Multimeric bicyclic peptide ligands
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 and activators of CD137. 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 mediated by CD137.
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 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 said at least two bicyclic peptide ligands are specific for CD137, and wherein each of said bicyclic peptide ligands comprises a core amino acid sequence selected from:
(SEQ ID NO: 23)
C i IEEGQYC ii FADPY(Nle)C iii ;
(SEQ ID NO: 24)
C i IKEGQYC ii FADPY(Nle)C iii ;
(SEQ ID NO: 25)
C i IEKGQYC ii FADPY(Nle)C iii ;
(SEQ ID NO: 26)
C i IEE(D-K)QYC ii FADPY(Nle)C iii ;
(SEQ ID NO: 27)
C i IEEGKYC ii FADPY(Nle)C iii ;
(SEQ ID NO: 28)
C i IEEGQYC ii KADPY(Nle)C iii ;
(SEQ ID NO: 29)
C i IEEGQYC ii FADKY(Nle)C iii ;
and
(SEQ ID NO: 30)
C i IEEGQYC ii FADPYKC iii ;
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively and Nle represents norleucine, or a pharmaceutically acceptable salt thereof.
2. The multimeric binding complex as defined in claim 1 , wherein each bicyclic peptide ligand is connected to a central hinge moiety by a spacer group.
3. The multimeric binding complex as defined in claim 1 , which comprises a compound of formula (I):
wherein CHM represents a central hinge moiety;
S 1 represents a spacer group;
Bicycle represents a bicyclic peptide ligand as defined in claim 1 ; and
m represents an integer selected from 2 to 10.
4. The multimeric binding complex as defined in claim 3 , wherein m represents 4 and CHM is a motif of formula (A):
wherein “-----” represents the point of attachment to each S 1 group.
5. The multimeric binding complex as defined in claim 3 , wherein m represents 3 and CHM is selected from a motif of formula (B), (C) or (D):
wherein “-----” represents the point of attachment to each S 1 group.
6. The multimeric binding complex as defined in claim 3 , wherein the spacer (S 1 ) is selected from any one of spacers S 1 A, S 1 B, S 1 C, S 1 D, S 1 E, S 1 F, S 1 G and S 1 H:
wherein “-----” represents the point of attachment to the CHM group; and
“ ” represents the point of attachment to the Bicycle group.
7. The multimeric binding complex as defined in claim 6 , wherein the spacer (S 1 ) is S 1 A:
and n=5, 10, or 23.
8. The multimeric binding complex as defined in claim 1 , wherein said at least two bicyclic peptide ligands are identical.
9. The multimeric binding complex as defined in claim 1 , said at least two bicyclic peptide ligands are different.
10. The multimeric binding complex as defined in claim 1 , wherein each of said bicyclic peptide ligands comprises N and C terminal modifications and comprises an amino acid sequence selected from:
(SEQ ID NO: 31)
A-C i IEEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 32)
Ac-A-C i IEEGQYC ii FADPY(Nle)C iii -Dap;
(SEQ ID NO: 33)
Ac-A-C i IKEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 34)
Ac-A-C i IEKGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 35)
Ac-A-C i IEE(D-K)QYC ii FADPY(Nle) iii -A;
(SEQ ID NO: 36)
Ac-A-C i IEEGKYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 37)
Ac-A-C i IEEGQYC ii KADPY(Nle)C iii -A;
(SEQ ID NO: 38)
Ac-A-C i IEEGQYC ii FADKY(Nle)C iii -A;
(SEQ ID NO: 39)
Ac-A-C i IEEGQYC ii FADPYKC iii -A;
(SEQ ID NO: 58)
A-C i IEEGQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 59)
Ac-C i [tBuAla]PK[D-A]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 60)
Ac-C i [tBuAla]PE[D-K]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 61)
Ac-A-C i IE[D-K]GQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 62)
Ac-A-C i IE[D-K]GQYC ii F[D-A]DPY[Nle]C iii -A;
and
(SEQ ID NO: 63)
[Ac]-[D-A]-[D-C i ][D-I][D-E][D-E]K[D-Q][D-Y][D-C ii ]
[D-F][D-A][D-D][D-P][D-Y][D-Nle][D-C iii ][D-A];
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively, Ac represents an N-terminal acetyl group, Dap represents diaminopropionic acid, tBuAla represents t-butyl-alanine and Nle represents norleucine, or a pharmaceutically acceptable salt thereof.
11. The multimeric binding complex as defined in claim 1 , wherein each of said bicyclic peptide ligands comprises attachment of a PYA moiety at the N-terminus, C-terminus or Lysine residues within said sequence and comprises an amino acid sequence selected from:
(SEQ ID NO: 40)
(PYA)-A-C i IEEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 41)
Ac-A-C i IEEGQYC ii FADPY(Nle)C iii -Dap(PYA);
(SEQ ID NO: 42)
Ac-A-C i IK(PYA)EGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 43)
Ac-A-C i IEK(PYA)GQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 44)
Ac-A-C i IEE(D-K)(PYA)QYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 45)
Ac-A-C i IEEGK(PYA)YC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 46)
Ac-A-C i IEEGQYC ii K(PYA)ADPY(Nle)C iii -A;
(SEQ ID NO: 47)
Ac-A-C i IEEGQYC ii FADK(PYA)Y(Nle)C iii -A;
(SEQ ID NO: 48)
Ac-A-C i IEEGQYC ii FADPYK(PYA)C iii -A;
(SEQ ID NO: 64)
(PYA)-A-C i IEEGQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 65)
Ac-C i [tBuAla]PK(PYA)[D-A]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 66)
Ac-C i [tBuAla]PE[D-K(PYA)]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 67)
Ac-A-CiIE[D-K(PYA)]GQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 68)
Ac-A-CiIE[K(PYA)]GQYC ii F[D-A]DPY[Nle]C iii -A;
and
(SEQ ID NO: 69)
[Ac]-[D-A][D-C i ][D-I][D-E][D-E][K(PYA)][D-Q][D-Y]
[D-C ii ][D-F][D-A][D-D][D-P][D-Y][D-Nle][D-C iii ][D-A];
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively, Ac represents an N-terminal acetyl group, Dap represents diaminopropionic acid, PYA represents propargyl-acid, tBuAla represents t-butyl-alanine and Nle represents norleucine, or a pharmaceutically acceptable salt thereof.
12. The multimeric binding complex as defined in claim 1 , wherein each of said bicyclic peptide ligands comprises attachment of a BCN moiety at the N-terminus or Lysine residues within said sequence and comprises an amino acid sequence selected from:
(SEQ ID NO: 49)
(BCN)-A-C i IEEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 50)
Ac-A-C i IK(BCN)EGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 51)
Ac-A-C i IEK(BCN)GQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 52)
Ac-A-C i IEE[(D-K)(BCN)]QYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 53)
Ac-A-C i IEEGK(BCN)YC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 54)
Ac-A-C i IEEGQYC ii K(BCN)ADPY(Nle)C iii -A ;
and
(SEQ ID NO: 55)
Ac-A-C i IEEGQYC ii FADPYK(BCN)C iii -A;
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively, Ac represents an N-terminal acetyl group, Nle represents norleucine and BCN represents:
or a pharmaceutically acceptable salt thereof.
13. The multimeric binding complex as defined in claim 1 , wherein said molecular scaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).
14. The multimeric binding complex as defined in claim 1 , wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, or ammonium salt.
15. A drug conjugate comprising the multimeric binding complex as defined in claim 1 , conjugated to one or more effector and/or functional groups.
16. A pharmaceutical composition which comprises the multimeric binding complex of claim 1 or the drug conjugate of claim 15 , in combination with one or more pharmaceutically acceptable excipients.
17. The multimeric binding complex as defined in claim 3 , wherein each of said bicyclic peptide ligands comprises N and C terminal modifications and comprises an amino acid sequence selected from:
(SEQ ID NO: 31)
A-C i IEEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 32)
Ac-A-C i IEEGQYC ii FADPY(Nle)C iii -Dap;
(SEQ ID NO: 33)
Ac-A-C i IKEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 34)
Ac-A-C i IEKGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 35)
Ac-A-C i IEE(D-K)QYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 36)
Ac-A-C i IEEGKYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 37)
Ac-A-C i IEEGQYC ii KADPY(Nle)C iii -A;
(SEQ ID NO: 38)
Ac-A-C i IEEGQYC ii FADKY(Nle)C iii -A;
(SEQ ID NO: 39)
Ac-A-C i IEEGQYC ii FADPYKC iii -A;
(SEQ ID NO: 58)
A-C i IEEGQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 59)
Ac-C i [tBuAla]PK[D-A]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 60)
Ac-C i [tBuAla]PE[D-K]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 61)
Ac-A-C i IE[D-K]GQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 62)
Ac-A-C i IE[D-K]GQYC ii F[D-A]DPY[Nle]C iii -A;
and
(SEQ ID NO: 63)
[Ac]-[D-A]-[D-C i ][D-I][D-E][D-E]K[D-Q][D-Y][D-C ii ][D-
F][D-A][D-D][D-P][D-Y][D-Nle][D-C iii ]-[D-A];
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively, Ac represents an N-terminal acetyl group, Dap represents diaminopropionic acid, tBuAla represents t-butyl-alanine and Nle represents norleucine, or a pharmaceutically acceptable salt thereof.
18. The multimeric binding complex as defined in claim 3 , wherein each of said bicyclic peptide ligands comprises attachment of a PYA moiety at the N-terminus, C-terminus or Lysine residues within said sequence and comprises an amino acid sequence selected from:
(SEQ ID NO: 40)
(PYA)-A-C i IEEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 41)
Ac-A-C i IEEGQYC ii FADPY(Nle)C iii -Dap(PYA);
(SEQ ID NO: 42)
Ac-A-C i IK(PYA)EGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 43)
Ac-A-C i IEK(PYA)GQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 44)
Ac-A-C i IEE(D-K)(PYA)QYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 45)
Ac-A-C i IEEGK(PYA)YC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 46)
Ac-A-C i IEEGQYC ii K(PYA)ADPY(Nle)C iii -A;
(SEQ ID NO: 47)
Ac-A-C i IEEGQYC ii FADK(PYA)Y(Nle)C iii -A;
(SEQ ID NO: 48)
Ac-A-C i IEEGQYC ii FADPYK(PYA)C iii -A;
(SEQ ID NO: 64)
(PYA)-A-C i IEEGQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 65)
Ac-C i [tBuAla]PK(PYA)[D-A]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 66)
Ac-C i [tBuAla]PE[D-K(PYA)]PYC ii FADPY[Nle]C iii -A;
(SEQ ID NO: 67)
Ac-A-C i IE[D-K(PYA)]GQYC ii F[D-A]DPY[Nle]C iii -A;
(SEQ ID NO: 68)
Ac-A-C i IE[K(PYA)]GQYC ii F[D-A]DPY[Nle]C iii -A;
and
(SEQ ID NO: 69)
[Ac]-[D-A]-[D-C i ][D-I][D-E][D-E][K(PYA)][D-Q][D-
Y][D-C ii ][D-F][D-A][D-D][D-P][D-Y][D-Nle][D-C iii ]-
[D-A];
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively, Ac represents an N-terminal acetyl group, Dap represents diaminopropionic acid, PYA represents propargyl-acid, tBuAla represents t-butyl-alanine and Nle represents norleucine, or a pharmaceutically acceptable salt thereof.
19. The multimeric binding complex as defined in claim 3 , wherein each of said bicyclic peptide ligands comprises attachment of a BCN moiety at the N-terminus or Lysine residues within said sequence and comprises an amino acid sequence selected from:
(SEQ ID NO: 49)
(BCN)-A-C i IEEGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 50)
Ac-A-C i IK(BCN)EGQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 51)
Ac-A-C i IEK(BCN)GQYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 52)
Ac-A-C i IEE[D-K)(BCN)]QYC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 53)
Ac-A-C i IEEGK(BCN)YC ii FADPY(Nle)C iii -A;
(SEQ ID NO: 54)
Ac-A-C i IEEGQYC ii K(BCN)ADPY(Nle)C iii -A;
and
(SEQ ID NO: 55)
Ac-A-C i IEEGQYC ii FADPYK(BCN)C iii -A;
wherein C i , C ii and C iii represent first, second and third cysteine residues, respectively, Ac represents an N-terminal acetyl group, Nle represents norleucine and BCN represents:
or a pharmaceutically acceptable salt thereof.
20. The multimeric binding complex as defined in claim 17 , wherein m represents 4 and CHM is a motif of formula (A):
wherein “-----” represents the point of attachment to each S 1 group.
21. The multimeric binding complex as defined in claim 17 , wherein m represents 3 and CHM is selected from a motif of formula (B), (C) or (D):
wherein “-----” represents the point of attachment to each S 1 group.
22. The multimeric binding complex as defined in claim 17 , wherein the spacer (S 1 ) is selected from any one of spacers S 1 A, S 1 B, S 1 C, S 1 D, S 1 E, S 1 F, S 1 G and S 1 H:
wherein “-----” represents the point of attachment to the CHM group; and
“ ” represents the point of attachment to the Bicycle group.
23. The multimeric binding complex as defined in claim 18 , wherein m represents 4 and CHM is a motif of formula (A):
wherein “-----” represents the point of attachment to each S 1 group.
24. The multimeric binding complex as defined in claim 18 , wherein m represents 3 and CHM is selected from a motif of formula (B), (C) or (D):
wherein “-----” represents the point of attachment to each S 1 group.
25. The multimeric binding complex as defined in claim 18 , wherein the spacer (S 1 ) is selected from any one of spacers S 1 A, S 1 B, S 1 C, S 1 D, S 1 E, S 1 F, S 1 G and S 1 H:
wherein “-----” represents the point of attachment to the CHM group; and
“ ” represents the point of attachment to the Bicycle group.
26. The multimeric binding complex as defined in claim 19 , wherein m represents 4 and CHM is a motif of formula (A):
wherein “-----” represents the point of attachment to each S 1 group.
27. The multimeric binding complex as defined in claim 19 , wherein m represents 3 and CHM is selected from a motif of formula (B), (C) or (D):
wherein “-----” represents the point of attachment to each S 1 group.
28. The multimeric binding complex as defined in claim 19 , wherein the spacer (S 1 ) is selected from any one of spacers S 1 A, S 1 B, S 1 C, S 1 D, S 1 E, S 1 F, S 1 G and S 1 H:
wherein “-----” represents the point of attachment to the CHM group; and
“ ” represents the point of attachment to the Bicycle group.
29. A drug conjugate comprising the multimeric binding complex as defined in claim 3 , conjugated to one or more effector and/or functional groups.