Claims

1. A method for providing a multispecific peptide ligand comprising a polypeptide covalently linked to a molecular scaffold at three or more amino acid residues and capable of binding to two or more separate targets, comprising the steps of:

(a) providing a first repertoire of polypeptides, each polypeptide comprising two or more reactive groups capable of covalent linkage to a molecular scaffold, and at least one loop which comprises a sequence of two or more amino acids subtended between two of said reactive groups;

(b) providing a second repertoire of polypeptides as described in (a);

(c) joining at least one loop of one or more members of the first repertoire to at least one loop of one or more members of the second repertoire to form at least one polypeptide comprising two loops, and

(d) conjugating the composite polypeptide(s) to a molecular scaffold at at least three amino acid positions.

2. A method according to claim 1, wherein said first and second repertoires are screened for binding to first and second targets to isolate said one or more members used in step (c).

3. A method according to claim 2, wherein said first and second repertoires are screened for binding by:

(i) conjugating the members of said first or second repertoire to a molecular scaffold, and screening for binding against said first or second target; or

(ii) crosslinking the members of said first or second repertoire through said reactive groups;

and screening for binding against said first or second targets.

4. A method according to claim 3, wherein the members of said first and/or second repertoires are conjugated to a molecular scaffold to form a single loop.

5. A method according to claim 3, wherein the members said first repertoire are conjugated to a molecular scaffold to form two or more loops, and wherein a single loop thereof is joined to at least one loop of the members of the second repertoire.

6. A method according to any preceding claim, wherein step (c) results in the formation of a third repertoire of peptide ligands.

7. A method according to claim 6, wherein one of said first and second repertoires is naive.

8. A method for providing a multispecific peptide ligand comprising a polypeptide covalently linked to a molecular scaffold at three or more amino acid residues and capable of binding to two or more separate targets, comprising the steps of:

(a) providing a first repertoire of polypeptides;

(b) conjugating said polypeptides to a molecular scaffold which binds to the polypeptides at two or more amino acid residues forming at least one loop, to form a first repertoire of polypeptide conjugates;

(c) screening said first repertoire for binding against a first target, and selecting members of the first repertoire which bind to the first target;

(d) repeating steps (a) to (c) with a second repertoire of polypeptides, yielding a second repertoire of polypeptide conjugates which bind to a second target;

(e) isolating loops from members of said first and said second repertoires, and combining them to form a third repertoire of polypeptide conjugates, wherein the polypeptides are bound to the molecular scaffold at at least three amino acids, and selecting molecules capable of binding both to the first and the second target.

9. A method according to any preceding claim, wherein polypeptide conjugates of one or more repertoires are treated with a protease.

10. A method according to claim 9, wherein said polypeptide conjugates of one or more repertoires are treated with a reducing agent and a protease.

11. A method according to any preceding claim, wherein one or more of the repertoires of polypeptides is encoded by one or more libraries of nucleic acid molecules.

12. A method according to claim 11, wherein the screening of said repertoires is performed using a genetic display system.

13. A method according to claim 12, wherein the genetic display system is phage display.

14. A method according to any preceding claim, wherein the multispecific peptide ligand is dual specific.

15. A method according to any preceding claim, wherein binding to the first and second targets by members of said third repertoire can occur simultaneously.

16. A method according to any of claims 1 to 14, wherein binding to the first and second targets by members of said third repertoire cannot occur simultaneously.

17. A method according to claim 16, wherein members of said third repertoire are screened against said first target, and those that bind are then screened against said second target.

18. A method according to claim 17, wherein after said first screening for binding to said first target, said third repertoire is amplified and then screened against said second target.

19. A multispecific peptide ligand comprising a polypeptide covalently linked to a molecular scaffold at at least three amino acid positions, which is capable of binding to two or more targets.

20. A multispecific ligand according to claim 19, which is capable of binding to two or more targets simultaneously.

21. A multispecific peptide ligand according to claim 19 or claim 20, obtainable by a method according to any one of claims 1 to 13.

22. A peptide ligand comprising a polypeptide covalently linked to a molecular scaffold, conjugated to one or more functional groups.

23. A peptide ligand according to any one of claims 19 to 21, conjugated to one or more functional groups.

24. A peptide ligand according to claim 22 or claim 23, wherein the functional group is attached at one or more positions selected from the Isl terminus of the polypeptide, the C terminus of the polypeptide, and the molecular scaffold.

25. A peptide ligand according to any one of claims 22 to 24, wherein the functional group is a ligand that binds to a molecule which extends the half-life of the peptide ligand in vivo.

26. A peptide ligand according to claim 25, wherein the molecule which extends the half-life of the peptide ligand in vivo is HSA or a cell matrix protein.

27. A peptide ligand according to claim 26, wherein the ligand that binds to a molecule which extends the half-life of the peptide ligand in vivo is an antibody or antibody fragment specific for HSA or a cell matrix protein.

28. A peptide ligand according to claim any one of claims 22 to 24, wherein the functional group is a ligand selected from the group consisting of a polypeptide covalently linked to a molecular scaffold, a chemical group of less than 1000 daltons, and an antibody or antibody fragment.

29. A peptide ligand according to any one of claims 22 to 24, wherein the functional group is an effector group.

30. A peptide ligand according to claim 29, wherein the effector group is an antibody Fc region.

31. A peptide ligand according to any one of claims 22 to 24, wherein the effector group is a cell penetrating peptide.

32. A peptide ligand according to claim 31, wherein the cell penetrating peptide is selected the group consisting of polyarginine, VP-22, Antennapedia, HIV-tat and penetratin.

33. A method for preparing a peptide ligand according to any one of claims 22 to 32, comprising the steps of

(a) producing the polypeptide;

(b) conjugating it with the molecular scaffold; and

(c) attaching said one or more functional groups to the N or C terminus of the polypeptide.

34. A method according to claim 33, wherein the functional group is FMOC.

35. A method for preparing a peptide ligand according to any one of claims 22 to 32. comprising the steps of

(a) producing the polypeptide;
(b) conjugating it with the molecular scaffold; and
(c) attaching said one or more functional groups to the molecular scaffold.

36. A peptide ligand according to any one of claims 19 to 32, which has a molecular weight of less than 3000 Dalton.

37. A peptide ligand according to any one of claims 19 to 32, which has a molecular weight of less than 5000 Dalton.

38. A peptide ligand according to any one of claims 19 to 32, wherein the length of the polypeptide loops subtended between any two adjacent points of attachment of the polypeptide to the molecular scaffold is between 0 and 9 amino acids, and the length of the polypeptide sequence between the first and last of said points of attachment is less than 27 amino acids.