Modulation of structured polypeptide specificity
The invention describes peptide ligands specific for human plasma Kallikrein.
1. A peptide ligand specific for human 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 loops of the peptide ligand comprise five amino acids and a first loop comprises the motif G r x W / F Px K / R G r (SEQ ID NO: 207), a second loop comprises the motif G r T / L H Q / T xLG r (SEQ ID NO: 208) and wherein the first G r of the second loop is the second G r of the first loop, wherein G r represents cysteine and x represents any amino acid, and wherein R may optionally be replaced with N-methyl arginine, homoarginine or guanidylphenylalanine, and/or P may optionally be replaced with azetidine carboxylic acid.
2. The peptide ligand according to claim 1 , wherein the first loop comprises the sequence G r xWPARG r (SEQ ID NO: 4), wherein G r represents cysteine and x represents any amino acid.
3. The peptide ligand according to claim 2 , wherein x is S or R.
4. The peptide ligand according to claim 1 , wherein the first loop comprises the sequence G r xWPSRG r (SEQ ID NO: 5), wherein G r represents cysteine and x represents any amino acid.
5. The peptide ligand according to claim 4 , wherein x is S or R.
6. The peptide ligand according to claim 1 , wherein the first loop comprises the sequence G r xFPFRG r (SEQ ID NO: 6), wherein G r represents cysteine and x represents any amino acid.
7. The peptide ligand according to claim 6 , wherein x is S or R.
8. The peptide ligand according to claim 1 , wherein the first loop comprises the sequence G r xFPYRG r (SEQ ID NO: 7), wherein G r represents cysteine and x represents any amino acid.
9. The peptide ligand according to claim 8 , wherein x is S or R.
10. The polypeptide ligand according to claim 1 , wherein the polypeptide comprises a first loop which comprises the motif G r xWPARG r (SEQ ID NO: 4), wherein P is replaced with azetidine carboxylic acid; and/or R is replaced with N-methyl arginine; or R is replaced with homoarginine; or R is replaced with guanidylphenylalanine.
11. The peptide ligand according to claim 10 , wherein x is S or R.
12. The polypeptide ligand according to claim 1 , wherein the polypeptide comprises a first loop which comprises the motif G r xFPYRG r (SEQ ID NO: 7), wherein R is replaced with N-methyl arginine; or R is replaced with homoarginine, or R is replaced with guanidylphenylalanine, and/or wherein P is replaced with azetidine carboxylic acid.
13. The peptide ligand according to claim 12 , wherein x is S or R.
14. The polypeptide ligand according to claim 1 , wherein the polypeptide comprises a first loop which comprises the motif G r xWPSRG r (SEQ ID NO: 5), wherein R is replaced with N-methyl arginine; or R is replaced with homoarginine, or R is replaced with guanidylphenylalanine, and/or wherein P is replaced with azetidine carboxylic acid.
15. The peptide ligand according to claim 14 , wherein x is S or R.
16. The polypeptide ligand according to claim 1 , wherein the polypeptide comprises a first loop which comprises the motif G r xFPFRG r (SEQ ID NO: 6), wherein R is replaced with N-methyl arginine; or R is replaced with homoarginine, or R is replaced with guanidylphenylalanine, and/or wherein P is replaced with azetidine carboxylic acid.
17. The peptide ligand according to claim 16 , wherein x is S or R.
18. The peptide ligand according to claim 1 , wherein R is replaced with N-methyl arginine.
19. The peptide ligand according to claim 1 , wherein R is replaced with homoarginine.
20. The peptide ligand according to claim 1 , wherein R is replaced with guanidylphenylalanine.
21. The peptide ligand according to claim 1 , wherein P is replaced with azetidine carboxylic acid.
22. The peptide ligand according to claim 1 , wherein x at position 2 of SEQ ID NO: 207 is S or R.
23. The peptide ligand according to claim 1 , wherein the polypeptide comprises two adjacent loops which comprise the motif G r x W / F Px K / R G r T / L H Q / T DLG r (SEQ ID NO: 8) wherein Gr represents cysteine.
24. The peptide ligand according to claim 1 , wherein the molecular scaffold comprises reactive groups for forming covalent bonds with the cysteine residues of the polypeptide, wherein the reactive groups are selected from the group consisting of alkyl halides and maleimides.
25. The peptide ligand according to claim 24 , wherein the molecular scaffold is tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, or tris-(maleimido)benzene.
26. The peptide ligand according to claim 24 , wherein the molecular scaffold is a tris(bromomethyl)benzene, or a derivative thereof.
27. The peptide ligand according to claim 26 , wherein the molecular scaffold is 1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene, 1,3,5-Tris(bromomethyl)benzene, or 1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.
28. The peptide ligand according to claim 27 , wherein the molecular scaffold is 1,3,5-Tris(bromomethyl)benzene.
29. The peptide ligand according to claim 1 , wherein the peptide ligand is attached to an antibody or a fragment thereof.
30. The peptide ligand according to claim 29 , 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.
31. The peptide ligand according to claim 29 , wherein the antibody or fragment thereof comprises an Fc region of an IgG molecule.
32. The peptide ligand according to claim 29 , wherein the antibody or fragment thereof comprises a hinge region of an antibody.
33. The peptide ligand according to claim 32 , wherein the hinge region comprises the region between the CH1 and CH2 domains of an IgG molecule.
34. The peptide ligand according to claim 1 , wherein the polypeptide comprises one of the polypeptides selected from:
ACNWPYRCLHTDLCA (SEQ ID NO. 47);
ACRWPARCTHQDLCA (SEQ ID NO. 57);
ACSWPARCTHQDLCA (SEQ ID NO. 58);
ACRWPARCLHQDLCA (SEQ ID NO. 60);
ACSWPARCLHQDLCA (SEQ ID NO. 62);
ACSWPAKCLHQDLCA (SEQ ID NO. 63); and
AC(N-MeS)WPARCLHQDLCA (SEQ ID NO. 66).
35. The peptide ligand according to claim 34 , wherein the molecular scaffold comprises reactive groups for forming covalent bonds with the cysteine residues of the polypeptide, wherein the reactive groups are selected from the group consisting of alkyl halides and maleimides.
36. The peptide ligand according to claim 35 , wherein the molecular scaffold is tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, or tris-(maleimido)benzene.
37. The peptide ligand according to claim 35 , wherein the molecular scaffold is a tris(bromomethyl)benzene, or a derivative thereof.
38. The peptide ligand according to claim 37 , wherein the molecular scaffold is 1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene, 1,3,5-Tris(bromomethyl)benzene, or 1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.
39. The peptide ligand according to claim 38 , wherein the molecular scaffold is 1,3,5-Tris(bromomethyl)benzene.
40. The peptide ligand according to claim 34 , wherein the peptide ligand is attached to an antibody or a fragment thereof.
41. The peptide ligand according to claim 40 , wherein the antibody or fragment thereof comprises an Fc region of an IgG molecule.
42. The peptide ligand according to claim 40 , 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.
43. The peptide ligand according to claim 40 , wherein the antibody or fragment thereof comprises a hinge region of an antibody.
44. The peptide ligand according to claim 43 , wherein the hinge region comprises the region between the CH1 and CH2 domains of an IgG molecule.
45. A Pharmaceutical composition comprising the peptide ligand according to claim 1 , and a pharmacologically appropriate carrier.
46. A Pharmaceutical composition comprising the peptide ligand according to claim 29 , and a pharmacologically appropriate carrier.
47. A Pharmaceutical composition comprising the peptide ligand according to claim 34 , and a pharmacologically appropriate carrier.
48. A Pharmaceutical composition comprising the peptide ligand according to claim 40 , and a pharmacologically appropriate carrier.