Note: Descriptions are shown in the official language in which they were submitted.
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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 Si 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
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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 COVI D-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 51 that is responsible for receptor binding
and a C-
terminal S2 domain responsible for fusion. CoV diversity is reflected in the
variable spike
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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 Si of S2
domain of the spike
protein (S protein). In a yet further embodiment, said peptide ligand binds to
the Si domain
of the spike protein (Si protein). VVithout being bound by theory it is
believed that binding to
the Si 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:
C,HHACõPILTGWC,,, (SEQ ID NO: 1);
C,PHAC,,PSLWGWC,,, (SEQ ID NO: 6);
C,LHACõPRLTHWCõ, (SEQ ID NO: 7);
C,LHACHQYLWGYC,,, (SEQ ID NO: 8);
C,SHACõPRLFGWC,,, (SEQ ID NO: 9);
C,QHACõPYLWDYCõ, (SEQ ID NO: 10);
C,PFACõHKLYGWC,,, (SEQ ID NO: 58);
C,MKACõPYLYGWCõ, (SEQ ID NO: 59);
C,RHACõTHLYGHC,,, (SEQ ID NO: 60);
C,PYACõTRLYGWC,,, (SEQ ID NO: 61);
C,SHACõPRLTGWCõ, (SEQ ID NO: 62);
C,LHSC,,PRLSGWC,,, (SEQ ID NO: 63);
C,RHSCõPILTGWC,,, (SEQ ID NO: 64);
C,GHSCõPVLWGWCõ, (SEQ ID NO: 65);
C,PHSCõPKLFGWC,,, (SEQ ID NO: 66);
C,THSCõPYLFGWC,,, (SEQ ID NO: 67);
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CiDWTCHYLTMMPCiii (SEQ ID NO: 118);
CiDWTCHYLRPLPCiii (SEQ ID NO: 119);
CiDWTCHYMSMKPCiii (SEQ ID NO: 120);
CiDWTCHYFRPLPCiii (SEQ ID NO: 121); and
CiDWTCHYISPMFDCiii (SEQ ID NO: 122);
wherein Ci, CH and CH; 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:
(SEQ ID NO: 1);
CilDHACHPSLWGWCiii (SEQ ID NO: 6);
CiLHACHPRLTHWCiii (SEQ ID NO: 7);
CiLHACHQYLWGYCiii (SEQ ID NO: 8);
CiSHACHPRLFGWCiii (SEQ ID NO: 9);
COHACHPYLWDYCiii (SEQ ID NO: 10);
CilDFACHHKLYGWCiii (SEQ ID NO: 58);
CilVIKACHPYLYGWCiii (SEQ ID NO: 59);
CiRHACHTHLYGHCiii (SEQ ID NO: 60);
CiPYACHTRLYGWCiii (SEQ ID NO: 61);
CiSHACHPRLTGWCiii (SEQ ID NO: 62);
(SEQ ID NO: 63);
CiRHSCHPILTGWCiii (SEQ ID NO: 64);
CiGHSCHPVLWGWCiii (SEQ ID NO: 65);
CilDHSCHPKLFGWCiii (SEQ ID NO: 66); and
CiTHSCHPYLFGWCiii (SEQ ID NO: 67);
wherein Ci, CH and CH; 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:
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A-(SEQ ID NO: 1)-A (herein referred to as B0Y15230);
A-(SEQ ID NO: 6)-A (herein referred to as B0Y15235);
A-(SEQ ID NO: 7)-A (herein referred to as B0Y15236);
A-(SEQ ID NO: 8)-A (herein referred to as B0Y15237);
A-(SEQ ID NO: 9)-A (herein referred to as B0Y15238);
A-(SEQ ID NO: 10)-A (herein referred to as B0Y15239);
A-(SEQ ID NO: 58)-A (herein referred to as B0Y15364);
A-(SEQ ID NO: 59)-A (herein referred to as B0Y15365);
A-(SEQ ID NO: 60)-A (herein referred to as B0Y15366);
A-(SEQ ID NO: 61)-A (herein referred to as B0Y15367);
A-(SEQ ID NO: 62)-A (herein referred to as B0Y15368);
A-(SEQ ID NO: 63)-A (herein referred to as B0Y15369);
A-(SEQ ID NO: 64)-A (herein referred to as B0Y15370);
A-(SEQ ID NO: 65)-A (herein referred to as BCY15371);
A-(SEQ ID NO: 66)-A (herein referred to as B0Y15372); and
A-(SEQ ID NO: 67)-A (herein referred to as B0Y15373).
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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 6)-A-[Sar6]-[KFI] (herein referred to as BCY15303); and
A-(SEQ ID NO: 63)-A-[Sar6]-[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).
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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:
C,DVVTCõYLNIYHECõ, (SEQ ID NO: 123);
C,DVVTCõYMDYLSNCõ, (SEQ ID NO: 124);
C,DVVTCõYLRIHEACõ, (SEQ ID NO: 125);
C,DVVTCõYMRINDACõ, (SEQ ID NO: 126); and
C,DWTCõYINIYNTCõ, (SEQ ID NO: 127);
wherein Cõ Cõ and Cõ, 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.
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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:
C,LTNDCõHSDIRYCõ, (SEQ ID NO: 29); and
C,ITNDCõHTSLIFCõ, (SEQ ID NO: 30);
wherein Cõ Cõ and Cõ, 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 (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 30)-A-[5ar6]-[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:
C,VDANCõKIKILQRMCõ, (SEQ ID NO: 3);
C,TSSVCõKIKELQRKCõ, (SEQ ID NO: 4);
C,RSLLCõEYLQRTDSCõ, (SEQ ID NO: 5);
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CiLTKSCHKIKMLQRVCiii (SEQ ID NO: 14);
CilVIQPSCHRVLQLQRVCiii (SEQ ID NO: 15);
CALPSCHRILHLQHRCiii (SEQ ID NO: 16);
Cil-IDAHCHKILELQHRCiii (SEQ ID NO: 17);
CiTSSHCHIRVLEEQRLCiii (SEQ ID NO: 18);
CiPIRDRCRTAWLYGLCiii (SEQ ID NO: 19);
CAEAGCHIRVKQLQQICiii (SEQ ID NO: 20);
CiTPSPCHIRVKELQRACiii (SEQ ID NO: 21);
CiSTANCHRILELQQLCiii (SEQ ID NO: 26);
CMGRLCHSTATDIRKCiii (SEQ ID NO: 44);
CiRQSQCOVVVVAIRSFCiii (SEQ ID NO: 48; herein referred to as B0Y16983
when cornplexed with TATB);
CiTDATCHSIKRLQRLCiii (SEQ ID NO: 49);
CiSPVSCHIDSGFKFGLCiii (SEQ ID NO: 50);
CiDSPWCHIRIRSLQRQCiii (SEQ ID NO: 68);
CiSVGACHIRVKLLQRVCiii (SEQ ID NO: 69);
CilVIFVPCHAVREILGLCiii (SEQ ID NO: 70);
CiSDLMCONYLQRTDSCiii (SEQ ID NO: 128);
CiNSYMCONYLQRTDSCiii (SEQ ID NO: 129);
CiTSYLCONYLQRTDSCiii (SEQ ID NO: 130); and
CiRSLMCONYLNQTDSCiii (SEQ ID NO: 131);
wherein Ci, Cu and Cu; 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:
CiVDANCHKIKILQRMCiii (SEQ ID NO: 3);
CiTSSVC;;KIKELQRKCiii (SEQ ID NO: 4);
CiRSLLCHEYLQRTDSCiii (SEQ ID NO: 5);
CiLTKSCHKIKMLQRVCiii (SEQ ID NO: 14);
CiMQPSCHRVLQLQRVCiii (SEQ ID NO: 15);
CALPSCHRILHLQHRCiii (SEQ ID NO: 16);
Cil-IDAHCHKILELQHRCiii (SEQ ID NO: 17);
CiTSSHCHIRVLEEQRLCiii (SEQ ID NO: 18);
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C,PRDRCõPTAWLYGLCõ, (SEQ ID NO: 19);
C,AEAGCõRVKQLQQICõ, (SEQ ID NO: 20);
C,TPSPCõRVKELQRACõ, (SEQ ID NO: 21);
C,STANCõRILELQQLCõ, (SEQ ID NO: 26);
C,VGRLC,,STATDIRKC,,, (SEQ ID NO: 44);
C,RQSQCõDVWVAIRSFCõ, (SEQ ID NO: 48; herein referred to as B0Y16983
when cornplexed with TATB);
C,TDATCõSIKRLQRLCõ, (SEQ ID NO: 49);
C,SPVSCõPSGFKFGLCõ, (SEQ ID NO: 50);
C,DSPWCõRIRSLQRQCõ, (SEQ ID NO: 68);
C,SVGACõRVKLLQRVCõ, (SEQ ID NO: 69); and
C,MFVPCõAVREILGLCõ, (SEQ ID NO: 70);
wherein Cõ Cõ and Cõ, 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 B0Y15334);
A-(SEQ ID NO: 15)-A (herein referred to as B0Y15244);
A-(SEQ ID NO: 16)-A (herein referred to as B0Y15245);
A-(SEQ ID NO: 17)-A (herein referred to as B0Y15246);
A-(SEQ ID NO: 18)-A (herein referred to as B0Y15247);
A-(SEQ ID NO: 19)-A (herein referred to as B0Y15248);
A-(SEQ ID NO: 20)-A (herein referred to as B0Y15249);
A-(SEQ ID NO: 21)-A (herein referred to as B0Y15250);
A-(SEQ ID NO: 26)-A (herein referred to as B0Y15255);
A-(SEQ ID NO: 48)-A (herein referred to as B0Y15354);
A-(SEQ ID NO: 48)-A (herein referred to as B0Y16534);
A-(SEQ ID NO: 48)-AK (herein referred to as B0Y16896);
A-(SEQ ID NO: 49)-A (herein referred to as B0Y15355); and
A-(SEQ ID NO: 50)-A (herein referred to as B0Y15356).
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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 (Fl), and comprises an amino acid sequence which is selected
from:
A-(SEQ ID NO: 3)-A-[Sar6]-[KFI] (herein referred to as BCY15301);
A-(SEQ ID NO: 15)-A-[Sar6]-[KFI] (herein referred to as BCY15307);
A-(SEQ ID NO: 17)-A-[Sar6]-[KFI] (herein referred to as BCY15308);
A-(SEQ ID NO: 19)-A-[Sar6]-[KFI] (herein referred to as BCY15309);
A-(SEQ ID NO: 48)-A-[Sar6]-[KFI] (herein referred to as BCY15324);
A-(SEQ ID NO: 49)-A-[Sar6]-[KFI] (herein referred to as BCY15325); and
A-(SEQ ID NO: 50)-A-[Sar6]-[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:
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A-(SEQ ID NO: 3)-A (herein referred to as B0Y15232);
A-(SEQ ID NO: 4)-A (herein referred to as B0Y15233);
A-(SEQ ID NO: 5)-A (herein referred to as B0Y15234);
A-(SEQ ID NO: 14)-A (herein referred to as B0Y15243);
A-(SEQ ID NO: 44)-A (herein referred to as B0Y15350);
A-(SEQ ID NO: 68)-A (herein referred to as B0Y15374);
A-(SEQ ID NO: 69)-A (herein referred to as B0Y15375); and
A-(SEQ ID NO: 70)-A (herein referred to as B0Y15376).
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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 3)-A-[Sar6]-[KFI] (herein referred to as BCY15300);
A-(SEQ ID NO: 5)-A-[Sar6]-[KFI] (herein referred to as BCY15302); and
A-(SEQ ID NO: 70)-A-[Sar6]-[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:
C,FDDWTCõYIQMCõ, (SEQ ID NO: 115);
wherein Cõ Cõ and Cõ, 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 (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 115)-A-[5ar6]-[KFI] (herein referred to as BCY15437).
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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:
C,TLMDPWCõLLKCõ, (SEQ ID NO: 71);
C,KIHDVVTCõLLRCõ, (SEQ ID NO: 72); and
C,IPLDVVTCõMIACõ, (SEQ ID NO: 79; herein referred to as B0Y18707 when
complexed with TATB);
wherein Cõ Cõ and Cõ, 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:
C,TLMDPWCõLLKCõ, (SEQ ID NO: 71);
C,KIHDVVTCõLLRCõ, (SEQ ID NO: 72); and
C,IPLDWTCõMIACõ, (SEQ ID NO: 79);
wherein Cõ Cõ and Cõ, 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
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additionally comprises N- and/or C-terminal additions and a labelling moiety,
such as
fluorescein (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 71)-A-[Sar6]-[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:
C,EYQGPHCõYRLYCõ, (SEQ ID NO: 11);
C,EYNGPYCõYRLYCõ, (SEQ ID NO: 132); and
C,EYVGPMCõYRLYCõ, (SEQ ID NO: 133);
wherein Cõ Cõ and Cõ, represent first, second and third cysteine residues,
respectively, or a
pharmaceutically acceptable salt thereof.
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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:
C,EYQGPHCõYRLYCõ, (SEQ ID NO: 11);
wherein Cõ Cõ and Cõ, 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 (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 11)-A-[5ar6]-[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.
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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:
CiEDHDVVVYCHSTCiii (SEQ ID NO: 2);
CAPWNYFRCHDLCiii (SEQ ID NO: 23);
CiLTPEDIWCHMLCiii (SEQ ID NO: 25);
CiENPVDIWCA/LCiii (SEQ ID NO: 28);
CiN/FTTVWDCHLACiii (SEQ ID NO: 46);
CYDPIDVWCHIVIMCiii (SEQ ID NO: 51);
CASYDDFWCA/LCiii (SEQ ID NO: 52);
CiDLTQHWTCHILCiii (SEQ ID NO: 53);
CiSEISDVWCHIVILCiii (SEQ ID NO: 54);
CiPTPVDIWCHMLCiii (SEQ ID NO: 55);
CiEQNGWIYCHSTCiii (SEQ ID NO: 73);
CiTDRSWIFCHSTCiii (SEQ ID NO: 74);
CilDNISWIYCHSTCiii (SEQ ID NO: 75);
CiDVC;;GLNAFNRCiii(SEQ ID NO: 117);
CiLDETWIYCHSTCiii (SEQ ID NO: 134);
CiPDETVVVYCHSTCiii (SEQ ID NO: 135);
CiESNDVVVYCHSTCiii (SEQ ID NO: 136);
CiEDNDVVVYCHSTCiii (SEQ ID NO: 137);
CiPDVSWIYCHSTCiii (SEQ ID NO: 138);
wherein Ci, Cu and Cu; 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:
CiEDHDVVVYCHSTCiii (SEQ ID NO: 2);
CAPWNYFRCHDLCiii (SEQ ID NO: 23);
CiLTPEDIWCHMLCiii (SEQ ID NO: 25);
CiENPVDIWCA/LCiii (SEQ ID NO: 28);
CiN/FTTVWDCHLACiii (SEQ ID NO: 46);
CYDPIDVWCHNIMCiii (SEQ ID NO: 51);
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CASYDDFWCA/LCiii (SEQ ID NO: 52);
CiDLTQHWTCHILCiii (SEQ ID NO: 53);
CiSEISDVWCHIVILCiii (SEQ ID NO: 54);
CiPTPVDIWCHIVILCiii (SEQ ID NO: 55);
CiEQNGWIYCHSTCiii (SEQ ID NO: 73);
CiTDRSWIFCHSTCiii (SEQ ID NO: 74);
CilDNISWIYCHSTCiii (SEQ ID NO: 75); and
CiDVC;;GLNAFNRCiii(SEQ ID NO: 117);
wherein Ci, Cu and Cu; 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:
CiEDHDVVVYCHSTCiii (SEQ ID NO: 2);
CAPWNYFRCHDLCiii (SEQ ID NO: 23);
CiLTPEDIWCHMLCiii (SEQ ID NO: 25);
CiENPVDIWCA/LCiii (SEQ ID NO: 28);
CiN/FTTVWDCHLACiii (SEQ ID NO: 46);
CYDPIDVWCA/IMCiii (SEQ ID NO: 51);
CASYDDFWCA/LCiii (SEQ ID NO: 52);
CiDLTQHWTCHILCiii (SEQ ID NO: 53);
CiSEISDVWCHIVILCiii (SEQ ID NO: 54);
CiPTPVDIWCHMLCiii (SEQ ID NO: 55);
CiEQNGWIYCHSTCiii (SEQ ID NO: 73);
CiTDRSWIFCHSTCiii (SEQ ID NO: 74); and
CilDNISWIYCHSTCiii (SEQ ID NO: 75);
wherein Ci, Cu and Cu; 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:
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A-(SEQ ID NO: 2)-A (herein referred to as B0Y15231);
Ac-(SEQ ID NO: 2) (herein referred to as B0Y16987);
A-(SEQ ID NO: 46)-A (herein referred to as B0Y15352);
A-(SEQ ID NO: 73)-A (herein referred to as B0Y15379);
A-(SEQ ID NO: 74)-A (herein referred to as B0Y15380);
A-(SEQ ID NO: 75)-A (herein referred to as B0Y15381);
A-(SEQ ID NO: 134)-A (herein referred to as B0Y17540);
A-(SEQ ID NO: 135)-A (herein referred to as B0Y17541);
A-(SEQ ID NO: 136)-A (herein referred to as B0Y17542);
A-(SEQ ID NO: 137)-A (herein referred to as B0Y17543); and
A-(SEQ ID NO: 138)-A (herein referred to as B0Y17544).
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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 2)-A-[Sar6]-[KFI] (herein referred to as BCY15299); and
A-(SEQ ID NO: 74)-A-[Sar6]-[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
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additionally comprises N- and/or C-terminal additions and a labelling moiety,
such as
fluorescein (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 117)-A-[Sar6]-[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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 23)-A-[Sar6]-[KFI] (herein referred to as BCY15311);
A-(SEQ ID NO: 25)-A-[Sar6]-[KFI] (herein referred to as BCY15312); and
A-(SEQ ID NO: 53)-A-[Sar6]-[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:
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CASPDNPVC;;RFYCiii (SEQ ID NO: 22; herein referred to as B0Y16534
when cornplexed with TATB);
CYNHANPVCHRYYCiii (SEQ ID NO: 24; herein referred to as B0Y16540
when cornplexed with TATB);
CiDLFLHELCOMPCiii (SEQ ID NO: 27);
CiNKQNWRYCHYLTCiii (SEQ ID NO: 31);
(SEQ ID NO: 56);
CYAPDNPVCiiIRMYCiii (SEQ ID NO: 57);
CiGILADPFCHLISCiii (SEQ ID NO: 76);
CYNHANPVCii[AgNYYCiii (SEQ ID NO: 89);
CASPDNPVCii[AgIDWYCiii (SEQ ID NO: 90);
CASPDNPVCii[Arg(Me)WYCiii (SEQ ID NO: 91);
CASPDNPVCii[HArgWYCiii (SEQ ID NO: 92);
CANPDNPVCiiIRFYCiii (SEQ ID NO: 93);
CiRNPDNPVCiiIRFYCiii (SEQ ID NO: 94);
(SEQ ID NO: 95);
CiVNKHNPVCiiIRFYCiii (SEQ ID NO: 96);
CiVNAENPVCiiIRFYCiii (SEQ ID NO: 97);
CONPGNPVCiiRFYCiii (SEQ ID NO: 98);
CiMNPDNPVC;;RFYCiii (SEQ ID NO: 99);
CYNQENPVCiiIRFYCiii (SEQ ID NO: 100);
CiNNPANPVC;;RFYCiii (SEQ ID NO: 101);
CFNIDNPVCiiIRFYCiii (SEQ ID NO: 102);
CiSNPENPVCiiIRFYCiii (SEQ ID NO: 103);
CiNINEDNPVCiiIRFYCiii (SEQ ID NO: 104);
CiNINEANPVC;;RFYCiii (SEQ ID NO: 105);
(SEQ ID NO: 106);
CANHDNPVCiiIRFYCiii (SEQ ID NO: 107);
CiKNYDNPVCiiIRFYCiii (SEQ ID NO: 108);
CiENMDNPVCiiIRFYCiii (SEQ ID NO: 109);
CiNINTDNPVCiiIRFYCiii (SEQ ID NO: 110);
CiLNVDNPVC;;RFYCiii (SEQ ID NO: 111);
CiLNPDNPVC;;RFYCiii (SEQ ID NO: 112);
CYNHANPVCii[HArg]YYCiii (SEQ ID NO: 113);
CYNHANPVCii[Arg(MeWYCiii (SEQ ID NO: 114);
CiMNPFFYDCHERTCiii (SEQ ID NO: 116);
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C,[AiNSPDNPVCõRFYCõ, (SEQ ID NO: 139);
C,AS[HyP]DNPVCõRFYCõ, (SEQ ID NO: 140);
C,AS[AiNDNPVCõRFYCõ, (SEQ ID NO: 141);
C,AS[PiNDNPVCõRFYCõ, (SEQ ID NO: 142);
C,ASPDN[PipWCõRFYCõ, (SEQ ID NO: 143);
C,ASPDN[44DFPWCõRFYCõ, (SEQ ID NO: 144);
C,ASPDN[4F1ProWCõRFYCõ, (SEQ ID NO: 145);
C,ASPDNPVCõR[1Nalr(Cõ, (SEQ ID NO: 146);
C,ASPDNPVCõR[2Nalr(Cõ, (SEQ ID NO: 147);
C,ASPDNPVCõR[2MePher(Cõ, (SEQ ID NO: 148);
C,ASPDNPVCõR[3MePher(Cõ, (SEQ ID NO: 149);
C,ASPDNPVCõR[4MePher(Cõ, (SEQ ID NO: 150);
C,ASPDNPVCõR[2CIPher(Cõ, (SEQ ID NO: 151);
C,ASPDNPVCõR[3CIPher(Cõ, (SEQ ID NO: 152);
C,ASPDNPVCõR[4CIPher(Cõ, (SEQ ID NO: 153);
C,ASPDNPVCõR[2FPher(Cõ, (SEQ ID NO: 154);
C,ASPDNPVCõR[3FPher(Cõ, (SEQ ID NO: 155);
C,ASPDNPVCõR[4FPher(Cõ, (SEQ ID NO: 156);
C,ASPDNPVCõR[26DiMeTyrr(Cõ, (SEQ ID NO: 157);
C[AiNSPDN[44DFPWCõR[4FPhe]YCõ, (SEQ ID NO: 158);
C,[AiNSPDN[44DFPWCõ[Arg(Me)][4FPhe]YCõ, (SEQ ID NO: 159);
C,[AiNSPDNPVCõR[4FPher(Cõ, (SEQ ID NO: 160);
C,[AiNSPDNPVCõ[Arg(Me)][4FPher(Cõ, (SEQ ID NO: 161);
C,[AiNNPDN[44DFPWCõR[4FPhe]YCõ, (SEQ ID NO: 162);
C,[AiNNPDN[44DFPWCõ[Arg(Me)][4FPhe]YCõ, (SEQ ID NO: 163);
C,[AiNNPDNPVCõR[4FPher(Cõ, (SEQ ID NO: 164); and
C,[AiNNPDNPVCõ[Arg(Me)][4FPher(Cõ, (SEQ ID NO: 165);
wherein Cõ Cõ and Cõ, represent first, second and third cysteine residues,
respectively, Aib
represents aminoisobutyric acid, Agb represents 2-amino-4-guanidinobutyric
acid, Arg(Me)
represents 8-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, 4F1Pro 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,
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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:
CASPDNPVCiiIRFYCiii (SEQ ID NO: 22; herein referred to as B0Y16534
when complexed with TATB);
CiYNHANPVCiiIRYYCiii (SEQ ID NO: 24; herein referred to as B0Y16540
when complexed with TATB);
CiDLFLHELCOMPCiii (SEQ ID NO: 27);
CiNKQNWRYCHYLTCiii (SEQ ID NO: 31);
Cil-IPWSALFCiiNYPCiii (SEQ ID NO: 56);
CiYAPDNPVCiiIRMYCiii (SEQ ID NO: 57);
CiGILADPFCHLISCiii (SEQ ID NO: 76);
CiYNHANPVCii[AgNYYCiii (SEQ ID NO: 89);
CASPDNPVCii[AgbWYCiii (SEQ ID NO: 90);
CASPDNPVCii[Arg(Me)WYCiii (SEQ ID NO: 91);
CiCASPDNPVCii[HArgWYCiii (SEQ ID NO: 92);
CANPDNPVCiiIRFYCiii (SEQ ID NO: 93);
CiRNPDNPVCiiIRFYCiii (SEQ ID NO: 94);
Cil-INPSNPVCiiRFYCiii (SEQ ID NO: 95);
CiVNKHNPVCiiIRFYCiii (SEQ ID NO: 96);
CiVNAENPVCiiIRFYCiii (SEQ ID NO: 97);
CONPGNPVCiiRFYCiii (SEQ ID NO: 98);
CiMNPDNPVC;;RFYCiii (SEQ ID NO: 99);
CiYNQENPVCiiIRFYCiii (SEQ ID NO: 100);
CiNNPANPVC;;RFYCiii (SEQ ID NO: 101);
CFNIDNPVCiiIRFYCiii (SEQ ID NO: 102);
CiSNPENPVCiiIRFYCiii (SEQ ID NO: 103);
CJVINEDNPVCiiIRFYCiii (SEQ ID NO: 104);
CJVINEANPVCiiIRFYCiii (SEQ ID NO: 105);
Cil-INLDNPVCiiIRFYCiii (SEQ ID NO: 106);
CANHDNPVCiiIRFYCiii (SEQ ID NO: 107);
CiKNYDNPVCiiIRFYCiii (SEQ ID NO: 108);
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CiENMDNPVCiiIRFYCiii (SEQ ID NO: 109);
CiMNTDNPVC;;RFYCiii (SEQ ID NO: 110);
Cil_NVDNPVC;;RFYCiii (SEQ ID NO: 111);
Cil_NPDNPVC;;RFYCiii (SEQ ID NO: 112);
CiYNHANPVCii[HArg]YYCiii (SEQ ID NO: 113);
CiYNHANPVCii[Arg(MeWYCiii (SEQ ID NO: 114); and
CiMNPFFYDCHERTCiii (SEQ ID NO: 116);
wherein Ci, Cu and Cu; represent first, second and third cysteine residues,
respectively, Agb
represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 8-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:
CASPDNPVCiiIRFYCiii (SEQ ID NO: 22; herein referred to as B0Y16534
when complexed with TATB);
CiYNHANPVCiiIRYYCiii (SEQ ID NO: 24; herein referred to as B0Y16540
when complexed with TATB);
CiDLFLHELCOMPCiii (SEQ ID NO: 27);
CiNKQNWRYCHYLTCiii (SEQ ID NO: 31);
(SEQ ID NO: 56);
CiYAPDNPVCiiIRMYCiii (SEQ ID NO: 57);
CiGILADPFCHLISCiii (SEQ ID NO: 76);
CiYNHANPVCii[AgNYYCiii (SEQ ID NO: 89);
CASPDNPVCii[AgbWYCiii (SEQ ID NO: 90);
CASPDNPVCii[Arg(Me)WYCiii (SEQ ID NO: 91);
CiCASPDNPVCii[HArgWYCiii (SEQ ID NO: 92);
CANPDNPVCiiIRFYCiii (SEQ ID NO: 93);
CiRNPDNPVCiiIRFYCiii (SEQ ID NO: 94);
CiFINPSNPVCiiRFYCiii (SEQ ID NO: 95);
CiVNKHNPVCiiIRFYCiii (SEQ ID NO: 96);
CiVNAENPVCiiIRFYCiii (SEQ ID NO: 97);
CONPGNPVCiiRFYCiii (SEQ ID NO: 98);
CiMNPDNPVC;;RFYCiii (SEQ ID NO: 99);
CiYNQENPVCiiIRFYCiii (SEQ ID NO: 100);
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C,NNPANPVCõRFYCõ, (SEQ ID NO: 101);
C,FNIDNPVCõRFYCõ, (SEQ ID NO: 102);
C,SNPENPVCõRFYCõ, (SEQ ID NO: 103);
C,MNEDNPVCõRFYCõ, (SEQ ID NO: 104);
C,MNEANPVCõRFYCõ, (SEQ ID NO: 105);
C,HNLDNPVCõRFYCõ, (SEQ ID NO: 106);
C,ANHDNPVCõRFYCõ, (SEQ ID NO: 107);
C,KNYDNPVCõRFYCõ, (SEQ ID NO: 108);
C,ENMDNPVCõRFYCõ, (SEQ ID NO: 109);
C,MNTDNPVCõRFYCõ, (SEQ ID NO: 110);
C,LNVDNPVCõRFYCõ, (SEQ ID NO: 111);
C,LNPDNPVCõRFYCõ, (SEQ ID NO: 112);
C,YNHANPVCõ[HArg]YYCõ, (SEQ ID NO: 113); and
C,YNHANPVCõ[Arg(MeWYCõ, (SEQ ID NO: 114);
wherein Cõ Cõ and Cõ, represent first, second and third cysteine residues,
respectively, Agb
represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 8-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);
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Ac-(SEQ ID NO: 93) (herein referred to as B0Y16903);
Ac-(SEQ ID NO: 94) (herein referred to as B0Y16905);
Ac-(SEQ ID NO: 95) (herein referred to as B0Y16906);
Ac-(SEQ ID NO: 96) (herein referred to as BCY16911);
Ac-(SEQ ID NO: 97) (herein referred to as B0Y16913);
Ac-(SEQ ID NO: 98) (herein referred to as B0Y16915);
Ac-(SEQ ID NO: 99) (herein referred to as B0Y16917);
Ac-(SEQ ID NO: 100) (herein referred to as B0Y16918);
Ac-(SEQ ID NO: 101) (herein referred to as B0Y16921);
Ac-(SEQ ID NO: 102) (herein referred to as B0Y16912);
Ac-(SEQ ID NO: 103) (herein referred to as B0Y16914);
Ac-(SEQ ID NO: 104) (herein referred to as B0Y16916);
Ac-(SEQ ID NO: 105) (herein referred to as B0Y16919);
Ac-(SEQ ID NO: 106) (herein referred to as B0Y16920);
Ac-(SEQ ID NO: 107) (herein referred to as B0Y16902);
Ac-(SEQ ID NO: 108) (herein referred to as B0Y16904);
Ac-(SEQ ID NO: 109) (herein referred to as B0Y16907);
Ac-(SEQ ID NO: 110) (herein referred to as B0Y16908);
Ac-(SEQ ID NO: 111) (herein referred to as B0Y16909);
Ac-(SEQ ID NO: 112) (herein referred to as BCY16910);
A-(SEQ ID NO: 113)-A (herein referred to as B0Y16543);
A-(SEQ ID NO: 114)-A (herein referred to as B0Y16542);
A-(SEQ ID NO: 116)-A (herein referred to as B0Y16207);
Ac-(SEQ ID NO: 116) (herein referred to as B0Y18698);
Ac-(SEQ ID NO: 139) (herein referred to as B0Y17279);
Ac-(SEQ ID NO: 140) (herein referred to as B0Y17281);
Ac-(SEQ ID NO: 141) (herein referred to as B0Y17282);
Ac-(SEQ ID NO: 142) (herein referred to as B0Y17283);
Ac-(SEQ ID NO: 143) (herein referred to as B0Y17287);
Ac-(SEQ ID NO: 144) (herein referred to as B0Y17289);
Ac-(SEQ ID NO: 145) (herein referred to as B0Y17294);
Ac-(SEQ ID NO: 146) (herein referred to as BCY17301);
Ac-(SEQ ID NO: 147) (herein referred to as B0Y17302);
Ac-(SEQ ID NO: 148) (herein referred to as B0Y17303);
Ac-(SEQ ID NO: 149) (herein referred to as B0Y17304);
Ac-(SEQ ID NO: 150) (herein referred to as B0Y17305);
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Ac-(SEQ ID NO: 151) (herein referred to as B0Y17306);
Ac-(SEQ ID NO: 152) (herein referred to as B0Y17307);
Ac-(SEQ ID NO: 153) (herein referred to as B0Y17308);
Ac-(SEQ ID NO: 154) (herein referred to as B0Y17309);
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 B0Y17313);
Ac-(SEQ ID NO: 158) (herein referred to as B0Y18340);
Ac-(SEQ ID NO: 159) (herein referred to as B0Y18341);
Ac-(SEQ ID NO: 160) (herein referred to as B0Y18342);
Ac-(SEQ ID NO: 161) (herein referred to as B0Y18343);
Ac-(SEQ ID NO: 162) (herein referred to as B0Y18344);
Ac-(SEQ ID NO: 163) (herein referred to as B0Y18345);
Ac-(SEQ ID NO: 164) (herein referred to as B0Y18346); and
Ac-(SEQ ID NO: 165) (herein referred to as B0Y18347).
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);
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Ac-(SEQ ID NO: 95) (herein referred to as B0Y16906);
Ac-(SEQ ID NO: 96) (herein referred to as BCY16911);
Ac-(SEQ ID NO: 97) (herein referred to as B0Y16913);
Ac-(SEQ ID NO: 98) (herein referred to as B0Y16915);
Ac-(SEQ ID NO: 99) (herein referred to as B0Y16917);
Ac-(SEQ ID NO: 100) (herein referred to as B0Y16918);
Ac-(SEQ ID NO: 101) (herein referred to as B0Y16921);
Ac-(SEQ ID NO: 102) (herein referred to as B0Y16912);
Ac-(SEQ ID NO: 103) (herein referred to as B0Y16914);
Ac-(SEQ ID NO: 104) (herein referred to as B0Y16916);
Ac-(SEQ ID NO: 105) (herein referred to as B0Y16919);
Ac-(SEQ ID NO: 106) (herein referred to as B0Y16920);
Ac-(SEQ ID NO: 107) (herein referred to as B0Y16902);
Ac-(SEQ ID NO: 108) (herein referred to as B0Y16904);
Ac-(SEQ ID NO: 109) (herein referred to as B0Y16907);
Ac-(SEQ ID NO: 110) (herein referred to as B0Y16908);
Ac-(SEQ ID NO: 111) (herein referred to as B0Y16909);
Ac-(SEQ ID NO: 112) (herein referred to as BCY16910);
A-(SEQ ID NO: 113)-A (herein referred to as B0Y16543); and
A-(SEQ ID NO: 114)-A (herein referred to as B0Y16542).
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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 22)-A-[Sar6]-[KFI] (herein referred to as BCY15310);
A-(SEQ ID NO: 27)-A-[Sar6]-[KFI] (herein referred to as BCY15313);
A-(SEQ ID NO: 56)-A-[Sar6]-[KFI] (herein referred to as BCY15328); and
A-(SEQ ID NO: 116)-A-[Sar6]-[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 (Fl), and comprises an amino acid sequence which is selected from:
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A-(SEQ ID NO: 22)-A-[Sar6]-[KFI] (herein referred to as BCY15310);
A-(SEQ ID NO: 27)-A-[Sar6]-[KFI] (herein referred to as B0Y15313); and
A-(SEQ ID NO: 56)-A-[Sar6]-[KFI] (herein referred to as B0Y15328).
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 (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 31)-A-[Sar6]-[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-(SEQ 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 (Fl), and comprises an amino acid sequence which is:
A-(SEQ ID NO: 76)-A-[Sar6]-[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.
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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:
C,TTSEKVKC,,LQRHPC,,, (SEQ ID NO: 32);
C,QPDMRIKCõLQRVACõ, (SEQ ID NO: 33);
C,SSNNRIKCõLQRVTCõ, (SEQ ID NO: 34);
C,KEKTTIGCõLMAGICõ, (SEQ ID NO: 35); and
C,NRPTSVYCõLQRGIC,,, (SEQ ID NO: 166);
wherein Cõ Cõ and Cõ, 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:
C,TTSEKVKC,,LQRHPC,,, (SEQ ID NO: 32);
C,QPDMRIKCõLQRVACõ, (SEQ ID NO: 33);
C,SSNNRIKCõLQRVTCõ, (SEQ ID NO: 34); and
C,KEKTTIGCõLMAGICõ, (SEQ ID NO: 35);
wherein Cõ Cõ and Cõ, 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
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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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 32)-A-[Sar6]-[KFI] (herein referred to as BCY15316); and
A-(SEQ ID NO: 33)-A-[Sar6]-[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:
C,GRDSSWIYCHSTC,,, (SEQ ID NO: 12);
C,RGTPAWKACõAlCõ, (SEQ ID NO: 13);
C,PFPSGFGTCõTFCõ, (SEQ ID NO: 36);
C,PYVAGRGTCõLLCõ, (SEQ ID NO: 37; herein referred to as BCY16312 when
complexed with TCMT);
C,PYPRGTGSC,,TFCõ, (SEQ ID NO: 38);
C,LYPPGKGTCõLLCõ, (SEQ ID NO: 39);
C,PSPAGRGTCõLLCõ, (SEQ ID NO: 40);
C,PATIGRGPCõTFCõ, (SEQ ID NO: 41);
C,PEANSVVVYC,,STC,,, (SEQ ID NO: 77);
C,APTSGWIYCHSTC,,, (SEQ ID NO: 78);
C,PYVAG[AgNGTO,,LLO,,, (SEQ ID NO: 60);
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CiPYVAG[Arg(MeNTCHLLCiii (SEQ ID NO: 81);
CiPYVAGRGTCHL[Cba]Ciii (SEQ ID NO: 82);
CiPYVAGRGTCii[Cba]LCiii (SEQ ID NO: 83);
CiPYVAGR[dA]TCHLLCiii (SEQ ID NO: 84);
CiPYVAG[HArg]GTCHLLCiii (SEQ ID NO: 85);
CiPYVAGRGTCHL[tBuAla]Ciii (SEQ ID NO: 86);
CiPYVAGRGTCii[tBuAla]LCiii (SEQ ID NO: 87);
CiPYVAG[Agb][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 88);
CiPYVAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 167; herein referred to as
BCY18111 when complexed with TCMT);
CiPYVPG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 168);
Ci[K(PYAUVAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 169);
Ci[HyP]YVAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 170);
CiPYVAGT[dA]TCHL[tBuAla]Ciii (SEQ ID NO: 171);
CiPYVAG[Agb][dA]TCHLLCiii (SEQ ID NO: 172);
CiPYVAG[Agb]GTCHL[tBuAla]Ciii (SEQ ID NO: 173);
CiPYV[HyP]G[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 174);
CiPY[K(PYMAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 175);
Ci[Oic]YVAG[HArg][dA]TC;;L[tBuAla]Ciii (SEQ ID NO: 176);
CiPY[B-Melle]AG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 177);
CiPY[tBuGly]AG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 178);
CiPYPAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 179);
CiP[44BPA]VAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 180);
CiP[2FPhe]VAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 181);
CiPY[Cba]AG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 182);
CiP[3FPhe]VAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 183);
Ci[55DMP]YVAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 184);
CiPYVAGQ[dA]TCHL[tBuAla]Ciii (SEQ ID NO: 185);
CiPYVAG[HArg][dS]TCHL[tBuAla]Ciii (SEQ ID NO: 186);
CiP[4tBuPhe]VAG[HArg][dA]TCHL[tBuAla]Ciii (SEQ ID NO: 187);
CiPYREGTGTCHLLCiii (SEQ ID NO: 188);
CiPYAPGNGTCHLLCiii (SEQ ID NO: 189);
CiPHPPGRGTCHLLCiii (SEQ ID NO: 190);
CiPYNAGTGTCHLLCiii (SEQ ID NO: 191);
CiPYSPGQGTCHLLCiii (SEQ ID NO: 192);
CiPYQPGSGTCHLLCiii (SEQ ID NO: 193);
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C,PFPPGMGTC,,LLC,,, (SEQ ID NO: 194);
C,PHQPGFGTC,,LLC,,, (SEQ ID NO: 195);
C,PYSPGSGTC,,LLC,,, (SEQ ID NO: 198);
C,PYLAGTGTC,,LLC,,, (SEQ ID NO: 197);
C,PWEAGKGTC,,LLC,,, (SEQ ID NO: 198);
C,PYAPGMGTC,,LLC,,, (SEQ ID NO: 199);
C,PHMPGSGTCõLLCõ, (SEQ ID NO: 200);
C,PYNKGEGTC,,LLC,,, (SEQ ID NO: 201);
C,PFKPGVGTC,,LLC,,, (SEQ ID NO: 202);
C,P[4tBuPheNAG[Orn][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 203);
C,[0ic][4tBuPheNAG[HArg][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 204);
C,P[4tBuPheNAG[Dab][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 205);
C,P[4tBuPheNAG[Dap][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 206);
C,P[4CF3PheNAG[HArg][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 207);
C,P[DMAPheNAG[HArg][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 208);
C,P[4tBuPhe]VAG[HArg][dDaNTCõL[tBuAla]Cõ, (SEQ ID NO: 209);
C,P[4tBuPheNAG[HSer][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 210);
C,P[4tBuPheNAG[Cit][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 211);
C[Oic][4tBuPheNAG[Orn][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 212);
C,P[4tBuPheNAG[HArg][dDaNTCõL[tBuAla]Cõ, (SEQ ID NO: 213); and
C,[0ic][4tBuPheNAG[Cit][dAriCõL[tBuAla]Cõ, (SEQ ID NO: 214);
wherein Cõ Cõ and Cõ, represent first, second and third cysteine residues,
respectively, Agb
represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents d-N methyl
arginine, B-
MeIle represents 8-methyl-isoleucine, 44BPA represents 4,4-biphenylalanine,
Cba
represents 8-cyclobutylalanine, 4CF3Phe represents 4-trifluoromethyl-
phenylalanine, Cit
represents citrulline, Dab represents diaminobutanoic acid, Dap represents
diaminopropionic
acid, DMAPhe represents 4-Dimethylamino- phenylalanine, 55DM P 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
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consists of 2 amino acids and the bicyclic peptide ligand comprises an amino
acid sequence
which is selected from:
C,GRDSSWIYCHSTC,,, (SEQ ID NO: 12);
C,RGTPAWKACõAlCõ, (SEQ ID NO: 13);
C,PFPSGFGTCõTFCõ, (SEQ ID NO: 36);
C,PYVAGRGTCõLLCõ, (SEQ ID NO: 37; herein referred to as B0Y16312 when
complexed with TCMT);
C,PYPRGTGSC,,TFCõ, (SEQ ID NO: 38);
C,LYPPGKGTCõLLCõ, (SEQ ID NO: 39);
C,PSPAGRGTCõLLCõ, (SEQ ID NO: 40);
C,PATIGRGPCõTFCõ, (SEQ ID NO: 41);
C,PEANSVVVYCHSTC,,, (SEQ ID NO: 77);
C,APTSGWIYCHSTC,,, (SEQ ID NO: 78);
C,PYVAG[AgNGTO,,LLO,,, (SEQ ID NO: 80);
C,PYVAG[Arg(MeNTOõLLOõ, (SEQ ID NO: 81);
C,PYVAGRGTOõL[Oba]Cw (SEQ ID NO: 82);
C,PYVAGRGTO,,[Oba]LC,,, (SEQ ID NO: 83);
C,PYVAGR[dAriCõLLOõ, (SEQ ID NO: 84);
C,PYVAG[HArg]GTOõLLOõ, (SEQ ID NO: 85);
C,PYVAGRGTOõL[tBuAla]Cõ, (SEQ ID NO: 86);
C,PYVAGRGTOõ[tBuAla]LCõ, (SEQ ID NO: 87); and
C,PYVAG[Agb][dAFCõL[tBuAla]Cõ, (SEQ ID NO: 88);
wherein Cõ Cõ and Cõ, represent first, second and third cysteine residues,
respectively, Agb
represents 2-amino-4-guanidinobutyric acid, Arg(Me) represents 8-N methyl
arginine, Cba
represents p-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).
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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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 12)-A-[Sar6]-[KFI] (herein referred to as BCY15305); and
A-(SEQ ID NO: 13)-A-[Sar6]-[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);
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Ac-(SEQ ID NO: 171)-[K(PYA)] (herein referred to as BCY18040);
Ac-(SEQ ID NO: 172) (herein referred to as B0Y18087);
Ac-(SEQ ID NO: 173) (herein referred to as B0Y18088);
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 B0Y18212);
Ac-(SEQ ID NO: 179)-[K(PYA)] (herein referred to as B0Y18351);
Ac-(SEQ ID NO: 180)-[K(PYA)] (herein referred to as B0Y18524);
Ac-(SEQ ID NO: 181)-[K(PYA)] (herein referred to as B0Y18527);
Ac-(SEQ ID NO: 182)-[K(PYA)] (herein referred to as B0Y18529);
Ac-(SEQ ID NO: 183)-[K(PYA)] (herein referred to as B0Y18661);
Ac-(SEQ ID NO: 184)-[K(PYA)] (herein referred to as B0Y18662);
Ac-(SEQ ID NO: 185) (herein referred to as B0Y19305);
Ac-(SEQ ID NO: 186) (herein referred to as B0Y19309);
Ac-(SEQ ID NO: 187)-[K(PYA)] (herein referred to as B0Y19378);
Ac-(SEQ ID NO: 188) (herein referred to as B0Y19533);
Ac-(SEQ ID NO: 189) (herein referred to as B0Y19534);
Ac-(SEQ ID NO: 190) (herein referred to as B0Y19535);
Ac-(SEQ ID NO: 191) (herein referred to as B0Y19536);
Ac-(SEQ ID NO: 192) (herein referred to as B0Y19537);
Ac-(SEQ ID NO: 193) (herein referred to as B0Y19538);
Ac-(SEQ ID NO: 194) (herein referred to as B0Y19539);
Ac-(SEQ ID NO: 195) (herein referred to as B0Y19541);
Ac-(SEQ ID NO: 196) (herein referred to as B0Y19542);
Ac-(SEQ ID NO: 197) (herein referred to as B0Y19543);
Ac-(SEQ ID NO: 198) (herein referred to as B0Y19544);
Ac-(SEQ ID NO: 199) (herein referred to as B0Y19545);
Ac-(SEQ ID NO: 200) (herein referred to as B0Y19546);
Ac-(SEQ ID NO: 201) (herein referred to as B0Y19547);
Ac-(SEQ ID NO: 202) (herein referred to as B0Y19548);
Ac-(SEQ ID NO: 203)-[K(PYA)] (herein referred to as B0Y19599);
Ac-(SEQ ID NO: 204)-[K(PYA)] (herein referred to as BCY19600);
Ac-(SEQ ID NO: 204)-[K(PYA)]-triazolyl-PEGio-amido-PIB (herein referred to
as BCY20014);
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Ac-(SEQ ID NO: 205)-[K(PYA)] (herein referred to as B0Y19638);
Ac-(SEQ ID NO: 206)-[K(PYA)] (herein referred to as B0Y19639);
Ac-(SEQ ID NO: 207)-[K(PYA)] (herein referred to as B0Y19640);
Ac-(SEQ ID NO: 208)-[K(PYA)] (herein referred to as B0Y19641);
Ac-(SEQ ID NO: 209)-[K(PYA)] (herein referred to as B0Y19654);
Ac-(SEQ ID NO: 210)-[K(PYA)] (herein referred to as B0Y19655);
Ac-(SEQ ID NO: 211)-[K(PYA)] (herein referred to as B0Y19658);
Ac-(SEQ ID NO: 212)-[K(PYA)] (herein referred to as B0Y19827);
Ac-(SEQ ID NO: 213)-[K(PYA)] (herein referred to as B0Y19990); and
Ac-(SEQ ID NO: 214)-[K(PYA)] (herein referred to as B0Y20268);
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).
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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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 37)-A-[Sar6]-[KFI] (herein referred to as BCY15318);
Ac-(SEQ ID NO: 37)-[Sar6]-[KFI] (herein referred to as BCY16323);
A-(SEQ ID NO: 38)-A-[Sar6]-[KFI] (herein referred to as BCY15319); and
Ac-(SEQ ID NO: 88)-A-[Sar6]-[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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 37)-A-[Sar6]-[KFI] (herein referred to as BCY15318);
A-(SEQ ID NO: 38)-A-[Sar6]-[KFI] (herein referred to as BCY15319); and
Ac-(SEQ ID NO: 88)-A-[Sar6]-[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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 37)-A-[Sar6]-[KFI] (herein referred to as BCY15318); and
A-(SEQ ID NO: 38)-A-[Sar6]-[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:
C,SNTWHVVTDCõLAECõ, (SEQ ID NO: 45); and
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C,NLWNGDPWCõLLRCõ, (SEQ ID NO: 47);
wherein Cõ Cõ and Cõ, 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-(SEQ ID NO: 45)-A (herein referred to as BCY15351); and
A-(SEQ 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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 45)-A-[5ar6]-[KFI] (herein referred to as BCY15322); and
A-(SEQ ID NO: 47)-A-[5ar6]-[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:
C,HQLMDIWDCõLRPDCõ, (SEQ ID NO: 42); and
C,LTAREKIQCõLQRRCõ, (SEQ ID NO: 43);
wherein Cõ Cõ and Cõ, 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
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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 (Fl), and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 42)-A-[Sar6]-[KFI] (herein referred to as BCY15320); and
A-(SEQ ID NO: 43)-A-[Sar6]-[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 Si
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 Si 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
Sambrook etal., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in
Molecular
Biology (1999) 4th ed., John Wiley & Sons, Inc.), which are incorporated
herein by reference.
Nomenclature
Numbering
When referring to amino acid residue positions within peptides of the
invention, cysteine
residues (Cõ Cõ and Cõ,) 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:
(SEQ ID NO: 1).
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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. Cõ Cõ,
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 13Ala-Sar10-
Ala tail would
be denoted as:
pAla-Sar10-A-(SEQ ID NO: X).
In versed 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 Cõ Cõ and
Cõ,), 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-PEG10-
amido-P1B (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.
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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 ll
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 ll 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.
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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, (+)-(1S)-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 Ca2+
and Mg2+, and
other cations such as Al3+ or Zn+. Examples of suitable organic cations
include, but are not
limited to, ammonium ion (i.e., NH4) and substituted ammonium ions (e.g.,
NH3R+, NH2R2+,
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NHR3+, NR4+). 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(CH3)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-
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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
Cõ,) 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.
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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
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 13-turn conformations (Tugyi et al (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;
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- 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 2H (D) and 3H (T), carbon, such as 1,,
L, 130 and 140, chlorine,
such as 3601, fluorine, such as 18F, iodine, such as 1231, 1251 and 1311,
nitrogen, such as 13N and
15N, oxygen, such as 150, 170 and 180, phosphorus, such as 32P, sulfur, such
as 35S, copper,
such as 640u, gallium, such as 67Ga or 68Ga, yttrium, such as 90Y and
lutetium, such as 177Lu,
and Bismuth, such as 213Bi.
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,
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luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The
radioactive isotopes
tritium, i.e. 3H (T), and carbon-14, i.e. 140, 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. 2H (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 110,
r 150 and 13N, 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.
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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, c43
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 1,1',1"-(1,3,5-
triazinane-1,3,5-
triAtriprop-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-triAtriprop-
2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA):
0 0
0
TATA.
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Thus, following cyclisation with the bicyclic peptides of the invention on the
Cõ Cõ, and Cõ,
cysteine residues, the molecular scaffold forms a tri-substituted 1,1',1"-
(1,3,5-triazinane-
1,3,5-triAtripropan-1-one derivative of TATA having the following structure:
0 0
0
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):
0 0
BrN Br
Br
0
TATB.
Thus, following cyclisation with the bicyclic peptides of the invention on the
Cõ Cõ, and Cõ,
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):
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CI
N
Cl
TCMT.
Thus, following cyclisation with the bicyclic peptides of the invention on the
Ci, Cu, and Cu;
cysteine residues, the molecular scaffold forms a tri-substituted 2,4,6-
tris(chloromomethyl)-s-
triazine derivative of TCMT having the following structure:
N N
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
(haps://onlinelibrarymiley.comidoilabs/10.1002/thic.201600612).
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
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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
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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)1Y(X),Y(X)nY(X)0, 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.
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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 al.
(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 al.
1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779),
or by
enzymes, for example using subtiligase as described in (Chang et al. Proc Natl
Acad Sci U S
A. 1994 Dec 20; 91(26):12544-8 or in Hikari et al 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.
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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
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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
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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 COVI D-19.
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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
(Fl). In a yet
further embodiment, the peptide ligand comprises any of the peptide ligands
described
herein which comprise a fluorescein (Fl) moiety.
Diagnostic Methods
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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
(Fl). In a yet
further embodiment, the peptide ligand comprises any of the peptide ligands
described
herein which comprise a fluorescein (Fl) moiety.
The invention is further described below with reference to the following
examples.
EXAMPLES
Materials and Methods
Peptide Synthesis
Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide
synthesiser
manufactured by Peptide Instruments and a Syro ll 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.
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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 MeCN:H20 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 NH41-1CO3 in H20.
The reaction
was allowed to proceed for -30 -60 min at RT, and lyophilised once the
reaction had
completed (judged by MALDI). Once completed, 1m1 of 1M L-cysteine
hydrochloride
monohydrate (Sigma) in H20 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 08 with a Gemini 018 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 (Kd) forwild-type (VVT) 51 domain
(ACROBiosystems, 51N-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 2.54pM. 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 Kd value. Where no significant assay window was
generated, data
was reported to show no binding at maximum concentration of protein. Where a
Kd was
generated above the top concentration of protein tested, the result was
flagged as Kd greater
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than maximum concentration of protein tested ¨ results associated with this
flag may be
displayed as Kd > x pM.
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
Bicyclic Peptide Ka (pM)
B0Y15318 0.7168
BOY 16312 0.7591
BOY 16313 0.3964
BOY 16314 0.518
BOY 16315 0.7775
BOY 16316 >1
BOY 16318 0.6018
BOY 16319 0.7258
BOY 16320 0.3605
BOY 16321 0.9906
BOY 16322 0.7221
BOY 16323 0.7125
BOY 16679 0.339
BOY 15310 0.1986
BOY 16538 0.2287
BOY 16534 0.3812
BOY 16535 >1
BOY 16536 0.424
BOY 16537 >0.963
BOY 16545 0.641
BOY 16544 0.921
BOY 16543 >1
BOY 16542 0.511
BOY 16541 >1
BOY 16540 >0.883
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BOY 15522 0.37
Example 2: Surface Plasm on 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
Bicyclic Peptide pKo
BOY 15576 6.88
BOY 16903 7.92
BOY 16905 7.98
BOY 16906 7.68
BOY 16911 8
BOY 16913 7.77
BOY 16915 7.81
BOY 16917 7.96
BOY 16918 7.94
BOY 16921 7.85
BOY 16912 7.72
BOY 16914 7.78
BOY 16916 7.75
BOY 16919 7.68
BOY 16920 7.69
BOY 16902 7.62
BOY 16904 7.24
BOY 16907 7.46
BOY 16908 7.47
BOY 16909 7.58
BOY 15251 7.65
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
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of the isolated Si, 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
Peptide Epitope Ka (pM) Tested COVID-19 S1
Number Spike Protein Mutant
B0Y15324 1 No binding at max conc N354D, D364Y
0.588 G4765
0.751 V483A
1.49 E484K
0.629 A475V
No binding at max conc L452R
No binding at max conc F490L
No binding at max conc K417N, E484K, N501Y
0.529 N439K
0.366 Y453F
0.715 N440K
1.26 N501Y
B0Y16679 2 0.294 N354D, D364Y
0.194 W436R
0.263 R4081
0.305 V367F
0.38 D614G
0.253 G4765
0.294 V483A
No binding at max conc E484K
0.134 A475V
No binding at max conc L452R
No binding at max conc F490L
No binding at max conc K417N, E484K, N501Y
0.19 N439K
0.52 Y453F
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0.13 N440K
0.402 N501Y
B0Y15299 3 No binding at max conc N354D, D364Y
0.944 G476S
0.988 V483A
0.475 E484K
0.71 A475V
0.548 L452R
0.441 F490L
0.676 K417N, E484K, N501Y
0.531 N439K
0.579 Y453F
0.539 N440K
0.935 N501Y
B0Y15437 4 0.167 Y144del
0.024 A222V
0.024 N234Q
0.015 A262S
0.017 P272L
0.043 HV69-70de1
0.019 L18F, D80A, D215G,
R2461
BCY15310 5 0.285 D614G
0.169 HV69-70de1, N501Y,
D614G
0.305 P681H
0.265 A570D
B0Y16298 9 Kd greater than max conc tested N354D, D364Y
1.31 G476S
Kd greater than max conc tested V483A
0.865 E484K
Kd greater than max conc tested A475V
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1.15 L452 R
1.22 F490L
Kd greater than max conc tested K417N, E484K, N501Y
Kd greater than max conc tested N439K
1.02 Y453F
1.17 N440K
Kd greater than max conc tested N501Y
B0Y16287 10 1.12 N354D, D364Y
0.058 G476S
0.069 V483A
0.055 E484K
0.054 A475V
0.053 L452 R
0.049 F490L
The effectiveness of the tested peptides from Table 3 against the many Si
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
0
t..)
o
t..)
Variant/Peptide BCY15324 BCY16679 BCY15299 BCY15437 BCY15310
BCY16298 BCY16287 t..)
4,.
cio
,o
o,
oo
G476S V 1 1 nt
nt 1 1
V483A I I I it
nt - 1
E484K I I nt
nt 1 I
A475V V i i nt
nt - 1
L452R I it
nt I i p
0
F490L V nt
nt 1 1 "
-
.3
K417N, E484K, N501Y i nt
nt - nt "
0
"
,
N439K V I 1 nt
nt ¨ nt ,
"
.3
Y453F I I vi nt
nt 1 nt
N440K 1 1 Si nt
nt i nt
N501Y V I 1 nt
nt - nt
W436R nt I nt nt
nt nt nt
od
R4081 nt 1 nt nt
nt nt nt n
1-i
V367F nt I nt nt
nt nt nt w
t..)
o
t..)
D614G nt I nt nt
I nt nt t..)
'a
u,
o
Y144del nt nt nt I
nt nt nt o
c..)
BIC-C-P2967PCT 65
A222V nt nt nt I
nt nt nt
0
N234Q nt nt nt 1
nt nt nt t..)
=
t..)
t..)
A262S nt nt it I
nt nt nt
.6.
oe
P272L nt nt nt 1
nt nt nt o,
oe
HV69-70de1 nt nt nt 1
nt nt nt
L18F, D80A, D215G, R246I nt nt nt 1
nt nt nt
HV69-70de1, N501Y, D614G nt nt nt nt
1 nt nt
P681 H nt nt nt nt
I it nt
P
A570D nt nt nt nt
I nt nt .
.3
Legend
.
,
0
nt = not tested
.
,
.3
I = measurable binding
¨ = Kd 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 A
slspike protein with only several instances of no binding observed within the
limits of the assay. g
to
,..,
=
,..,
,..,
-a
u,
=
=
,...,
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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 pl/min flow rate with appropriate
association and
dissociation periods. Bicycles were assayed at concentrations between no
greater than
10000nM 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 KD) were derived from the sensorgrams by applying 1:1 Binding or
Steady-State
analysis, using the Biacore TM 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
BCY Number Geomean Spike Protein
Average KD (nM) Construct
BCY15251 69.1 Biotinylated 2019-nCoV
BCY15343 2240 (COVID-19) 51 protein
BCY15444 2180 (16-685), His,Avitag
BCY15445 482
BCY15446 1060
BCY15576 89.9
BCY16207 8300
BCY16591 545
BCY16886 154
BCY16887 95.3
BCY16889 44.8
BCY16895 43.7
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B0Y16902 24.0
B0Y16903 11.1
B0Y16904 58.2
B0Y16905 10.0
B0Y16906 17.8
B0Y16907 34.4
B0Y16908 33.9
B0Y16909 26.3
BCY16910 22.6
BCY16911 12.6
B0Y16912 19.0
B0Y16913 15.5
B0Y16914 16.5
B0Y16915 17.9
B0Y16916 17.9
B0Y16917 10.7
B0Y16918 11.3
B0Y16919 20.8
B0Y16920 20.3
B0Y16921 14.0
B0Y16926 2470
B0Y16927 18.8
B0Y16930 59.1
B0Y16933 25.3
B0Y16940 207
B0Y16941 83.9
B0Y16942 92.0
B0Y16946 78.1
B0Y16948 9.79
B0Y17279 55.2
B0Y17281 105
B0Y17282 99.5
B0Y17283 225
B0Y17287 1390
B0Y17289 44.1
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B0Y17294 76.3
BCY17301 77.0
B0Y17302 187
B0Y17303 109
B0Y17304 122
B0Y17305 67.3
B0Y17306 37.9
B0Y17307 73.3
B0Y17308 127
B0Y17309 95.3
BCY17310 86.5
BCY17311 33.6
B0Y17313 1350
B0Y17359 2250
B0Y17540 1810
B0Y17541 245
B0Y17542 574
B0Y17543 178
B0Y17544 350
B0Y17547 933
B0Y17548 1610
B0Y18024 800
B0Y18025 898
B0Y18026 1160
B0Y18027 1660
BCY18040 2220
B0Y18086 1710
B0Y18087 1220
B0Y18088 793
BCY18109 945
BCY18110 829
BCY18111 914
BCY18115 224
BCY18211 813
B0Y18212 1060
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B0Y18340 20.4
B0Y18341 66.7
B0Y18342 31.3
B0Y18343 71.5
B0Y18344 12.7
B0Y18345 34.7
B0Y18346 20.6
B0Y18347 50.3
B0Y18351 717
B0Y18524 102
B0Y18527 1720
B0Y18529 1240
B0Y18654 1280
B0Y18661 1600
B0Y18662 2610
B0Y18698 8900
B0Y18707 2440
B0Y19305 3500
B0Y19309 220
B0Y19378 70.9
B0Y19599 44.5
BCY19600 0.588
B0Y19638 11.9
B0Y19639 34.4
B0Y19640 350
B0Y19641 199
B0Y19654 384
B0Y19655 98.5
B0Y19658 61.0
B0Y19827 0.485
B0Y19990 66.1
BCY20014 1.38
B0Y20268 0.462
B0Y16591 458 Biotinylated SARS-
B0Y19309 186 CoV-2 (COVID-19) S
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B0Y19533 6510 protein RBD,
B0Y19534 5230 His,Avitag TM
B0Y19535 565
B0Y19536 7570
B0Y19537 2650
B0Y19538 2100
B0Y19539 2080
B0Y19541 2170
B0Y19542 1950
B0Y19543 2940
B0Y19544 6650
B0Y19545 1570
B0Y19546 3520
B0Y19547 9160
B0Y19548 2990
B0Y19599 61.6
BCY19600 0.834
B0Y19638 29.5
B0Y19639 72.0
B0Y19654 115
B0Y19655 169
B0Y19658 96.6
BCY20014 0.379
BCY19600 457 Biotinylated SARS-
BCY20014 192 CoV-2 S protein RBD
(K417T,E484K,N501Y),
His,Avitag TM
BCY20014 311 Biotinylated SARS-
CoV-2 S protein RBD
(K417N,E484K,N501Y),
His,Avitag TM
BCY19600 30100 Biotinylated SARS-
BCY20014 7620 CoV-2 Spike RBD
(L452R, E484Q),
His,Avitag TM
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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 (CSGVV).
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 pl
pseudovirus-containing supernatant was mixed with 2 pl dilutions of bicyclic
peptide and
incubated for 40 min at RT. 10 pl of this mixture was added to cells. 72 h
later, cell entry was
detected through the expression of GFP by visualisation on an lncucyte 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
BCY Number Geomean IC50 (nM) Pseudovirus Construct
BCY19600 183 VVild-Type
Example 6: SARS-CoV-2 Cytopathic 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 pl 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 pl
per well was added to the wells containing compounds. Plates were incubated
for 72 h at
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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
BCY Number ICso (nM)
BCY16591 21400
BCY18024 29000
BCY20014 168
