WO2024009108A1 - Anti-infective bicyclic peptide ligands - Google Patents

Anti-infective bicyclic peptide ligands Download PDF

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Publication number
WO2024009108A1
WO2024009108A1 PCT/GB2023/051798 GB2023051798W WO2024009108A1 WO 2024009108 A1 WO2024009108 A1 WO 2024009108A1 GB 2023051798 W GB2023051798 W GB 2023051798W WO 2024009108 A1 WO2024009108 A1 WO 2024009108A1
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WIPO (PCT)
Prior art keywords
seq
referred
peptide ligand
derivative
protein
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Application number
PCT/GB2023/051798
Other languages
French (fr)
Inventor
Michael Skynner
Maximilian HARMAN
Katie Gaynor
Katerine VAN RIETSCHOTEN
Liuhong CHEN
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Bicycletx Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority claimed from GBGB2209990.7A external-priority patent/GB202209990D0/en
Priority claimed from GBGB2210376.6A external-priority patent/GB202210376D0/en
Priority claimed from GBGB2301857.5A external-priority patent/GB202301857D0/en
Application filed by Bicycletx Limited filed Critical Bicycletx Limited
Publication of WO2024009108A1 publication Critical patent/WO2024009108A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • the invention describes peptides which are high affinity binders of the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2).
  • S protein spike protein
  • SARS-CoV- 2 severe acute respiratory syndrome coronavirus 2
  • the invention also includes multimeric binding complexes comprising said bicyclic peptide ligands.
  • the invention also includes pharmaceutical compositions comprising said polypeptides or said multimeric binding complexes and to the use of said polypeptides, multimeric binding complexes and pharmaceutical compositions 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.
  • Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 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.
  • a peptide ligand specific for the spike protein (S protein) of 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
  • CVVNGTIRYCALC (SEQ ID NO: 2);
  • CIVDDHIVYCGSKQC (SEQ ID NO: 19); CIKDGLLVYCGSYQC (SEQ ID NO: 20);
  • CMNPFFYDCERTC SEQ ID NO: 22
  • BCY18697 when complexed with a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues;
  • CMNPFFYDCDHIC SEQ ID NO: 23
  • CMNPFFYDCHEQC SEQ ID NO: 24
  • CMNPFFYDCKWC SEQ ID NO: 25
  • CMNPFFYDCEDRC SEQ ID NO: 26
  • CMNPFFYDCEEIC SEQ ID NO: 27
  • CMNPFFYDCENPC SEQ ID NO: 28
  • CMNPFFYDCETVC SEQ ID NO: 29
  • CMNPFFYDCEYVC SEQ ID NO: 30
  • CMNPFYYDCEEVC (SEQ ID NO: 31);
  • CMNPFF[4FPhe]DCERTC SEQ ID NO: 34
  • CEDNDWVYCSTC SEQ ID NO: 35
  • CDWTCYLRPLPC (SEQ ID NO: 36);
  • CMFVPCATRVALGLC SEQ ID NO: 51
  • CMFVPCAVREELGLC SEQ ID NO: 52
  • CEINASLPCTFTC SEQ ID NO: 69
  • CDPDPYDSSCWTC (SEQ ID NO: 71);
  • CDWDWHVCAIMNESC SEQ ID NO: 72
  • CTPLDATFCFSKC (SEQ ID NO: 73);
  • CEEDWHICQIHGYDC SEQ ID NO: 74
  • CNNPFCEYHIC (SEQ ID NO: 77);
  • CTNIMCEFMFC (SEQ ID NO: 81);
  • CDGPDWHSCMVSC (SEQ ID NO: 87);
  • CDHYHCPWLALGGSC SEQ ID NO: 90
  • CELDEWLCIIGHLDC SEQ ID NO: 91
  • CMEFAANCEDIYDDC SEQ ID NO: 92
  • Hse(Me) represents homoserine-methyl
  • HyP represents hydroxyproline
  • tBuAla represents t-butyl-alanine
  • HArg represents homoarginine
  • 4FPhe represents 4- fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • a multimeric binding complex which comprises either: (i) at least two bicyclic peptide ligands of the invention, wherein said peptide ligands may be the same or different; or (ii) at least one bicyclic peptide ligand of the invention, which may be the same or different, in combination with one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be the same or different.
  • S protein spike protein
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • a pharmaceutical composition comprising the peptide ligand or the multimeric binding complex as defined herein in combination with one or more pharmaceutically acceptable excipients.
  • the peptide ligand or the multimeric binding complex 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.
  • peptide ligand specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
  • CVVNGTIRYCALC (SEQ ID NO: 2);
  • CMNPFFYDCERTC SEQ ID NO: 22
  • BCY18697 when complexed with a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues;
  • CMNPFFYDCDHIC SEQ ID NO: 23
  • CMNPFFYDCHEQC SEQ ID NO: 24
  • CMNPFFYDCKWC SEQ ID NO: 25
  • CMNPFFYDCEDRC SEQ ID NO: 26
  • CMNPFFYDCEEIC SEQ ID NO: 27
  • CMNPFFYDCENPC SEQ ID NO: 28
  • CMNPFFYDCETVC SEQ ID NO: 29
  • CMNPFFYDCEYVC SEQ ID NO: 30
  • CMNPFYYDCEEVC (SEQ ID NO: 31);
  • CMNPFF[4FPhe]DCERTC SEQ ID NO: 34
  • CEDNDWVYCSTC SEQ ID NO: 35
  • CDWTCYLRPLPC SEQ ID NO: 36
  • CMFVPCAARHELGLC SEQ ID NO: 41
  • CMFVPCATRQMLGLC SEQ ID NO: 50
  • CMFVPCATRVALGLC SEQ ID NO: 51
  • BCY18090 when complexed with a derivative of TCMT which has the following structure: wherein * denotes the point of attachment of the three cysteine residues;
  • BCY18092 when complexed with a derivative of TCMT which has the following structure: wherein * denotes the point of attachment of the three cysteine residues;
  • BCY18094 when complexed with a derivative of TCMT which has the following structure: wherein * denotes the point of attachment of the three cysteine residues; CHPVCSVPAIGLLC (SEQ ID NO: 68);
  • CEINASLPCTFTC SEQ ID NO: 69
  • CDPDPYDSSCWTC (SEQ ID NO: 71);
  • CDWDWHVCAIMNESC SEQ ID NO: 72
  • CTPLDATFCFSKC (SEQ ID NO: 73);
  • CEEDWHICQIHGYDC SEQ ID NO: 74
  • CNNPFCEYHIC (SEQ ID NO: 77);
  • CTNIMCEFMFC (SEQ ID NO: 81);
  • CDGPDWHSCMVSC (SEQ ID NO: 87);
  • CDHYHCPWLALGGSC SEQ ID NO: 90
  • CELDEWLCIIGHLDC SEQ ID NO: 91
  • CMEFAANCEDIYDDC SEQ ID NO: 92
  • Hse(Me) represents homoserine-methyl
  • HyP represents hydroxyproline
  • tBuAla represents t-butyl-alanine
  • HArg represents homoarginine
  • 4FPhe represents 4- fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • a peptide ligand specific for the spike protein (S protein) of 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
  • CVVNGTIRYCALC (SEQ ID NO: 2);
  • CMNPFFYDCERTC SEQ ID NO: 22
  • BCY18697 when complexed with a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues;
  • CMNPFFYDCDHIC SEQ ID NO: 23
  • CMNPFFYDCHEQC SEQ ID NO: 24
  • CMNPFFYDCKWC SEQ ID NO: 25
  • CMNPFFYDCEDRC SEQ ID NO: 26
  • CMNPFFYDCEEIC SEQ ID NO: 27
  • CMNPFFYDCENPC SEQ ID NO: 28
  • CMNPFFYDCETVC SEQ ID NO: 29
  • CMNPFFYDCEYVC SEQ ID NO: 30
  • CMNPFYYDCEEVC (SEQ ID NO: 31);
  • CMNPFF[4FPhe]DCERTC SEQ ID NO: 34
  • CEDNDWVYCSTC SEQ ID NO: 35
  • CDWTCYLRPLPC (SEQ ID NO: 36); wherein the three cysteine residues within each peptide ligand represent the three reactive groups and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, and 4FPhe represents 4-fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • the spike protein is a large type I transmembrane protein of SARS-CoV-2. This protein is highly glycosylated as it contains 21 to 35 N-glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive “corona”, or crown-like appearance.
  • the ectodomain of all CoV spike proteins share the same organization in two domains: a N-terminal domain named S1 that is responsible for receptor binding and a C- terminal S2 domain responsible for fusion.
  • CoV diversity is reflected in the variable spike proteins (S proteins), which have evolved into forms differing in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion.
  • said peptide ligand is specific for the S2 domain of the spike protein (S2 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
  • CVVNGTIRYCALC (SEQ ID NO: 2);
  • CTPNGCTDLWRYIAC SEQ ID NO: 9
  • CSDEFCSAWWGFNEC SEQ ID NO: 10
  • CIKDGLLVYCGSYQC SEQ ID NO: 20
  • CIKDGVLIYCGGPMC SEQ ID NO: 21
  • the three cysteine residues within each peptide ligand represent the three reactive groups, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TATA which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:
  • A-(SEQ ID NO: 1)-A (herein referred to as BCY18948);
  • A-(SEQ ID NO: 8)-A (herein referred to as BCY19894);
  • A-(SEQ ID NO: 9)-A (herein referred to as BCY19896);
  • A-(SEQ ID NO: 10)-A (herein referred to as BCY19899);
  • A-(SEQ ID NO: 11)-A (herein referred to as BCY19900);
  • A-(SEQ ID NO: 12)-A (herein referred to as BCY19901);
  • BCY22415 Ac-(SEQ ID NO: 12)-[K(PYA)]
  • A-(SEQ ID NO: 13)-A (herein referred to as BCY19902); and A-(SEQ ID NO: 14)-A (herein referred to as BCY19903); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, 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: 2)-A (herein referred to as BCY18950);
  • A-(SEQ ID NO: 3)-A (herein referred to as BCY18953);
  • A-(SEQ ID NO: 4)-A (herein referred to as BCY18956);
  • A-(SEQ ID NO: 5)-A (herein referred to as BCY18957);
  • A-(SEQ ID NO: 6)-A (herein referred to as BCY18958);
  • A-(SEQ ID NO: 7)-A (herein referred to as BCY18961);
  • BCY19916 A-(SEQ ID NO: 17)-A (herein referred to as BCY19916);
  • A-(SEQ ID NO: 18)-A (herein referred to as BCY19919);
  • A-(SEQ ID NO: 19)-A (herein referred to as BCY19921);
  • A-(SEQ ID NO: 20)-A (herein referred to as BCY19923); and A-(SEQ ID NO: 21)-A (herein referred to as BCY19924); or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TCMT which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, 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: 15)-A (herein referred to as BCY19904);
  • BCY19905 A-(SEQ ID NO: 16)-A (herein referred to as BCY19905);
  • BCY22416 Ac-(SEQ ID NO: 16)-K (herein referred to as BCY22416); or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:
  • BCY17367 A-(SEQ ID NO: 41)-A (herein referred to as BCY17367);
  • A-(SEQ ID NO: 42)-A (herein referred to as BCY17368);
  • A-(SEQ ID NO: 43)-A (herein referred to as BCY17369);
  • A-(SEQ ID NO: 44)-A (herein referred to as BCY17370);
  • A-(SEQ ID NO: 45)-A (herein referred to as BCY17371);
  • A-(SEQ ID NO: 46)-A (herein referred to as BCY17372);
  • BCY17373 A-(SEQ ID NO: 47)-A (herein referred to as BCY17373); A-(SEQ ID NO: 48)-A (herein referred to as BCY17375);
  • A-(SEQ ID NO: 49)-A (herein referred to as BCY17376);
  • A-(SEQ ID NO: 50)-A (herein referred to as BCY17377);
  • A-(SEQ ID NO: 51)-A (herein referred to as BCY17378);
  • A-(SEQ ID NO: 52)-A (herein referred to as BCY17380);
  • A-(SEQ ID NO: 53)-A (herein referred to as BCY17381);
  • A-(SEQ ID NO: 54)-A (herein referred to as BCY17382);
  • A-(SEQ ID NO: 55)-A (herein referred to as BCY17383);
  • A-(SEQ ID NO: 56)-A (herein referred to as BCY17384);
  • A-(SEQ ID NO: 57)-A (herein referred to as BCY17385);
  • A-(SEQ ID NO: 58)-A (herein referred to as BCY17387);
  • A-(SEQ ID NO: 59)-A (herein referred to as BOY 17615);
  • BCY17617 A-(SEQ ID NO: 60)-A (herein referred to as BCY17617); and A-(SEQ ID NO: 61)-A (herein referred to as BCY17618); or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
  • CMNPFFYDCERTC SEQ ID NO: 22
  • BCY18697 when complexed with a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues);
  • CMNPFFYDCDHIC SEQ ID NO: 23
  • CMNPFFYDCHEQC SEQ ID NO: 24
  • CMNPFFYDCKWC SEQ ID NO: 25
  • CMNPFFYDCEDRC SEQ ID NO: 26
  • CMNPFFYDCEEIC SEQ ID NO: 27
  • CMNPFFYDCENPC SEQ ID NO: 28
  • CMNPFFYDCETVC SEQ ID NO: 29
  • CMNPFFYDCEYVC SEQ ID NO: 30
  • CMNPFYYDCEEVC SEQ ID NO: 31
  • CMNPFF[4FPhe]DCERTC SEQ ID NO: 34
  • CEDNDWVYCSTC SEQ ID NO: 35
  • Hse(Me) represents homoserine-methyl
  • HyP represents hydroxyproline
  • 4FPhe represents 4-fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, 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: 22)-A (herein referred to as BCY16207);
  • BCY18696 Ac-A-(SEQ ID NO: 22)-A
  • BCY18698 Ac-(SEQ ID NO: 22) (herein referred to as BCY18698);
  • A-(SEQ ID NO: 23)-A (herein referred to as BCY18144);
  • A-(SEQ ID NO: 24)-A (herein referred to as BCY18145);
  • A-(SEQ ID NO: 25)-A (herein referred to as BCY18146);
  • A-(SEQ ID NO: 26)-A (herein referred to as BCY18147);
  • BCY18148 A-(SEQ ID NO: 27)-A (herein referred to as BCY18148);
  • A-(SEQ ID NO: 28)-A (herein referred to as BCY18149);
  • A-(SEQ ID NO: 29)-A (herein referred to as BCY18150);
  • BCY22405 Ac-(SEQ ID NO: 29) (herein referred to as BCY22405);
  • A-(SEQ ID NO: 30)-A (herein referred to as BCY18151);
  • BCY18152 A-(SEQ ID NO: 31)-A (herein referred to as BCY18152); A-(SEQ ID NO: 32)-A (herein referred to as BCY18700);
  • BCY18702 A-(SEQ ID NO: 33)-A (herein referred to as BCY18702);
  • A-(SEQ ID NO: 34)-A (herein referred to as BCY18703); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:
  • BCY22412 Ac-(SEQ ID NO: 35)-[K(PYA)]
  • A-(SEQ ID NO: 42)-A (herein referred to as BCY17368);
  • A-(SEQ ID NO: 44)-A (herein referred to as BCY17370);
  • A-(SEQ ID NO: 45)-A (herein referred to as BCY17371);
  • A-(SEQ ID NO: 47)-A (herein referred to as BCY17373);
  • A-(SEQ ID NO: 49)-A (herein referred to as BCY17376);
  • A-(SEQ ID NO: 50)-A (herein referred to as BCY17377);
  • A-(SEQ ID NO: 51)-A (herein referred to as BCY17378);
  • A-(SEQ ID NO: 53)-A (herein referred to as BCY17381);
  • BCY17384 A-(SEQ ID NO: 56)-A (herein referred to as BCY17384);
  • A-(SEQ ID NO: 58)-A (herein referred to as BCY17387); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:
  • said peptide ligand is specific for the N-Terminal Domain (NTD) of the S1 domain of the spike protein (S1 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is:
  • CDWTCYLRPLPC (SEQ ID NO: 36); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • said peptide ligand is specific for the N-Terminal Domain (NTD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:
  • BCY22414 Ac-(SEQ ID NO: 36)-[K(PYA)]
  • BCY22417 Ac-(SEQ ID NO: 36)-K (herein referred to as BCY22417); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • the peptide ligand specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 has a molecular scaffold which is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, 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: 68)-A (herein referred to as BCY16129);
  • BCY161278 A-(SEQ ID NO: 69)-A (herein referred to as BCY16128);
  • A-(SEQ ID NO: 70)-A (herein referred to as BCY16127);
  • BCY16126 A-(SEQ ID NO: 71)-A (herein referred to as BCY16126);
  • BCY16125 A-(SEQ ID NO: 72)-A (herein referred to as BCY16125);
  • A-(SEQ ID NO: 73)-A (herein referred to as BCY16124);
  • A-(SEQ ID NO: 74)-A (herein referred to as BCY16123);
  • A-(SEQ ID NO: 75)-A (herein referred to as BCY16122);
  • A-(SEQ ID NO: 76)-A (herein referred to as BCY16121);
  • A-(SEQ ID NO: 77)-A (herein referred to as BCY16120);
  • A-(SEQ ID NO: 78)-A (herein referred to as BCY16119);
  • A-(SEQ ID NO: 79)-A (herein referred to as BCY16118);
  • A-(SEQ ID NO: 80)-A (herein referred to as BCY16117);
  • BCY16116 A-(SEQ ID NO: 81)-A (herein referred to as BCY16116);
  • A-(SEQ ID NO: 82)-A (herein referred to as BCY16115);
  • A-(SEQ ID NO: 83)-A (herein referred to as BCY16114);
  • A-(SEQ ID NO: 84)-A (herein referred to as BCY16113); A-(SEQ ID NO: 85)-A (herein referred to as BCY16112);
  • A-(SEQ ID NO: 86)-A (herein referred to as BCY16111);
  • A-(SEQ ID NO: 87)-A (herein referred to as BCY16110);
  • A-(SEQ ID NO: 88)-A (herein referred to as BCY16109);
  • BCY16108 A-(SEQ ID NO: 89)-A (herein referred to as BCY16108);
  • A-(SEQ ID NO: 90)-A (herein referred to as BCY16107);
  • BCY16106 A-(SEQ ID NO: 91)-A
  • BCY16105 A-(SEQ ID NO: 92)-A
  • the peptide ligand specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 has a molecular scaffold which is a derivative of TCMT which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand and comprises an amino acid sequence which is selected from:
  • BCY18094 (SEQ ID NO: 67) (herein referred to as BCY18094); or a modified derivative and/or pharmaceutically acceptable salt thereof.
  • a multimeric binding complex which comprises either: (i) at least two bicyclic peptide ligands of the invention, wherein said peptide ligands may be the same or different; or (ii) at least one bicyclic peptide ligand of the invention, which may be the same or different, in combination with one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be the same or different.
  • the multimeric binding complexes comprise at least two (e.g. two or three) bicyclic peptides which are the same (i.e. homomultimers).
  • the multimeric binding complexes comprise three bicyclic peptides which are the same (i.e. homotrimers). In an alternative embodiment, the multimeric binding complexes comprise at least two (e.g. two or three) bicyclic peptides which are different (i.e. heteromultimers). In a further embodiment, the multimeric binding complexes comprise two bicyclic peptides, each of which are different (i.e. heterodimers). In an alternative embodiment, the multimeric binding complexes comprise three bicyclic peptides, each of which are different (i.e. heterotri mers).
  • references herein to “one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” refer to any bicyclic peptide which is capable of binding to the spike protein of SARS-CoV-2.
  • suitable bicyclic peptides include those in PCT/GB2022/050031 , PCT/GB2022/050036, and PCT/GB2022/050037, the bicyclic peptides of which are herein incorporated by reference.
  • said “one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” is selected from one or more of: BCY16592, BCY18579, BCY18654, and/or BCY19600.
  • BCY16592 is a bicyclic peptide ligand having the sequence Ac- (CPYVAG[Agb][dA]TCL[tBuAla]C)-[K(PYA)] (SEQ ID NO: 37) and a molecular scaffold which is a derivative of TCMT which has the following structure: wherein * denotes the point of attachment of the three cysteine residues, and Agb represents 2-amino-4-guanidinobutyric acid, tBuAla represents t-butyl-alanine, and PYA represents pentynoic acid.
  • BCY18579 is a bicyclic peptide ligand having the sequence Ac-(CANPDNPVCRFYC)-K (SEQ ID NO: 38) and a molecular scaffold which is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues.
  • BCY18654 is a bicyclic peptide ligand having the sequence Ac-(CIPLDWTCMIAC)-K (SEQ ID NO: 39) and a molecular scaffold which is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues.
  • BCY19600 is a bicyclic peptide ligand having the sequence Ac-(CIPLDWTCMIAC)-K (SEQ ID NO: 39) and a molecular scaffold which is a derivative of TATB which has the following structure: wherein * denotes the point of attachment of the three cysteine residues.
  • BCY19600 is a bicyclic peptide ligand having the sequence Ac-
  • the multimeric binding complex comprises a linker between said bicyclic peptide ligands.
  • the linker comprises a PEG containing linker.
  • the linker when the multimeric binding complex comprises two bicyclic peptide ligands, the linker is a linear linker which is: wherein n represents an integer from 1 to 30, such as 10 or 24 and * represents the point of attachment to each bicyclic peptide ligand.
  • the linker when the multimeric binding complex comprises three bicyclic peptide ligands, the linker is a branched linker selected from: and
  • the multimeric binding complex comprises two differing bicyclic peptides (i.e. heterodimers). Examples of such heterodimers are shown in Table A below:
  • the multimeric binding complex comprises three identical bicyclic peptides (i.e. homotrimers). Examples of such homotrimers are shown in Table B below:
  • the multimeric binding complex comprises three differing bicyclic peptides (i.e. heterotrimers). Examples of such heterotrimers are shown in Table C below:
  • cysteine residues (C) 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:
  • 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.
  • an N-terminal pAla-Sar10-Ala tail would be denoted as:
  • a peptide ligand refers to a peptide covalently bound to a molecular scaffold.
  • 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.
  • the peptides comprise at least three cysteine residues and form at least two loops on the scaffold.
  • 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.
  • a half-life extending moiety is a polyethylene glycol (PEG) moiety, such as triazolyl-PEGw- amido-PIB (wherein PIB represents 4(4-iodophenyl)butyrate).
  • 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:
  • Certain ligands demonstrate cross-reactivity across Lipid II from different bacterial species and hence are able to treat infections caused by multiple species of bacteria.
  • Other ligands may be highly specific for the Lipid II of certain bacterial species which may be advantageous for treating an infection without collateral damage to the beneficial flora of the patient;
  • 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;
  • references to peptide ligands include the salt forms of said ligands.
  • the salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.
  • 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 may be formed with a wide variety of acids, both inorganic and organic.
  • 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.
  • D-glucuronic D-glucuronic
  • glutamic e.g. L-glutamic
  • a-oxoglutaric glycolic, hippuric
  • hydrohalic acids e.g. hydrobromic, hydrochloric, hydriodic
  • isethionic lactic (e.g.
  • salts consist 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.
  • a salt may be formed with an organic or inorganic base, generating a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Li + , Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + .
  • Suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NHsR + , NH2R2 + , NHRs + , NR 4 + ).
  • Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CHs)4 + .
  • 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 of the peptide ligands as defined herein are within the scope of the present invention.
  • 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 surrog
  • the modified derivative comprises an N-terminal and/or C-terminal modification.
  • the modified derivative comprises an N- terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry.
  • said N-terminal or C- terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.
  • the modified derivative comprises an N-terminal modification.
  • the N-terminal modification comprises an N-terminal acetyl group.
  • the N-terminal cysteine group (the group referred to herein as Ci) 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.
  • 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.
  • the modified derivative comprises a C-terminal modification.
  • the C-terminal modification comprises an amide group.
  • the C-terminal cysteine group 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.
  • the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues.
  • 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.
  • 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.
  • 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.
  • the modified derivative comprises the addition of a spacer group.
  • the modified derivative comprises the addition of a spacer group to the N-terminal cysteine and/or the C-terminal cysteine.
  • the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues.
  • 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).
  • 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 p-turn conformations (Tugyi et a/ (2005) PNAS, 102(2), 413-418).
  • 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).
  • 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.
  • isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as 2 H (D) and 3 H (T), carbon, such as 11 C, 13 C and 14 C, chlorine, such as 36 CI, fluorine, such as 18 F, iodine, such as 123 l, 125 l and 131 l, nitrogen, such as 13 N and 15 N, oxygen, such as 15 O, 17 O and 18 O, phosphorus, such as 32 P, sulfur, such as 35 S, copper, such as 64 Cu, gallium, such as 67 Ga or 68 Ga, yttrium, such as 90 Y and lutetium, such as 177 Lu, and Bismuth, such as 213 Bi.
  • hydrogen such as 2 H (D) and 3 H (T)
  • carbon such as 11 C, 13 C and 14 C
  • chlorine such as 36 CI
  • fluorine such as 18 F
  • iodine such as 123 l, 125 l and
  • Certain isotopically-labelled peptide ligands of the invention are useful in drug and/or substrate tissue distribution studies.
  • the peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors.
  • the detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc.
  • the radioactive isotopes tritium, i.e. 3 H (T), and carbon-14, i.e. 14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
  • Substitution with heavier isotopes such as deuterium, i.e. 2 H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
  • 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.
  • the molecular scaffold may be a small molecule, such as a small organic molecule.
  • the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.
  • the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds.
  • the molecular scaffold may comprise chemical groups which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.
  • the molecular scaffold of the invention contains chemical groups that allow functional groups of the polypeptide of the encoded library of the invention to form covalent links with the molecular scaffold.
  • Said chemical groups are selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.
  • Scaffold reactive groups that could be used on the molecular scaffold to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloalkanes).
  • scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides, a unsaturated carbonyl containing compounds and a-halomethylcarbonyl containing compounds.
  • maleimides which may be used as molecular scaffolds in the invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris- (maleimido)benzene.
  • the molecular scaffold is selected from 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5- triyl)triprop-2-en-1-one (also known as triacryloylhexahydro-s-triazine; TATA), 1 ,3,5- tris(bromoacetyl) hexahydro-1 , 3, 5-triazine (TATB) and 2,4,6-tris(chloromethyl)-s-triazine (TCMT).
  • 1 ,1',1"-(1 ,3,5-triazinane-1 ,3,5- triyl)triprop-2-en-1-one also known as triacryloylhexahydro-s-triazine; TATA
  • TATA triacryloylhexahydro-s-triazine
  • TATB 5-triazine
  • TCMT 2,4,6-tris(chloromethyl)-s-triazine
  • the molecular scaffold is 1 , 1 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop- 2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA):
  • the molecular scaffold forms a tri-substituted 1 ,1',1"-(1,3,5-triazinane-1,3,5-triyl)tripropan-1- one derivative of TATA having the following structure: wherein * denotes the point of attachment of the three cysteine residues.
  • the molecular scaffold is 1 ,3,5-tris(bromoacetyl) hexahydro-1, 3, 5-triazine (TATB):
  • 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.
  • the molecular scaffold is 2,4,6-tris(chloromomethyl)-s-triazine (TCMT):
  • the molecular scaffold forms a tri-substituted 2,4,6-tris(chloromomethyl)-s-triazine derivative of TCMT having the following structure: wherein * denotes the point of attachment of the three cysteine residues.
  • 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.
  • reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine.
  • Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group.
  • the amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold/molecular core.
  • the polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.
  • 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.
  • a single product isomer is also advantageous if a specific member of a library of the invention is synthesized.
  • the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.
  • 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.
  • At least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups.
  • the use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core.
  • Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at least three bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved.
  • 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.
  • amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid.
  • 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.
  • a polypeptide with three reactive groups has the sequence (X)iY(X)mY(X) n Y(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 2 and 8 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.
  • thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions.
  • 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.
  • thiol mediated 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.
  • 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).
  • 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.
  • lysines and analogues
  • Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus.
  • 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).
  • 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.
  • 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.
  • composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.
  • the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers.
  • 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.
  • the peptide ligands of the invention can be administered to any patient in accordance with standard techniques.
  • Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intraderma
  • 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.
  • compositions containing the present peptide ligands or a cocktail thereof can be administered for therapeutic treatments.
  • 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.
  • the peptide ligands described herein may be used extracorporeal ly or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.
  • Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
  • the bicyclic peptides of the invention have specific utility as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding agents.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • 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.
  • the selected polypeptides may be used diagnostically or therapeutically (including extracorporeal ly) 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).
  • 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.
  • a method of suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2 which comprises administering to a patient in need thereof the peptide ligand as defined herein.
  • references herein to “disease or disorder mediated by infection of SARS-CoV-2” include: respiratory disorders, such as a respiratory disorder mediated by an inflammatory response within the lung, in particular COVID-19.
  • references herein to the term “suppression” refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment” involves administration of the protective composition after disease symptoms become manifest. Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available.
  • bicyclic peptide ligands of the invention also find utility as agents for screening for other SARS-CoV-2 binding agents.
  • 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.
  • step (c) incubating said peptide ligand from step (a) with a test compound and SARS- CoV-2;
  • step (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.
  • the peptide ligand comprises a reporter moiety for ease of detecting binding.
  • the reporter moiety comprises fluorescein (Fl).
  • the peptide ligand comprises any of the peptide ligands described herein which comprise a fluorescein (Fl) moiety.
  • bicyclic peptide ligands of the invention also find utility as agents for diagnosing infection of SARS-CoV-2.
  • 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.
  • a method of diagnosing SARS-CoV-2 infection comprising the following steps: a) obtaining a biological sample from an individual;
  • step (b) incubating a peptide ligand as defined herein with the biological sample obtained in step (a);
  • the peptide ligand comprises a reporter moiety for ease of detecting binding.
  • the reporter moiety comprises fluorescein (Fl).
  • the peptide ligand comprises any of the peptide ligands described herein which comprise a fluorescein (Fl) moiety.
  • Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc-amino acids were employed (Sigma, Merck), with appropriate side chain protecting groups: where applicable standard coupling conditions were used in each case, followed by deprotection using standard methodology.
  • peptides were purified using HPLC and following isolation they were modified with the required molecular scaffold (namely, TATA, TATB or TCMT).
  • linear peptide was diluted with 50:50 MeC k W up to ⁇ 35 mL, -500 pL of 100 mM scaffold in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed for -30 -60 min at RT, and lyophilised once the reaction had completed (judged by MALDI). Once completed, 1 ml of 1 M L-cysteine hydrochloride monohydrate (Sigma) in H2O was added to the reaction for ⁇ 60 min at RT to quench any excess TATA, TATB or TCMT.
  • the modified peptide was purified as above, while replacing the Luna C8 with a Gemini C18 column (Phenomenex), and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct scaffold-modified material were pooled, lyophilised and kept at -20°C for storage.
  • 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 DI PEA (20 mol equiv) was added. The reaction was monitored by LC/MS until complete.
  • the multimeric binding complexes of the invention may be prepared in accordance with analogous methodology to that described in WO 2019/162682, PCT/GB2022/050031 , PCT/GB2022/050036, and PCT/GB2022/050037.
  • SPR analysis was performed on a Biacore 8K+ (Cytiva). Briefly, SARS-CoV-2 Spike Trimer glycoprotein was immobilized on a Series S Sensor Chip CM5 using standard primary amine- coupling chemistry at 25°C with PBS, 0.05 % P20, 1 % DMSO, pH 7.4 as the running buffer. The carboxymethyl dextran surface was activated with a 7 min injection of a 1 :1 v/v ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 pLmin -1 .
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxy succinimide
  • ACE2 protein was diluted to 40 nM in 10 mM sodium acetate (pH 5.0) and captured with 200 s contact time at 10 pLmin -1 . Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5. Surface densities of 1200 RU were achieved.
  • an 8-point titration of bicyclic peptide underwent multi cycle kinetic evaluation at 25 °C, flow rate of 50 pLmin -1 , with association time of 60 seconds and dissociation time of up to 400 seconds. A maximum concentration of 20000 nM bicyclic peptide was used. Data were solvent corrected for DMSO bulk effects. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using Biacore Insight Evaluation Software. Data were fitted using the 1 :1 binding model or steadystate affinity model where appropriate.
  • SPR analysis was performed on a Biacore 8K+ (Cytiva). Briefly, SARS-CoV-2 Spike Trimer glycoprotein was immobilized on a Series S Sensor Chip CM5 using standard primary amine- coupling chemistry at 25°C with PBS, 0.05% P20, 1 % DMSO, pH 7.4 as the running buffer. The carboxymethyl dextran surface was activated with a 7 min injection of a 1 :1 v/v ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 pLmin -1 .
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N-hydroxy succinimide
  • SARS-CoV-2 Spike Trimer glycoprotein protein was diluted to 40 nM in 10 mM sodium acetate (pH 5.0) and captured with 200 s contact time at 10 pLmin -1 . Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5. Surface densities of 1200 Rll were achieved.
  • an 8-point titration of bicyclic peptide underwent multi cycle kinetic evaluation at 25 °C, flow rate of 50 pLmin -1 , with association time of 60 seconds and dissociation time of up to 400 seconds. A maximum concentration of 20000 nM bicyclic peptide was used. Data were solvent corrected for DMSO bulk effects. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using Biacore Insight Evaluation Software. Data were fitted using the steady-state affinity model.
  • SPR assays were performed on a Biacore T200 (Cytiva) with Series S Streptavidin (SA) sensor chips (Cytiva) in running buffer 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.05 % (v/v) P20, 2% DMSO, pH 7.4. His, AvitagTM biotinylated SARS-CoV-2 S1 protein (ACROBiosystems) protein was captured using standard methodlogy to generate 2700-3700 Rll.
  • SA Streptavidin
  • a 5- or 8-point titration of bicyclic peptide underwent respectively single or multi cycle kinetic evaluation at 25 °C, flow rate of 30 pLmin -1 , with association time of 120 seconds and dissociation time of up to 300 seconds.
  • a maximum concentration of 10000 nM bicyclic peptide was used.
  • Regeneration of the surface was performed using 1 mM HCI (30 seconds at 30 pL/min) followed by a stabilization period of 30 seconds. Each injection was followed by an additional wash with 50 % (v/v) DMSO/H2O, to limit carry-over. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using the Biacore T200 Evaluation Software (version 3.1). Data were fitted using the steady-state affinity model.
  • Replication deficient SARS-CoV-2 pseudotyped HIV-1 virions were prepared similarly as described in Mallery et al (2021) Sci Adv 7(11). Briefly, virions were produced in HEK 293T cells by transfection with 1 pg of the plasmid encoding SARS CoV-2 Spike protein (pCAGGS- SpikeAc19), 1 pg pCRV GagPol and 1.5 pg GFP-encoding plasmid (CSGW). Viral supernatants were filtered through a 0.45 pm syringe filter at 48 h and 72 h post-transfection and pelleted for 2 h at 28,000 x g. Pelleted virions were drained and then resuspended in DM EM (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 bicycle 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 Incucyte S3 live cell imaging system (Sartorius). The percent of cell entry was quantified as GFP positive areas of cells over the total area covered by cells. Entry inhibition by the Bicyclic peptide was calculated as percent virus infection relative to virus only control.
  • BCY16257 ACDEKAWWCQLAYVDCA[Sar6][KBiot] (SEQ ID NO: 93) - the C-terminally biotinylated form of BCY16113;
  • BCY16274 ACDPDPYDSSCWTCA[Sar6][KBiot] (SEQ ID NO: 94) - the C-terminally biotinylated form of BCY16126; were incubated on separate plates with poly(Histidine) tagged SARS-CoV-2 Spike Trimer Glycoprotein, along with duplicate titrations of multiple unmodified bicyclic peptides. Finally, 20 pg/mL of AlphaScreen Streptavidin donor beads (Perkin Elmer) and 20 pg/mL Nickel- chelate AlphaLISA Acceptor beads (Perkin Elmer) were added and incubated.
  • SPR analysis was performed on a Biacore 8K+ (Cytiva). Briefly, SARS-CoV-2 Spike Trimer Glycoprotein was immobilized on a Series S Sensor Chip CM5 using standard primary amine-coupling chemistry at 25°C with PBS, 0.05 % P20, 1 % DMSO, pH 7.4 as the running buffer. The carboxymethyl dextran surface was activated with a 7 min injection of a 1 :1 v/v ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N- hydroxy succinimide (NHS) at a flow rate of 10 pLmin' 1 .
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • NHS N- hydroxy succinimide
  • Spike Trimer Glycoprotein was diluted to 40 nM in 10 mM sodium acetate (pH 5.0) and captured with 200 s contact time at 10 pLmin' 1 . Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5. Surface densities ranging 870 - 1500 Rll were achieved.
  • an 8-point titration of bicyclic peptide underwent multi cycle kinetic evaluation at 25 °C, flow rate of 50 pLmin' 1 , with association time of 60 seconds and dissociation time of up to 120 seconds. A maximum concentration of 20000 nM bicyclic peptide was used. Data were solvent corrected for DMSO bulk effects. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using Biacore Insight Evaluation Software. Data were fitted using steady-state affinity model where appropriate.
  • 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).
  • Spike Glycoprotein domain S1 (ACROBiosystems - S1 N-C82E8) protein was captured to generate 3000-4000 Rll.
  • 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 a multiple cycle kinetic format.

Abstract

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 the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2). The invention also includes multimeric binding complexes comprising said bicyclic peptide ligands. The invention also includes pharmaceutical compositions comprising said polypeptides or said multimeric binding complexes and to the use of said polypeptides, multimeric binding complexes and pharmaceutical compositions 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.

Description

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 the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2). The invention also includes multimeric binding complexes comprising said bicyclic peptide ligands. The invention also includes pharmaceutical compositions comprising said polypeptides or said multimeric binding complexes and to the use of said polypeptides, multimeric binding complexes and pharmaceutical compositions in suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2.
BACKGROUND OF THE INVENTION
Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and spread globally, resulting in a pandemic. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include fatigue, muscle pain, diarrhea, sore throat, loss of smell, and abdominal pain. The time from exposure to onset of symptoms is typically around five days but may range from two to fourteen days. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure. As of 6 January 2021 , more than 86 million cases have been reported globally, resulting in more than 1.8 million deaths.
The virus is primarily spread between people during close contact, often via droplets produced by coughing, sneezing, or talking. While these droplets are produced when breathing out, they usually fall to the ground or onto surfaces rather than being infectious over long distances. People may also become infected by touching a contaminated surface and then their face. The virus can survive on surfaces for up to 72 hours. It is most contagious during the first three days after the onset of symptoms, although spread may be possible before symptoms appear and in later stages of the disease.
Currently, there is no vaccine or specific antiviral treatment for COVID-19. Management involves treatment of symptoms, supportive care, isolation, and experimental measures. The World Health Organization (WHO) declared the 2019-2020 coronavirus outbreak a Public Health Emergency of International Concern (PHEIC) on 30 January 2020 and a pandemic on 11 March 2020. Local transmission of the disease has been recorded in many countries across all six WHO regions.
There is therefore a great need to provide an effective prophylactic and/or therapeutic treatment intended to avoid or ameliorate the symptoms associated with infection of SARS- CoV-2, such as COVID-19.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a peptide ligand specific for the spike protein (S protein) of 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CLPAGCTDLWRYIQC (SEQ ID NO: 1);
CVVNGTIRYCALC (SEQ ID NO: 2);
CIVRGEIRWCGGPEC (SEQ ID NO: 3);
CIQNGVLKYCAQC (SEQ ID NO: 4);
CIKDNQILYCATC (SEQ ID NO: 5);
CIINNHVVYCATC (SEQ ID NO: 6);
CIRDGGIQYCALC (SEQ ID NO: 7);
CLPNGCTDLERYIKC (SEQ ID NO: 8);
CTPNGCTDLWRYIAC (SEQ ID NO: 9);
CSDEFCSAWWGFNEC (SEQ ID NO: 10);
CSDAFCSAWWGFNQC (SEQ ID NO: 11);
CSSKFCDAWWNFNRC (SEQ ID NO: 12);
CSDDFCSAWWGFNHC (SEQ ID NO: 13);
CSNKFCDAWWNFNRC (SEQ ID NO: 14);
CFPAPWLGLCTPC (SEQ ID NO: 15);
CFPEPWLGLCTPC (SEQ ID NO: 16);
CIVNGEIKYCADC (SEQ ID NO: 17);
CIKNDELVYCGGPKC (SEQ ID NO: 18);
CIVDDHIVYCGSKQC (SEQ ID NO: 19); CIKDGLLVYCGSYQC (SEQ ID NO: 20);
CIKDGVLIYCGGPMC (SEQ ID NO: 21);
CMNPFFYDCERTC (SEQ ID NO: 22) herein referred to as BCY18697 when complexed with a derivative of TATB which has the following structure:
Figure imgf000004_0001
wherein * denotes the point of attachment of the three cysteine residues;
CMNPFFYDCDHIC (SEQ ID NO: 23);
CMNPFFYDCHEQC (SEQ ID NO: 24);
CMNPFFYDCKWC (SEQ ID NO: 25);
CMNPFFYDCEDRC (SEQ ID NO: 26);
CMNPFFYDCEEIC (SEQ ID NO: 27);
CMNPFFYDCENPC (SEQ ID NO: 28);
CMNPFFYDCETVC (SEQ ID NO: 29);
CMNPFFYDCEYVC (SEQ ID NO: 30);
CMNPFYYDCEEVC (SEQ ID NO: 31);
C[Hse(Me)]NPFFYDCERTC (SEQ ID NO: 32);
CMN[HyP]FFYDCERTC (SEQ ID NO: 33);
CMNPFF[4FPhe]DCERTC (SEQ ID NO: 34);
CEDNDWVYCSTC (SEQ ID NO: 35);
CDWTCYLRPLPC (SEQ ID NO: 36);
CMFVPCAARHELGLC (SEQ ID NO: 41);
CMFVPCAARVELGLC (SEQ ID NO: 42);
CMFVPCAIRQTLGLC (SEQ ID NO: 43);
CMFVPCATRHELGLC (SEQ ID NO: 44);
CMFVPCATRHQLGLC (SEQ ID NO: 45);
CMFVPCATRHSLGLC (SEQ ID NO: 46);
CMFVPCATRLALGLC (SEQ ID NO: 47);
CMFVPCATRLQLGLC (SEQ ID NO: 48);
CMFVPCATRQELGLC (SEQ ID NO: 49);
CMFVPCATRQMLGLC (SEQ ID NO: 50);
CMFVPCATRVALGLC (SEQ ID NO: 51); CMFVPCAVREELGLC (SEQ ID NO: 52);
CMFVPCAVRHALGLC (SEQ ID NO: 53);
CMFVPCAVRHSLGLC (SEQ ID NO: 54);
CMFVPCAVRKDLGLC (SEQ ID NO: 55);
CMFVPCAVRQTLGLC (SEQ ID NO: 56);
CMFTPCHVREILGLC (SEQ ID NO: 57);
CMGVPCKVREILGLC (SEQ ID NO: 58);
CMFVPCAVREIL[dA]LC (SEQ ID NO: 59);
C[Hse(Me)]FVPCAVREILGLC (SEQ ID NO: 60);
CMFV[HyP]CAVREILGLC (SEQ ID NO: 61);
Ac-CPYVAGR[dA]TCLLC (SEQ ID NO: 62) herein referred to as BCY18089 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000005_0001
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAGR[dA]TCL[tBuAla]C (SEQ ID NO: 63) herein referred to as BCY18090 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000005_0002
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg]GTCLLC (SEQ ID NO: 64) herein referred to as BCY18091 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000006_0001
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg][dA]TCLLC (SEQ ID NO: 65) herein referred to as BCY18092 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000006_0002
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg]GTCL[tBuAla]C (SEQ ID NO: 66) herein referred to as BCY18093 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000006_0003
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAGRGTCL[tBuAla]C (SEQ ID NO: 67) herein referred to as BCY18094 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000006_0004
wherein * denotes the point of attachment of the three cysteine residues; CHPVCSVPAIGLLC (SEQ ID NO: 68);
CEINASLPCTFTC (SEQ ID NO: 69);
CEFYYANCEDVLPWC (SEQ ID NO: 70);
CDPDPYDSSCWTC (SEQ ID NO: 71);
CDWDWHVCAIMNESC (SEQ ID NO: 72);
CTPLDATFCFSKC (SEQ ID NO: 73);
CEEDWHICQIHGYDC (SEQ ID NO: 74);
CNEDWHSCMIADSDC (SEQ ID NO: 75);
CWDSSCWAHMDKC (SEQ ID NO: 76);
CNNPFCEYHIC (SEQ ID NO: 77);
CTTLDKIFCFSSC (SEQ ID NO: 78);
CFGEDWHTCSIYC (SEQ ID NO: 79);
CMDWHVCMLNDTLFC (SEQ ID NO: 80);
CTNIMCEFMFC (SEQ ID NO: 81);
CINPYCEHHIYLEHC (SEQ ID NO: 82);
CNMDCYHLPFTSMYC (SEQ ID NO: 83);
CDEKAWWCQLAYVDC (SEQ ID NO: 84);
CHQAYGMCSIFPEWC (SEQ ID NO: 85);
CKESSWYCQMWDIQC (SEQ ID NO: 86);
CDGPDWHSCMVSC (SEQ ID NO: 87);
CQNLHPLCGVLESHMC (SEQ ID NO: 88);
CWNSEDWHACQIC (SEQ ID NO: 89);
CDHYHCPWLALGGSC (SEQ ID NO: 90);
CELDEWLCIIGHLDC (SEQ ID NO: 91); and CMEFAANCEDIYDDC (SEQ ID NO: 92); wherein the three cysteine residues within each peptide ligand represent the three reactive groups and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, tBuAla represents t-butyl-alanine, HArg represents homoarginine, and 4FPhe represents 4- fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
According to a further aspect of the invention, there is provided a multimeric binding complex which comprises either: (i) at least two bicyclic peptide ligands of the invention, wherein said peptide ligands may be the same or different; or (ii) at least one bicyclic peptide ligand of the invention, which may be the same or different, in combination with one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be the same or different. According to a further aspect of the invention, there is provided a pharmaceutical composition comprising the peptide ligand or the multimeric binding complex 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 or the multimeric binding complex 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 peptide ligand specific for the spike protein (S protein) of 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CLPAGCTDLWRYIQC (SEQ ID NO: 1);
CVVNGTIRYCALC (SEQ ID NO: 2);
CIVRGEIRWCGGPEC (SEQ ID NO: 3);
CIQNGVLKYCAQC (SEQ ID NO: 4);
CIKDNQILYCATC (SEQ ID NO: 5);
CIINNHVVYCATC (SEQ ID NO: 6);
CIRDGGIQYCALC (SEQ ID NO: 7);
CLPNGCTDLERYIKC (SEQ ID NO: 8);
CTPNGCTDLWRYIAC (SEQ ID NO: 9);
CSDEFCSAWWGFNEC (SEQ ID NO: 10);
CSDAFCSAWWGFNQC (SEQ ID NO: 11);
CSSKFCDAWWNFNRC (SEQ ID NO: 12);
CSDDFCSAWWGFNHC (SEQ ID NO: 13);
CSNKFCDAWWNFNRC (SEQ ID NO: 14);
CFPAPWLGLCTPC (SEQ ID NO: 15);
CFPEPWLGLCTPC (SEQ ID NO: 16);
CIVNGEIKYCADC (SEQ ID NO: 17);
CIKNDELVYCGGPKC (SEQ ID NO: 18); CIVDDHIVYCGSKQC (SEQ ID NO: 19);
CIKDGLLVYCGSYQC (SEQ ID NO: 20);
CIKDGVLIYCGGPMC (SEQ ID NO: 21);
CMNPFFYDCERTC (SEQ ID NO: 22) herein referred to as BCY18697 when complexed with a derivative of TATB which has the following structure:
Figure imgf000009_0001
wherein * denotes the point of attachment of the three cysteine residues;
CMNPFFYDCDHIC (SEQ ID NO: 23);
CMNPFFYDCHEQC (SEQ ID NO: 24);
CMNPFFYDCKWC (SEQ ID NO: 25);
CMNPFFYDCEDRC (SEQ ID NO: 26);
CMNPFFYDCEEIC (SEQ ID NO: 27);
CMNPFFYDCENPC (SEQ ID NO: 28);
CMNPFFYDCETVC (SEQ ID NO: 29);
CMNPFFYDCEYVC (SEQ ID NO: 30);
CMNPFYYDCEEVC (SEQ ID NO: 31);
C[Hse(Me)]NPFFYDCERTC (SEQ ID NO: 32);
CMN[HyP]FFYDCERTC (SEQ ID NO: 33);
CMNPFF[4FPhe]DCERTC (SEQ ID NO: 34);
CEDNDWVYCSTC (SEQ ID NO: 35); and CDWTCYLRPLPC (SEQ ID NO: 36); CMFVPCAARHELGLC (SEQ ID NO: 41);
CMFVPCAARVELGLC (SEQ ID NO: 42);
CMFVPCAIRQTLGLC (SEQ ID NO: 43);
CMFVPCATRHELGLC (SEQ ID NO: 44);
CMFVPCATRHQLGLC (SEQ ID NO: 45);
CMFVPCATRHSLGLC (SEQ ID NO: 46);
CMFVPCATRLALGLC (SEQ ID NO: 47);
CMFVPCATRLQLGLC (SEQ ID NO: 48);
CMFVPCATRQELGLC (SEQ ID NO: 49);
CMFVPCATRQMLGLC (SEQ ID NO: 50); CMFVPCATRVALGLC (SEQ ID NO: 51);
CMFVPCAVREELGLC (SEQ ID NO: 52);
CMFVPCAVRHALGLC (SEQ ID NO: 53);
CMFVPCAVRHSLGLC (SEQ ID NO: 54);
CMFVPCAVRKDLGLC (SEQ ID NO: 55);
CMFVPCAVRQTLGLC (SEQ ID NO: 56);
CMFTPCHVREILGLC (SEQ ID NO: 57);
CMGVPCKVREILGLC (SEQ ID NO: 58);
CMFVPCAVREIL[dA]LC (SEQ ID NO: 59);
C[Hse(Me)]FVPCAVREILGLC (SEQ ID NO: 60);
CMFV[HyP]CAVREILGLC (SEQ ID NO: 61);
Ac-CPYVAGR[dA]TCLLC (SEQ ID NO: 62) herein referred to as BCY18089 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000010_0001
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAGR[dA]TCL[tBuAla]C (SEQ ID NO: 63) herein referred to as
BCY18090 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000010_0002
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg]GTCLLC (SEQ ID NO: 64) herein referred to as BCY18091 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000011_0001
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg][dA]TCLLC (SEQ ID NO: 65) herein referred to as
BCY18092 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000011_0002
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg]GTCL[tBuAla]C (SEQ ID NO: 66) herein referred to as BCY18093 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000011_0003
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAGRGTCL[tBuAla]C (SEQ ID NO: 67) herein referred to as
BCY18094 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000011_0004
wherein * denotes the point of attachment of the three cysteine residues; CHPVCSVPAIGLLC (SEQ ID NO: 68);
CEINASLPCTFTC (SEQ ID NO: 69);
CEFYYANCEDVLPWC (SEQ ID NO: 70);
CDPDPYDSSCWTC (SEQ ID NO: 71);
CDWDWHVCAIMNESC (SEQ ID NO: 72);
CTPLDATFCFSKC (SEQ ID NO: 73);
CEEDWHICQIHGYDC (SEQ ID NO: 74);
CNEDWHSCMIADSDC (SEQ ID NO: 75);
CWDSSCWAHMDKC (SEQ ID NO: 76);
CNNPFCEYHIC (SEQ ID NO: 77);
CTTLDKIFCFSSC (SEQ ID NO: 78);
CFGEDWHTCSIYC (SEQ ID NO: 79);
CMDWHVCMLNDTLFC (SEQ ID NO: 80);
CTNIMCEFMFC (SEQ ID NO: 81);
CINPYCEHHIYLEHC (SEQ ID NO: 82);
CNMDCYHLPFTSMYC (SEQ ID NO: 83);
CDEKAWWCQLAYVDC (SEQ ID NO: 84);
CHQAYGMCSIFPEWC (SEQ ID NO: 85);
CKESSWYCQMWDIQC (SEQ ID NO: 86);
CDGPDWHSCMVSC (SEQ ID NO: 87);
CQNLHPLCGVLESHMC (SEQ ID NO: 88);
CWNSEDWHACQIC (SEQ ID NO: 89);
CDHYHCPWLALGGSC (SEQ ID NO: 90);
CELDEWLCIIGHLDC (SEQ ID NO: 91); and CMEFAANCEDIYDDC (SEQ ID NO: 92); wherein the three cysteine residues within each peptide ligand represent the three reactive groups and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, tBuAla represents t-butyl-alanine, HArg represents homoarginine, and 4FPhe represents 4- fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
According to one particular aspect of the invention which may be mentioned, there is provided a peptide ligand specific for the spike protein (S protein) of 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CLPAGCTDLWRYIQC (SEQ ID NO: 1);
CVVNGTIRYCALC (SEQ ID NO: 2);
CIVRGEIRWCGGPEC (SEQ ID NO: 3);
CIQNGVLKYCAQC (SEQ ID NO: 4);
CIKDNQILYCATC (SEQ ID NO: 5);
CIINNHVVYCATC (SEQ ID NO: 6);
CIRDGGIQYCALC (SEQ ID NO: 7);
CLPNGCTDLERYIKC (SEQ ID NO: 8);
CTPNGCTDLWRYIAC (SEQ ID NO: 9);
CSDEFCSAWWGFNEC (SEQ ID NO: 10);
CSDAFCSAWWGFNQC (SEQ ID NO: 11);
CSSKFCDAWWNFNRC (SEQ ID NO: 12);
CSDDFCSAWWGFNHC (SEQ ID NO: 13);
CSNKFCDAWWNFNRC (SEQ ID NO: 14);
CFPAPWLGLCTPC (SEQ ID NO: 15);
CFPEPWLGLCTPC (SEQ ID NO: 16);
CIVNGEIKYCADC (SEQ ID NO: 17);
CIKNDELVYCGGPKC (SEQ ID NO: 18);
CIVDDHIVYCGSKQC (SEQ ID NO: 19);
CIKDGLLVYCGSYQC (SEQ ID NO: 20);
CIKDGVLIYCGGPMC (SEQ ID NO: 21);
CMNPFFYDCERTC (SEQ ID NO: 22) herein referred to as BCY18697 when complexed with a derivative of TATB which has the following structure:
Figure imgf000013_0001
wherein * denotes the point of attachment of the three cysteine residues;
CMNPFFYDCDHIC (SEQ ID NO: 23);
CMNPFFYDCHEQC (SEQ ID NO: 24);
CMNPFFYDCKWC (SEQ ID NO: 25);
CMNPFFYDCEDRC (SEQ ID NO: 26); CMNPFFYDCEEIC (SEQ ID NO: 27);
CMNPFFYDCENPC (SEQ ID NO: 28);
CMNPFFYDCETVC (SEQ ID NO: 29);
CMNPFFYDCEYVC (SEQ ID NO: 30);
CMNPFYYDCEEVC (SEQ ID NO: 31);
C[Hse(Me)]NPFFYDCERTC (SEQ ID NO: 32);
CMN[HyP]FFYDCERTC (SEQ ID NO: 33);
CMNPFF[4FPhe]DCERTC (SEQ ID NO: 34);
CEDNDWVYCSTC (SEQ ID NO: 35); and
CDWTCYLRPLPC (SEQ ID NO: 36); wherein the three cysteine residues within each peptide ligand represent the three reactive groups and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, and 4FPhe represents 4-fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
The spike protein (S protein) is a large type I transmembrane protein of SARS-CoV-2. This protein is highly glycosylated as it contains 21 to 35 N-glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive “corona”, or crown-like appearance. The ectodomain of all CoV spike proteins share the same organization in two domains: a N-terminal domain named S1 that is responsible for receptor binding and a C- terminal S2 domain responsible for fusion. CoV diversity is reflected in the variable spike proteins (S proteins), which have evolved into forms differing in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion.
In one embodiment, said peptide ligand is specific for the S2 domain of the spike protein (S2 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CLPAGCTDLWRYIQC (SEQ ID NO: 1);
CVVNGTIRYCALC (SEQ ID NO: 2);
CIVRGEIRWCGGPEC (SEQ ID NO: 3);
CIQNGVLKYCAQC (SEQ ID NO: 4);
CIKDNQILYCATC (SEQ ID NO: 5);
CIINNHVVYCATC (SEQ ID NO: 6);
CIRDGGIQYCALC (SEQ ID NO: 7);
CLPNGCTDLERYIKC (SEQ ID NO: 8);
CTPNGCTDLWRYIAC (SEQ ID NO: 9); CSDEFCSAWWGFNEC (SEQ ID NO: 10);
CSDAFCSAWWGFNQC (SEQ ID NO: 11);
CSSKFCDAWWNFNRC (SEQ ID NO: 12);
CSDDFCSAWWGFNHC (SEQ ID NO: 13);
CSNKFCDAWWNFNRC (SEQ ID NO: 14);
CFPAPWLGLCTPC (SEQ ID NO: 15);
CFPEPWLGLCTPC (SEQ ID NO: 16);
CIVNGEIKYCADC (SEQ ID NO: 17);
CIKNDELVYCGGPKC (SEQ ID NO: 18);
CIVDDHIVYCGSKQC (SEQ ID NO: 19);
CIKDGLLVYCGSYQC (SEQ ID NO: 20); and CIKDGVLIYCGGPMC (SEQ ID NO: 21); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a further embodiment, said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000015_0001
wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 1)-A (herein referred to as BCY18948);
A-(SEQ ID NO: 8)-A (herein referred to as BCY19894);
A-(SEQ ID NO: 9)-A (herein referred to as BCY19896);
A-(SEQ ID NO: 10)-A (herein referred to as BCY19899);
A-(SEQ ID NO: 11)-A (herein referred to as BCY19900);
A-(SEQ ID NO: 12)-A (herein referred to as BCY19901);
Ac-(SEQ ID NO: 12)-[K(PYA)] (herein referred to as BCY22415);
A-(SEQ ID NO: 13)-A (herein referred to as BCY19902); and A-(SEQ ID NO: 14)-A (herein referred to as BCY19903); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a yet further embodiment, said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TATB which has the following structure:
Figure imgf000016_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 2)-A (herein referred to as BCY18950);
A-(SEQ ID NO: 3)-A (herein referred to as BCY18953);
A-(SEQ ID NO: 4)-A (herein referred to as BCY18956);
Ac-(SEQ ID NO: 4)-K (herein referred to as BCY22419);
A-(SEQ ID NO: 5)-A (herein referred to as BCY18957);
A-(SEQ ID NO: 6)-A (herein referred to as BCY18958);
A-(SEQ ID NO: 7)-A (herein referred to as BCY18961);
A-(SEQ ID NO: 17)-A (herein referred to as BCY19916);
A-(SEQ ID NO: 18)-A (herein referred to as BCY19919);
A-(SEQ ID NO: 19)-A (herein referred to as BCY19921);
A-(SEQ ID NO: 20)-A (herein referred to as BCY19923); and A-(SEQ ID NO: 21)-A (herein referred to as BCY19924); or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a still yet further embodiment, said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TCMT which has the following structure:
Figure imgf000017_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 15)-A (herein referred to as BCY19904);
A-(SEQ ID NO: 16)-A (herein referred to as BCY19905); and
Ac-(SEQ ID NO: 16)-K (herein referred to as BCY22416); or a modified derivative and/or pharmaceutically acceptable salt thereof.
In an alternative embodiment, said peptide ligand is specific for the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000017_0002
wherein * denotes the point of attachment of the three cysteine residues, 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: 41)-A (herein referred to as BCY17367);
A-(SEQ ID NO: 42)-A (herein referred to as BCY17368);
A-(SEQ ID NO: 43)-A (herein referred to as BCY17369);
A-(SEQ ID NO: 44)-A (herein referred to as BCY17370);
A-(SEQ ID NO: 45)-A (herein referred to as BCY17371);
A-(SEQ ID NO: 46)-A (herein referred to as BCY17372);
A-(SEQ ID NO: 47)-A (herein referred to as BCY17373); A-(SEQ ID NO: 48)-A (herein referred to as BCY17375);
A-(SEQ ID NO: 49)-A (herein referred to as BCY17376);
A-(SEQ ID NO: 50)-A (herein referred to as BCY17377);
A-(SEQ ID NO: 51)-A (herein referred to as BCY17378);
A-(SEQ ID NO: 52)-A (herein referred to as BCY17380);
A-(SEQ ID NO: 53)-A (herein referred to as BCY17381);
A-(SEQ ID NO: 54)-A (herein referred to as BCY17382);
A-(SEQ ID NO: 55)-A (herein referred to as BCY17383);
A-(SEQ ID NO: 56)-A (herein referred to as BCY17384);
A-(SEQ ID NO: 57)-A (herein referred to as BCY17385);
A-(SEQ ID NO: 58)-A (herein referred to as BCY17387);
A-(SEQ ID NO: 59)-A (herein referred to as BOY 17615);
A-(SEQ ID NO: 60)-A (herein referred to as BCY17617); and A-(SEQ ID NO: 61)-A (herein referred to as BCY17618); or a modified derivative and/or pharmaceutically acceptable salt thereof.
In an alternative embodiment, said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CMNPFFYDCERTC (SEQ ID NO: 22); herein referred to as BCY18697 when complexed with a derivative of TATB which has the following structure:
Figure imgf000018_0001
wherein * denotes the point of attachment of the three cysteine residues);
CMNPFFYDCDHIC (SEQ ID NO: 23);
CMNPFFYDCHEQC (SEQ ID NO: 24);
CMNPFFYDCKWC (SEQ ID NO: 25);
CMNPFFYDCEDRC (SEQ ID NO: 26);
CMNPFFYDCEEIC (SEQ ID NO: 27);
CMNPFFYDCENPC (SEQ ID NO: 28);
CMNPFFYDCETVC (SEQ ID NO: 29);
CMNPFFYDCEYVC (SEQ ID NO: 30); CMNPFYYDCEEVC (SEQ ID NO: 31);
C[Hse(Me)]NPFFYDCERTC (SEQ ID NO: 32);
CMN[HyP]FFYDCERTC (SEQ ID NO: 33);
CMNPFF[4FPhe]DCERTC (SEQ ID NO: 34); and CEDNDWVYCSTC (SEQ ID NO: 35); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, and 4FPhe represents 4-fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a further embodiment, said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATB which has the following structure:
Figure imgf000019_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 22)-A (herein referred to as BCY16207);
Ac-A-(SEQ ID NO: 22)-A (herein referred to as BCY18696);
Ac-(SEQ ID NO: 22) (herein referred to as BCY18698);
A-(SEQ ID NO: 23)-A (herein referred to as BCY18144);
A-(SEQ ID NO: 24)-A (herein referred to as BCY18145);
A-(SEQ ID NO: 25)-A (herein referred to as BCY18146);
A-(SEQ ID NO: 26)-A (herein referred to as BCY18147);
A-(SEQ ID NO: 27)-A (herein referred to as BCY18148);
A-(SEQ ID NO: 28)-A (herein referred to as BCY18149);
A-(SEQ ID NO: 29)-A (herein referred to as BCY18150);
Ac-(SEQ ID NO: 29) (herein referred to as BCY22405);
Ac-(SEQ ID NO: 29)-[K(PYA)] (herein referred to as BCY22413);
A-(SEQ ID NO: 30)-A (herein referred to as BCY18151);
A-(SEQ ID NO: 31)-A (herein referred to as BCY18152); A-(SEQ ID NO: 32)-A (herein referred to as BCY18700);
A-(SEQ ID NO: 33)-A (herein referred to as BCY18702); and
A-(SEQ ID NO: 34)-A (herein referred to as BCY18703); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a further embodiment, said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000020_0001
wherein * denotes the point of attachment of the three cysteine residues, and 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: 35)-[K(PYA)] (herein referred to as BCY22412);
A-(SEQ ID NO: 42)-A (herein referred to as BCY17368);
A-(SEQ ID NO: 44)-A (herein referred to as BCY17370);
A-(SEQ ID NO: 45)-A (herein referred to as BCY17371);
A-(SEQ ID NO: 47)-A (herein referred to as BCY17373);
A-(SEQ ID NO: 49)-A (herein referred to as BCY17376);
A-(SEQ ID NO: 50)-A (herein referred to as BCY17377);
A-(SEQ ID NO: 51)-A (herein referred to as BCY17378);
A-(SEQ ID NO: 53)-A (herein referred to as BCY17381);
A-(SEQ ID NO: 56)-A (herein referred to as BCY17384); and
A-(SEQ ID NO: 58)-A (herein referred to as BCY17387); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof. In a further embodiment, said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000021_0001
wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:
Ac-(SEQ ID NO: 35)-[K(PYA)] (herein referred to as BCY22412); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In an alternative embodiment, said peptide ligand is specific for the N-Terminal Domain (NTD) of the S1 domain of the spike protein (S1 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is:
CDWTCYLRPLPC (SEQ ID NO: 36); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a further embodiment, said peptide ligand is specific for the N-Terminal Domain (NTD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATB which has the following structure:
Figure imgf000021_0002
wherein * denotes the point of attachment of the three cysteine residues, and 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: 36)-[K(PYA)] (herein referred to as BCY22414); and
Ac-(SEQ ID NO: 36)-K (herein referred to as BCY22417); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a further embodiment, the peptide ligand specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a molecular scaffold which is a derivative of TATB which has the following structure:
Figure imgf000022_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 68)-A (herein referred to as BCY16129);
A-(SEQ ID NO: 69)-A (herein referred to as BCY16128);
A-(SEQ ID NO: 70)-A (herein referred to as BCY16127);
A-(SEQ ID NO: 71)-A (herein referred to as BCY16126);
A-(SEQ ID NO: 72)-A (herein referred to as BCY16125);
A-(SEQ ID NO: 73)-A (herein referred to as BCY16124);
A-(SEQ ID NO: 74)-A (herein referred to as BCY16123);
A-(SEQ ID NO: 75)-A (herein referred to as BCY16122);
A-(SEQ ID NO: 76)-A (herein referred to as BCY16121);
A-(SEQ ID NO: 77)-A (herein referred to as BCY16120);
A-(SEQ ID NO: 78)-A (herein referred to as BCY16119);
A-(SEQ ID NO: 79)-A (herein referred to as BCY16118);
A-(SEQ ID NO: 80)-A (herein referred to as BCY16117);
A-(SEQ ID NO: 81)-A (herein referred to as BCY16116);
A-(SEQ ID NO: 82)-A (herein referred to as BCY16115);
A-(SEQ ID NO: 83)-A (herein referred to as BCY16114);
A-(SEQ ID NO: 84)-A (herein referred to as BCY16113); A-(SEQ ID NO: 85)-A (herein referred to as BCY16112);
A-(SEQ ID NO: 86)-A (herein referred to as BCY16111);
A-(SEQ ID NO: 87)-A (herein referred to as BCY16110);
A-(SEQ ID NO: 88)-A (herein referred to as BCY16109);
A-(SEQ ID NO: 89)-A (herein referred to as BCY16108);
A-(SEQ ID NO: 90)-A (herein referred to as BCY16107);
A-(SEQ ID NO: 91)-A (herein referred to as BCY16106); and A-(SEQ ID NO: 92)-A (herein referred to as BCY16105); or a modified derivative and/or pharmaceutically acceptable salt thereof.
In a further embodiment, the peptide ligand specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a molecular scaffold which is a derivative of TCMT which has the following structure:
Figure imgf000023_0001
wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand and comprises an amino acid sequence which is selected from:
(SEQ ID NO: 62) (herein referred to as BCY18089);
(SEQ ID NO: 63) (herein referred to as BCY18090);
(SEQ ID NO: 64) (herein referred to as BCY18091);
(SEQ ID NO: 65) (herein referred to as BCY18092);
(SEQ ID NO: 66) (herein referred to as BCY18093); and
(SEQ ID NO: 67) (herein referred to as BCY18094); or a modified derivative and/or pharmaceutically acceptable salt thereof.
Multimeric Binding Complexes
According to a further aspect of the invention, there is provided a multimeric binding complex which comprises either: (i) at least two bicyclic peptide ligands of the invention, wherein said peptide ligands may be the same or different; or (ii) at least one bicyclic peptide ligand of the invention, which may be the same or different, in combination with one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be the same or different. In one embodiment, the multimeric binding complexes comprise at least two (e.g. two or three) bicyclic peptides which are the same (i.e. homomultimers). In a further embodiment, the multimeric binding complexes comprise three bicyclic peptides which are the same (i.e. homotrimers). In an alternative embodiment, the multimeric binding complexes comprise at least two (e.g. two or three) bicyclic peptides which are different (i.e. heteromultimers). In a further embodiment, the multimeric binding complexes comprise two bicyclic peptides, each of which are different (i.e. heterodimers). In an alternative embodiment, the multimeric binding complexes comprise three bicyclic peptides, each of which are different (i.e. heterotri mers).
References herein to “one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” refer to any bicyclic peptide which is capable of binding to the spike protein of SARS-CoV-2. Examples of suitable bicyclic peptides include those in PCT/GB2022/050031 , PCT/GB2022/050036, and PCT/GB2022/050037, the bicyclic peptides of which are herein incorporated by reference. In one embodiment, said “one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” is selected from one or more of: BCY16592, BCY18579, BCY18654, and/or BCY19600.
BCY16592 is a bicyclic peptide ligand having the sequence Ac- (CPYVAG[Agb][dA]TCL[tBuAla]C)-[K(PYA)] (SEQ ID NO: 37) and a molecular scaffold which is a derivative of TCMT which has the following structure:
Figure imgf000024_0001
wherein * denotes the point of attachment of the three cysteine residues, and Agb represents 2-amino-4-guanidinobutyric acid, tBuAla represents t-butyl-alanine, and PYA represents pentynoic acid.
BCY18579 is a bicyclic peptide ligand having the sequence Ac-(CANPDNPVCRFYC)-K (SEQ ID NO: 38) and a molecular scaffold which is a derivative of TATB which has the following structure:
Figure imgf000025_0001
wherein * denotes the point of attachment of the three cysteine residues.
BCY18654 is a bicyclic peptide ligand having the sequence Ac-(CIPLDWTCMIAC)-K (SEQ ID NO: 39) and a molecular scaffold which is a derivative of TATB which has the following structure:
Figure imgf000025_0002
wherein * denotes the point of attachment of the three cysteine residues. BCY19600 is a bicyclic peptide ligand having the sequence Ac-
(C[Oic][4tBuPhe]VAG[HArg][dA]TCL[tBuAla]C)-[K(PYA)] (SEQ ID NO: 40) and a molecular scaffold which is a derivative of TCMT which has the following structure:
Figure imgf000025_0003
wherein * denotes the point of attachment of the three cysteine residues, HArg represents homoarginine, tBuAla represents t-butyl-alanine, 4tBuPhe represents 4-t-butyl-phenylalanine, Oic represents octahydroindolecarboxylic acid, and PYA represents pentynoic acid. In one embodiment, the multimeric binding complex comprises a linker between said bicyclic peptide ligands. In a further embodiment, the linker comprises a PEG containing linker.
In one embodiment, when the multimeric binding complex comprises two bicyclic peptide ligands, the linker is a linear linker which is:
Figure imgf000026_0001
wherein n represents an integer from 1 to 30, such as 10 or 24 and * represents the point of attachment to each bicyclic peptide ligand. In an alternative embodiment, when the multimeric binding complex comprises three bicyclic peptide ligands, the linker is a branched linker selected from:
Figure imgf000026_0002
and
Figure imgf000027_0001
wherein n represents an integer from 1 to 30, such as 10 or 24 and * represents the point of attachment to each bicyclic peptide ligand. In one embodiment, the multimeric binding complex comprises two differing bicyclic peptides (i.e. heterodimers). Examples of such heterodimers are shown in Table A below:
Table A: Exemplified Heterodimeric Binding Complexes of the Invention
Figure imgf000028_0001
In an alternative embodiment, the multimeric binding complex comprises three identical bicyclic peptides (i.e. homotrimers). Examples of such homotrimers are shown in Table B below:
Table B: Exemplified Homotrimeric Binding Complexes of the Invention
Figure imgf000028_0002
Figure imgf000029_0001
In an alternative embodiment, the multimeric binding complex comprises three differing bicyclic peptides (i.e. heterotrimers). Examples of such heterotrimers are shown in Table C below:
Table C: Exemplified Heterotrimeric Binding Complexes of the Invention
Figure imgf000030_0001
Figure imgf000030_0002
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry.
Standard techniques are used for molecular biology, genetic and biochemical methods (see Sam brook et a/., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel etal., 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 (C) 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:
C-L1-P2-A3-G4-C-T5-D6-L7-W8-R9-Y10-I11-Q12-C (SEQ ID NO: 1).
For the purpose of this description, all bicyclic peptides are assumed to be cyclised with TATA, TATB or TCMT and yielding a tri-substituted structure. Cyclisation with TATA, TATB or TCMT occurs on the first, second and third reactive groups.
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 pAla-Sar10-Ala tail would be denoted as:
PAIa-Sar10-A-(SEQ ID NO: X).
Inversed Peptide Sequences
In light of the disclosure in Nair etal (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 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-PEGw- amido-PIB (wherein PIB represents 4(4-iodophenyl)butyrate).
Advantages of the Peptide Ligands
Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:
Species cross-reactivity. Certain ligands demonstrate cross-reactivity across Lipid II from different bacterial species and hence are able to treat infections caused by multiple species of bacteria. Other ligands may be highly specific for the Lipid II of certain bacterial species which may be advantageous for treating an infection without collateral damage to the beneficial flora of the patient;
Protease stability. Bicyclic peptide ligands should ideally demonstrate stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a bicycle lead candidate can be developed in animal models as well as administered with confidence to humans;
Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra/inter-molecular H-bonding, which is important for formulation and absorption purposes;
An optimal plasma half-life in the circulation. Depending upon the clinical indication and treatment regimen, it may be required to develop a bicyclic peptide for short exposure in an acute illness management setting, or develop a bicyclic peptide with enhanced retention in the circulation, and is therefore optimal for the management of more chronic disease states. Other factors driving the desirable plasma half-life are requirements of sustained exposure for maximal therapeutic efficiency versus the accompanying toxicology due to sustained exposure of the agent; and
Selectivity. Pharmaceutically Acceptable Salts
It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.
The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.
Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1 ,2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1 ,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L- pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L- tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.
If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO'), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li+, Na+ and K+, alkaline earth metal cations such as 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., NHsR+, NH2R2+, NHRs+, 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(CHs)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 phenolreactive reagents so as to functionalise said amino acids, and introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide-bearing moieties, respectively. In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N- terminal modification using suitable amino-reactive chemistry, and/or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C- terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.
In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (the group referred to herein as Ci) 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 is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.
In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.
Alternatively, non-natural amino acids may be used having constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Ca- disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid. In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine 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 p-turn conformations (Tugyi et a/ (2005) PNAS, 102(2), 413-418).
In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential proteolytic attack site(s).
It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:
Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved;
Incorporating charged groups that exploit long-range ionic interactions, leading to faster on rates and to higher affinities (see for example Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and
Incorporating additional constraint into the peptide, by for example constraining side chains of amino acids correctly such that loss in entropy is minimal upon target binding, constraining the torsional angles of the backbone such that loss in entropy is minimal upon target binding and introducing additional cyclisations in the molecule for identical reasons.
(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et a/, 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 11C, 13C and 14C, chlorine, such as 36CI, fluorine, such as 18F, iodine, such as 123l, 125l and 131l, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, phosphorus, such as 32P, sulfur, such as 35S, copper, such as 64Cu, gallium, such as 67Ga or 68Ga, yttrium, such as 90Y and 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, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. 3H (T), and carbon-14, i.e. 14C, 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 11C, 18F, 15O 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. 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, a 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- triyl)triprop-2-en-1-one (also known as triacryloylhexahydro-s-triazine; TATA), 1 ,3,5- tris(bromoacetyl) hexahydro-1 , 3, 5-triazine (TATB) and 2,4,6-tris(chloromethyl)-s-triazine (TCMT).
In a further embodiment, the molecular scaffold is 1 , 1 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop- 2-en-1-one (also known as triacryloylhexahydro-s-triazine (TATA):
Figure imgf000039_0001
TATA. Thus, following cyclisation with the bicyclic peptides of the invention on the cysteine residues, the molecular scaffold forms a tri-substituted 1 ,1',1"-(1,3,5-triazinane-1,3,5-triyl)tripropan-1- one derivative of TATA having the following structure:
Figure imgf000040_0001
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):
Figure imgf000040_0002
TATB.
Thus, following cyclisation with the bicyclic peptides of the invention on the 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:
Figure imgf000040_0003
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):
Figure imgf000041_0001
Thus, following cyclisation with the bicyclic peptides of the invention on the three cysteine residues, the molecular scaffold forms a tri-substituted 2,4,6-tris(chloromomethyl)-s-triazine derivative of TCMT having the following structure:
Figure imgf000041_0002
wherein * denotes the point of attachment of the three cysteine residues.
Reactive Groups
The molecular scaffold of the invention may be bonded to the polypeptide via functional or reactive groups on the polypeptide. These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. Such reactive groups may be a cysteine side chain, a [Dap(Me)] group, a lysine side chain, or an N-terminal amine group or any other suitable reactive group. Details may be found in WO 2009/098450. In one embodiment, the reactive groups are all cysteine residues.
Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold/molecular core. The polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.
In a preferred embodiment, polypeptides with three reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is favourable for several reasons. The nucleic acids of the compound libraries encode only the primary sequences of the polypeptide but not the isomeric state of the molecules that are formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer that was isolated in a screening or selection process. The formation of a single product isomer is also advantageous if a specific member of a library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.
In another embodiment of the invention, polypeptides with four reactive groups are generated. Reaction of said polypeptides with a molecular scaffold/molecular core having a tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomer can be determined by chemically synthesizing both isomers, separating the two isomers and testing both isomers for binding to a target ligand.
In one embodiment of the invention, at least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core. Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at least three bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved.
In another embodiment, the reactive groups of the polypeptide of the invention are reacted with molecular linkers wherein said linkers are capable to react with a molecular scaffold so that the linker will intervene between the molecular scaffold and the polypeptide in the final bonded state.
In some embodiments, amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid. Excluded from these exchangeable amino acids are the ones harbouring functional groups for cross-linking the polypeptides to a molecular core, such that the loop sequences alone are exchangeable. The exchangeable polypeptide sequences have either random sequences, constant sequences or sequences with random and constant amino acids. The amino acids with reactive groups are either located in defined positions within the polypeptide, since the position of these amino acids determines loop size.
In one embodiment, a polypeptide with three reactive groups has the sequence (X)iY(X)mY(X)nY(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 2 and 8 defining the length of intervening polypeptide segments, which may be the same or different, and I and o are numbers between 0 and 20 defining the length of flanking polypeptide segments.
Alternatives to thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively, these techniques may be used in modification or attachment of further moieties (such as small molecules of interest which are distinct from the molecular scaffold) to the polypeptide after they have been selected or isolated according to the present invention - in this embodiment then clearly the attachment need not be covalent and may embrace non-covalent attachment. These methods may be used instead of (or in combination with) the thiol mediated methods by producing phage that display proteins and peptides bearing unnatural amino acids with the requisite chemical reactive groups, in combination small molecules that bear the complementary reactive group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection/isolation phase. Further details can be found in WO 2009/098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.
Synthesis
The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large-scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et 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.
Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.
Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.
Pharmaceutical Compositions
According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.
Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically- acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).
The compounds of the invention can be used alone or in combination with another agent or agents.
The compounds of the invention can also be used in combination with biological therapies such as nucleic acid based therapies, antibodies, bacteriophage or phage lysins.
The route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. Preferably, the pharmaceutical compositions according to the invention will be administered parenterally. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.
The peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that levels may have to be adjusted upward to compensate.
The compositions containing the present peptide ligands or a cocktail thereof can be administered for therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically- effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 10 pg to 250 mg of selected peptide ligand per kilogram of body weight, with doses of between 100 pg to 25 mg/kg/dose being more commonly used.
A composition containing a peptide ligand according to the present invention may be utilised in therapeutic settings to treat a microbial infection or to provide prophylaxis to a subject at risk of infection e.g. undergoing surgery, chemotherapy, artificial ventilation or other condition or planned intervention. In addition, the peptide ligands described herein may be used extracorporeal ly or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques. Therapeutic Uses
The bicyclic peptides of the invention have specific utility as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding agents.
Polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. In some applications, such as vaccine applications, the ability to elicit an immune response to predetermined ranges of antigens can be exploited to tailor a vaccine to specific diseases and pathogens.
Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides may be used diagnostically or therapeutically (including extracorporeal ly) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).
According to a further aspect of the invention, there is provided a peptide ligand as defined herein, for use in suppressing or treating a disease or disorder mediated by infection of SARS- CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2.
According to a further aspect of the invention, there is provided a method of suppressing or treating a disease or disorder mediated by infection of SARS-CoV-2 or for providing prophylaxis to a subject at risk of infection of SARS-CoV-2, which comprises administering to a patient in need thereof the peptide ligand as defined herein.
References herein to “disease or disorder mediated by infection of SARS-CoV-2” include: respiratory disorders, such as a respiratory disorder mediated by an inflammatory response within the lung, in particular COVID-19.
References herein to the term "suppression" refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after disease symptoms become manifest. Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available.
Screening Methods
It will be appreciated that the bicyclic peptide ligands of the invention also find utility as agents for screening for other SARS-CoV-2 binding agents.
For example, screening for a SARS-CoV-2 binding agent may typically involve incubating a bicyclic peptide ligand of the invention with SARS-CoV-2 in the presence and absence of a test compound and assessing a difference in the degree of binding, such that a difference in binding will result from competition of the test compound with the bicyclic peptide ligand of the invention for binding to SARS-CoV-2.
Thus, according to a further aspect of the invention, there is provided a method of screening for a compound which binds to SARS-CoV-2 wherein said method comprises the following steps:
(a) incubating a peptide ligand as defined herein with SARS-CoV-2;
(b) measuring the binding activity of said peptide ligand;
(c) incubating said peptide ligand from step (a) with a test compound and SARS- CoV-2;
(d) measuring the binding activity of said peptide ligand; and
(e) comparing the binding activity in steps (b) and (d), such that a difference in binding activity of said peptide ligand is indicative of the test compound binding to SARS-CoV- 2.
In one embodiment, the peptide ligand comprises a reporter moiety for ease of detecting binding. In a further embodiment, the reporter moiety comprises fluorescein (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
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 II synthesiser by MultiSynTech. Standard Fmoc-amino acids were employed (Sigma, Merck), with appropriate side chain protecting groups: where applicable standard coupling conditions were used in each case, followed by deprotection using standard methodology.
Alternatively, peptides were purified using HPLC and following isolation they were modified with the required molecular scaffold (namely, TATA, TATB or TCMT). For this, linear peptide was diluted with 50:50 MeC k W up to ~35 mL, -500 pL of 100 mM scaffold in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed for -30 -60 min at RT, and lyophilised once the reaction had completed (judged by MALDI). Once completed, 1 ml of 1 M L-cysteine hydrochloride monohydrate (Sigma) in H2O was added to the reaction for ~60 min at RT to quench any excess TATA, TATB or TCMT.
Following lyophilisation, the modified peptide was purified as above, while replacing the Luna C8 with a Gemini C18 column (Phenomenex), and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct scaffold-modified material were pooled, lyophilised and kept at -20°C for storage.
All amino acids, unless noted otherwise, were used in the L- configurations.
In some cases peptides are converted to activated disulfides prior to coupling with the free thiol group of a toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2- pyridylthio)pentanoate) (100mM) in dry DMSO (1.25 mol equiv) was added to a solution of peptide (20mM) in dry DMSO (1 mol equiv). The reaction was well mixed and DI PEA (20 mol equiv) was added. The reaction was monitored by LC/MS until complete.
Mutimeric Binding Complex Synthesis
The multimeric binding complexes of the invention may be prepared in accordance with analogous methodology to that described in WO 2019/162682, PCT/GB2022/050031 , PCT/GB2022/050036, and PCT/GB2022/050037.
BIOLOGICAL DATA
Example 1 : Surface Plasmon Resonance (SPR) Assay of Spike Protein Binders
SPR analysis was performed on a Biacore 8K+ (Cytiva). Briefly, SARS-CoV-2 Spike Trimer glycoprotein was immobilized on a Series S Sensor Chip CM5 using standard primary amine- coupling chemistry at 25°C with PBS, 0.05 % P20, 1 % DMSO, pH 7.4 as the running buffer. The carboxymethyl dextran surface was activated with a 7 min injection of a 1 :1 v/v ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 pLmin-1. ACE2 protein was diluted to 40 nM in 10 mM sodium acetate (pH 5.0) and captured with 200 s contact time at 10 pLmin-1. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5. Surface densities of 1200 RU were achieved. To determine the affinity for the Spike glycoprotein, an 8-point titration of bicyclic peptide underwent multi cycle kinetic evaluation at 25 °C, flow rate of 50 pLmin-1, with association time of 60 seconds and dissociation time of up to 400 seconds. A maximum concentration of 20000 nM bicyclic peptide was used. Data were solvent corrected for DMSO bulk effects. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using Biacore Insight Evaluation Software. Data were fitted using the 1 :1 binding model or steadystate affinity model where appropriate.
Selected bicyclic peptides of the invention were tested in the above-mentioned SPR assay and the results are shown in Table 1:
Table 1 : SPR Assay Results for Selected Bicyclic Peptides of the Invention
Figure imgf000051_0001
Example 2: Surface Plasmon Resonance (SPR) Assay of Receptor-Binding Domain Binders
SPR analysis was performed on a Biacore 8K+ (Cytiva). Briefly, SARS-CoV-2 Spike Trimer glycoprotein was immobilized on a Series S Sensor Chip CM5 using standard primary amine- coupling chemistry at 25°C with PBS, 0.05% P20, 1 % DMSO, pH 7.4 as the running buffer. The carboxymethyl dextran surface was activated with a 7 min injection of a 1 :1 v/v ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxy succinimide (NHS) at a flow rate of 10 pLmin-1. SARS-CoV-2 Spike Trimer glycoprotein protein was diluted to 40 nM in 10 mM sodium acetate (pH 5.0) and captured with 200 s contact time at 10 pLmin-1. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5. Surface densities of 1200 Rll were achieved. To determine the affinity for the Spike glycoprotein, an 8-point titration of bicyclic peptide underwent multi cycle kinetic evaluation at 25 °C, flow rate of 50 pLmin-1, with association time of 60 seconds and dissociation time of up to 400 seconds. A maximum concentration of 20000 nM bicyclic peptide was used. Data were solvent corrected for DMSO bulk effects. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using Biacore Insight Evaluation Software. Data were fitted using the steady-state affinity model.
Alternatively, SPR assays were performed on a Biacore T200 (Cytiva) with Series S Streptavidin (SA) sensor chips (Cytiva) in running buffer 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 0.05 % (v/v) P20, 2% DMSO, pH 7.4. His, Avitag™ biotinylated SARS-CoV-2 S1 protein (ACROBiosystems) protein was captured using standard methodlogy to generate 2700-3700 Rll. To determine the affinity for the Spike glycoprotein, a 5- or 8-point titration of bicyclic peptide underwent respectively single or multi cycle kinetic evaluation at 25 °C, flow rate of 30 pLmin-1, with association time of 120 seconds and dissociation time of up to 300 seconds. A maximum concentration of 10000 nM bicyclic peptide was used. Regeneration of the surface was performed using 1 mM HCI (30 seconds at 30 pL/min) followed by a stabilization period of 30 seconds. Each injection was followed by an additional wash with 50 % (v/v) DMSO/H2O, to limit carry-over. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using the Biacore T200 Evaluation Software (version 3.1). Data were fitted using the steady-state affinity model.
Selected bicyclic peptides of the invention were tested in the above-mentioned SPR assay and the results are shown in Table 2: Table 2: SPR Assay Results for Selected Bicyclic Peptides of the Invention
Figure imgf000053_0001
Example 3: Pseudovirus Neutralization Assay
Replication deficient SARS-CoV-2 pseudotyped HIV-1 virions were prepared similarly as described in Mallery et al (2021) Sci Adv 7(11). Briefly, virions were produced in HEK 293T cells by transfection with 1 pg of the plasmid encoding SARS CoV-2 Spike protein (pCAGGS- SpikeAc19), 1 pg pCRV GagPol and 1.5 pg GFP-encoding plasmid (CSGW). Viral supernatants were filtered through a 0.45 pm syringe filter at 48 h and 72 h post-transfection and pelleted for 2 h at 28,000 x g. Pelleted virions were drained and then resuspended in DM EM (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 bicycle 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 Incucyte S3 live cell imaging system (Sartorius). The percent of cell entry was quantified as GFP positive areas of cells over the total area covered by cells. Entry inhibition by the Bicyclic peptide was calculated as percent virus infection relative to virus only control.
Certain multimeric binding complexes of the invention were tested in the above assay and the results shown in Table 3:
Table 3: Pseudovirus Neutralization Assay Results for Selected Multimeric Binding Complexes of the Invention
Figure imgf000054_0001
Example 4: Spike-ACE2 AlphaScreen Inhibition Assay
Avi-tagged tagged SARS-CoV-2 Spike Receptor Binding Domain (RBD, ACROBiosystems - SPD-C82E9) or S1 domain (ACROBiosystems - S1 N-C82E8) at 0.25 nM or 1.0 nM, respectively, were incubated with 0.25 nM or 1 nM respectively, of hACE2 Fc-fusion (ACROBiosystems - AC2-H5257) and a duplicate titration of monomeric or multimeric untagged Bicycles. Finally, 20 pg/mL Streptavidin AlphaScreen donor beads (Perkin Elmer) alongside 20 pg/mL AlphaScreen Protein A acceptor beads (Perkin Elmer) were added and incubated. Plates were read on a Pherastar FS/FSX (BMG Labtech) using excitation wavelength 680 nm, emission wavelength 615 nm. Data was normalized to beads alone (low) and no competitor (high) reference averages. The normalized data was used to generate a four parameter logistic curve fit in Dotmatics. Where no IC50 was generated, this was reported as greater than the top concentration tested.
Certain peptide ligands of the invention were tested in the above assay and the results shown in Table 4:
Table 4: Spike-ACE2 AlphaScreen Inhibition Assay Results for Selected Bicyclic Peptides of the Invention
Figure imgf000055_0001
Figure imgf000056_0001
Example 5: Bicyclic Peptide-Spike AlphaScreen Competition Assay
Representative non-competitive biotinylated Bicyclic peptides:
BCY16257: ACDEKAWWCQLAYVDCA[Sar6][KBiot] (SEQ ID NO: 93) - the C-terminally biotinylated form of BCY16113; and
BCY16274: ACDPDPYDSSCWTCA[Sar6][KBiot] (SEQ ID NO: 94) - the C-terminally biotinylated form of BCY16126; were incubated on separate plates with poly(Histidine) tagged SARS-CoV-2 Spike Trimer Glycoprotein, along with duplicate titrations of multiple unmodified bicyclic peptides. Finally, 20 pg/mL of AlphaScreen Streptavidin donor beads (Perkin Elmer) and 20 pg/mL Nickel- chelate AlphaLISA Acceptor beads (Perkin Elmer) were added and incubated. Plates were read on a Pherastar FS/FSX (BMG Labtech) using excitation wavelength 680 nm, emission wavelength 615 nm. Data was normalized to beads alone (low) and no competitor (high) reference averages. The normalized data was used to generate a four-parameter logistic curve fit in Dotmatics. Where no IC50 was generated at the top concentration tested, this was used to define epitope boundaries. In the case of generically weak binders, Surface Plasmon Resonance data confirming binding is also presented.
Certain peptide ligands of the invention were tested in the above assay and the results shown in Table 5: Table 5: Bicyclic Peptide-Spike AlphaScreen Competition Assay Results for Selected Bicyclic Peptides of the Invention
Figure imgf000057_0001
Example 6: Surface Plasmon Resonance (SPR) Assays
Method 1
SPR analysis was performed on a Biacore 8K+ (Cytiva). Briefly, SARS-CoV-2 Spike Trimer Glycoprotein was immobilized on a Series S Sensor Chip CM5 using standard primary amine-coupling chemistry at 25°C with PBS, 0.05 % P20, 1 % DMSO, pH 7.4 as the running buffer. The carboxymethyl dextran surface was activated with a 7 min injection of a 1 :1 v/v ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N- hydroxy succinimide (NHS) at a flow rate of 10 pLmin'1. Spike Trimer Glycoprotein was diluted to 40 nM in 10 mM sodium acetate (pH 5.0) and captured with 200 s contact time at 10 pLmin'1. Residual activated groups were blocked with a 7 min injection of 1 M ethanolamine pH 8.5. Surface densities ranging 870 - 1500 Rll were achieved. To determine the affinity for the Spike Trimer Glycoprotein, an 8-point titration of bicyclic peptide underwent multi cycle kinetic evaluation at 25 °C, flow rate of 50 pLmin'1, with association time of 60 seconds and dissociation time of up to 120 seconds. A maximum concentration of 20000 nM bicyclic peptide was used. Data were solvent corrected for DMSO bulk effects. All data were double reference corrected against the reference flow cell and matched buffer blanks. Data processing and kinetic fitting were performed using Biacore Insight Evaluation Software. Data were fitted using steady-state affinity model where appropriate.
Certain peptide ligands of the invention were tested in Method 1 of the above assay and the results shown in Table 6:
Table 6: SPR Assay Results for Selected Bicyclic Peptides of the Invention
Figure imgf000058_0001
Figure imgf000059_0001
Method 2
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). Spike Glycoprotein domain S1 (ACROBiosystems - S1 N-C82E8) protein was captured to generate 3000-4000 Rll. 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 a multiple 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. Affinity constants (KD) were derived from the sensorgrams by applying Steady-State analysis, using the Biacore™ T200 Evaluation Software (version 3.1).
Certain peptide ligands of the invention were tested in Method 2 of the above assay and the results shown in Table 7:
Table 7: SPR Assay Results for Selected Bicyclic Peptides of the Invention
Figure imgf000060_0001

Claims

1. A peptide ligand specific for the spike protein (S protein) of 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, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CLPAGCTDLWRYIQC (SEQ ID NO: 1);
CVVNGTIRYCALC (SEQ ID NO: 2);
CIVRGEIRWCGGPEC (SEQ ID NO: 3);
CIQNGVLKYCAQC (SEQ ID NO: 4);
CIKDNQILYCATC (SEQ ID NO: 5);
CIINNHVVYCATC (SEQ ID NO: 6);
CIRDGGIQYCALC (SEQ ID NO: 7);
CLPNGCTDLERYIKC (SEQ ID NO: 8);
CTPNGCTDLWRYIAC (SEQ ID NO: 9);
CSDEFCSAWWGFNEC (SEQ ID NO: 10);
CSDAFCSAWWGFNQC (SEQ ID NO: 11);
CSSKFCDAWWNFNRC (SEQ ID NO: 12);
CSDDFCSAWWGFNHC (SEQ ID NO: 13);
CSNKFCDAWWNFNRC (SEQ ID NO: 14);
CFPAPWLGLCTPC (SEQ ID NO: 15);
CFPEPWLGLCTPC (SEQ ID NO: 16);
CIVNGEIKYCADC (SEQ ID NO: 17);
CIKNDELVYCGGPKC (SEQ ID NO: 18);
CIVDDHIVYCGSKQC (SEQ ID NO: 19);
CIKDGLLVYCGSYQC (SEQ ID NO: 20);
CIKDGVLIYCGGPMC (SEQ ID NO: 21);
CMNPFFYDCERTC (SEQ ID NO: 22) herein referred to as BCY18697 when complexed with a derivative of TATB which has the following structure:
Figure imgf000062_0001
wherein * denotes the point of attachment of the three cysteine residues;
CMNPFFYDCDHIC (SEQ ID NO: 23);
CMNPFFYDCHEQC (SEQ ID NO: 24);
CMNPFFYDCKWC (SEQ ID NO: 25);
CMNPFFYDCEDRC (SEQ ID NO: 26);
CMNPFFYDCEEIC (SEQ ID NO: 27);
CMNPFFYDCENPC (SEQ ID NO: 28);
CMNPFFYDCETVC (SEQ ID NO: 29);
CMNPFFYDCEYVC (SEQ ID NO: 30);
CMNPFYYDCEEVC (SEQ ID NO: 31);
C[Hse(Me)]NPFFYDCERTC (SEQ ID NO: 32);
CMN[HyP]FFYDCERTC (SEQ ID NO: 33);
CMNPFF[4FPhe]DCERTC (SEQ ID NO: 34);
CEDNDWVYCSTC (SEQ ID NO: 35);
CDWTCYLRPLPC (SEQ ID NO: 36);
CMFVPCAARHELGLC (SEQ ID NO: 41);
CMFVPCAARVELGLC (SEQ ID NO: 42);
CMFVPCAIRQTLGLC (SEQ ID NO: 43);
CMFVPCATRHELGLC (SEQ ID NO: 44);
CMFVPCATRHQLGLC (SEQ ID NO: 45);
CMFVPCATRHSLGLC (SEQ ID NO: 46);
CMFVPCATRLALGLC (SEQ ID NO: 47);
CMFVPCATRLQLGLC (SEQ ID NO: 48);
CMFVPCATRQELGLC (SEQ ID NO: 49);
CMFVPCATRQMLGLC (SEQ ID NO: 50);
CMFVPCATRVALGLC (SEQ ID NO: 51);
CMFVPCAVREELGLC (SEQ ID NO: 52);
CMFVPCAVRHALGLC (SEQ ID NO: 53);
CMFVPCAVRHSLGLC (SEQ ID NO: 54);
CMFVPCAVRKDLGLC (SEQ ID NO: 55); CMFVPCAVRQTLGLC (SEQ ID NO: 56);
CMFTPCHVREILGLC (SEQ ID NO: 57);
CMGVPCKVREILGLC (SEQ ID NO: 58);
CMFVPCAVREIL[dA]LC (SEQ ID NO: 59);
C[Hse(Me)]FVPCAVREILGLC (SEQ ID NO: 60);
CMFV[HyP]CAVREILGLC (SEQ ID NO: 61);
Ac-CPYVAGR[dA]TCLLC (SEQ ID NO: 62) herein referred to as BCY18089 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000063_0001
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAGR[dA]TCL[tBuAla]C (SEQ ID NO: 63) herein referred to as BCY18090 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000063_0002
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg]GTCLLC (SEQ ID NO: 64) herein referred to as BCY18091 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000063_0003
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg][dA]TCLLC (SEQ ID NO: 65) herein referred to as BCY18092 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000064_0001
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAG[HArg]GTCL[tBuAla]C (SEQ ID NO: 66) herein referred to as BCY18093 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000064_0002
wherein * denotes the point of attachment of the three cysteine residues;
Ac-CPYVAGRGTCL[tBuAla]C (SEQ ID NO: 67) herein referred to as BCY18094 when complexed with a derivative of TCMT which has the following structure:
Figure imgf000064_0003
wherein * denotes the point of attachment of the three cysteine residues;
CHPVCSVPAIGLLC (SEQ ID NO: 68);
CEINASLPCTFTC (SEQ ID NO: 69);
CEFYYANCEDVLPWC (SEQ ID NO: 70);
CDPDPYDSSCWTC (SEQ ID NO: 71);
CDWDWHVCAIMNESC (SEQ ID NO: 72);
CTPLDATFCFSKC (SEQ ID NO: 73);
CEEDWHICQIHGYDC (SEQ ID NO: 74);
CNEDWHSCMIADSDC (SEQ ID NO: 75);
CWDSSCWAHMDKC (SEQ ID NO: 76);
CNNPFCEYHIC (SEQ ID NO: 77); CTTLDKIFCFSSC (SEQ ID NO: 78);
CFGEDWHTCSIYC (SEQ ID NO: 79);
CMDWHVCMLNDTLFC (SEQ ID NO: 80);
CTNIMCEFMFC (SEQ ID NO: 81);
CINPYCEHHIYLEHC (SEQ ID NO: 82);
CNMDCYHLPFTSMYC (SEQ ID NO: 83);
CDEKAWWCQLAYVDC (SEQ ID NO: 84);
CHQAYGMCSIFPEWC (SEQ ID NO: 85);
CKESSWYCQMWDIQC (SEQ ID NO: 86);
CDGPDWHSCMVSC (SEQ ID NO: 87);
CQNLHPLCGVLESHMC (SEQ ID NO: 88);
CWNSEDWHACQIC (SEQ ID NO: 89);
CDHYHCPWLALGGSC (SEQ ID NO: 90);
CELDEWLCIIGHLDC (SEQ ID NO: 91); and CMEFAANCEDIYDDC (SEQ ID NO: 92); wherein the three cysteine residues within each peptide ligand represent the three reactive groups and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, tBuAla represents t-butyl-alanine, HArg represents homoarginine, and 4FPhe represents 4- fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
2. The peptide ligand according to claim 1 , wherein said peptide ligand is specific for the S2 domain of the spike protein (S2 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CLPAGCTDLWRYIQC (SEQ ID NO: 1);
CVVNGTIRYCALC (SEQ ID NO: 2);
CIVRGEIRWCGGPEC (SEQ ID NO: 3);
CIQNGVLKYCAQC (SEQ ID NO: 4);
CIKDNQILYCATC (SEQ ID NO: 5);
CIINNHVVYCATC (SEQ ID NO: 6);
CIRDGGIQYCALC (SEQ ID NO: 7);
CLPNGCTDLERYIKC (SEQ ID NO: 8);
CTPNGCTDLWRYIAC (SEQ ID NO: 9);
CSDEFCSAWWGFNEC (SEQ ID NO: 10);
CSDAFCSAWWGFNQC (SEQ ID NO: 11);
CSSKFCDAWWNFNRC (SEQ ID NO: 12);
CSDDFCSAWWGFNHC (SEQ ID NO: 13); CSNKFCDAWWNFNRC (SEQ ID NO: 14);
CFPAPWLGLCTPC (SEQ ID NO: 15);
CFPEPWLGLCTPC (SEQ ID NO: 16);
CIVNGEIKYCADC (SEQ ID NO: 17);
CIKNDELVYCGGPKC (SEQ ID NO: 18);
CIVDDHIVYCGSKQC (SEQ ID NO: 19);
CIKDGLLVYCGSYQC (SEQ ID NO: 20); and
CIKDGVLIYCGGPMC (SEQ ID NO: 21); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, or a modified derivative and/or pharmaceutically acceptable salt thereof.
3. The peptide ligand according to claim 1 or claim 2, wherein said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000066_0001
wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:
A-(SEQ ID NO: 1)-A (herein referred to as BCY18948);
A-(SEQ ID NO: 8)-A (herein referred to as BCY19894);
A-(SEQ ID NO: 9)-A (herein referred to as BCY19896);
A-(SEQ ID NO: 10)-A (herein referred to as BCY19899);
A-(SEQ ID NO: 11)-A (herein referred to as BCY19900);
A-(SEQ ID NO: 12)-A (herein referred to as BCY19901);
Ac-(SEQ ID NO: 12)-[K(PYA)] (herein referred to as BCY22415);
A-(SEQ ID NO: 13)-A (herein referred to as BCY19902); and A-(SEQ ID NO: 14)-A (herein referred to as BCY19903); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
4. The peptide ligand according to claim 1 or claim 2, wherein said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TATB which has the following structure:
Figure imgf000067_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 2)-A (herein referred to as BCY18950);
A-(SEQ ID NO: 3)-A (herein referred to as BCY18953);
A-(SEQ ID NO: 4)-A (herein referred to as BCY18956);
Ac-(SEQ ID NO: 4)-K (herein referred to as BCY22419);
A-(SEQ ID NO: 5)-A (herein referred to as BCY18957);
A-(SEQ ID NO: 6)-A (herein referred to as BCY18958);
A-(SEQ ID NO: 7)-A (herein referred to as BCY18961);
A-(SEQ ID NO: 17)-A (herein referred to as BCY19916);
A-(SEQ ID NO: 18)-A (herein referred to as BCY19919);
A-(SEQ ID NO: 19)-A (herein referred to as BCY19921);
A-(SEQ ID NO: 20)-A (herein referred to as BCY19923); and A-(SEQ ID NO: 21)-A (herein referred to as BCY19924); or a modified derivative and/or pharmaceutically acceptable salt thereof.
5. The peptide ligand according to claim 1 or claim 2, wherein said peptide ligand is specific for the S2 domain of the spike protein (S2 protein), the molecular scaffold is a derivative of TCMT which has the following structure:
Figure imgf000068_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 15)-A (herein referred to as BCY19904);
A-(SEQ ID NO: 16)-A (herein referred to as BCY19905);
Ac-(SEQ ID NO: 16)-K (herein referred to as BCY22416); or a modified derivative and/or pharmaceutically acceptable salt thereof.
6. The peptide ligand according to claim 1, wherein said peptide ligand is specific for the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000068_0002
wherein * denotes the point of attachment of the three cysteine residues, 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: 41)-A (herein referred to as BCY17367);
A-(SEQ ID NO: 42)-A (herein referred to as BCY17368);
A-(SEQ ID NO: 43)-A (herein referred to as BCY17369);
A-(SEQ ID NO: 44)-A (herein referred to as BCY17370);
A-(SEQ ID NO: 45)-A (herein referred to as BCY17371);
A-(SEQ ID NO: 46)-A (herein referred to as BCY17372);
A-(SEQ ID NO: 47)-A (herein referred to as BCY17373); A-(SEQ ID NO: 48)-A (herein referred to as BCY17375);
A-(SEQ ID NO: 49)-A (herein referred to as BCY17376);
A-(SEQ ID NO: 50)-A (herein referred to as BCY17377);
A-(SEQ ID NO: 51)-A (herein referred to as BCY17378);
A-(SEQ ID NO: 52)-A (herein referred to as BCY17380);
A-(SEQ ID NO: 53)-A (herein referred to as BCY17381);
A-(SEQ ID NO: 54)-A (herein referred to as BCY17382);
A-(SEQ ID NO: 55)-A (herein referred to as BCY17383);
A-(SEQ ID NO: 56)-A (herein referred to as BCY17384);
A-(SEQ ID NO: 57)-A (herein referred to as BCY17385);
A-(SEQ ID NO: 58)-A (herein referred to as BCY17387);
A-(SEQ ID NO: 59)-A (herein referred to as BOY 17615);
A-(SEQ ID NO: 60)-A (herein referred to as BCY17617); and A-(SEQ ID NO: 61)-A (herein referred to as BCY17618); or a modified derivative and/or pharmaceutically acceptable salt thereof.
7. The peptide ligand according to claim 1 , wherein said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is selected from:
CMNPFFYDCERTC (SEQ ID NO: 22); herein referred to as BCY18697 when complexed with a derivative of TATB which has the following structure:
Figure imgf000069_0001
wherein * denotes the point of attachment of the three cysteine residues);
CMNPFFYDCDHIC (SEQ ID NO: 23);
CMNPFFYDCHEQC (SEQ ID NO: 24);
CMNPFFYDCKWC (SEQ ID NO: 25);
CMNPFFYDCEDRC (SEQ ID NO: 26);
CMNPFFYDCEEIC (SEQ ID NO: 27);
CMNPFFYDCENPC (SEQ ID NO: 28);
CMNPFFYDCETVC (SEQ ID NO: 29);
CMNPFFYDCEYVC (SEQ ID NO: 30); CMNPFYYDCEEVC (SEQ ID NO: 31);
C[Hse(Me)]NPFFYDCERTC (SEQ ID NO: 32);
CMN[HyP]FFYDCERTC (SEQ ID NO: 33);
CMNPFF[4FPhe]DCERTC (SEQ ID NO: 34); and CEDNDWVYCSTC (SEQ ID NO: 35); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, and wherein Hse(Me) represents homoserine-methyl, HyP represents hydroxyproline, and 4FPhe represents 4-fluoro-phenylalanine, or a modified derivative and/or pharmaceutically acceptable salt thereof.
8. The peptide ligand according to claim 1 or claim 7, wherein said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATB which has the following structure:
Figure imgf000070_0001
wherein * denotes the point of attachment of the three cysteine residues, 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: 22)-A (herein referred to as BCY16207);
Ac-A-(SEQ ID NO: 22)-A (herein referred to as BCY18696);
Ac-(SEQ ID NO: 22) (herein referred to as BCY18698);
A-(SEQ ID NO: 23)-A (herein referred to as BCY18144);
A-(SEQ ID NO: 24)-A (herein referred to as BCY18145);
A-(SEQ ID NO: 25)-A (herein referred to as BCY18146);
A-(SEQ ID NO: 26)-A (herein referred to as BCY18147);
A-(SEQ ID NO: 27)-A (herein referred to as BCY18148);
A-(SEQ ID NO: 28)-A (herein referred to as BCY18149);
A-(SEQ ID NO: 29)-A (herein referred to as BCY18150);
Ac-(SEQ ID NO: 29) (herein referred to as BCY22405);
Ac-(SEQ ID NO: 29)-[K(PYA)] (herein referred to as BCY22413);
A-(SEQ ID NO: 30)-A (herein referred to as BCY18151);
A-(SEQ ID NO: 31)-A (herein referred to as BCY18152); A-(SEQ ID NO: 32)-A (herein referred to as BCY18700);
A-(SEQ ID NO: 33)-A (herein referred to as BCY18702); and
A-(SEQ ID NO: 34)-A (herein referred to as BCY18703); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
9. The peptide ligand according to claim 1 or claim 7, wherein said peptide ligand is specific for the receptor-binding domain (RBD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATA which has the following structure:
Figure imgf000071_0001
wherein * denotes the point of attachment of the three cysteine residues, and 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: 35)-[K(PYA)] (herein referred to as BCY22412);
A-(SEQ ID NO: 42)-A (herein referred to as BCY17368);
A-(SEQ ID NO: 44)-A (herein referred to as BCY17370);
A-(SEQ ID NO: 45)-A (herein referred to as BCY17371);
A-(SEQ ID NO: 47)-A (herein referred to as BCY17373);
A-(SEQ ID NO: 49)-A (herein referred to as BCY17376);
A-(SEQ ID NO: 50)-A (herein referred to as BCY17377);
A-(SEQ ID NO: 51)-A (herein referred to as BCY17378);
A-(SEQ ID NO: 53)-A (herein referred to as BCY17381);
A-(SEQ ID NO: 56)-A (herein referred to as BCY17384); and
A-(SEQ ID NO: 58)-A (herein referred to as BCY17387); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof. 10. The peptide ligand according to claim 1, wherein said peptide ligand is specific for the N-Terminal Domain (NTD) of the S1 domain of the spike protein (S1 protein) and the bicyclic peptide ligand comprises an amino acid sequence which is:
CDWTCYLRPLPC (SEQ ID NO: 36); wherein the three cysteine residues within each peptide ligand represent the three reactive groups, or a modified derivative and/or pharmaceutically acceptable salt thereof.
11. The peptide ligand according to claim 1 or claim 10, wherein said peptide ligand is specific for the N-Terminal Domain (NTD) of the S1 domain of the spike protein (S1 protein), the molecular scaffold is a derivative of TATB which has the following structure:
Figure imgf000072_0001
wherein * denotes the point of attachment of the three cysteine residues, and 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: 36)-[K(PYA)] (herein referred to as BCY22414); and Ac-(SEQ ID NO: 36)-K (herein referred to as BCY22417); wherein PYA represents pentynoic acid, or a modified derivative and/or pharmaceutically acceptable salt thereof.
12. The peptide ligand according to claim 1, which is specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a molecular scaffold which is a derivative of TATB which has the following structure:
Figure imgf000072_0002
wherein * denotes the point of attachment of the three cysteine residues, 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: 68)-A (herein referred to as BCY16129);
A-(SEQ ID NO: 69)-A (herein referred to as BCY16128);
A-(SEQ ID NO: 70)-A (herein referred to as BCY16127);
A-(SEQ ID NO: 71)-A (herein referred to as BCY16126);
A-(SEQ ID NO: 72)-A (herein referred to as BCY16125);
A-(SEQ ID NO: 73)-A (herein referred to as BCY16124);
A-(SEQ ID NO: 74)-A (herein referred to as BCY16123);
A-(SEQ ID NO: 75)-A (herein referred to as BCY16122);
A-(SEQ ID NO: 76)-A (herein referred to as BCY16121);
A-(SEQ ID NO: 77)-A (herein referred to as BCY16120);
A-(SEQ ID NO: 78)-A (herein referred to as BCY16119);
A-(SEQ ID NO: 79)-A (herein referred to as BCY16118);
A-(SEQ ID NO: 80)-A (herein referred to as BCY16117);
A-(SEQ ID NO: 81)-A (herein referred to as BCY16116);
A-(SEQ ID NO: 82)-A (herein referred to as BCY16115);
A-(SEQ ID NO: 83)-A (herein referred to as BCY16114);
A-(SEQ ID NO: 84)-A (herein referred to as BCY16113);
A-(SEQ ID NO: 85)-A (herein referred to as BCY16112);
A-(SEQ ID NO: 86)-A (herein referred to as BCY16111);
A-(SEQ ID NO: 87)-A (herein referred to as BCY16110);
A-(SEQ ID NO: 88)-A (herein referred to as BCY16109);
A-(SEQ ID NO: 89)-A (herein referred to as BCY16108);
A-(SEQ ID NO: 90)-A (herein referred to as BCY16107);
A-(SEQ ID NO: 91)-A (herein referred to as BCY16106); and
A-(SEQ ID NO: 92)-A (herein referred to as BCY16105); or a modified derivative and/or pharmaceutically acceptable salt thereof.
13. The peptide ligand according to claim 1, which is specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a molecular scaffold which is a derivative of TCMT which has the following structure:
Figure imgf000073_0001
wherein * denotes the point of attachment of the three cysteine residues, and the bicyclic peptide ligand and comprises an amino acid sequence which is selected from: (SEQ ID NO: 62) (herein referred to as BCY18089);
(SEQ ID NO: 63) (herein referred to as BCY18090);
(SEQ ID NO: 64) (herein referred to as BCY18091);
(SEQ ID NO: 65) (herein referred to as BCY18092);
(SEQ ID NO: 66) (herein referred to as BCY18093); and
(SEQ ID NO: 67) (herein referred to as BCY18094); or a modified derivative and/or pharmaceutically acceptable salt thereof.
14. The peptide ligand according to any one of claims 1 to 13, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium and ammonium salt.
15. A multimeric binding complex which comprises either: (i) at least two bicyclic peptide ligands according to any one of claims 1 to 14, wherein said peptide ligands may be the same or different; or (ii) at least one bicyclic peptide ligand according to any one of claims 1 to 14, which may be the same or different, in combination with one or more bicyclic peptide ligands specific for the spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be the same or different.
16. The multimeric binding complex according to claim 15, which is selected from: BCY22420, BCY22431 , BCY22421 , BCY22422, BCY22423, BCY22424, BCY22425, BCY22426, BCY22427, BCY22428, BCY22429, BCY22430, BCY22432, BCY22433, and BCY24540.
17. A pharmaceutical composition which comprises the peptide ligand of any one of claims 1 to 14 or the multimeric binding complex of claim 15 or claim 16, in combination with one or more pharmaceutically acceptable excipients.
18. The pharmaceutical composition according to claim 17, which additionally comprises one or more therapeutic agents.
19. The peptide ligand according to any of claims 1 to 14, or the multimeric binding complex of claim 15 or claim 16, or the pharmaceutical composition as defined in claim 17 or claim 18, 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, such as COVID-19.
PCT/GB2023/051798 2022-07-07 2023-07-07 Anti-infective bicyclic peptide ligands WO2024009108A1 (en)

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