Method of identifying polypeptide monobodies which bind to target proteins and use thereof

A fibronectin type III (Fn3) polypeptide monobody comprising the formula βA-LAB-βB-LBC-βC-LCD-βD-LDE-βE-LEF-βF-LFG-βG, wherein:

• βA, βB, βC, βD, βE, βF, and βG are, respectively, β-strand domain sequences A through G of a tenth Fn3 domain of fibronectin;
• and
• LAB, LBC, LCD, LDE, LEF, and LFG are, respectively, loop region sequences AB, BC, CD, DE, EF, and FG, wherein at least one loop region sequence selected from the group of AB, BC, and FG varies from a corresponding loop region present in the tenth Fn3 domain of fibronectin by deletion of up to all but one amino acid residue, insertion of two to about 25 amino acid residues, or replacement of two to all amino acid residues;
• and
• wherein the polypeptide monobody exhibits estrogen receptor binding activity via interaction of the at least one loop region sequence with the estrogen receptor.

A fibronectin type III (Fn3) polypeptide monobody derived from the amino acid sequence of SEQ ID NO:

• 2 or SEQ ID NO:
• 3, the polypeptide monobody comprising:
• at least two adjacent Fn3 β-strand domain sequences selected from the group of A (residues 9-14 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), B (residues 17-21 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), C (residues 31-38 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), D (residues 46-50 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), E (residues 55-59 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), F (residues 67-75 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), and G (residues 88-94 of SEQ ID NO:
• 2 and SEQ ID NO:
• 3), with an associated loop region sequence linked between each pair of adjacent β-strand domain sequences, each of the loop region sequences being selected from the group of loops AB, BC, CD, DE, EF, and FG;
• and
• optionally, an N-terminal tail of at least about 2 to about 33 amino acids, a C-terminal tail of at least about 2 to about 25 amino acids, or both;
• wherein at least one loop region sequence is modified from the corresponding loop region of SEQ ID NO:
• 2 or SEQ ID NO:3, the at least one loop region sequence being selected from the group of (i) the BC loop region sequence comprising the amino acid sequence of SEQ ID NO:
• 23, (ii) the FG loop region sequence comprising the amino acid sequence of SEQ ID NO:
• 67, and (iii) the AB loop region sequence comprising the amino acid sequence of SEQ ID NO:
• 34;
• and
• wherein the polypeptide monobody exhibits estrogen receptor binding activity via interaction of the at least one loop region sequence with the estrogen receptor.