Multispecific peptides
The invention relates to 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.
1. A multispecific peptide ligand comprising:
(a) a nucleic acid encoding the multispecific polypeptide;
(b) the multispecific peptide displayed on a phage display system, wherein the multispecific polypeptide comprises the sequence (X)IC(X)mC(X)nC(X)o (SEQ ID No. 26), wherein C represents the amino acid cysteine, X represents a random amino acid, m and n are numbers between 1 and 20 defining the length of intervening polypeptide segments and I and o are numbers between 0 and 20 defining the length of the flanking polypeptide segments;
(c) the multispecific peptide ligand encoded by the nucleic acid is linked to the nucleic acid by the phage; and
(d) the multispecific peptide ligand is attached to tris-(bromomethyl)benzene (TBMB) by three discrete covalent bonds between said TBMB and said multispecific peptide ligand via said C amino acid residues, and form at least one loop which comprises a sequence of two or more amino acids subtended between two of said C amino acid residues, wherein the multispecific peptide ligand is capable of binding to two or more targets, thus forming the multispecific peptide ligand.