Fibronectin Type III Repeat Based Protein Scaffolds with Alternative Binding Surfaces

• US 20130079243A1
• Filed: 09/27/2012
• Published: 03/28/2013
• Est. Priority Date: 09/27/2011
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Diem, Michael, Jacobs, Steven
• Granted Patent: US 9,200,273 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: G01N 2333/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

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.

Claims

1. A method of making 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, comprising

a. providing a reference FN3 domain polypeptide having the amino acid sequence at least 80% identical to that of SEQ ID NO:

27;

b. 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.

2. The method of claim 1, wherein 1, 2, 3 or 4 residues in the C beta-strand are mutated with the proviso that S30 is not mutated (residue numbering according to SEQ ID NO:

27).

3. The method of claim 2, wherein the C beta-strand residues L32, Q34 and Q36 are mutated (residue numbering according to SEQ ID NO:

27).

4. The method of claim 3, wherein 1, 2, 3 or 4 residues in the F beta-strand are mutated with the proviso that E66 is not mutated (residue numbering according to SEQ ID NO:

27).

5. The method of claim 4, wherein the F-beta strand residues T68, S70 and Y72 are mutated (residue numbering according to SEQ ID NO:

27).

6. The method of claim 5, wherein 1, 2, 3 or 4 residues in the CD loop residues are mutated with the proviso that G42 and E43 are not mutated (residues numbering according to SEQ ID NO:

27).

7. The method of claim 6, wherein the residues S38, E39, K40 and V41 in the CD loop are mutated.

8. The method of claim 7, wherein 1, 2, 3 or 4 residues in the FG loop are mutated with the proviso that the residues K75, G76, G77 and S80 are not mutated (residue numbering according to SEQ ID NO:

27).

9. The method of claim 8, wherein the residues H78, R79 and N81 in the FG loop are mutated (residue numbering according to SEQ ID NO:

27).

10. The method of claim 9, wherein the reference FN3 domain 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.

11. A library produced by the method of claim 1.

12. The library of claim 11, wherein the library comprises an amino acid sequence of SEQ ID NO:

28.

13. A method of obtaining a protein scaffold comprising a fibronectin module of type III (FN3) domain having a diversified C-CD-F-FG alternative that specifically binds to a target molecule, comprising contacting or panning the library of claim 11 with the target molecule and isolating a protein scaffold specifically binding to the target molecule with a predefined affinity.