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US-20260124312-A1 - BICYCLIC PEPTIDE LIGANDS SPECIFIC FOR EPHA2

US20260124312A1US 20260124312 A1US20260124312 A1US 20260124312A1US-20260124312-A1

Abstract

The present invention relates to polypeptides which are covalently bound to non-aromatic 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 Eph receptor tyrosine kinase A2 (EphA2). The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour).

Inventors

  • Liuhong Chen
  • Philip Huxley
  • Silvia PAVAN
  • Katerine Van Rietschoten

Assignees

  • BICYCLETX LIMITED

Dates

Publication Date
20260507
Application Date
20250825
Priority Date
20171219

Claims (20)

  1. 1 - 33 . (canceled)
  2. 34 . A peptide ligand specific for EphA2 comprising a polypeptide comprising the amino acid sequence: or a pharmaceutically acceptable salt thereof, and a non-aromatic molecular scaffold which forms covalent bonds with the polypeptide; wherein each of C i , C ii and C iii is an amino acid comprising a reactive group capable of forming a covalent bond to the molecular scaffold; wherein X 1 and X 2 each represent the amino acid residues between the cysteine residues in SEQ ID NO: 1: (SEQ ID NO: 1) C(HyP)LVNPLCLHP(D-Asp)W(HArg)C, wherein D-Asp is D-aspartate; HArg is homoarginine; and HyP is hydroxyproline.
  3. 35 . The peptide ligand of claim 34 , wherein C i , C ii and C iii are each independently cysteine or a non-natural amino acid.
  4. 36 . The peptide ligand of claim 34 , wherein C i , C ii and C iii are each independently cysteine or a non-natural amino acid which is isoelectronic and/or isosteric with cysteine.
  5. 37 . The peptide ligand of claim 34 , wherein C i , C ii and C iii are each independently cysteine or a non-natural amino acid which is isoelectronic with cysteine.
  6. 38 . The peptide ligand of claim 34 , comprising a C-terminal amide group.
  7. 39 . The peptide ligand of claim 34 , comprising a N-terminal spacer group.
  8. 40 . The peptide ligand of claim 34 , wherein the polypeptide is cyclised with the molecular scaffold such that at least two polypeptide loops are formed on the molecular scaffold, and wherein the molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).
  9. 41 . The peptide ligand of claim 34 , wherein the peptide ligand is the free acid or a pharmaceutically acceptable salt selected from sodium, potassium, calcium, and ammonium salts.
  10. 42 . A drug conjugate comprising the peptide ligand of claim 34 , conjugated to one or more effector and/or functional groups.
  11. 43 . A drug conjugate comprising the peptide ligand of claim 34 , conjugated to a cytotoxic agent, a radiochelator, a metal chelator, or a chromophore
  12. 44 . The drug conjugate of claim 42 , wherein the peptide ligand is conjugated to a cytotoxic agent.
  13. 45 . The drug conjugate of claim 44 , wherein said cytotoxic agent is selected from monomethyl auristatin E (MMAE) and DM1.
  14. 46 . The drug conjugate of claim 44 comprising a linker between said peptide ligand and said cytotoxic agent.
  15. 47 . The drug conjugate of claim 46 , wherein the cytotoxic agent is MMAE and the linker is selected from: -Val-Cit-, -Trp-Cit-, -Val-Lys-, -D-Trp-Cit-, -Ala-Ala-Asn-, D-Ala-Phe-Lys-, and -Glu-Pro-Cit-Gly-hPhe-Tyr-Leu-(SEQ ID NO: 98).
  16. 48 . The drug conjugate of claim 46 , wherein the cytotoxic agent is MMAE and the linker is Val-Cit, the Val-Cit-MMAE moiety having the structure:
  17. 49 . The drug conjugate of claim 48 , wherein said drug conjugate is obtainable by coupling the peptide ligand with Compound 8:
  18. 50 . A drug conjugate comprising: a peptide ligand comprising a polypeptide cyclised with a molecular scaffold such that at least two polypeptide loops are formed on the molecular scaffold, wherein the molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA); wherein the polypeptide comprises the amino acid sequence: wherein C i , C ii and C iii are each independently cysteine or a non-natural amino acid which is isoelectronic with cysteine and is capable of forming a covalent bond to the molecular scaffold; wherein X 1 and X 2 each represent the amino acid residues between the cysteine residues in SEQ ID NO: 1: C(HyP)LVNPLCLHP(D-Asp)W(HArg)C (SEQ ID NO: 1), wherein D-Asp is D-aspartate; HArg is homoarginine; and HyP is hydroxyproline; the peptide ligand comprising a N-terminal spacer group; the peptide ligand being conjugated via a linker to a cytotoxic agent; wherein the cytotoxic agent is MMAE and the linker is Val-Cit, the Val-Cit-MMAE moiety having the structure: a pharmaceutically acceptable salt thereof.
  19. 51 . The drug conjugate of claim 50 , wherein the polypeptide comprises a C-terminal amide group.
  20. 52 . A pharmaceutical composition which comprises the peptide ligand of claim 34 , in combination with one or more pharmaceutically acceptable excipients.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 18/313,983, filed May 8, 2023, which is a continuation of U.S. application Ser. No. 16/771,186, filed Jun. 9, 2020, which is a U.S. national stage application under 35 U.S.C. § 371 of International Application No. PCT/GB2018/053678, filed Dec. 19, 2018, which claims priority to United Kingdom Application No. 1721259.8, filed Dec. 19, 2017, United Kingdom Application No. 1804102.0, filed Mar. 14, 2018, and United Kingdom Application No. 1818603.1, filed Nov. 14, 2018, the entire contents of each of which are hereby incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (B170170055US02-SEQ-PJH.xml; Size: 141,887 bytes; and Date of Creation: Aug. 22, 2025) are herein incorporated by reference in its entirety. FIELD OF THE INVENTION The present invention relates to polypeptides which are covalently bound to non-aromatic 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 Eph receptor tyrosine kinase A2 (EphA2). The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder characterised by overexpression of EphA2 in diseased tissue (such as a tumour). BACKGROUND OF THE INVENTION Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355 Å2) (Xiong et al. (2002), Science 296 (5565). 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 Å2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10). Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8. (MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin and actinomycin. Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005). ChemBioChem). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example TATA (1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one, Heinis et al. Angew Chem, Int Ed. 2014; 53:1602-1606). Phage display-based combinatorial approaches have been developed to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule scaffold. SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a peptide ligand specific for EphA2 comprising a polypeptide comprising at least three cysteine residues, sepa