pamidzumab, ipilimumab, avistuzumab, devaluzumab, alemtuzumab, or Pieribulab.

Examples

The following examples illustrate the invention described above; however, it is 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 to those skilled in the relevant art

Example 1: analysis and research of transcriptional patterns

Atlas analysis study using BCY12491

For transcription and Immunohistochemical (IHC) analysis, 6-8 week old female huCD137-C57B/6J mice (Biocytogen) were subcutaneously implanted 1X 10 ⁶ And MC38 cells. When the average tumor volume reached about 240mm ³ At this time, mice were randomized into treatment groups and either intravenously treated with vehicle (25 mM histidine, 10% sucrose, pH 7), 15mg/kg BCY12491, 15mg/kg BCY13626 (non-binding control), or intraperitoneally treated with 2mg/kg anti-CD 137 antibody Wu Ruilu mab. Treatment tumor growth was monitored via calliper measurements at Q3D dosing with three doses, and tumor tissue was harvested 1 hour after the last dose on day 6. A portion of tumor tissue was used for RNA isolation for transcriptional analysis and a portion of tumor tissue was used for formalin-fixed stone Wax embedded (FFPE) samples were prepared for IHC analysis. RNA was isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcribed using the nCounter Mouse PanCancer IO group (Nanostring) from 100ng RNA/tumor. Data were analyzed using nSolver analysis software with 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 antibodies (Abcam, # ab 217444) and Ventana Discovery OmniMap anti-rabbit-HRP kit (Ventana # 7604310).

The data is shown in fig. 1.

The discovery is as follows: transcriptional analysis showed a significant increase in immune cell scores such as cytotoxicity cell score, T cell score and macrophage cell score in tumor tissue following EPhA2 heteroconcatemeric bicyclic peptide complex BCY12491 treatment when compared to tumors from vehicle treated mice. anti-CD 137 antibody treatment also significantly increased the cytotoxic cell score and T cell score in tumor tissue, but to a lesser extent than BCY12491. No change in immune cell score was observed in tumor tissue from non-binding control (BCY 13626) treated animals. IHC analysis of cd8+ cells in tumor tissue demonstrated strong infiltration of cd8+ cells in tumors from mice treated with BCY12491 when compared to tumors from mice treated with vehicle or non-binding BCY 13626. Some increase in cd8+ cell infiltration was also observed in tumors from mice treated with anti-CD 137 antibodies. These changes in immune cell scores and cd8+ cells in tumor tissues indicate that the agonism of EphA2/CD137 heterotandem bicyclic peptide complex BCY12491 on CD137 in tumor tissues causes significant modulation (increase) of tumor infiltrating immune cells and immune responses.

Studies using BT7480

For transcription and Immunohistochemical (IHC) analysis, 6-8 week old female huCD137-C57B/6J mice (Biocytogen) were subcutaneously implanted 1X 10 ⁶ And MC38#13 cells (MC 38 cells engineered to express anserin-4). When the average tumor volume reached about 255mm ³ At this time, mice were randomized into treatment groups to receive vehicle, BT7480 (BCY 00011863)) Non-binding BCY control BCY00012797 (BCY 12797) or an αcd137 antibody (Wu Ruilu mab analog). BT7480 and its non-binding controls were administered intravenously at 5mg/kg (in 25mM histidine HCl, 10% sucrose (pH 7; vehicle) at 0h and 24h, and Wu Ruilu mab analogs were administered intraperitoneally at 2mg/kg dose and schedule in PBS BIW (0 h,72 h). Tumors from BT7480 treated mice were harvested at 24h (after 0h dosing), 48h (24 h after last 0h and 24h dosing), 96h (72 h after last 0h and 24h dosing), and 144h (120 h after last 0h and 24h dosing). Tumors from αcd 137-treated mice were harvested 144h after the start of treatment. Tumors from vehicle-treated mice were harvested 24h and 144h after 0h dosing (120 h after the last 0h and 24h dosing). RNA was isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcribed using the nCounter Mouse PanCancer IO group (Nanostring) from 100ng RNA/tumor. Data were analyzed using nSolver analysis software with advanced analysis probe set ns_mm_io_360_v1.0 (Nanostring).

The data are shown in fig. 2-4.

The discovery is as follows: transcriptional analysis showed a significant early (24 hour time point) increase in mRNA of some T cell chemotactic chemokines/cytokines (such as Ccl1, ccl17, and Ccl24, etc., which are thought to be secreted by bone marrow cells, resulting in recruitment of T cells in the chemokine secretion site). Transcriptional analysis also showed a significant increase in immune cell scores such as cytotoxicity cell scores and macrophage cell scores in tumor tissue following BT7480 treatment when compared to tumors from vehicle treated mice. Macrophage cell score began to increase 24h after BT7480 administration, increasing significantly from 24h vehicle reading to 48 h. On the other hand, when the cytotoxic cell score increased significantly compared to the vehicle-treated tumor at 144h, the cytotoxic cell score began to increase 48 hours after the start of treatment and increased up to 144h. Superimposing normalized mRNA counts of Ccl1, ccl17, and Ccl24 in response to the cytotoxicity cell score of BT7480 confirms how increases in the transcription of Ccl1, ccl17, and Ccl24 precede increases in the cytotoxicity cell score.

Superimposing the macrophage and cytotoxic cell scores in response to BT7480 demonstrates how the increase in cell score of the macrophage precedes the increase in cytotoxic cell score.

Transcriptional analysis showed a trend of increased or significant increase in mRNA for several different immune checkpoints including CTLA-4 (CTLA 4), PD-1 (Pdcd 1), PD-L1 (Cd 274), LAG3 (LAG 3), TIM3 (Havcr 2), PD-L2 (Pdcd 1lg 2) and TIGIT (TIGIT), supporting the concept of BT7480 in combination with checkpoint inhibitors.

Example 2: efficacy study using BCY12491 and palbociclizumab combination

For tumor growth analysis, 6-8 week old female huCD137/huPD-1-C57B/6J mice (Biocytogen) were subcutaneously implanted 1X 10 ⁶ And MC38 cells. When the average tumor volume reached about 92mm ³ At this time, mice were randomized into treatment groups and treated intravenously with vehicle (25 mM histidine, 10% sucrose, pH 7), 5mg/kg BCY12491 (0, 24 h), or intraperitoneally with 3mg/kg anti-PD-1 antibody, palbociclizumab or a combination of BCY12491 and palbociclizumab. Combination therapy was given on three different dosing schedules: BCY12491 and palbocizumab therapy initiated at day 0, BCY12491 therapy initiated at day 1 and palbocizumab therapy initiated at day 5, or palbocizumab therapy initiated at day 0 and BCY12491 therapy initiated at day 5. Treatment was given four times a week for four doses and tumor growth was monitored via calliper measurements.

The data are shown in fig. 5 and 6.

The discovery is as follows: BCY12491 and pamidzumab monotherapy and combinations thereof both showed significant antitumor activity when compared to vehicle controls (all p < 0.0001, mixed effect analysis compared to Dunnett post test, comparison of therapeutic with vehicle at D18). In addition, the combination treatment was more effective than either of the monotherapy (p < 0.0001), the mixed effect analysis and the Dunnett post test, the combination was compared to the monotherapy at D20), resulting in a complete response in all treated animals by day 22. In comparison, these treatment regimens and schedules caused a 2/10 complete response in the BCY12491 single drug therapy treatment population and a 3/10 complete response in the palbociclizumab single drug therapy treatment population. Alternate sequencing of BCY12491 in combination with palbociclib (BCY 12491 treatment beginning on day 0 and palbociclib treatment beginning on day 5, or vice versa) also produced significant anti-tumor activity (both x p < 0.0001, mixed effect analysis compared the therapeutic agent to vehicle at D18 with Dunnett post test), both schedules to day 42 induced a 9/10 complete response (BCY 12491 treatment beginning on day 0 and palbociclib treatment beginning on day 5) and an 8/10 complete response (palbociclib treatment beginning on day 0 and BCY12491 treatment beginning on day 5) in treated mice.

Example 3: efficacy study using BCY11864 and anti-PD-1 combinations

For tumor growth analysis, 6-8 week old female Balb/c-huCD137 mice (Gemphamatech) were subcutaneously implanted with 3×10+e5 CT26#7 cells (CT 26 cells engineered to overexpress zonin-4). When the average tumor volume reached about 80mm ³ At this time, mice were randomized into treatment groups and treated intravenously with vehicle (25 mM histidine, 10% sucrose, pH 7), 10mg/kg BCY11864 (0, 24 h), or intraperitoneally with 10mg/kg anti-PD-1 antibody (RMP 1-14) or a combination of BCY11864 and anti-PD-1 antibody. Weekly administration and treatment, and tumor growth was monitored via calliper measurements. With > 2000mm ³ Tumor animals were sacrificed when they had reached the humane endpoint. The study was terminated at day 66 after the start of treatment, when only 2 animals (all in the combination treatment group) remained in the study (one complete responder, one tumor size still regressing).

The data is shown in fig. 7.

The discovery is as follows: addition of BCY11864 to anti-PD-1 monotherapy was significant (p=0.004, mantel-Cox log rank test, which compares anti-PD-1 with the anti-PD-1+bcy11864 combination group) increased survival of CT26# 7-bearing mice (measured as reaching the humane endpoint (i.e., tumor volume > 2000mm ³ ) Time of (d) a).

Example 4 efficacy study Using 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 subcutaneously implanted with 1X 10+e6 MC38#13 cells (MC 38 cells engineered to overexpress petin-4). When the average tumor volume reaches about 100mm ³ At this time, mice were randomized into treatment groups and were treated intraperitoneally with vehicle (25 mM histidine, 10% sucrose, pH 7), 1mg/kg BT7480, 5mg/kg anti-PD-1 (RMP 1-14), 5mg/kg anti-Ctla-4 (9H 10), or a combination of BT 7480/anti-PD-1 and BT 7480/anti-Ctla-4. Twice weekly (BIW) treatments were given for 2 weeks and tumor growth was monitored via calliper measurements until day 33 after initiation of treatment. With > 2000mm ³ Tumor animals were sacrificed when they had reached the humane endpoint.

The data are shown in fig. 8 and 9.

The discovery is as follows: adding BT7480 to anti-PD-1 monotherapy increases the complete response rate (CR) from 0/8 (in the BT7480 and anti-PD-1 monotherapy groups) to 2/8 in the BT 7480/anti-PD-1 combination therapy group. By day 33 after initiation of treatment, adding BT7480 to anti-Ctla-4 monotherapy increased the complete response rate (CR) from 0/8 or 1/8 (in BT7480 and anti-Ctla-4 monotherapy groups, respectively) to 4/8 in BT 7480/anti-Ctla-4 combination therapy group. Furthermore, the addition of BT7480 to anti-CTLA-4 monotherapy significantly (p=0.0499, mantel-Cox log rank test, comparison of anti-CTLA-4 with anti-CTLA-4+bt7480 combination group) increased survival of mice carrying MC38#13 (measured as reaching the humane endpoint (i.e. tumor volume > 2000mm ³ ) Time of (d) a).

Example 5 transcriptional profiling study Using BT7455

For transcriptional profiling of the effect of BT7455 in the immune tumor microenvironment, 6-8 week old female huCD137-C57B/6J mice (Biocytogen) were subcutaneously implanted with 1X 10+E6 MC38 cells. When the average tumor volume reached about 350mm ³ At this time, mice were randomized into treatment groups to receive vehicle, BT7455, αcd137 antibody (Wu Ruilu mab analog) or αpd-1 antibody. BT7455 was administered intravenously at 8mg/kg (in 25mM histidine HCl, 10% sucrose (pH 7; vehicle)) at 0h and 24h, and 2mg/kg (Wu Ruilu mab analogue) or 10mg/kg (alpha PD-1 antibody) in PBS at 0hWu Ruilu monoclonal antibody analogs and alpha PD-1 antibodies. Tumors from mice treated with vehicle, BT7455, wu Ruilu mab analogs and αpd-1 antibodies were harvested 24h, 48h and 144h after initiation of treatment. RNA was isolated from tumor tissue using the RNAeasy kit (Qiagen) and transcribed using the nCounter Mouse PanCancer IO group (Nanostring) from 100ng RNA/tumor. Data were analyzed using nSolver analysis software with advanced analysis probe set ns_mm_io_360_v1.0 (Nanostring).

The data are shown in fig. 10-13.

The discovery is as follows: transcriptional analysis showed a significant increase in mRNA for several different immune checkpoints including CTLA-4 (CTLA 4), PD-1 (Pdcd 1), PD-L1 (Cd 274), LAG3 (LAG 3), TIM3 (Havcr 2), PD-L2 (Pdcd 1lg 2) and TIGIT (TIGIT) following BT7455 treatment, supporting the concept of BT7455 in combination with checkpoint inhibitors. Transcriptional analysis also showed a significant early (24 to 48 hour time point) increase in mRNA of some T cell chemotactic chemokines/cytokines (such as Ccl1, ccl17, and Ccl24, etc., which are thought to be secreted by bone marrow cells, resulting in recruitment of T cells in the chemokine secretion site). Transcriptional analysis also showed a significant increase in immune cell scores (such as cytotoxic cell scores) in tumor tissue following BT7455 treatment when compared to tumors from vehicle or anti-PD-1 or anti-CD 137 treated mice. BT7455 treatment triggers a significant early (48 hours) modulation of several gene sets, including those associated with cytokine and chemokine signaling, cytotoxicity, apoptosis, and NK- κb signaling gene sets.

Claims

1. 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 pharmaceutically acceptable salt thereof and an immunooncology agent, wherein the heterotandem bicyclic peptide complex comprises:

(b) One or more CD 137-binding peptide ligands;

2. The method of claim 1, wherein the heterotandem bicyclic peptide complex comprises:

(b) One or more CD 137-binding peptide ligands;

3. The method of claim 1 or 2, wherein the heterotandem bicyclic peptide complex comprises:

(b) Two or more CD137 binding peptide ligands.

4. The method of claim 1 or 2, wherein the heterotandem bicyclic peptide complex comprises:

(b) Two or more CD137 binding peptide ligands.

5. The method of any one of claims 1 to 4, wherein the CD 137-binding peptide ligand comprises an amino acid sequence selected from the group consisting of:

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)；

(SEQ ID NO: 11) -A (referred to herein as BCY 14601);

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)；

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 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

wherein [ MerPro] _i 、C _i 、C _ii 、C _iii And [ Cysam ]] _iii Represents a first (i), a second (ii) and a third (iii) reactive group selected from cysteine, merPro and Cysam, nle represents norleucine, tbu ala represents tert-leucine, tbu represents noriButyl-alanine, PYA stands for 4-pentynoic acid, aad for alpha-L-aminoadipic acid, merPro for 3-mercaptopropionic acid, cysam for cysteamine, NMeAla for N-methyl-alanine.

6. The method of any one of claims 1 to 5, wherein the CD 137-binding peptide ligand comprises the amino acid sequence of:

C _i [tBuAla]PE[D-Lys(PYA)]PYC _ii FADPY[Nle]C _iii (SEQ ID NO:11)；

7. The method of any one of claims 1 to 6, wherein the CD 137-binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

Ac-A- (SEQ ID NO: 5) -Dap (referred to herein as BCY 7732);

Ac-A- (SEQ ID NO: 5) -Dap (PYA) (referred to herein as BCY 7741);

ac- (SEQ ID NO: 6) -Dap (referred to herein as BCY 9172);

ac- (SEQ ID NO: 6) -Dap (PYA) (referred to herein as BCY 11014);

Ac-A- (SEQ ID NO: 7) -Dap (referred to herein as BCY 8045);

ac- (SEQ ID NO: 8) -A (referred to herein as BCY 8919);

ac- (SEQ ID NO: 9) -A (referred to herein as BCY 8920);

ac- (SEQ ID NO: 10) -A (referred to herein as BCY 8927);

ac- (SEQ ID NO: 11) -A (referred to herein as BCY 8928);

Ac-A- (SEQ ID NO: 12) -A (referred to herein as BCY 7744);

ac- (SEQ ID NO: 60) -Dap (PYA) (referred to herein as BCY 11144);

Ac-A- (SEQ ID NO: 61) -K (referred to herein as BCY 11613);

ac- (SEQ ID NO: 62) -Dap (PYA) (referred to herein as BCY 12023);

ac- (SEQ ID NO: 63) (referred to herein as BCY 12149);

ac- (SEQ ID NO: 64) (referred to herein as BCY 12143);

Ac- (SEQ ID NO: 65) (referred to herein as BCY 12147);

ac- (SEQ ID NO: 66) (referred to herein as BCY 12145);

ac- (SEQ ID NO: 67) (referred to herein as BCY 12146);

ac- (SEQ ID NO: 68) (referred to herein as BCY 12150);

ac- (SEQ ID NO: 69) (referred to herein as BCY 12352);

ac- (SEQ ID NO: 72) - [1, 2-diaminoethane ] (referred to herein as BCY 12358);

ac- (SEQ ID NO: 75) (referred to herein as BCY 12381);

ac- (SEQ ID NO: 76) (referred to herein as BCY 12382);

ac- (SEQ ID NO: 77) -K (referred to herein as BCY 12357);

ac- (SEQ ID NO: 78) - [ dA ] (referred to herein as BCY 13095);

[ Ac ] - (SEQ ID NO: 78) -K (referred to herein as BCY 13389);

ac- (SEQ ID NO: 79) - [ dA ] (referred to herein as BCY 13096); and

ac- (SEQ ID NO: 80) (referred to herein as BCY 13097);

8. The method of any one of claims 1 to 7, wherein the CD 137-binding bicyclic peptide ligand comprises an N-terminal and a C-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

ac- (SEQ ID NO: 11) -A (referred to herein as BCY 8928);

wherein Ac represents acetyl.

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

10. The method of claim 9, wherein the ansa-4-binding bicyclic peptide ligand comprises an amino acid sequence selected from the group consisting of:

C _i P[1Nal][dK]C _ii M[HArg]DWSTP[HyP]WC _iii (SEQ ID NO:14)；

C _i P[1Nal][dK]C _ii M[HArg]DWSTP[HyP]W[Cysam] _iii (SEQ ID NO:16)；

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)；

11. The method of claim 9 or 10, wherein the ansa-4-binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

SEQ ID NO. 1 (referred to herein as BCY 8116);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 1) (referred to herein as BCY 8846);

[ PYA ] - (SEQ ID NO: 1) (referred to herein as BCY 11015);

[ PYA ] - [ B-Ala ] - (SEQ ID NO: 1) (referred to herein as BCY 11016);

[PYA]-[B-Ala]-[Sar ₁₀ ]- (SEQ ID NO: 2) (referred to herein as BCY 11942);

ac- (SEQ ID NO: 3) (referred to herein as BCY 8831);

SEQ ID NO. 4 (referred to herein as BCY 11414);

[ PYA ] - [ B-Ala ] - (SEQ ID NO: 14) (referred to herein as BCY 11143);

palmitic acid-yGlu-yGlu- (SEQ ID NO: 14) (referred to herein as BCY 12371);

ac- (SEQ ID NO: 14) (referred to herein as BCY 12024);

ac- (SEQ ID NO: 16) (referred to herein as BCY 12364);

ac- (SEQ ID NO: 18) (referred to herein as BCY 12366); and

ac- (SEQ ID NO: 19) (referred to herein as BCY 12367);

wherein PYA represents 4-pentynoic acid, B-Ala represents beta-alanine, sar ₁₀ Representing 10 musclesAn amino acid unit.

12. The method of any one of claims 9 to 11, wherein the ansa-4-binding bicyclic peptide ligand comprises SEQ ID No. 1 (referred to herein as BCY 8116).

13. The method according to any one of claims 9 to 12, wherein the heterotandem bicyclic peptide complex is selected from the heterotandem bicyclic peptide complexes listed in tables a and B, such as BCY11027, BCY11863 and BCY11864, or a pharmaceutically acceptable salt thereof.

14. The method of any one of claims 1 to 8, wherein the component present on the cancer cell is EphA2 and the first peptide ligand comprises an EphA 2-binding bicyclic peptide ligand.

15. The method of claim 14, wherein the EphA 2-binding bicyclic peptide ligand comprises an amino acid sequence selected from the group consisting of:

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][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][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

Wherein [ MerPro] _i 、C _i 、C _ii 、C _iii And [ Cysam ]] _iii Represents a first (i), a second (ii) and a 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 denotes 3, 3-diphenylalanine, cba denotes β -cyclobutylalanine, 1Nal denotes 1-naphthylalanine, hGlu denotes homoglutamic acid, thi denotes thienyl-alanine, 4ThiAz denotes β - (4-thiazolyl) -alanine, his1Me denotes N1-methyl-L-histidine, his3Me denotes N3-methyl-L-histidine, 3Thi denotes 3-thienyl alanine, palmitoyl-Glu-LysN ₃ [PYA]The representation is:

(palmitoyl-Glu-LysN) ₃ )[PYA]，

[ K (PYA- (palmitoyl-Glu-LysN) ₃ )]The representation is:

[ K (PYA (palmitoyl-Glu-LysN) ₃ ))]，

16. The method of claim 14 or 15, wherein EphA 2-binding bicyclic peptide ligand comprises the amino acid sequence of:

C _i [HyP]LVNPLC _ii LHP[dD]W[HArg]C _iii (SEQ ID NO:24)；

wherein C is _i 、C _ii 、C _iii Represents a first (i), a second (ii) and a third (iii) cysteine group, hyP represents trans-4-hydroxy-L-proline, and HArg represents homoarginine.

17. The method of any one of claims 14 to 16, wherein EphA 2-binding bicyclic peptide ligand comprises the amino acid sequence of:

C _i [HyP]LVNPLC _ii LEP[d1Nal]WTC _iii (SEQ ID NO:44)；

wherein C is _i 、C _ii 、C _iii Represents the first (i), second (ii) and third (iii) cysteine groups, hyP represents trans-4-hydroxy-L-proline, and 1Nal represents 1-naphthylalanine.

18. The method of any one of claims 14 to 17, wherein EphA 2-binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

a- [ HArg ] -D- (SEQ ID NO: 24) (referred to herein as BCY 9594);

[ PYA ] -A- [ HArg ] -D- (SEQ NO: 24) (referred to herein as BCY 11813);

Ac-A- [ HArg ] -D- (SEQ ID NO: 24) -K (referred to herein as BCY 12734);

[ NMeAla ] - [ HArg ] -D- (SEQ ID NO: 24) (referred to herein as BCY 13121);

[ Ac ] - (SEQ ID NO: 24) -L [ dH ] G [ dK ] (referred to herein as BCY 13125);

Ac-A- [ HArg ] -D- (SEQ ID NO: 26) (referred to herein as BCY 11815);

Ac-A- [ HArg ] -D- (SEQ ID NO: 27) (referred to herein as BCY 11816);

Ac-A- [ HArg ] -D- (SEQ ID NO: 28) (referred to herein as BCY 11817);

Ac-A- [ HArg ] -D- (SEQ ID NO: 29) (referred to herein as BCY 12735);

(palmitoyl-Glu-LysN) ₃ )[PYA]A[HArg]D- (SEQ ID NO: 29) (referred to herein as BCY 14327);

Ac-A- [ HArg ] -D- (SEQ ID NO: 30) (referred to herein as BCY 12736);

Ac-A- [ HArg ] -D- (SEQ ID NO: 31) (referred to herein as BCY 12737); a- [ HArg ] -D- (SEQ ID NO: 32) (referred to herein as BCY 12738);

a- [ HArg ] -E- (SEQ ID NO: 32) (referred to herein as BCY 12739);

a- [ HArg ] -D- (SEQ ID NO: 33) (referred to herein as BCY 12854);

a- [ HArg ] -D- (SEQ ID NO: 34) (referred to herein as BCY 12855);

a- [ HArg ] -D- (SEQ ID NO: 35) (referred to herein as BCY 12856);

a- [ HArg ] -D- (SEQ ID NO: 35) - [ dA ] (referred to herein as BCY 12857);

(SEQ ID NO: 35) - [ dA ] (referred to herein as BCY 12861);

[ NMeAla ] - [ HArg ] -D- (SEQ ID NO: 35) (referred to herein as BCY 13122); [ dA ] -ED- (SEQ ID NO: 35) (referred to herein as BCY 13126);

[ dA ] - [ dA ] -D- (SEQ ID NO: 35) (referred to herein as BCY 13127);

AD- (SEQ ID NO: 35) (referred to herein as BCY 13128);

a- [ HArg ] -D- (SEQ ID NO: 36) (referred to herein as BCY 12858);

a- [ HArg ] -D- (SEQ ID NO: 37) (referred to herein as BCY 12859);

ac- (SEQ ID NO: 37) - [ dK ] (referred to herein as BCY 13120);

a- [ HArg ] -D- (SEQ ID NO: 38) (referred to herein as BCY 12862);

a- [ HArg ] -D- (SEQ ID NO: 39) (referred to herein as BCY 12863);

[ dA ] - [ HArg ] -D- (SEQ ID NO: 39) - [ dA ] (referred to herein as BCY 12864); (SEQ ID NO: 40) - [ dA ] (referred to herein as BCY 12865);

a- [ HArg ] -D- (SEQ ID NO: 41) (referred to herein as BCY 12866);

a- [ HArg ] -D- (SEQ ID NO: 42) (referred to herein as BCY 13116);

a- [ HArg ] -D- (SEQ ID NO: 43) (referred to herein as BCY 13117);

a- [ HArg ] -D- (SEQ ID NO: 44) (referred to herein as BCY 13118);

[ dA ] - [ HArg ] -D- (SEQ ID NO: 46) - [ dA ] (referred to herein as BCY 13123); [ D1Nal ] - [ HArg ] -D- (SEQ ID NO: 46) - [ dA ] (referred to herein as BCY 13124); a- [ HArg ] -D- (SEQ ID NO: 47) (referred to herein as BCY 13130);

A- [ HArg ] -D- (SEQ ID NO: 48) (referred to herein as BCY 13131);

a- [ HArg ] -D- (SEQ ID NO: 49) (referred to herein as BCY 13132);

a- [ HArg ] -D- (SEQ ID NO: 50) (referred to herein as BCY 13134);

a- [ HArg ] -D- (SEQ ID NO: 51) (referred to herein as BCY 13135);

(SEQ ID NO: 154) - [ dK ] (referred to herein as BCY 13129);

a [ HArg ] D- (SEQ ID NO: 155) (referred to herein as BCY 13133);

a [ HArg ] D- (SEQ ID NO: 156) (referred to herein as BCY 13917);

a [ HArg ] D- (SEQ ID NO: 157) (referred to herein as BCY 13918);

a [ HArg ] D- (SEQ ID NO: 158) (referred to herein as BCY 13919);

a [ HArg ] D- (SEQ ID NO: 159) (referred to herein as BCY 13920);

a [ HArg ] D- (SEQ ID NO: 160) (referred to herein as BCY 13922);

a [ HArg ] D- (SEQ ID NO: 161) (referred to herein as BCY 13923);

a [ HArg ] D- (SEQ ID NO: 162) (referred to herein as BCY 14047);

a [ HArg ] D- (SEQ ID NO: 163) (referred to herein as BCY 14048); and

a [ HArg ] D- (SEQ ID NO: 164) (referred to herein as BCY 14313);

(palmitoyl-Glu-LysN) ₃ )[PYA]。

19. The method of any one of claims 14 to 18, wherein EphA 2-binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

a- [ HArg ] -D- (SEQ ID NO: 24) (referred to herein as BCY 9594);

wherein HArg represents homoarginine.

20. The method of any one of claims 14 to 19, wherein the EphA 2-binding bicyclic peptide ligand optionally comprises an N-terminal modification and comprises the following or a pharmaceutically acceptable salt thereof:

a- [ HArg ] -D- (SEQ ID NO: 44) (referred to herein as BCY 13118);

wherein HArg represents homoarginine.

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

22. The method of any one of claims 1 to 21, wherein the molecular scaffold is 1,1',1"- (1, 3, 5-triazin-1, 3, 5-triyl) trip-2-en-1-one (TATA).

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

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

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

26. The method of any one of claims 1-25, wherein the heterotandem bicyclic peptide complex or pharmaceutically acceptable salt thereof and the immunooncology agent are administered within 1, 2, 3, 4, 5, 6, or 7 days of each other.

27. A method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of BT7480 or a pharmaceutically acceptable salt thereof and an immunooncology agent.

28. The method of claim 27, wherein the immunooncology agent is a checkpoint inhibitor.

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

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

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

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

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

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

35. The method of any one of claims 27-34, wherein BT7480 or a pharmaceutically acceptable salt thereof and the immunooncology agent are administered simultaneously or sequentially.

36. The method of any one of claims 27-35, wherein BT7480 or a pharmaceutically acceptable salt thereof and the immunooncology agent are administered within 1, 2, 3, 4, 5, 6, or 7 days of each other.

37. The method of any one of claims 27-36, wherein BT7480 or a pharmaceutically acceptable salt thereof is administered via intravenous infusion.

38. The method of any one of claims 27-37, wherein BT7480 or a pharmaceutically acceptable salt thereof is administered at a frequency of once a week.

39. The method of any one of claims 27-37, wherein BT7480 or a pharmaceutically acceptable salt thereof is administered twice a week.

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

Use of bt7480 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer, wherein the medicament is used in combination with a checkpoint inhibitor.

42. The use according to claim 41, wherein the medicament further comprises histidine.

43. The use of claim 41 or 42, wherein the medicament further comprises sucrose.

44. The use of any one of claims 41 to 43, wherein the medicament is a formulation at about pH7 comprising BT7480 or a pharmaceutically acceptable salt thereof, histidine, sucrose and water.

45. The method according to any one of claims 27 to 40 or the use according to any one of claims 41 to 44, wherein the cancer is a high-stalk protein-4 expressing cancer.

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

47. The method or use according to claim 46, wherein the solid tumor is a sarcoma, carcinoma or lymphoma.

48. A method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of BT7455 or a pharmaceutically acceptable salt thereof and an immunooncology agent.

49. The method of claim 48, wherein the immunooncology agent is a checkpoint inhibitor.

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

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

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

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

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

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

56. The method of any one of claims 48 to 55, wherein BT7455 or a pharmaceutically acceptable salt thereof and said immunooncology agent are administered simultaneously or sequentially.

57. The method of any one of claims 48 to 56, wherein BT7455 or a pharmaceutically acceptable salt thereof and said immunooncology agent are administered within 1, 2, 3, 4, 5, 6, or 7 days of each other.

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

59. The method of any one of claims 48 to 58, wherein BT7455 or a pharmaceutically acceptable salt thereof is administered at a frequency of once a week.

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

Use of bt7455 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating cancer, wherein the medicament is used in combination with a checkpoint inhibitor.

62. The use according to claim 61, wherein the medicament further comprises histidine.

63. The use according to claim 61 or 62, wherein the medicament further comprises sucrose.

64. The use according to any one of claims 61 to 63, wherein the medicament is a formulation at about pH7 comprising BT7455 or a pharmaceutically acceptable salt thereof, histidine, sucrose and water.

65. The method of any one of claims 48 to 60 or the use of any one of claims 61 to 64, wherein the cancer is a high EphA22 expression cancer.