Fibronectin type III repeat based protein scaffolds with alternative binding surfaces
Protein scaffolds and scaffold libraries based on a fibronectin type III (FN3) repeat with an alternative binding surface design, isolated nucleic acids encoding the protein scaffolds, vectors, host cells, and methods of making thereof are useful in the generation of therapeutic molecules and treatment and diagnosis of diseases and disorders.
1. A method of obtaining a protein scaffold that specifically binds to a target molecule, comprising:
a. contacting or panning a library of fibronectin module of type III (FN3) domains having a diversified C-CD-F-FG alternative surface formed by a C beta-strand, a CD loop, an F beta-strand, and an FG loop with the target molecule, and
b. isolating a protein scaffold specifically binding to the target molecule with a predefined affinity, wherein the library is formed by providing a reference FN3 domain polypeptide having the amino acid sequence at least 80% identical to that of SEQ ID NO: 27 and introducing diversity into the reference FN3 domain polypeptide by mutating at least one C beta-strand residue and at least one F beta-strand residue to form the FN3 domain library having the diversified C-CD-F-FG alternative surface, wherein the at least one residue in the C beta-strand is mutated with the proviso that S30 is not mutated (residue numbering according to SEQ ID NO: 27), wherein the at least one residue in the F beta-strand is mutated with the proviso that E66 is not mutated (residue numbering according to SEQ ID NO: 27), wherein each of the C beta-strand residues L32, Q34 and Q36 are mutated (residue numbering according to SEQ ID NO: 27) or each of the F-beta strand residues T68, S70 and Y72 are mutated (residue numbering according to SEQ ID NO: 27).
2. The method of claim 1 , wherein 1, 2, 3 or 4 residues in the CD loop residues in the reference FN3 domain polypeptide are mutated with the proviso that G42 and E43are not mutated (residues numbering according to SEQ ID NO: 27).
3. The method of claim 2 , wherein the residues S38, E39, K40 and V41 in the CD loop in the reference FN3 domain polypeptide are mutated.
4. The method of claim 3 , wherein 1, 2, 3 or 4 residues in the FG loop in the reference FN3 domain polypeptide are mutated with the proviso that the residues K75, G76, G77 and S80 are not mutated (residue numbering according to SEQ ID NO: 27).
5. The method of claim 4 , wherein the residues H78, R79 and N81 in the FG loop in the reference FN3 domain polypeptide are mutated (residue numbering according to SEQ ID NO: 27).
6. The method of claim 5 , wherein the reference FN3 domain polypepetide comprises an amino acid sequence of SEQ ID NO: 27, optionally comprising at least one substitution at amino acid positions 11, 14, 17, 37, 46, 73, or 86.
7. The method of claim 6 , wherein the reference FN3 domain polypeptide comprises an amino acid sequence of SEQ ID NO: 28.