ANTI-INFECTIVE BICYCLIC PEPTIDE LIGANDS

FIELD OF THE INVENTION

The present invention relates to polypeptides which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), particularly the spike protein S1 of SARS-CoV-2. The invention also includes pharmaceutical compositions comprising said polypeptides and to the use of said polypeptides in suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2.

BACKGROUND OF THE INVENTION

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and spread globally, resulting in a pandemic. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include fatigue, muscle pain, diarrhea, sore throat, loss of smell, and abdominal pain. The time from exposure to onset of symptoms is typically around five days but may range from two to fourteen days. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure. As of 6 January 2021, more than 86 million cases have been reported globally, resulting in more than 1.8 million deaths.

The virus is primarily spread between people during close contact, often via droplets produced by coughing, sneezing, or talking. While these droplets are produced when breathing out, they usually fall to the ground or onto surfaces rather than being infectious over long distances. People may also become infected by touching a contaminated surface and then their face. The virus can survive on surfaces for up to 72 hours. It is most contagious during the first three days after the onset of symptoms, although spread may be possible before symptoms appear and in later stages of the disease.

Currently, there is no vaccine or specific antiviral treatment for COVID-19. Management involves treatment of symptoms, supportive care, isolation, and experimental measures.

The World Health Organization (WHO) declared the 2019-2020 coronavirus outbreak a Public Health Emergency of International Concern (PHEIC) on 30 January 2020 and a pandemic on 11 March 2020. Local transmission of the disease has been recorded in many countries across all six WHO regions.

There is therefore a great need to provide an effective prophylactic and/or therapeutic treatment intended to avoid or ameliorate the symptoms associated with infection of SARS- CoV-2, such as COVID-19.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a peptide ligand specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising the peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided the peptide ligand as defined herein for use in suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided a peptide ligand specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

In one embodiment, said peptide ligand is specific for the spike protein of SARS-CoV-2. The spike protein (S protein) is a large type I transmembrane protein of SARS-CoV-2. This protein is highly glycosylated as it contains 21 to 35 N-glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive “corona”, or crown-like appearance. The ectodomain of all CoV spike proteins share the same organization in two domains: a N-terminal domain named S1 that is responsible for receptor binding and a C- terminal S2 domain responsible for fusion. CoV diversity is reflected in the variable spike proteins (S proteins), which have evolved into forms differing in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion.

In a further embodiment, said peptide ligand binds to either the S1 of S2 domain of the spike protein (S protein). In a yet further embodiment, said peptide ligand binds to the S1 domain of the spike protein (S1 protein). Without being bound by theory it is believed that binding to the S1 domain of SARS-CoV-2, namely the receptor binding domain of SARS-CoV-2, will prevent the virus from binding to its target (thought to be ACE2 bound to the surface of lung airway cells) to enter tissue and cause disease.

In one embodiment, said loop sequences comprise 2, 3, 4, 5, 6, 7 or 8 amino acids.

In one embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 6 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 6 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiH H ACiiPI LT GWCiii (SEQ ID NO: 1);

CiPHACiiPSLWGWCiii (SEQ ID NO: 6);

CiLHACiiPRLTHWCiii (SEQ ID NO: 7);

CiLHACiiQYLWGYCiii (SEQ ID NO: 8);

CiSHACiiPRLFGWCiii (SEQ ID NO: 9);

CiQHACiiPYLWDYCiii (SEQ ID NO: 10);

CiPFACiiHKLYGWCiii (SEQ ID NO: 58);

CiMKACiiPYLYGWCiii (SEQ ID NO: 59);

CiRHACiiTHLYGHCiii (SEQ ID NO: 60);

CiPYACiiTRLYGWCiii (SEQ ID NO: 61);

CiSHACiiPRLTGWCiii (SEQ ID NO: 62);

CiLHSCiiPRLSGWCiii (SEQ ID NO: 63);

CiRHSCiiPILTGWCiii (SEQ ID NO: 64);

CGHSCiiPVLWGWCiii (SEQ ID NO: 65);

CiPHSCiiPKLFGWCiii (SEQ ID NO: 66);

CiTHSCiiPYLFGWCiii (SEQ ID NO: 67); CiDWTCiiYLTMMPCiii (SEQ ID NO: 118);

CiDWTCiiYLRPLPCiii (SEQ ID NO: 119);

CiDWTCiYMSMKPCiii (SEQ ID NO: 120);

CiDWTCiYFRPLPCiii (SEQ ID NO: 121); and CiDWTCiYISPMFDCiii (SEQ ID NO: 122); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 6 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiH H ACiiPI LT GWCiii (SEQ ID NO: 1);

CiPHACiiPSLWGWCiii (SEQ ID NO: 6);

CiLHACiiPRLTHWCiii (SEQ ID NO: 7);

CiLHACiiQYLWGYCiii (SEQ ID NO: 8);

CiSHACiiPRLFGWCiii (SEQ ID NO: 9);

CiQHACiiPYLWDYCiii (SEQ ID NO: 10);

CiPFACiiHKLYGWCiii (SEQ ID NO: 58);

CiMKACiiPYLYGWCiii (SEQ ID NO: 59);

CiRHACiiTHLYGHCiii (SEQ ID NO: 60);

CiPYACiiTRLYGWCiii (SEQ ID NO: 61);

CiSHACiiPRLTGWCiii (SEQ ID NO: 62);

CiLHSCiiPRLSGWCiii (SEQ ID NO: 63);

CiRHSCiiPILTGWCiii (SEQ ID NO: 64);

CGHSCiiPVLWGWCiii (SEQ ID NO: 65);

CiPHSCiiPKLFGWCiii (SEQ ID NO: 66); and CiTHSCiiPYLFGWCiii (SEQ ID NO: 67); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 6 amino acids, the molecular scaffold is TATA and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 1)-A (herein referred to as BCY15230);

A-(SEQ ID NO: 6)-A (herein referred to as BCY15235);

A-(SEQ ID NO: 7)-A (herein referred to as BCY15236);

A-(SEQ ID NO: 8)-A (herein referred to as BCY15237);

A-(SEQ ID NO: 9)-A (herein referred to as BCY15238);

A-(SEQ ID NO: 10)-A (herein referred to as BCY15239);

A-(SEQ ID NO: 58)-A (herein referred to as BCY15364);

A-(SEQ ID NO: 59)-A (herein referred to as BCY15365);

A-(SEQ ID NO: 60)-A (herein referred to as BCY15366);

A-(SEQ ID NO: 61)-A (herein referred to as BCY15367);

A-(SEQ ID NO: 62)-A (herein referred to as BCY15368);

A-(SEQ ID NO: 63)-A (herein referred to as BCY15369);

A-(SEQ ID NO: 64)-A (herein referred to as BCY15370);

A-(SEQ ID NO: 65)-A (herein referred to as BCY15371);

A-(SEQ ID NO: 66)-A (herein referred to as BCY15372); and A-(SEQ ID NO: 67)-A (herein referred to as BCY15373).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 6 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 6)-A-[Sar _e ]-[KFI] (herein referred to as BCY15303); and A-(SEQ ID NO: 63)-A-[Sar _e ]-[KFI] (herein referred to as BCY15329).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 6 amino acids, the molecular scaffold is TATB and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 118)-A (herein referred to as BCY15444);

A-(SEQ ID NO: 119)-A (herein referred to as BCY16927);

A-(SEQ ID NO: 120)-A (herein referred to as BCY16930);

A-(SEQ ID NO: 121)-A (herein referred to as BCY16933); and A-(SEQ ID NO: 122)-A (herein referred to as BCY16940). In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 7 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 7 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiDWTCiiYLNIYHECiii (SEQ ID NO: 123);

CiDWTCiYMDYLSNCiii (SEQ ID NO: 124);

CiDWTCiYLRIHEACiii (SEQ ID NO: 125);

CiDWTCiYMRINDACiii (SEQ ID NO: 126); and CiDWTCiYINIYNTCiii (SEQ ID NO: 127); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 7 amino acids, the molecular scaffold is TATB and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 123)-A (herein referred to as BCY15445);

A-(SEQ ID NO: 124)-A (herein referred to as BCY16941);

A-(SEQ ID NO: 125)-A (herein referred to as BCY16942); and A-(SEQ ID NO: 126)-A (herein referred to as BCY16946).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 3 amino acids and the other of which consists of 7 amino acids, the molecular scaffold is TCMT and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:

A-(SEQ ID NO: 127)-A (herein referred to as BCY16948).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 6 amino acids. In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 6 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiLTNDCiiHSDIRYCiii (SEQ ID NO: 29); and CilTNDCiiHTSLIFCiii (SEQ ID NO: 30); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 6 amino acids, the molecular scaffold is TCMT and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 29)-A (herein referred to as BCY15335); and A-(SEQ ID NO: 30)-A (herein referred to as BCY15336).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 6 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEQ ID NO: 30)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15314).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiVDANCiiKI Kl LQRMCiii (SEQ ID NO: 3);

CiTSSVCiiKI KELQRKCiii (SEQ ID NO: 4);

CiRSLLCiiEYLQRTDSCiii (SEQ ID NO: 5); CiLTKSCiiKI KM LQRVCiii (SEQ ID NO: 14);

CiMQPSCiiRVLQLQRVCiii (SEQ ID NO: 15);

CiALPSCiiRILHLQHRCiii (SEQ ID NO: 16);

CiH DAHCiiKI LELQH RCiii (SEQ ID NO: 17);

CiTSSHCiiRVLEEQRLCiii (SEQ ID NO: 18);

CiPRDRCiiPTAWLYGLCiii (SEQ ID NO: 19);

CAEAGCiiRVKQLQQICiii (SEQ ID NO: 20);

CiTPSPCiiRVKELQRACiii (SEQ ID NO: 21);

CiSTANCiiRILELQQLCiii (SEQ ID NO: 26);

CiVGRLCiiSTATDIRKCiii (SEQ ID NO: 44);

CiRQSQCiiDWWAIRSFCiii (SEQ ID NO: 48; herein referred to as BCY16983 when complexed with TATB);

CiTDATCiiSI KRLQRLCiii (SEQ ID NO: 49);

CiSPVSCiiPSGFKFGLCiii (SEQ ID NO: 50);

CiDSPWCiiRI RSLQRQCiii (SEQ ID NO: 68);

CiSVGACiiRVKLLQRVCiii (SEQ ID NO: 69);

CiMFVPCiAVREILGLCiii (SEQ ID NO: 70);

CiSDLMCiiWYLQRTDSCiii (SEQ ID NO: 128);

CiNSYMCiiWYLQRTDSCiii (SEQ ID NO: 129);

CTSYLCiiWYLQRTDSCiii (SEQ ID NO: 130); and CiRSLMCiiWYLNQTDSCiii (SEQ ID NO: 131); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiVDANCiiKI Kl LQRMCiii (SEQ ID NO: 3);

CiTSSVCiiKI KELQRKCiii (SEQ ID NO: 4);

CiRSLLCiiEYLQRTDSCiii (SEQ ID NO: 5);

CiLTKSCiiKI KM LQRVCiii (SEQ ID NO: 14);

CiMQPSCiiRVLQLQRVCiii (SEQ ID NO: 15);

CiALPSCiiRILHLQHRCiii (SEQ ID NO: 16);

CiH DAHCiiKI LELQH RCiii (SEQ ID NO: 17);

CiTSSHCiiRVLEEQRLCiii (SEQ ID NO: 18); CiPRDRCiiPTAWLYGLCiii (SEQ ID NO: 19);

CAEAGCiiRVKQLQQICiii (SEQ ID NO: 20);

CiTPSPCiiRVKELQRACiii (SEQ ID NO: 21);

CiSTANCiiRILELQQLCiii (SEQ ID NO: 26);

CiVGRLCiiSTATDIRKCiii (SEQ ID NO: 44);

CiRQSQCiiDWWAIRSFCiii (SEQ ID NO: 48; herein referred to as BCY16983 when complexed with TATB);

CiTDATCiiSI KRLQRLCiii (SEQ ID NO: 49);

CiSPVSCiiPSGFKFGLCiii (SEQ ID NO: 50);

CiDSPWCiiRI RSLQRQCiii (SEQ ID NO: 68);

CiSVGACiiRVKLLQRVCiii (SEQ ID NO: 69); and CiM FVPCiAVREI LGLCiii (SEQ ID NO: 70); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids, the molecular scaffold is TATB and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 3)-A (herein referred to as BCY15334);

A-(SEQ ID NO: 15)-A (herein referred to as BCY15244);

A-(SEQ ID NO: 16)-A (herein referred to as BCY15245);

A-(SEQ ID NO: 17)-A (herein referred to as BCY15246);

A-(SEQ ID NO: 18)-A (herein referred to as BCY15247);

A-(SEQ ID NO: 19)-A (herein referred to as BCY15248);

A-(SEQ ID NO: 20)-A (herein referred to as BCY15249);

A-(SEQ ID NO: 21)-A (herein referred to as BCY15250);

A-(SEQ ID NO: 26)-A (herein referred to as BCY15255);

A-(SEQ ID NO: 48)-A (herein referred to as BCY15354);

A-(SEQ ID NO: 48)-A (herein referred to as BCY16534);

A-(SEQ ID NO: 48)-AK (herein referred to as BCY16896);

A-(SEQ ID NO: 49)-A (herein referred to as BCY15355); and A-(SEQ ID NO: 50)-A (herein referred to as BCY15356). In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids, the molecular scaffold is TATB, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 3)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15301);

A-(SEQ ID NO: 15)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15307);

A-(SEQ ID NO: 17)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15308);

A-(SEQ ID NO: 19)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15309);

A-(SEQ ID NO: 48)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15324);

A-(SEQ ID NO: 49)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15325); and

A-(SEQ ID NO: 50)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15326).

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 3)-A (herein referred to as BCY15232);

A-(SEQ ID NO: 4)-A (herein referred to as BCY15233);

A-(SEQ ID NO: 5)-A (herein referred to as BCY15234);

A-(SEQ ID NO: 14)-A (herein referred to as BCY15243);

A-(SEQ ID NO: 44)-A (herein referred to as BCY15350);

A-(SEQ ID NO: 68)-A (herein referred to as BCY15374);

A-(SEQ ID NO: 69)-A (herein referred to as BCY15375);

A-(SEQ ID NO: 70)-A (herein referred to as BCY15376);

A-(SEQ ID NO: 128)-A (herein referred to as BCY16886);

A-(SEQ ID NO: 129)-A (herein referred to as BCY16887);

A-(SEQ ID NO: 130)-A (herein referred to as BCY16889); and

A-(SEQ ID NO: 131)-A (herein referred to as BCY16895).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 3)-A (herein referred to as BCY15232);

A-(SEQ ID NO: 4)-A (herein referred to as BCY15233);

A-(SEQ ID NO: 5)-A (herein referred to as BCY15234);

A-(SEQ ID NO: 14)-A (herein referred to as BCY15243);

A-(SEQ ID NO: 44)-A (herein referred to as BCY15350);

A-(SEQ ID NO: 68)-A (herein referred to as BCY15374);

A-(SEQ ID NO: 69)-A (herein referred to as BCY15375); and A-(SEQ ID NO: 70)-A (herein referred to as BCY15376).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 3)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15300);

A-(SEQ ID NO: 5)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15302); and A-(SEQ ID NO: 70)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15330).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 3 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is: CiFDDWTCiYIQMCiii (SEQ ID NO: 115); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEC ID NO: 115)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15437). In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiTLMDPWCiiLLKCiii (SEQ ID NO: 71);

CiKIHDWTCiiLLRCiii (SEQ ID NO: 72); and

CilPLDWTCiiMIACiii (SEQ ID NO: 79; herein referred to as BCY18707 when complexed with TATB); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiTLMDPWCiiLLKCiii (SEQ ID NO: 71);

CiKIHDWTCiiLLRCiii (SEQ ID NO: 72); and CilPLDWTCiiMIACiii (SEQ ID NO: 79); wherein Q, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATA and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 71)-A (herein referred to as BCY15377); and A-(SEQ ID NO: 72)-A (herein referred to as BCY15378).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEQ ID NO: 71)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15331).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATB, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

Ac-(SEQ ID NO: 79) (herein referred to as BCY16991);

A-(SEQ ID NO: 79)-A (herein referred to as BCY15446);

A-(SEQ ID NO: 79)-AK (herein referred to as BCY16994); and Ac-(SEQ ID NO: 79)-K (herein referred to as BCY18654).

In a yet further alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATB, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

Ac-(SEQ ID NO: 79) (herein referred to as BCY16991);

A-(SEQ ID NO: 79)-A (herein referred to as BCY15446); and A-(SEQ ID NO: 79)-AK (herein referred to as BCY16994).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiEYQGPHCiYRLYCiii (SEQ ID NO: 11);

CiEYNGPYCiYRLYCiii (SEQ ID NO: 132); and CiEYVGPMCiYRLYCiii (SEQ ID NO: 133); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof. In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is:

CiEYQGPHCiiYRLYCiii (SEQ ID NO: 11); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 11)-A (herein referred to as BCY15240);

A-(SEQ ID NO: 132)-A (herein referred to as BCY17547); and A-(SEQ ID NO: 133)-A (herein referred to as BCY17548).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:

A-(SEQ ID NO: 11)-A (herein referred to as BCY15240).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEQ ID NO: 11)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15304).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids. In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiEDHDWVYCiiSTCiii (SEQ ID NO: 2);

CAPWNYFRCiiDLCiii (SEQ ID NO: 23);

CiLTPEDIWCiiMLCiii (SEQ ID NO: 25);

CiENPVDIWCiiVLCiii (SEQ ID NO: 28);

CiVFTTVWDCiiLACiii (SEQ ID NO: 46);

CiYDPIDVWCiiMMCiii (SEQ ID NO: 51);

CASYDDFWCiiVLCiii (SEQ ID NO: 52);

CiDLTQHWTCiilLCiii (SEQ ID NO: 53);

CiS E I S DVWCiiM LCiii (SEQ ID NO: 54);

CiPTPVDIWCiiMLCiii (SEQ ID NO: 55);

CiEQNGWIYCiiSTCiii (SEQ ID NO: 73);

CiTDRSWI FCiiSTCiii (SEQ ID NO: 74);

CiPNISWIYCiiSTCiii (SEQ ID NO: 75);

CiDVCiiGLNAFNRCiii (SEQ ID NO: 117);

CiLDETWIYCiiSTCiii (SEQ ID NO: 134);

CiPDETWVYCiiSTCiii (SEQ ID NO: 135);

CiESNDWVYCiiSTCiii (SEQ ID NO: 136);

CiEDNDWVYCiiSTCiii (SEQ ID NO: 137);

CiPDVSWIYCiiSTCiii (SEQ ID NO: 138); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiEDHDWVYCiiSTCiii (SEQ ID NO: 2);

CAPWNYFRCiiDLCiii (SEQ ID NO: 23);

CiLTPEDIWCiiMLCiii (SEQ ID NO: 25);

CiENPVDIWCiiVLCiii (SEQ ID NO: 28);

CiVFTTVWDCiiLACiii (SEQ ID NO: 46);

CiYDPIDVWCiiMMCiii (SEQ ID NO: 51); CASYDDFWCiiVLCiii (SEQ ID NO: 52);

CiDLTQHWTCiilLCiii (SEQ ID NO: 53);

CiSEISDVWCiiMLCiii (SEQ ID NO: 54);

CiPTPVDIWCiiMLCiii (SEQ ID NO: 55);

CiEQNGWIYCiiSTCiii (SEQ ID NO: 73);

CiTDRSWI FCiiSTCiii (SEQ ID NO: 74);

CiPNISWIYCiiSTCiii (SEQ ID NO: 75); and CiDVCiiGLNAFNRCiii (SEQ ID NO: 117); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiEDHDWVYCiiSTCiii (SEQ ID NO: 2);

CAPWNYFRCiiDLCiii (SEQ ID NO: 23);

CiLTPEDIWCiiMLCiii (SEQ ID NO: 25);

CiENPVDIWCiiVLCiii (SEQ ID NO: 28);

CiVFTTVWDCiiLACiii (SEQ ID NO: 46);

CYDPIDVWCiiMMCiii (SEQ ID NO: 51);

CASYDDFWCiiVLCiii (SEQ ID NO: 52);

CiDLTQHWTCiilLCiii (SEQ ID NO: 53);

CiSEISDVWCiiMLCiii (SEQ ID NO: 54);

CiPTPVDIWCiiMLCiii (SEQ ID NO: 55);

CiEQNGWIYCiiSTCiii (SEQ ID NO: 73);

CiTDRSWI FCiiSTCiii (SEQ ID NO: 74); and CiPNISWIYCiiSTCiii (SEQ ID NO: 75); wherein O, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 2)-A (herein referred to as BCY15231);

Ac-(SEQ ID NO: 2) (herein referred to as BCY16987);

A-(SEQ ID NO: 46)-A (herein referred to as BCY15352);

A-(SEQ ID NO: 73)-A (herein referred to as BCY15379);

A-(SEQ ID NO: 74)-A (herein referred to as BCY15380);

A-(SEQ ID NO: 75)-A (herein referred to as BCY15381);

A-(SEQ ID NO: 134)-A (herein referred to as BCY17540);

A-(SEQ ID NO: 135)-A (herein referred to as BCY17541);

A-(SEQ ID NO: 136)-A (herein referred to as BCY17542);

A-(SEQ ID NO: 137)-A (herein referred to as BCY17543); and A-(SEQ ID NO: 138)-A (herein referred to as BCY17544).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 2)-A (herein referred to as BCY15231);

Ac-(SEQ ID NO: 2) (herein referred to as BCY16987);

A-(SEQ ID NO: 46)-A (herein referred to as BCY15352);

A-(SEQ ID NO: 73)-A (herein referred to as BCY15379);

A-(SEQ ID NO: 74)-A (herein referred to as BCY15380); and A-(SEQ ID NO: 75)-A (herein referred to as BCY15381).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 2)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15299); and A-(SEQ ID NO: 74)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15332).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEQ ID NO: 117)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY16287).

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATB, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 23)-A (herein referred to as BCY15252);

A-(SEQ ID NO: 25)-A (herein referred to as BCY15254);

A-(SEQ ID NO: 28)-A (herein referred to as BCY15257);

A-(SEQ ID NO: 51)-A (herein referred to as BCY15357);

A-(SEQ ID NO: 52)-A (herein referred to as BCY15358);

A-(SEQ ID NO: 53)-A (herein referred to as BCY15359);

A-(SEQ ID NO: 54)-A (herein referred to as BCY15360); and A-(SEQ ID NO: 55)-A (herein referred to as BCY15361).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATB, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 23)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15311);

A-(SEQ ID NO: 25)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15312); and A-(SEQ ID NO: 53)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15327).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from: CiASPDNPVCiiRFYCiii (SEQ ID NO: 22; herein referred to as BCY16534 when complexed with TATB);

CYNHANPVCiiRYYCiii (SEQ ID NO: 24; herein referred to as BCY16540 when complexed with TATB);

CiDLFLHELCiiDMPCiii (SEQ ID NO: 27);

CiNKQNWRYCiYLTCiii (SEQ ID NO: 31);

CiHPWSALFCiiNYPCiii (SEQ ID NO: 56);

CYAPDNPVCiiRMYCiii (SEQ ID NO: 57);

CiGILADPFCiiLISCiii (SEQ ID NO: 76);

CYNHANPVCii[Agb]YYCiii (SEQ ID NO: 89);

CASPDNPVCii[Agb]FYCiii (SEQ ID NO: 90);

CASPDNPVCii[Arg(Me)]FYCiii (SEQ ID NO: 91);

CASPDNPVCii[HArg]FYCiii (SEQ ID NO: 92);

CANPDNPVCiiRFYCiii (SEQ ID NO: 93);

CiRNPDNPVCiiRFYCiii (SEQ ID NO: 94);

CiHNPSNPVCiiRFYCiii (SEQ ID NO: 95);

CiVNKHNPVCiiRFYCiii (SEQ ID NO: 96);

CiVNAENPVCiiRFYCiii (SEQ ID NO: 97);

CiQNPGNPVCiiRFYCiii (SEQ ID NO: 98);

CiMNPDNPVCiiRFYCiii (SEQ ID NO: 99);

CYNQENPVCiiRFYCiii (SEQ ID NO: 100);

CiNNPANPVCiiRFYCiii (SEQ ID NO: 101);

CiFNIDNPVCiiRFYCiii (SEQ ID NO: 102);

CiSNPENPVCiiRFYCiii (SEQ ID NO: 103);

CiMNEDNPVCiiRFYCiii (SEQ ID NO: 104);

CiMNEANPVCiiRFYCiii (SEQ ID NO: 105);

CiHNLDNPVCiiRFYCiii (SEQ ID NO: 106);

CANHDNPVCiiRFYCiii (SEQ ID NO: 107);

CiKNYDNPVCiiRFYCiii (SEQ ID NO: 108);

CiENMDNPVCiiRFYCiii (SEQ ID NO: 109);

CiMNTDNPVCiiRFYCiii (SEQ ID NO: 110);

CiLNVDNPVCiiRFYCiii (SEQ ID NO: 111);

CiLNPDNPVCiiRFYCiii (SEQ ID NO: 112);

CYNHANPVCii[HArg]YYCiii (SEQ ID NO: 113);

CYNHANPVCii[Arg(Me)]YYCiii (SEQ ID NO: 114);

CiMNPFFYDCiiERTCiii (SEQ ID NO: 116); Ci[Aib]SPDNPVCiiRFYCiii (SEQ ID NO: 139);

CiAS[HyP]DNPVCiiRFYCiii (SEQ ID NO: 140);

CAS[Aib]DNPVCiiRFYCiii (SEQ ID NO: 141);

CAS[Pip]DNPVCiiRFYCiii (SEQ ID NO: 142);

CASPDN[Pip]VCiiRFYCiii (SEQ ID NO: 143);

CASPDN[44DFP]VCiiRFYCiii (SEQ ID NO: 144);

CASPDN[4FIPro]VCiiRFYCiii (SEQ ID NO: 145);

CASPDNPVCiiR[1Nal]YCiii (SEQ ID NO: 146);

CASPDNPVCiiR[2Nal]YCiii (SEQ ID NO: 147);

CASPDNPVCiiR[2MePhe]YCiii (SEQ ID NO: 148);

CASPDNPVCiiR[3MePhe]YCiii (SEQ ID NO: 149);

CASPDNPVCiiR[4MePhe]YCiii (SEQ ID NO: 150);

CASPDNPVCiiR[2CIPhe]YCiii (SEQ ID NO: 151);

CASPDNPVCiiR[3CIPhe]YCiii (SEQ ID NO: 152);

CASPDNPVCiiR[4CIPhe]YCiii (SEQ ID NO: 153);

CASPDNPVCiiR[2FPhe]YCiii (SEQ ID NO: 154);

CASPDNPVCiiR[3FPhe]YCiii (SEQ ID NO: 155);

CASPDNPVCiiR[4FPhe]YCiii (SEQ ID NO: 156);

CASPDNPVCiiR[26DiMeTyr]YCiii (SEQ ID NO: 157);

C[Aib]SPDN[44DFP]VCiiR[4FPhe]YCiii (SEQ ID NO: 158);

Ci[Aib]SPDN[44DFP]VCii[Arg(Me)][4FPhe]YCiii (SEQ ID NO: 159);

Ci[Aib]SPDNPVCiiR[4FPhe]YCiii (SEQ ID NO: 160);

Ci[Aib]SPDNPVCii[Arg(Me)][4FPhe]YCiii (SEQ ID NO: 161);

Ci[Aib]NPDN[44DFP]VCiiR[4FPhe]YCiii (SEQ ID NO: 162);

Ci[Aib]NPDN[44DFP]VCii[Arg(Me)][4FPhe]YCiii (SEQ ID NO: 163);

Ci[Aib]NPDNPVCiiR[4FPhe]YCiii (SEQ ID NO: 164); and

Ci[Aib]NPDNPVCii[Arg(Me)][4FPhe]YCiii (SEQ ID NO: 165); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, Aib represents aminoisobutyric acid, Agb represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 5-N methyl arginine, 2CIPhe represents 2-chloro-phenylalanine, 3CIPhe represents 3-chloro-phenylalanine, 4CIPhe represents 4-chloro-phenylalanine, 44DFP represents 4,4-difluoroproline, 26DiMeTyr represents 2,6-dimethyl-tyrosine, 2FPhe represents 2-fluoro-phenylalanine, 3FPhe represents 3-fluoro-phenylalanine, 4FPhe represents 4-fluoro-phenylalanine, 4FIPro represents 4-fluoro-proline, HArg represents homoarginine, HyP represents hydroxyproline, 2MePhe represents 2-methyl-phenylalanine, 3MePhe represents 3-methyl-phenylalanine, 4MePhe represents 4-methyl-phenylalanine, 1Nal represents 1-naphthylalanine, 2Nal represents 2-naphthylalanine, Pip represents pipecolic acid, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiASPDNPVCiiRFYCiii (SEQ ID NO: 22; herein referred to as BCY16534 when complexed with TATB);

CYNHANPVCiiRYYCiii (SEQ ID NO: 24; herein referred to as BCY16540 when complexed with TATB);

CiDLFLHELCiiDMPCiii (SEQ ID NO: 27);

CiNKQNWRYCiYLTCiii (SEQ ID NO: 31);

CiHPWSALFCiiNYPCiii (SEQ ID NO: 56);

CYAPDNPVCiiRMYCiii (SEQ ID NO: 57);

CiGILADPFCiiLISCiii (SEQ ID NO: 76);

CYNHANPVCii[Agb]YYCiii (SEQ ID NO: 89);

CASPDNPVCii[Agb]FYCiii (SEQ ID NO: 90);

CASPDNPVCii[Arg(Me)]FYCiii (SEQ ID NO: 91);

CiCASPDNPVCii[HArg]FYCiii (SEQ ID NO: 92);

CANPDNPVCiiRFYCiii (SEQ ID NO: 93);

CiRNPDNPVCiiRFYCiii (SEQ ID NO: 94);

CiHNPSNPVCiiRFYCiii (SEQ ID NO: 95);

CiVNKHNPVCiiRFYCiii (SEQ ID NO: 96);

CiVNAENPVCiiRFYCiii (SEQ ID NO: 97);

CiQNPGNPVCiiRFYCiii (SEQ ID NO: 98);

CiMNPDNPVCiiRFYCiii (SEQ ID NO: 99);

CYNQENPVCiiRFYCiii (SEQ ID NO: 100);

CiNNPANPVCiiRFYCiii (SEQ ID NO: 101);

CiFNIDNPVCiiRFYCiii (SEQ ID NO: 102);

CiSNPENPVCiiRFYCiii (SEQ ID NO: 103);

CiMNEDNPVCiiRFYCiii (SEQ ID NO: 104);

CiMNEANPVCiiRFYCiii (SEQ ID NO: 105);

CiHNLDNPVCiiRFYCiii (SEQ ID NO: 106);

CANHDNPVCiiRFYCiii (SEQ ID NO: 107);

CiKNYDNPVCiiRFYCiii (SEQ ID NO: 108); CiENMDNPVCiiRFYCiii (SEQ ID NO: 109);

CiMNTDNPVCiiRFYCiii (SEQ ID NO: 110);

CiLNVDNPVCiiRFYCiii (SEQ ID NO: 111);

CiLNPDNPVCiiRFYCiii (SEQ ID NO: 112);

CYNHANPVCii[HArg]YYCiii (SEQ ID NO: 113);

CYNHANPVCii[Arg(Me)]YYCiii (SEQ ID NO: 114); and CiMNPFFYDCiiERTCiii (SEQ ID NO: 116); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, Agb represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 5-N methyl arginine, HArg represents homoarginine, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiASPDNPVCiiRFYCiii (SEQ ID NO: 22; herein referred to as BCY16534 when complexed with TATB);

CYNHANPVCiiRYYCiii (SEQ ID NO: 24; herein referred to as BCY16540 when complexed with TATB);

CiDLFLHELCiiDMPCiii (SEQ ID NO: 27);

CiNKQNWRYCiYLTCiii (SEQ ID NO: 31);

CiHPWSALFCiiNYPCiii (SEQ ID NO: 56);

CYAPDNPVCiiRMYCiii (SEQ ID NO: 57);

CiGILADPFCiiLISCiii (SEQ ID NO: 76);

CYNHANPVCii[Agb]YYCiii (SEQ ID NO: 89);

CASPDNPVCii[Agb]FYCiii (SEQ ID NO: 90);

CASPDNPVCii[Arg(Me)]FYCiii (SEQ ID NO: 91);

CiCASPDNPVCii[HArg]FYCiii (SEQ ID NO: 92);

CANPDNPVCiiRFYCiii (SEQ ID NO: 93);

CiRNPDNPVCiiRFYCiii (SEQ ID NO: 94);

CiHNPSNPVCiiRFYCiii (SEQ ID NO: 95);

CiVNKHNPVCiiRFYCiii (SEQ ID NO: 96);

CiVNAENPVCiiRFYCiii (SEQ ID NO: 97);

CiQNPGNPVCiiRFYCiii (SEQ ID NO: 98);

CiMNPDNPVCiiRFYCiii (SEQ ID NO: 99);

CYNQENPVCiiRFYCiii (SEQ ID NO: 100); CiNNPANPVCiiRFYCiii (SEQ ID NO: 101);

CiFNIDNPVCiiRFYCiii (SEQ ID NO: 102);

CiSNPENPVCiiRFYCiii (SEQ ID NO: 103);

CiMNEDNPVCiiRFYCiii (SEQ ID NO: 104);

CiMNEANPVCiiRFYCiii (SEQ ID NO: 105);

CiHNLDNPVCiiRFYCiii (SEQ ID NO: 106);

CANHDNPVCiiRFYCiii (SEQ ID NO: 107);

CiKNYDNPVCiiRFYCiii (SEQ ID NO: 108);

CiENMDNPVCiiRFYCiii (SEQ ID NO: 109);

CiMNTDNPVCiiRFYCiii (SEQ ID NO: 110);

CiLNVDNPVCiiRFYCiii (SEQ ID NO: 111);

CiLNPDNPVCiiRFYCiii (SEQ ID NO: 112);

CYNHANPVCii[HArg]YYCiii (SEQ ID NO: 113); and

CYNHANPVCii[Arg(Me)]YYCiii (SEQ ID NO: 114); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, Agb represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 5-N methyl arginine, HArg represents homoarginine, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATB, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 22)-A (herein referred to as BCY15251);

Ac-A-(SEQ ID NO: 22)-A (herein referred to as BCY16538);

Ac-(SEQ ID NO: 22) (herein referred to as BCY15576);

Ac-A-(SEQ ID NO: 24)-A (herein referred to as BCY16545);

Ac-(SEQ ID NO: 24) (herein referred to as BCY16544);

A-(SEQ ID NO: 24)-A (herein referred to as BCY15522);

A-(SEQ ID NO: 27)-A (herein referred to as BCY15256);

A-(SEQ ID NO: 56)-A (herein referred to as BCY15362);

A-(SEQ ID NO: 57)-A (herein referred to as BCY15363);

A-(SEQ ID NO: 89)-A (herein referred to as BCY16541);

A-(SEQ ID NO: 90)-A (herein referred to as BCY16535);

A-(SEQ ID NO: 91)-A (herein referred to as BCY16536);

A-(SEQ ID NO: 92)-A (herein referred to as BCY16537); Ac-(SEQ ID NO: 93) (herein referred to as BCY16903); Ac-(SEQ ID NO: 94) (herein referred to as BCY16905); Ac-(SEQ ID NO: 95) (herein referred to as BCY16906); Ac-(SEQ ID NO: 96) (herein referred to as BCY16911); Ac-(SEQ ID NO: 97) (herein referred to as BCY16913); Ac-(SEQ ID NO: 98) (herein referred to as BCY16915); Ac-(SEQ ID NO: 99) (herein referred to as BCY16917); Ac-(SEQ ID NO: 100) (herein referred to as BCY16918); Ac-(SEQ ID NO: 101) (herein referred to as BCY16921); Ac-(SEQ ID NO: 102) (herein referred to as BCY16912); Ac-(SEQ ID NO: 103) (herein referred to as BCY16914); Ac-(SEQ ID NO: 104) (herein referred to as BCY16916); Ac-(SEQ ID NO: 105) (herein referred to as BCY16919); Ac-(SEQ ID NO: 106) (herein referred to as BCY16920); Ac-(SEQ ID NO: 107) (herein referred to as BCY16902); Ac-(SEQ ID NO: 108) (herein referred to as BCY16904); Ac-(SEQ ID NO: 109) (herein referred to as BCY16907); Ac-(SEQ ID NO: 110) (herein referred to as BCY16908); Ac-(SEQ ID NO: 111) (herein referred to as BCY16909); Ac-(SEQ ID NO: 112) (herein referred to as BCY16910); A-(SEQ ID NO: 113)-A (herein referred to as BCY16543); A-(SEQ ID NO: 114)-A (herein referred to as BCY16542); A-(SEQ ID NO: 116)-A (herein referred to as BCY16207); Ac-(SEQ ID NO: 116) (herein referred to as BCY18698); Ac-(SEQ ID NO: 139) (herein referred to as BCY17279); Ac-(SEQ ID NO: 140) (herein referred to as BCY17281); Ac-(SEQ ID NO: 141) (herein referred to as BCY17282); Ac-(SEQ ID NO: 142) (herein referred to as BCY17283); Ac-(SEQ ID NO: 143) (herein referred to as BCY17287); Ac-(SEQ ID NO: 144) (herein referred to as BCY17289); Ac-(SEQ ID NO: 145) (herein referred to as BCY17294); Ac-(SEQ ID NO: 146) (herein referred to as BCY17301); Ac-(SEQ ID NO: 147) (herein referred to as BCY17302); Ac-(SEQ ID NO: 148) (herein referred to as BCY17303); Ac-(SEQ ID NO: 149) (herein referred to as BCY17304); Ac-(SEQ ID NO: 150) (herein referred to as BCY17305); Ac-(SEQ ID NO: 151) (herein referred to as BCY17306); Ac-(SEQ ID NO: 152) (herein referred to as BCY17307); Ac-(SEQ ID NO: 153) (herein referred to as BCY17308); Ac-(SEQ ID NO: 154) (herein referred to as BCY17309); Ac-(SEQ ID NO: 155) (herein referred to as BCY17310); Ac-(SEQ ID NO: 156) (herein referred to as BCY17311); Ac-(SEQ ID NO: 157) (herein referred to as BCY17313); Ac-(SEQ ID NO: 158) (herein referred to as BCY18340); Ac-(SEQ ID NO: 159) (herein referred to as BCY18341); Ac-(SEQ ID NO: 160) (herein referred to as BCY18342); Ac-(SEQ ID NO: 161) (herein referred to as BCY18343); Ac-(SEQ ID NO: 162) (herein referred to as BCY18344); Ac-(SEQ ID NO: 163) (herein referred to as BCY18345); Ac-(SEQ ID NO: 164) (herein referred to as BCY18346); and Ac-(SEQ ID NO: 165) (herein referred to as BCY18347).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATB, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 22)-A (herein referred to as BCY15251);

Ac-A-(SEQ ID NO: 22)-A (herein referred to as BCY16538);

Ac-(SEQ ID NO: 22) (herein referred to as BCY15576);

Ac-A-(SEQ ID NO: 24)-A (herein referred to as BCY16545);

Ac-(SEQ ID NO: 24) (herein referred to as BCY16544);

A-(SEQ ID NO: 24)-A (herein referred to as BCY15522);

A-(SEQ ID NO: 27)-A (herein referred to as BCY15256);

A-(SEQ ID NO: 56)-A (herein referred to as BCY15362);

A-(SEQ ID NO: 57)-A (herein referred to as BCY15363);

A-(SEQ ID NO: 89)-A (herein referred to as BCY16541);

A-(SEQ ID NO: 90)-A (herein referred to as BCY16535);

A-(SEQ ID NO: 91)-A (herein referred to as BCY16536);

A-(SEQ ID NO: 92)-A (herein referred to as BCY16537);

Ac-(SEQ ID NO: 93) (herein referred to as BCY16903);

Ac-(SEQ ID NO: 94) (herein referred to as BCY16905); Ac-(SEQ ID NO: 95) (herein referred to as BCY16906);

Ac-(SEQ ID NO: 96) (herein referred to as BCY16911);

Ac-(SEQ ID NO: 97) (herein referred to as BCY16913);

Ac-(SEQ ID NO: 98) (herein referred to as BCY16915);

Ac-(SEQ ID NO: 99) (herein referred to as BCY16917);

Ac-(SEQ ID NO: 100) (herein referred to as BCY16918);

Ac-(SEQ ID NO: 101) (herein referred to as BCY16921);

Ac-(SEQ ID NO: 102) (herein referred to as BCY16912);

Ac-(SEQ ID NO: 103) (herein referred to as BCY16914);

Ac-(SEQ ID NO: 104) (herein referred to as BCY16916);

Ac-(SEQ ID NO: 105) (herein referred to as BCY16919);

Ac-(SEQ ID NO: 106) (herein referred to as BCY16920);

Ac-(SEQ ID NO: 107) (herein referred to as BCY16902);

Ac-(SEQ ID NO: 108) (herein referred to as BCY16904);

Ac-(SEQ ID NO: 109) (herein referred to as BCY16907);

Ac-(SEQ ID NO: 110) (herein referred to as BCY16908);

Ac-(SEQ ID NO: 111) (herein referred to as BCY16909);

Ac-(SEQ ID NO: 112) (herein referred to as BCY16910);

A-(SEQ ID NO: 113)-A (herein referred to as BCY16543); and A-(SEQ ID NO: 114)-A (herein referred to as BCY16542).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATB, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 22)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15310);

A-(SEQ ID NO: 27)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15313);

A-(SEQ ID NO: 56)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15328); and A-(SEQ ID NO: 116)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY16298).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATB, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 22)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15310);

A-(SEQ ID NO: 27)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15313); and A-(SEQ ID NO: 56)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15328).

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TCMT, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:

A-(SEQ ID NO: 31)-A (herein referred to as BCY15315).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEC ID NO: 31)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15313).

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:

A-(SEC ID NO: 76)-A (herein referred to as BCY15382).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is:

A-(SEC ID NO: 76)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15333).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 5 amino acids. In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 5 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiTTSEKVKCiiLQRHPCiii (SEQ ID NO: 32);

CiQPDMRIKCiiLQRVACiii (SEQ ID NO: 33);

CiSSN N Rl KCiiLQRVTCiii (SEQ ID NO: 34);

CiKEKTTIGCiiLMAGICiii (SEQ ID NO: 35); and CiNRPTSVYCiiLQRGICiii (SEQ ID NO: 166); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 5 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiTTSEKVKCiiLQRHPCiii (SEQ ID NO: 32);

CiQPDMRIKCiiLQRVACiii (SEQ ID NO: 33);

CiSSN NRI KCiiLQRVTCiii (SEQ ID NO: 34); and CiKEKTTIGCiiLMAGICiii (SEQ ID NO: 35); wherein O, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 5 amino acids, the molecular scaffold is TCMT, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 32)-A (herein referred to as BCY15338);

A-(SEQ ID NO: 33)-A (herein referred to as BCY15339);

A-(SEQ ID NO: 34)-A (herein referred to as BCY15340);

A-(SEQ ID NO: 35)-A (herein referred to as BCY15341); and A-(SEQ ID NO: 166)-A (herein referred to as BCY17359).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 5 amino acids, the molecular scaffold is TCMT, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 32)-A (herein referred to as BCY15338);

A-(SEQ ID NO: 33)-A (herein referred to as BCY15339);

A-(SEQ ID NO: 34)-A (herein referred to as BCY15340); and A-(SEQ ID NO: 35)-A (herein referred to as BCY15341).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 7 amino acids and the other of which consists of 5 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 32)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15316); and A-(SEQ ID NO: 33)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15317).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiGRDSSWIYCiiSTCiii (SEQ ID NO: 12);

CiRGTPAWKACiAICiii (SEQ ID NO: 13);

CiPFPSGFGTCiiTFCiii (SEQ ID NO: 36);

CiPYVAGRGTCiiLLCiii (SEQ ID NO: 37; herein referred to as BCY16312 when complexed with TCMT);

CiPYPRGTGSCiTFCiii (SEQ ID NO: 38);

CiLYPPGKGTCiiLLCiii (SEQ ID NO: 39);

CiPSPAGRGTCiiLLCiii (SEQ ID NO: 40);

CiPATIGRGPCiiTFCiii (SEQ ID NO: 41);

CiPEANSWVYCiiSTCiii (SEQ ID NO: 77);

CAPTSGWIYCiiSTCiii (SEQ ID NO: 78);

CiPYVAG[Agb]GTCiiLLCiii (SEQ ID NO: 80); CiPYVAG[Arg(Me)]GTCiiLLCiii (SEQ ID NO: 81);

CiPYVAGRGTCiiL[Cba]Ciii (SEQ ID NO: 82);

CiPYVAGRGTCii[Cba]LCiii (SEQ ID NO: 83);

CiPYVAGR[dA]TCiiLLCiii (SEQ ID NO: 84);

CiPYVAG[HArg]GTCiiLLCiii (SEQ ID NO: 85);

CiPYVAGRGTCiiL[tBuAla]Ciii (SEQ ID NO: 86);

CiPYVAGRGTCii[tBuAla]LCiii (SEQ ID NO: 87); CiPYVAG[Agb][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 88);

CiPYVAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 167; herein referred to as BCY18111 when complexed with TCMT);

CiPYVPG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 168); Ci[K(PYA)]YVAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 169); Ci[HyP]YVAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 170); CiPYVAGT[dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 171);

CiPYVAG[Agb][dA]TCiiLLCiii (SEQ ID NO: 172); CiPYVAG[Agb]GTCiiL[tBuAla]Ciii (SEQ ID NO: 173); CiPYV[HyP]G[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 174); CiPY[K(PYA)]AG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 175); Ci[Oic]YVAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 176); CiPY[B-Melle]AG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 177); CiPY[tBuGly]AG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 178); CiPYPAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 179); CiP[44BPA]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 180); CiP[2FPhe]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 181); CiPY[Cba]AG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 182); CiP[3FPhe]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 183); Ci[55DMP]YVAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 184); CiPYVAGQ[dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 185); CiPYVAG[HArg][dS]TCiiL[tBuAla]Ciii (SEQ ID NO: 186); CiP[4tBuPhe]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 187); CiPYREGTGTCiiLLCiii (SEQ ID NO: 188);

CiPYAPGNGTCiiLLCiii (SEQ ID NO: 189);

CiPHPPGRGTCiiLLCiii (SEQ ID NO: 190);

CiPYNAGTGTCiiLLCiii (SEQ ID NO: 191);

CiPYSPGQGTCiiLLCiii (SEQ ID NO: 192);

CiPYQPGSGTCiiLLCiii (SEQ ID NO: 193); CiPFPPGMGTCiiLLCiii (SEQ ID NO: 194);

CiPHQPGFGTCiiLLCiii (SEQ ID NO: 195);

CiPYSPGSGTCiiLLCiii (SEQ ID NO: 196);

CiPYLAGTGTCiiLLCiii (SEQ ID NO: 197);

CiPWEAGKGTCiiLLCiii (SEQ ID NO: 198);

CiPYAPGMGTCiiLLCiii (SEQ ID NO: 199);

CiPHMPGSGTCiiLLCiii (SEQ ID NO: 200);

CiPYN KGEGTCiiLLCiii (SEQ ID NO: 201);

CiPFKPGVGTCiiLLCiii (SEQ ID NO: 202);

CiP[4tBuPhe]VAG[Orn][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 203);

Ci[Oic][4tBuPhe]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 204);

CiP[4tBuPhe]VAG[Dab][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 205);

CiP[4tBuPhe]VAG[Dap][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 206);

CiP[4CF3Phe]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 207);

CiP[DMAPhe]VAG[HArg][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 208);

CiP[4tBuPhe]VAG[HArg][dDab]TCiiL[tBuAla]Ciii (SEQ ID NO: 209);

CiP[4tBuPhe]VAG[HSer][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 210);

CiP[4tBuPhe]VAG[Cit][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 211);

C[Oic][4tBuPhe]VAG[Orn][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 212);

CiP[4tBuPhe]VAG[HArg][dDap]TCiiL[tBuAla]Ciii (SEQ ID NO: 213); and

Ci[Oic][4tBuPhe]VAG[Cit][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 214); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, Agb represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents d-N methyl arginine, B- Melle represents b-methyl-isoleucine, 44BPA represents 4,4-biphenylalanine, Cba represents b-cyclobutylalanine, 4CF3Phe represents 4-trifluoromethyl-phenylalanine, Cit represents citrulline, Dab represents dia inobutanoic acid, Dap represents diaminopropionic acid, DMAPhe represents 4-Dimethylamino- phenylalanine, 55DMP represents 5,5- Dimethyl-L-proline, 2FPhe represents 2-fluoro-phenylalanine, 3FPhe represents 3-fluoro- phenylalanine, HArg represents homoarginine, HSer represents homoserine, HyP represents hydroxyproline, Oic represents octahydroindolecarboxylic acid, Orn represents ornithine, PYA represents pentynoic acid, tBuAla represents t-butyl-alanine, tBuGly represents t-butyl-glycine, 4tBuPhe represents 4-t-butyl-phenylalanine, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiGRDSSWIYCiiSTCiii (SEQ ID NO: 12);

CiRGTPAWKACiiAICiii (SEQ ID NO: 13);

CiPFPSGFGTCiiTFCiii (SEQ ID NO: 36);

CiPYVAGRGTCiiLLCiii (SEQ ID NO: 37; herein referred to as BCY16312 when complexed with TCMT);

CiPYPRGTGSCiiTFCiii (SEQ ID NO: 38);

CiLYPPGKGTCiiLLCiii (SEQ ID NO: 39);

CiPSPAGRGTCiiLLCiii (SEQ ID NO: 40);

CiPATIGRGPCiiTFCiii (SEQ ID NO: 41);

CiPEANSWVYCiiSTCiii (SEQ ID NO: 77);

CAPTSGWIYCiiSTCiii (SEQ ID NO: 78);

CiPYVAG[Agb]GTCiiLLCiii (SEQ ID NO: 80);

CiPYVAG[Arg(Me)]GTCiiLLCiii (SEQ ID NO: 81);

CiPYVAGRGTCiiL[Cba]Ciii (SEQ ID NO: 82);

CiPYVAGRGTCii[Cba]LCiii (SEQ ID NO: 83);

CiPYVAGR[dA]TCiiLLCiii (SEQ ID NO: 84);

CiPYVAG[HArg]GTCiiLLCiii (SEQ ID NO: 85);

CiPYVAGRGTCiiL[tBuAla]Ciii (SEQ ID NO: 86);

CiPYVAGRGTCii[tBuAla]LCiii (SEQ ID NO: 87); and

CiPYVAG[Agb][dA]TCiiL[tBuAla]Ciii (SEQ ID NO: 88); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, Agb represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 5-N methyl arginine, Cba represents b-cyclobutylalanine, HArg represents homoarginine, tBuAla represents t-butyl- alanine, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 12)-A (herein referred to as BCY15241);

A-(SEQ ID NO: 13)-A (herein referred to as BCY15242);

A-(SEQ ID NO: 77)-A (herein referred to as BCY15383); and

A-(SEQ ID NO: 78)-A (herein referred to as BCY15384). In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 12)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15305); and

A-(SEQ ID NO: 13)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15306.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 36)-A (herein referred to as BCY15342);

A-(SEQ ID NO: 37)-A (herein referred to as BCY15343);

Ac-A-(SEQ ID NO: 37)-A (herein referred to as BCY16322);

Ac-(SEQ ID NO: 37) (herein referred to as BCY16926);

A-(SEQ ID NO: 38)-A (herein referred to as BCY15344);

A-(SEQ ID NO: 39)-A (herein referred to as BCY15345);

A-(SEQ ID NO: 40)-A (herein referred to as BCY15346);

A-(SEQ ID NO: 41)-A (herein referred to as BCY15347);

A-(SEQ ID NO: 80)-A (herein referred to as BCY16313);

Ac-(SEQ ID NO: 80) (herein referred to as BCY18086);

A-(SEQ ID NO: 81)-A (herein referred to as BCY16314);

A-(SEQ ID NO: 82)-A (herein referred to as BCY16315);

A-(SEQ ID NO: 83)-A (herein referred to as BCY16316);

A-(SEQ ID NO: 84)-A (herein referred to as BCY16318);

A-(SEQ ID NO: 85)-A (herein referred to as BCY16319);

A-(SEQ ID NO: 86)-A (herein referred to as BCY16320);

A-(SEQ ID NO: 87)-A (herein referred to as BCY16321);

Ac-(SEQ ID NO: 88) (herein referred to as BCY16591);

Ac-(SEQ ID NO: 167) (herein referred to as BCY18024);

Ac-(SEQ ID NO: 168)-[K(PYA)] (herein referred to as BCY18025);

Ac-(SEQ ID NO: 169) (herein referred to as BCY18026);

Ac-(SEQ ID NO: 170)-[K(PYA)] (herein referred to as BCY18027); Ac-(SEQ ID NO: 171)-[K(PYA)] (herein referred to as BCY18040);

Ac-(SEQ ID NO: 172) (herein referred to as BCY18087);

Ac-(SEQ ID NO: 173) (herein referred to as BCY18088);

Ac-(SEQ ID NO: 174)-[K(PYA)] (herein referred to as BCY18109);

Ac-(SEQ ID NO: 175) (herein referred to as BCY18110);

Ac-(SEQ ID NO: 176)-[K(PYA)] (herein referred to as BCY18115);

Ac-(SEQ ID NO: 177)-[K(PYA)] (herein referred to as BCY18211);

Ac-(SEQ ID NO: 178)-[K(PYA)] (herein referred to as BCY18212);

Ac-(SEQ ID NO: 179)-[K(PYA)] (herein referred to as BCY18351);

Ac-(SEQ ID NO: 180)-[K(PYA)] (herein referred to as BCY18524);

Ac-(SEQ ID NO: 181)-[K(PYA)] (herein referred to as BCY18527);

Ac-(SEQ ID NO: 182)-[K(PYA)] (herein referred to as BCY18529);

Ac-(SEQ ID NO: 183)-[K(PYA)] (herein referred to as BCY18661);

Ac-(SEQ ID NO: 184)-[K(PYA)] (herein referred to as BCY18662);

Ac-(SEQ ID NO: 185) (herein referred to as BCY19305);

Ac-(SEQ ID NO: 186) (herein referred to as BCY19309);

Ac-(SEQ ID NO: 187)-[K(PYA)] (herein referred to as BCY19378);

Ac-(SEQ ID NO: 188) (herein referred to as BCY19533);

Ac-(SEQ ID NO: 189) (herein referred to as BCY19534);

Ac-(SEQ ID NO: 190) (herein referred to as BCY19535);

Ac-(SEQ ID NO: 191) (herein referred to as BCY19536);

Ac-(SEQ ID NO: 192) (herein referred to as BCY19537);

Ac-(SEQ ID NO: 193) (herein referred to as BCY19538);

Ac-(SEQ ID NO: 194) (herein referred to as BCY19539);

Ac-(SEQ ID NO: 195) (herein referred to as BCY19541);

Ac-(SEQ ID NO: 196) (herein referred to as BCY19542);

Ac-(SEQ ID NO: 197) (herein referred to as BCY19543);

Ac-(SEQ ID NO: 198) (herein referred to as BCY19544);

Ac-(SEQ ID NO: 199) (herein referred to as BCY19545);

Ac-(SEQ ID NO: 200) (herein referred to as BCY19546);

Ac-(SEQ ID NO: 201) (herein referred to as BCY19547);

Ac-(SEQ ID NO: 202) (herein referred to as BCY19548);

Ac-(SEQ ID NO: 203)-[K(PYA)] (herein referred to as BCY19599);

Ac-(SEQ ID NO: 204)-[K(PYA)] (herein referred to as BCY19600);

Ac-(SEQ ID NO: 204)-[K(PYA)]-triazolyl-PEG ₁₀ -amido-PIB (herein referred to as BCY20014); Ac-(SEQ ID NO: 205)-[K(PYA)] (herein referred to as BCY19638);

Ac-(SEQ ID NO: 206)-[K(PYA)] (herein referred to as BCY19639);

Ac-(SEQ ID NO: 207)-[K(PYA)] (herein referred to as BCY19640);

Ac-(SEQ ID NO: 208)-[K(PYA)] (herein referred to as BCY19641);

Ac-(SEQ ID NO: 209)-[K(PYA)] (herein referred to as BCY19654);

Ac-(SEQ ID NO: 210)-[K(PYA)] (herein referred to as BCY19655);

Ac-(SEQ ID NO: 211)-[K(PYA)] (herein referred to as BCY19658);

Ac-(SEQ ID NO: 212)-[K(PYA)] (herein referred to as BCY19827);

Ac-(SEQ ID NO: 213)-[K(PYA)] (herein referred to as BCY19990); and Ac-(SEQ ID NO: 214)-[K(PYA)] (herein referred to as BCY20268); wherein PYA represents pentynoic acid and PIB represents 4(4-iodophenyl)butyrate.

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 36)-A (herein referred to as BCY15342);

A-(SEQ ID NO: 37)-A (herein referred to as BCY15343);

Ac-A-(SEQ ID NO: 37)-A (herein referred to as BCY16322);

Ac-(SEQ ID NO: 37) (herein referred to as BCY16926);

A-(SEQ ID NO: 38)-A (herein referred to as BCY15344);

A-(SEQ ID NO: 39)-A (herein referred to as BCY15345);

A-(SEQ ID NO: 40)-A (herein referred to as BCY15346);

A-(SEQ ID NO: 41)-A (herein referred to as BCY15347);

A-(SEQ ID NO: 80)-A (herein referred to as BCY16313);

A-(SEQ ID NO: 81)-A (herein referred to as BCY16314);

A-(SEQ ID NO: 82)-A (herein referred to as BCY16315);

A-(SEQ ID NO: 83)-A (herein referred to as BCY16316);

A-(SEQ ID NO: 84)-A (herein referred to as BCY16318);

A-(SEQ ID NO: 85)-A (herein referred to as BCY16319);

A-(SEQ ID NO: 86)-A (herein referred to as BCY16320);

A-(SEQ ID NO: 87)-A (herein referred to as BCY16321); and Ac-(SEQ ID NO: 88) (herein referred to as BCY16591). In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 37)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15318);

Ac-(SEQ ID NO: 37)-[Sar ₆ ]-[KFI] (herein referred to as BCY16323);

A-(SEQ ID NO: 38)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15319); and Ac-(SEQ ID NO: 88)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY16679).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 37)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15318);

A-(SEQ ID NO: 38)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15319); and Ac-(SEQ ID NO: 88)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY16679).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 37)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15318); and A-(SEQ ID NO: 38)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15319).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 3 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 3 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiSNTWHWTDCiiLAECiii (SEQ ID NO: 45); and CiNLWNGDPWCiiLLRCiii (SEQ ID NO: 47); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEC ID NO: 45)-A (herein referred to as BCY15351); and A-(SEC ID NO: 47)-A (herein referred to as BCY15353).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 3 amino acids, the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEC ID NO: 45)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15322); and A-(SEC ID NO: 47)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15323).

In an alternative embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 4 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 4 amino acids and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

CiHQLM Dl WDCiiLRPDCiii (SEQ ID NO: 42); and CiLTAREKIQCiiLQRRCiii (SEQ ID NO: 43); wherein C,, C _M and C represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 4 amino acids, the molecular scaffold is TCMT, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 42)-A (herein referred to as BCY15348); and A-(SEQ ID NO: 43)-A (herein referred to as BCY15349).

In a still yet further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 8 amino acids and the other of which consists of 2 amino acids, the molecular scaffold is TCMT, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 42)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15320); and A-(SEQ ID NO: 43)-A-[Sar ₆ ]-[KFI] (herein referred to as BCY15321).

In one particular embodiment, the peptide ligand of the invention is selected from BCY15324, BCY16679, BCY15299, BCY15437, BCY15310, BCY16298 and BCY16287. These peptide ligands are believed to bind to 7 different epitopes of the S1 spike protein (referred to internally as Epitopes 1, 2, 3, 4, 5, 9 and 10, respectively). More crucially, the peptide ligands of this embodiment provide the significant advantage of being able to bind to a variety of differing mutant/variants of COVID-19 S1 spike protein as evidenced in the data presented herein as Example 3.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sam brook et ai, Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et ai, Short Protocols in Molecular Biology (1999) 4 ^th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within peptides of the invention, cysteine residues (Ci, C _M and C _m ) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within peptides of the invention is referred to as below:

Ci- H i - H ₂ -A ₃ -Cii- P ₄ - 15- L ₆ -T ₇ -G ₈ - W ₉ -Ciii (SEQ ID NO: 1). For the purpose of this description, all bicyclic peptides are assumed to be cyclised with TATA, TATB or TCMT and yielding a tri-substituted structure. Cyclisation with TATA, TATB or TCMT occurs on the first, second and third reactive groups (i.e. C,, C _M , Cm).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal bAIq-BqM 0-Ala tail would be denoted as: bAIq-BqMO-A-^EO ID NO: X).

Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus becomes C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalently bound to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e. cysteine residues) which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide is bound to the scaffold. In the present case, the peptides comprise at least three cysteine residues (referred to herein as C,, C _M and C _m ), and form at least two loops on the scaffold.

Half-Life Extending Moieties

In one embodiment, the peptide ligand may additionally comprise a half-life extending moiety in order to extend and improve the half-life of the resultant peptide ligand. One such example of a half-life extending moiety is a polyethylene glycol (PEG) moiety, such as triazolyl-PEGio- amido-PIB (wherein PIB represents 4(4-iodophenyl)butyrate). BCY20014 is an example of a bicyclic peptide ligand of the invention described herein which contains this half-life extending moiety. Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

Species cross-reactivity. Certain ligands demonstrate cross-reactivity across Lipid II from different bacterial species and hence are able to treat infections caused by multiple species of bacteria. Other ligands may be highly specific for the Lipid II of certain bacterial species which may be advantageous for treating an infection without collateral damage to the beneficial flora of the patient;

Protease stability. Bicyclic peptide ligands should ideally demonstrate stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a bicycle lead candidate can be developed in animal models as well as administered with confidence to humans;

Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra/inter-molecular H-bonding, which is important for formulation and absorption purposes;

An optimal plasma half-life in the circulation. Depending upon the clinical indication and treatment regimen, it may be required to develop a bicyclic peptide for short exposure in an acute illness management setting, or develop a bicyclic peptide with enhanced retention in the circulation, and is therefore optimal for the management of more chronic disease states. Other factors driving the desirable plasma half-life are requirements of sustained exposure for maximal therapeutic efficiency versus the accompanying toxicology due to sustained exposure of the agent; and

Selectivity.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands. The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1, 2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L- lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1 , 5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L- pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L- tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO ), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li ⁺ , Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 ⁺ ) and substituted ammonium ions (e.g., NHsR ⁺ , NH2R2 ⁺ , NHR ₃ ⁺ , NFV). 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, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with an alanine, replacement 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; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively.

In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N- terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C- terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.

In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (the group referred to herein as C,) is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target.

In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group (the group referred to herein as C _m ) is synthesized as an amide during peptide synthesis leading to a molecule which is C- terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca- disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid. In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (C,) and/or the C-terminal cysteine (C _m ).

In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues.

In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

In one embodiment, the modified derivative comprises replacement 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 a propensity of D-amino acids to stabilise b-turn conformations (Tugyi et a/ (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved; Incorporating charged groups that exploit long-range ionic interactions, leading to faster on rates and to higher affinities (see for example Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

Incorporating additional constraint into the peptide, by for example constraining side chains of amino acids correctly such that loss in entropy is minimal upon target binding, constraining the torsional angles of the backbone such that loss in entropy is minimal upon target binding and introducing additional cyclisations in the molecule for identical reasons.

(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ² H (D) and ³ H (T), carbon, such as ¹¹ C, ¹³ C and ¹⁴ C, chlorine, such as ³⁶ CI, fluorine, such as ¹⁸ F, iodine, such as ¹²³ l, ¹²⁵ l and ¹³¹ l, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ 0, ¹⁷ 0 and ¹⁸ 0, phosphorus, such as ³² P, sulfur, such as ³⁵ S, copper, such as ⁶⁴ Cu, gallium, such as ⁶⁷ Ga or ⁶⁸ Ga, yttrium, such as ⁹⁰ Y and lutetium, such as ¹⁷⁷ Lu, and Bismuth, such as ²¹³ Bi.

Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³ H (T), and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ² H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹ C, ¹⁸ F, ¹⁵ 0 and ¹³ N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

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

Molecular Scaffold

Molecular scaffolds are described in, for example, WO 2009/098450 and references cited therein, particularly WO 2004/077062 and WO 2006/078161.

As noted 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 reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.

The molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides. The molecular scaffold of the invention contains chemical groups that allow functional groups of the polypeptide of the encoded library of the invention to form covalent links with the molecular scaffold. Said chemical groups are selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

Scaffold reactive groups that could be used on the molecular scaffold to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloalkanes).

Examples include bromomethylbenzene or iodoacetamide. Other scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides, ab unsaturated carbonyl containing compounds and a-halomethylcarbonyl containing compounds. Examples of maleimides which may be used as molecular scaffolds in the invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris- (maleimido)benzene.

In one embodiment, the molecular scaffold is selected from 1,T,1"-(1,3,5-triazinane-1,3,5- triyl)triprop-2-en-1-one (also known as triacryloylhexahydro-s-triazine; TATA), 1,3,5- tris(bromoacetyl) hexahydro-1,3,5-triazine (TATB) and 2,4,6-tris(chloromethyl)-s-triazine (TCMT).

In a further embodiment, the molecular scaffold is 1 , 1 ', 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop- 2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA):

TATA. Thus, following cyclisation with the bicyclic peptides of the invention on the C,, C _M , and Cm cysteine residues, the molecular scaffold forms a tri-substituted 1,T,1"-(1,3,5-triazinane- 1,3,5-triyl)tripropan-1-one derivative of TATA having the following structure: wherein * denotes the point of attachment of the three cysteine residues.

In an alternative embodiment, the molecular scaffold is 1 ,3,5-tris(bromoacetyl) hexahydro-1, 3,5-triazine (TATB):

Thus, following cyclisation with the bicyclic peptides of the invention on the C,, C _M , and C _m cysteine residues, the molecular scaffold forms a tri-substituted 1,3,5-tris(bromoacetyl) hexahydro-1, 3,5-triazine derivative of TATB having the following structure: wherein * denotes the point of attachment of the three cysteine residues.

In an alternative embodiment, the molecular scaffold is 2,4,6-tris(chloromomethyl)-s-triazine (TCMT):

TCMT.

Thus, following cyclisation with the bicyclic peptides of the invention on the C,, C _M , and Cm cysteine residues, the molecular scaffold forms a tri-substituted 2,4,6-tris(chloromomethyl)-s- triazine derivative of TCMT having the following structure: wherein * denotes the point of attachment of the three cysteine residues.

Details of chemistryrelated to TCMT and the use of the corresponding bromide and its use in the formation of cyclic peptides are described in van de Langemheen et al (2016)

ChemBioChem 10.1002/cbic.201600612

(https://onlinelibrarv.wilev.com/doi/abs/10.1002/cbic.201 600612).

Reactive Groups

The molecular scaffold of the invention may be bonded to the polypeptide via functional or reactive groups on the polypeptide. These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. Such reactive groups may be a cysteine side chain, a [Dap(Me)] group, a lysine side chain, or an N-terminal amine group or any other suitable reactive group. Details may be found in WO 2009/098450. In one embodiment, the reactive groups are all cysteine residues.

Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold/molecular core.

The polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.

In a preferred embodiment, polypeptides with three reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is favourable for several reasons. The nucleic acids of the compound libraries encode only the primary sequences of the polypeptide but not the isomeric state of the molecules that are formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer that was isolated in a screening or selection process. The formation of a single product isomer is also advantageous if a specific member of a library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.

In another embodiment of the invention, polypeptides with four reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomer can be determined by chemically synthesizing both isomers, separating the two isomers and testing both isomers for binding to a target ligand.

In one embodiment of the invention, at least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core. Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at least three bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved.

In another embodiment, the reactive groups of the polypeptide of the invention are reacted with molecular linkers wherein said linkers are capable to react with a molecular scaffold so that the linker will intervene between the molecular scaffold and the polypeptide in the final bonded state.

In some embodiments, amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid. Excluded from these exchangeable amino acids are the ones harbouring functional groups for cross-linking the polypeptides to a molecular core, such that the loop sequences alone are exchangeable. The exchangeable polypeptide sequences have either random sequences, constant sequences or sequences with random and constant amino acids. The amino acids with reactive groups are either located in defined positions within the polypeptide, since the position of these amino acids determines loop size.

In one embodiment, an polypeptide with three reactive groups has the sequence (X)iY(X) _m Y(X)nY(X) _o , wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 3 and 6 defining the length of intervening polypeptide segments, which may be the same or different, and I and o are numbers between 0 and 20 defining the length of flanking polypeptide segments.

Alternatives to thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively these techniques may be used in modification or attachment of further moieties (such as small molecules of interest which are distinct from the molecular scaffold) to the polypeptide after they have been selected or isolated according to the present invention - in this embodiment then clearly the attachment need not be covalent and may embrace non-covalent attachment. These methods may be used instead of (or in combination with) the thiol mediated methods by producing phage that display proteins and peptides bearing unnatural amino acids with the requisite chemical reactive groups, in combination small molecules that bear the complementary reactive group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection/isolation phase. Further details can be found in WO 2009/098450 or Heinis, et al. , Nat Chem Biol 2009, 5 (7), 502-7. Synthesis

The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et ai. (supra).

Thus, the invention also relates to manufacture of polypeptides selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide made by chemical synthesis.

Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities.

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

Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptide to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TATA, TATB or TCMT) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptide, forming a disulfide -linked bicyclic peptide-peptide conjugate. Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically- acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The compounds of the invention can be used alone or in combination with another agent or agents.

The compounds of the invention can also be used in combination with biological therapies such as nucleic acid based therapies, antibodies, bacteriophage or phage lysins.

The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. Preferably, the pharmaceutical compositions according to the invention will be administered parenterally. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

The peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktail thereof can be administered for therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 10 pg to 250 mg of selected peptide ligand per kilogram of body weight, with doses of between 100 pg to 25 mg/kg/dose being more commonly used.

A composition containing a peptide ligand according to the present invention may be utilised in therapeutic settings to treat a microbial infection or to provide prophylaxis to a subject at risk of infection e.g. undergoing surgery, chemotherapy, artificial ventilation or other condition or planned intervention. In addition, the peptide ligands described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding agents.

Polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. In some applications, such as vaccine applications, the ability to elicit an immune response to predetermined ranges of antigens can be exploited to tailor a vaccine to specific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

According to a further aspect of the invention, there is provided a peptide ligand as defined herein, for use in suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2.

According to a further aspect of the invention, there is provided a method of suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2, which comprises administering to a patient in need thereof the peptide ligand as defined herein.

References herein to “disease or disorder mediated by infection of SARS-CoV-2” include: respiratory disorders, such as a respiratory disorder mediated by an inflammatory response within the lung, in particular COVID-19. References herein to the term "suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available.

Screening Methods

It will be appreciated that the bicyclic peptide ligands of the invention also find utility as agents for screening for other SARS-CoV-2 binding agents.

For example, screening for a SARS-CoV-2 binding agent may typically involve incubating a bicyclic peptide ligand of the invention with SARS-CoV-2 in the presence and absence of a test compound and assessing a difference in the degree of binding, such that a difference in binding will result from competition of the test compound with the bicyclic peptide ligand of the invention for binding to SARS-CoV-2.

Thus, according to a further aspect of the invention, there is provided a method of screening for a compound which binds to SARS-CoV-2 wherein said method comprises the following steps:

(a) incubating a peptide ligand as defined herein with SARS-CoV-2;

(b) measuring the binding activity of said peptide ligand;

(c) incubating said peptide ligand from step (a) with a test compound and SARS-

CoV-2;

(d) measuring the binding activity of said peptide ligand; and

(e) comparing the binding activity in steps (b) and (d), such that a difference in binding activity of said peptide ligand is indicative of the test compound binding to SARS- CoV-2.

In one embodiment, the peptide ligand comprises a reporter moiety for ease of detecting binding. In a further embodiment, the reporter moiety comprises fluorescein (FI). In a yet further embodiment, the peptide ligand comprises any of the peptide ligands described herein which comprise a fluorescein (FI) moiety.

Diagnostic Methods It will be appreciated that the bicyclic peptide ligands of the invention also find utility as agents for diagnosing infection of SARS-CoV-2.

For example, diagnosis of SARS-CoV-2 infection may typically involve incubating a bicyclic peptide ligand of the invention with SARS-CoV-2 in the presence and absence of a test compound and assessing a difference in the degree of binding, such that a difference in binding will result from competition of the test compound with the bicyclic peptide ligand of the invention for binding to SARS-CoV-2.

Thus, according to a further aspect of the invention, there is provided a method of diagnosing SARS-CoV-2 infection wherein said method comprises the following steps: a) obtaining a biological sample from an individual;

(b) incubating a peptide ligand as defined herein with the biological sample obtained in step (a); and

(c) detecting binding of said peptide ligand to SARS-CoV-2 , such that a detection of measurable binding activity is indicative of a diagnosis of SARS-CoV-2 infection.

In one embodiment, the peptide ligand comprises a reporter moiety for ease of detecting binding. In a further embodiment, the reporter moiety comprises fluorescein (FI). In a yet further embodiment, the peptide ligand comprises any of the peptide ligands described herein which comprise a fluorescein (FI) moiety.

The invention is further described below with reference to the following examples.

EXAMPLES

Materials and Methods

Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc-amino acids were employed (Sigma, Merck), with appropriate side chain protecting groups: where applicable standard coupling conditions were used in each case, followed by deprotection using standard methodology. Alternatively, peptides were purified using HPLC and following isolation they were modified with the required molecular scaffold (namely, TATA, TATB or TCMT). For this, linear peptide was diluted with 50:50 MeCNLFhO up to ~35 ml_, -500 pl_ of 100 mM scaffold in acetonitrile was added, and the reaction was initiated with 5 ml_ of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed for -30 -60 min at RT, and lyophilised once the reaction had completed (judged by MALDI). Once completed, 1ml of 1M L-cysteine hydrochloride monohydrate (Sigma) in H2O was added to the reaction for -60 min at RT to quench any excess TATA, TATB or TCMT.

Following lyophilisation, the modified peptide was purified as above, while replacing the Luna C8 with a Gemini C18 column (Phenomenex), and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct scaffold-modified material were pooled, lyophilised and kept at -20°C for storage.

All amino acids, unless noted otherwise, were used in the L- configurations.

In some cases peptides are converted to activated disulfides prior to coupling with the free thiol group of a toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2- pyridylthio)pentanoate) (100mM) in dry DMSO (1.25 mol equiv) was added to a solution of peptide (20mM) in dry DMSO (1 mol equiv). The reaction was well mixed and DIPEA (20 mol equiv) was added. The reaction was monitored by LC/MS until complete.

BIOLOGICAL DATA

Example 1 : Affinity determination by fluorescence polarization (FP) direct binding.

Bicycles labelled with fluorescein (tracers) were screened in a fluorescence polarisation direct binding assay to determine affinity (K _d ) forwild-type (WT) S1 domain (ACROBiosystems, S1N-C82E8) of the SARS-CoV-2 Spike Protein. Tracers were added at 1 nM final to a titration of individual SARS-CoV-2 Spike Protein variants in assay buffer (PBS + 0.01 % Tween20, pH7.4) to a maximum of 2.54mM. Fluorescence was measured at 485/520/520 on a BMG PHERAstar FSX plate reader. Where appropriate, SARS-CoV-2 Spike protein variants alone parallel and perpendicular intensities were subtracted before mP was calculated. Subsequently, mP data was fit to non-linear regression analysis in Dotmatics to generate a K _d value. Where no significant assay window was generated, data was reported to show no binding at maximum concentration of protein. Where a K _d was generated above the top concentration of protein tested, the result was flagged as K _d greater than maximum concentration of protein tested - results associated with this flag may be displayed as K _d > x mM.

Selected bicyclic peptides of the invention were tested in the above mentioned direct binding assay and the results are shown in Table 1:

Table 1 : Direct Binding Assay Results for Selected Bicyclic Peptides of the

Invention

Example 2: Surface Plasmon Resonance (SPR) Assay

Selected bicyclic peptides of the invention were tested in an SPR assay in accordance with standard procedures known to those skilled in the art and the results are shown in Table 2:

Table 2: SPR Assay Results for Selected Bicyclic Peptides of the Invention

Example 3: Affinity determination by fluorescence polarization (FP) direct binding using mutated variants of Spike Protein

This experiment was conducted in the same manner as described in Example 1 to assess binding of selected bicyclic peptides of the invention against a selection of mutated variants of the isolated S1, S1-NTD and S1-RBD domains of spike protein. The results are shown in Table 3 below.

Table 3: Direct Binding Assay Results for Selected Bicyclic Peptides of the Invention

The effectiveness of the tested peptides from Table 3 against the many S1 spike protein mutants may be summarised in Table 4:

BIC-C-P2967PCT 64

Table 4: Summary of Binding Status of Tested Bicyclic Peptides to S1 Spike Protein Variants

BIC-C-P2967PCT 65

Legend nt = not tested = measurable binding ~ = K _d greater than maximum concentration tested - binding observed shaded cell = no binding at maximum concentration

The results of Example 3 demonstrate that the tested bicyclic peptides demonstrated excellent binding activity to the mutated variants of the S1 spike protein with only several instances of no binding observed within the limits of the assay.

Example 4: Affinity determination by Surface Plasmon Resonance (SPR) against multiple Spike Protein constructs

SPR assays were performed on a Biacore T200 (Cytiva) with Series S Streptavidin (SA) sensor chips (Cytiva) in assay buffer (pH7.4, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.05 % (v/v) Surfactant P20, 2% DMSO) or alternatively nickle chloride activated Sesies S

Sensor Chip NTA (Cytiva) in assay buffer (PBS-P+ (Cytiva), 1% DMSO). Spike protein contructs (ACROBiosystems) protein was captured to generate 3000-4000 RU. Peptide binding was performed at 25 °C, using a 30 mI/min flow rate with appropriate association and dissociation periods. Bicycles were assayed at concentrations between no greater than IOOOOhM in either a multiple or single cycle kinetic format. All data were double- referenced against blank injections and reference surface (treated with assay buffer) using standard processing procedures. Regeneration of streptavidin surface was performed using 1mM HCI (30 s at 30 pL/min) followed by a stabilization period of 30 s. Each injection was followed by an additional wash with 50% DMSO, to reduce Bicycle carry-over. Kinetic values (k„, k, and K _D ) were derived from the sensorgrams by applying 1:1 Binding or Steady-State analysis, using the Biacore™ T200 Evaluation Software (version 3.1).

Selected bicyclic peptides of the invention were tested in the above mentioned SPR assay and the results are shown in Table 5:

Table 5: SPR Assay Results for Selected Bicyclic Peptides of the Invention Example 5: Pseudovirus Neutralisation Assay

Replication deficient SARS-CoV-2 pseudotyped HIV-1 virions were prepared similarly as described in Mallery et al (2021) Sci Adv 7(11). Briefly, virions were produced in HEK 293T cells by transfection with 1 pg of the plasmid encoding SARS CoV-2 Spike protein (pCAGGS-SpikeAc19), 1 pg pCRV GagPol and 1.5 pg GFP-encoding plasmid (CSGW). Viral supernatants were filtered through a 0.45 pm syringe filter at 48 h and 72 h post transfection and pelleted for 2 h at 28,000 x g. Pelleted virions were drained and then resuspended in DMEM (Gibco).

HEK 293T-hACE2-TMPRSS2 cells were prepared as described in Papa et al (2021) PLoS Pathog 17(1): p. e1009246. Cells were plated into 96-well plates at a density of 2 x 103 cells per well in Free style 293T expression media and allowed to attach overnight. 18 pi pseudovirus-containing supernatant was mixed with 2 pi dilutions of bicyclic peptide and incubated for 40 min at RT. 10 pi of this mixture was added to cells. 72 h later, cell entry was detected through the expression of GFP by visualisation on an Incucyte S3 live cell imaging system (Sartorius). The percent of cell entry was quantified as GFP positive areas of cells over the total area covered by cells. Entry inhibition by the Bicycle was calculated as percent virus infection relative to virus only control.

Selected bicyclic peptides of the invention were tested in the above mentioned Pseudovirus Neutralisation Assay and the results are shown in Table 6:

Table 6: Pseudovirus Neutralisation Assay Results for Selected Bicyclic

Peptides of the Invention

Example 6: SARS-CoV-2 Cvtopathic Effect (CPE)

A549_ACE_TMPRSS2 cells were seeded in 96-well plates and cultured overnight. The following day, 4-fold serial dilutions of the bicyclic peptides were prepared in medium and 60 pi of the diluted compounds starting from a maximum concentration of 30, 15, 10, 3, 1 , or 0.1 pM were added to the plates with cells. After 3h pre-incubation, cells were infected with SARS-CoV-2 GLA-1 at MOI 0.04 PFU/cell. One dose of 522 PFU of the virus in 60 pi per well was added to the wells containing compounds. Plates were incubated for 72 h at 37°C, fixed and stained when the cytopathic effect (CPE) was visible. Plates were scanned in a plate reader to quantitate the levels of CPE.

Selected bicyclic peptides of the invention were tested in the above mentioned Cytopathic Effect Assay and the results are shown in Table 7:

Table 7: Cytopathic Effect Assay Results for Selected Bicyclic Peptides of the

Invention