ANTI-INFECTIVE BICYCLIC PEPTIDE LIGANDS

Abstract

The present invention relates to multimers of 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 also describes the multimerization of polypeptides through various chemical linkers and hinges of various lengths and rigidity using different sites of attachments within polypeptides. In particular, the invention describes multimers of peptides which are high affinity binders of ACE2. The invention also includes pharmaceutical compositions comprising said polypeptides and to the use of said polypeptides in suppressing or treating a disease or disorder mediated by ACE2, such as infection of COVID-19 or for providing prophylaxis to a subject at risk of infection of COVID-19.

Claims

1. A multimeric binding complex which comprises at least two bicyclic peptide ligands, wherein said peptide ligands may be the same or different, each of which comprises a peptide ligand specific for ACE2 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.

2. The multimeric binding complex according to claim 1, wherein said loop sequences comprise 4, 5, 6 or 8 amino acids.

3. The multimeric binding complex according to claim 1 or claim 2, wherein said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 4 amino acids and the other of which consists of 8 amino acids, such as: wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from: TABLE-US-00013 (SEQIDNO:1) C.sub.iHKFPC.sub.iiRDPQQYLFC.sub.iii; (SEQIDNO:2) C.sub.iTSPMC.sub.iiYVLKHQNRC.sub.iii; (SEQIDNO:3) C.sub.iTRPWC.sub.iiHSLLPRATC.sub.iii; (SEQIDNO:4) C.sub.iGRQFC.sub.iiHTLMPRHLC.sub.iii; (SEQIDNO:5) C.sub.iVRSHC.sub.iiSSLLPRIHC.sub.i; (SEQIDNO:9) C.sub.iAPILC.sub.iiRWAERQGYC.sub.iii; (SEQIDNO:10) C.sub.iNAVLC.sub.iiSWARANSFC.sub.iii; (SEQIDNO:11) C.sub.iNAVLC.sub.iiS[1Nal]ARANSFC.sub.ii; (SEQIDNO:12) C.sub.iNAVLC.sub.iiS[2Nal]ARANSFC.sub.iii; (SEQIDNO:13) C.sub.iiNAVLC.sub.iiSW[A.sub.ib]RANSFC.sub.iii; (SEQIDNO:14) C.sub.iNAVLC.sub.iiSWA[HArg]ANSFC.sub.iii; and (SEQIDNO:19) C.sub.iNSYTC.sub.iiHYIKHILG[Agb]C.sub.ii; wherein C.sub.i, C.sub.ii and C.sub.iii represent first, second and third cysteine residues, respectively, 1Nal represents 1-naphthylalanine, 2Nal represents 2-naphthylalanine, Aib represents aminoisobutyric acid, Agb represents 2-amino-4-guanidinobutyric acid, HArg represents homoarginine, or a pharmaceutically acceptable salt thereof, in particular: wherein the molecular scaffold is TATA and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 1)-A (herein referred to as BCY15296); A-(SEQ ID NO: 2)-A (herein referred to as BCY15295); A-(SEQ ID NO: 3)-A (herein referred to as BCY15293); A-(SEQ ID NO: 4)-A (herein referred to as BCY15292); A-(SEQ ID NO: 5)-A (herein referred to as BCY15291); A-(SEQ ID NO: 9)-A (herein referred to as BCY15425); A-(SEQ ID NO: 10)-A (herein referred to as BCY15429); A-(SEQ ID NO: 11)-A (herein referred to as BCY16866); A-(SEQ ID NO: 12)-A (herein referred to as BCY16867); A-(SEQ ID NO: 13)-A (herein referred to as BCY16872); A-(SEQ ID NO: 14)-A (herein referred to as BCY16874); Ac-(SEQ ID NO: 14)-[K(PYA)] (herein referred to as BCY18784); and Ac-(SEQ ID NO: 19) (herein referred to as BCY18748); wherein PYA represents pentynoic acid; or wherein the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 1)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15288); A-(SEQ ID NO: 2)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15287); A-(SEQ ID NO: 3)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15285); A-(SEQ ID NO: 4)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15284); and A-(SEQ ID NO: 5)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15283).

4. The multimeric binding complex according to claim 1 or claim 2, wherein said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 4 amino acids, such as: wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from: TABLE-US-00014 (SEQIDNO:15) C.sub.iLELYQC.sub.iiWRGKC.sub.iii; (SEQIDNO:16) C.sub.iPSQYKC.sub.iiWRGKC.sub.iii; and (SEQIDNO:17) C.sub.iLEVYKC.sub.iiWRGKC.sub.iii; wherein C.sub.i, C.sub.ii and C.sub.iii represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof, in particular: wherein the molecular scaffold is TATA and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 15)-A (herein referred to as BCY15426); A-(SEQ ID NO: 16)-A (herein referred to as BCY15427); and A-(SEQ ID NO: 17)-A (herein referred to as BCY15428).

5. The multimeric binding complex according to claim 1 or claim 2, wherein said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 5 amino acids and the other of which consists of 8 amino acids, such as: wherein the bicyclic peptide ligand comprises an amino acid sequence which is: TABLE-US-00015 (SEQIDNO:18) C.sub.iAN[A.sub.ib]VLC.sub.iiSWARANSFC.sub.iii; wherein C.sub.i, C.sub.ii and C.sub.iii represent first, second and third cysteine residues, respectively, Aib represents aminoisobutyric acid, or a pharmaceutically acceptable salt thereof, in particular: wherein the molecular scaffold is TATA and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is: A-(SEQ ID NO: 18)-A (herein referred to as BCY16871).

6. The multimeric binding complex according to claim 1 or claim 2, wherein said loop sequences comprise three reactive groups separated by two loop sequences one of which consists of 6 amino acids and the other of which consists of 4 amino acids, such as: wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from: TABLE-US-00016 (SEQIDNO:6) C.sub.iGREELPC.sub.iiRIKLC.sub.iii; and (SEQIDNO:7) C.sub.iLRSYNLC.sub.iiPRINC.sub.iii; wherein C.sub.i, C.sub.ii and C.sub.iii represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof, in particular: wherein the molecular scaffold is TATA, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 6)-A (herein referred to as BCY15298); and A-(SEQ ID NO: 7)-A (herein referred to as BCY15294); or wherein the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is selected from: A-(SEQ ID NO: 6)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15290); and A-(SEQ ID NO: 7)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15286).

7. The multimeric binding complex according to claim 1 or claim 2, wherein said loop sequences comprise three reactive groups separated by two loop sequences both of which consist of 6 amino acids, such as: wherein the bicyclic peptide ligand comprises an amino acid sequence which is: TABLE-US-00017 (SEQIDNO:8) C.sub.iHRDFPRC.sub.iiTWETQWC.sub.iii; wherein C.sub.i, C.sub.ii and C.sub.iii represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof, in particular: wherein the molecular scaffold is TATA and the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is: A-(SEQ ID NO: 8)-A (herein referred to as BCY15297); or wherein the molecular scaffold is TATA, the bicyclic peptide additionally comprises N- and/or C-terminal additions and a labelling moiety, such as fluorescein (FI), and comprises an amino acid sequence which is: A-(SEQ ID NO: 8)-A-[Sar.sub.6]-[KFI] (herein referred to as BCY15289).

8. The multimeric binding complex according to any one of claims 1 to 7, which is selected from: BCY17345, BCY17346, BCY17347, BCY19071, BCY19147, and BCY19148.

9. The multimeric binding complex according to any one of claims 1 to 8, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium and ammonium salt.

10. A pharmaceutical composition which comprises the multimeric binding complex of any one of claims 1 to 9, in combination with one or more pharmaceutically acceptable excipients.

11. The pharmaceutical composition according to claim 10, which additionally comprises one or more therapeutic agents.

12. The multimeric binding complex according to any of claims 1 to 9, or the pharmaceutical composition as defined in claim 10 or claim 11, 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.

Description

EXAMPLES

Materials and Methods

Peptide Synthesis

[0189] 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.

[0190] Alternatively, peptides were purified using HPLC and following isolation they were modified with the required molecular scaffold (namely, TATA). For this, linear peptide was diluted with 50:50 MeCN:H.sub.2O up to ?35 mL, ?500 ?L of 100 mM scaffold in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M NH.sub.4HCO.sub.3 in H.sub.2O. 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 1M L-cysteine hydrochloride monohydrate (Sigma) in H.sub.2O was added to the reaction for ?60 min at RT to quench any excess TATA.

[0191] 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.

[0192] All amino acids, unless noted otherwise, were used in the L-configurations.

[0193] 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) (100 mM) in dry DMSO (1.25 mol equiv) was added to a solution of peptide (20 mM) in dry DMSO (1 mol equiv). The reaction was well mixed and DIPEA (20 mol equiv) was added. The reaction was monitored by LC/MS until complete.

Mutimeric Binding Complex Synthesis

[0194] The multimeric complexes of the invention may be prepared in accordance with analogous methodology to that described in WO 2019/162682.

BIOLOGICAL DATA

1. Pseudoviral Neutralisation Assay

[0195] 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 ?g of the plasmid encoding SARS CoV-2 Spike protein (pCAGGS-SpikeAc19), 1 ?g pCRV GagPol and 1.5 ?g GFP-encoding plasmid (CSGVV). Viral supernatants were filtered through a 0.45 pm syringe filter at 48 h and 72 h post-transfection and pelleted for 2 h at 28,000?g. Pelleted virions were drained and then resuspended in DMEM (Gibco).

[0196] 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 ?l pseudovirus-containing supernatant was mixed with 2 ?l dilutions of bicycle peptide and incubated for 40 min at RT. 10 ?l 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.

[0197] Certain ACE2 monomers and multimeric binding complexes of the invention were tested in the above assay and the results shown in Table 4:

TABLE-US-00012 TABLE 4 Pseudoviral Neutralisation Assay Results for Selected Bicyclic Peptides and Multimeric Binding Complexes of the Invention BCY Number Geomean IC.sub.50 (nM) BCY15429 (Parent Monomer) 1280 BCY17345 6.96 BCY17346 12 BCY17347 3.75 BCY19071 24 BCY19147 17.7 BCY19148 10.1