Polypeptide ligands specific for plasma kallikrein
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 specific for the human and rat protease plasma kallikrein and are modified in one or two peptide loops to enhance potency and/or protease resistance.
1. A peptide ligand specific for plasma kallikrein comprising a polypeptide comprising at least three cysteine residues, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises a peptide sequence of:
(SEQ ID NO: 6)
-C i -(N) a -U-J-P-J-R-C ii -V-Y-Y-P-D-I-C iii -,
or a modified derivative, or pharmaceutically acceptable salt, thereof;
wherein:
C i , C ii and C iii represent first, second and third cysteine residues, respectively;
subscript “a” represents an integer selected from 0 or 1;
J represents a non-polar aromatic amino acid residue selected from F, W and Y;
U represents a polar, uncharged amino acid residue selected from N, C, Q, M, S and T.
2. The peptide ligand as defined in claim 1 , wherein the peptide comprises a sequence of:
(SEQ ID NO: 53)
-Ci-(N) a -N/S-F-P-F/Y-R-C ii -V-Y-Y-P-D-I-C iii -.
3. The peptide ligand as defined in claim 1 , wherein the peptide comprises a sequence of:
(SEQ ID NO: 54)
-C i -N-N-F-P-F-R-C ii -V-Y-Y-P-D-I-C iii -(06-261);
or
(SEQ ID NO: 55)
-C i -S-F-P-Y-R-C ii -V-Y-Y-P-D-I-C iii -(06-550).
4. The peptide ligand as defined in claim 1 , wherein the modified derivative includes 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; 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 α-carbon of one or more amino acid residues with another chemical group, and post-synthetic bioorthogonal modification of amino acids.
5. The peptide ligand as defined in claim 4 , wherein the modified derivative comprises an N-terminal modification.
6. The peptide ligand as defined in claim 5 , wherein the N-terminal modification comprises an N-terminal acetyl group.
7. The peptide ligand as defined in claim 5 , wherein the N-terminal modification comprises capping an N-terminal cysteine group with acetic anhydride.
8. The peptide ligand as defined in claim 4 , wherein the modified derivative comprises a C-terminal modification.
9. The peptide ligand as defined in claim 8 , wherein the C-terminal modification comprises a C-terminal amide group.
10. The peptide ligand as defined in claim 8 , wherein the C-terminal modification comprises amidation of a C-terminal cysteine group.
11. The peptide ligand as defined in claim 4 , wherein the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues.
12. The peptide ligand as defined in claim 11 , which is a non-natural derivative comprising a peptide having a sequence selected from:
(SEQ ID NO: 56)
-C i -S-F-P-Y-[hR]-C ii -V-Y-Y-P-D-I-C iii -
((06-550) HArg5);
(SEQ ID NO: 57)
-C i -S-F-[Aze]-Y-R-C ii -V-Y-Y-P-D-I-C iii -;
(SEQ ID NO: 58)
-C i -S-F-[Aze]-Y-[hR]-C ii -V-Y-Y-P-D-I-C iii -
((06-550) Aze3 HArg5);
(SEQ ID NO: 59)
-C i -S-F-P-Y-[NMeR]-C ii -V-Y-Y-P-D-I-C iii -
((06-550) NMeArg5);
(SEQ ID NO: 60)
-C i -S-F-[Aze]-Y-[NMeR]-C ii -V-Y-Y-P-D-I-C iii -
((06-550) Aze3 NMeArg5);
(SEQ ID NO: 61)
-C i -N-T-W-N-P-W-C ii -P-W-D-A-P-L-C iii -A-Sar 3 -
(D-Arg) 2 ((06-259-02 (Sar 3 -(D-Arg) 2 );
(SEQ ID NO: 62)
-C i -S-F-P-Y-R-C ii -V-Y-Y-P-D-I-C iii -A-Sar 3 -((D-Arg) 2
(06-550)-Sar 3 -(DArg) 2 );
and
(SEQ ID NO: 63)
-C i -S-F-[Aze]-Y-[hR]-C ii -V-Y-Y-P-D-I-C iii -A-Sar 3 -
(D-Arg) 2 ((06-550)-Sar 3 -(DArg) 2 Aze3 HArg5),
wherein Aze represents an L-azetidine carboxylic acid residue, hR and HArg represents an L-homoarginine residue, NMeR and NMeArg represents an N-α-methyl arginine residue, Sar 3 represents 3 sarcosine spacers and (D-Arg) 2 represents 2 D-arginine residues.
13. The peptide ligand according to claim 12 , wherein the peptide ligand is attached to an antibody or a fragment thereof.
14. The peptide ligand according to claim 13 , wherein the antibody fragment is selected from an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to the one or more constant region domains.
15. The peptide ligand according to claim 13 , wherein the antibody or a fragment thereof comprises a hinge region of an antibody.
16. The peptide ligand according to claim 15 , wherein the hinge region comprises the region between the CH1 and CH2 domains of an IgG molecule.
17. The peptide ligand according to claim 13 , wherein the antibody or fragment thereof comprises an Fc region of an IgG molecule.
18. The peptide ligand as defined in claim 11 , wherein the modified derivative comprises replacement of a proline residue with an L-azetidine carboxylic acid residue, and/or replacement of an arginine residue with an N-α-methyl arginine or L-homoarginine residue.
19. A peptide ligand as defined in claim 4 , comprising a peptide sequence of:
(SEQ ID NO: 63)
-C i -S-F-[Aze]-Y-[hR]-C ii -V-Y-Y-P-D-I-C iii -A-Sar 3 -
(D-Arg) 2 ((06-550)-Sar 3 -(DArg) 2 Aze3 HArg5),
wherein Sar 3 represents 3 sarcosine spacers, (D-Arg) 2 represents 2 D-arginine residues, Aze represents an L-azetidine carboxylic acid residue and hR and HArg represents an L-homoarginine residue.
20. The peptide ligand as defined in claim 1 , wherein the pharmaceutically acceptable salt is selected from a hydrochloride or acetate salt.
21. The peptide ligand as defined in claim 1 , which is specific for human, rat and/or rabbit plasma kallikrein.
22. The peptide ligand as defined in claim 21 , which is specific for human plasma kallikrein.
23. The peptide ligand according to claim 1 , wherein the peptide ligand is attached to an antibody or a fragment thereof.
24. The peptide ligand according to claim 23 , wherein the antibody fragment is selected from an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to the one or more constant region domains.
25. The peptide ligand according to claim 23 , wherein the antibody or a fragment thereof comprises a hinge region of an antibody.
26. The peptide ligand according to claim 25 , wherein the hinge region comprises the region between the CH1 and CH2 domains of an IgG molecule.
27. The peptide ligand according to claim 23 , wherein the antibody or fragment thereof comprises an Fc region of an IgG molecule.
28. A pharmaceutical composition comprising a peptide ligand as defined in claim 1 in combination with one or more pharmaceutically acceptable excipients.
29. The peptide ligand as defined in claim 4 , wherein the replacement of one or more amino acid residues with one or more non-natural amino acid residues comprises replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids, and/or replacement of one or more hydrophobic amino acid residues with other non-natural isosteric or isoelectronic amino acids.
30. The peptide ligand as defined in claim 4 , wherein the post-synthetic bioorthogonal modification of amino acids comprises modification of cysteine, lysine, glutamate, and/or tyrosine with suitable amine, thiol, carboxylic acid, and/or phenol-reactive reagents.
31. A method of treating an ophthalmic disorder comprising administering to a patient in need thereof a peptide ligand as defined in claim 1 .
32. The method as defined in claim 31 , wherein said ophthalmic disorder is an oedema in the eye.
33. The method as defined in claim 31 , wherein said ophthalmic disorder is a disorder related to impaired retinal vessel permeability.
34. The method as defined in claim 31 , wherein said ophthalmic disorder is an angioneurotic oedema.
35. A method of treating an ophthalmic disorder comprising administering to a patient in need thereof a peptide ligand as defined in claim 12 .
36. The method as defined in claim 35 , wherein said ophthalmic disorder is an oedema in the eye.
37. The method as defined in claim 35 , wherein said ophthalmic disorder is a disorder related to impaired retinal vessel permeability.
38. The method as defined in claim 35 , wherein said ophthalmic disorder is an angioneurotic oedema.
39. A method for inhibiting plasma kallikrein activity in a subject, the method comprising administering a peptide ligand as defined in claim 1 .
40. A method for inhibiting plasma kallikrein activity in a subject, the method comprising administering a peptide ligand as defined in claim 12 .
41. A method for inhibiting plasma kallikrein activity in the eye of a subject, the method comprising administering to the eye of the subject a peptide ligand as defined in claim 1 .
42. A method for inhibiting plasma kallikrein activity in the eye of a subject, the method comprising administering to the eye of the subject a peptide ligand as defined in claim 12 .