Protein scaffolds for antibody mimics and other binding proteins

• US 6,818,418 B1
• Filed: 02/29/2000
• Issued: 11/16/2004
• Est. Priority Date: 12/10/1998
• Status: Expired due to Term

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Compound Therapeutics, Inc. (Bristol-Myers Squibb Company)
• Inventors: Kuimelis, Robert G., Lipovsek, Dasa, Wagner, Richard W.
• Primary Examiner(s): Tate, Christopher R.
• Assistant Examiner(s): AUDET, MAURY A
• Application Number:
  US09/515,260
• Time in Patent Office: 1,722 Days
• Field of Search: 435/7.1, 435/6, 435/7.4, 435/7.6, 435/7.8, 435/7.92, 435/69.1, 436/501, 514/2, 530/350, 530/810, 530/811, 530/812, 530/866
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2318/20 Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

Disclosed herein are proteins that include a fibronectin type III domain having at least one randomized loop. Also disclosed herein are nucleic acids encoding such proteins and the use of such proteins in diagnostic methods and in methods for evolving novel compound-binding species and their ligands.

Claims

1. A method for obtaining a scaffold-based protein that binds to a compound, said method comprising:

(a) contacting a compound with a library of scaffold-based proteins under conditions that allow binding to form a compound-scaffold-based protein complex, wherein the scaffold is derived from the tenth module of human fibronectin type III (¹⁰Fn3), said tenth module having the amino acid sequence, VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT,

said library comprising scaffold-based proteins having at least three randomized loops and being characterized by their ability to bind to a compound that is not bound by said human ¹⁰Fn3; and

(b) obtaining, from said complex, a scaffold-based protein that binds to said compound and that has at least one amino acid alteration in each of three loops relative to the human ¹⁰Fn3 sequence.

3. The method of claim 1, said method further comprising further randomizing at least one loop of said human fibronectin type III domain of said protein obtained in step (b) and repeating said steps (a) and (b) using said further randomized protein.

5. The method of claim 1 or 2, wherein said compound is a protein.

6. The method of claim 1 or 2, wherein at least one of said randomized loops is extended in length relative to the corresponding loop of human ¹⁰Fn3.

7. The method of claim 1 or 2, wherein said ¹⁰Fn3 lacks an integrin-binding motif.

8. The method of claim 1 or 2, wherein said protein is covalently bound to a nucleic acid.

9. The method of claim 8, wherein said nucleic acid encodes said protein.

10. The method of claim 8, wherein said nucleic acid is RNA.

11. The method of claim 1, wherein said compound is immobilized on a solid support.

13. The method of claim 11 or 12, wherein said solid support is a column or microchip.

2. A method for obtaining a compound that binds to a scaffold-based protein, said method comprising:

(a) contacting a scaffold-based protein with a candidate compound under conditions that allow binding to form a compound-scaffold-based protein complex, wherein the scaffold is derived from the tenth module of human fibronectin type III (¹⁰Fn3), said tenth module having the amino acid sequence, VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT,

said scaffold-based protein having at least one amino acid alteration in each of three loops relative to the human ¹⁰Fn3 sequence, said scaffold-based protein being characterized by its ability to bind to a compound that is not bound by said human ¹⁰Fn3; and

(b) obtaining, from said complex, a compound that binds to said scaffold-based protein.

4. The method of claim 2, said method further comprising modifying said compound obtained in step (b) and repeating said steps (a) and (b) using said further modified compound.

12. The method of claim 2, wherein said scaffold-based protein is immobilized on a solid support.

14. A method for detecting a compound in a sample, said method comprising:

(a) contacting said sample with a scaffold-based protein which binds to said compound under conditions that allow binding to form a compound-scaffold-based protein complex, wherein the scaffold is derived from the tenth module of human fibronectin type III (¹⁰Fn3), said tenth module having the amino acid sequence, VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT,

said scaffold-based protein having at least one amino acid alteration in each of three loops relative to the human ¹⁰Fn3 sequence, said scaffold-based protein being characterized by its ability to bind to a compound that is not bound by said human ¹⁰Fn3; and

(b) detecting said complex, thereby detecting said compound in said sample.

15. The method of claim 14, wherein said scaffold-based protein is immobilized on a solid support.

16. The method of claim 15, wherein said scaffold-based protein is immobilized on said solid support as part of an array.

17. The method of claim 15, wherein said solid support is a chip or bead.

18. The method of claim 14, wherein said scaffold-based protein is covalently bound to a nucleic acid.

19. The method of claim 18, wherein said nucleic acid encodes and scaffold-based protein.

20. The method of claim 19, wherein said nucleic acid is RNA.

21. The method of claim 14, wherein said compound is a protein.

22. The method of claim 14, wherein said compound is detected by radiography, fluorescence detection, mass spectroscopy, or surface plasmon resonance.