Alphabody libraries and methods for producing the same

A single-chain Alphabody library comprising at least 100 different-sequence single-chain Alphabody polypeptides, each of said single-chain Alphabody polypeptides having the general formula HRS1-L1-HRS2-L2-HRS3, wherein

• each of HRS1, HRS2 and HRS3 is independently a heptad repeat sequence (HRS) consisting of 2 to 7 consecutive heptad repeat units, at least 50% of all heptad a- and d-positions are occupied by isoleucine residues, each HRS starts and ends with an aliphatic or aromatic amino acid residue located at either a heptad a- or d-position, and HRS1, HRS2 and HRS3 together form a triple-stranded, alpha-helical, coiled coil structure;
• and
• each of L1 and L2 is independently a linker fragment, which covalently connects HRS1 to HRS2 and HRS2 to HRS3, respectively, and consisting of at least 4 amino acid residues, preferably at least 50% of which are selected from the group proline, glycine, serine,
• wherein said Alphabody polypeptides differ from each other in at least one of a defined set of 5 to 20 variegated amino acid residue positions, and wherein at least 70% but not all of said variegated amino acid residue positions are located either:
• (i) at heptad e-positions in a first alpha-helix of the Alphabody polypeptides and at heptad g-positions in a second alpha-helix, and optionally at heptad b-positions in said first alpha-helix of the Alphabody polypeptides and/or at heptad c-positions in said second alpha-helix of the Alphabody polypeptides,
• or
• (ii) at heptad b-, c- and f-positions in one alpha-helix of the Alphabody polypeptides, or
• (iii) at positions in a linker fragment connecting two consecutive alpha-helices of the Alphabody polypeptides.