Protein scaffolds for antibody mimics and other binding proteins

• US 7,115,396 B2
• Filed: 12/03/2003
• Issued: 10/03/2006
• Est. Priority Date: 12/10/1998
• Status: Expired due to Term

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Compound Therapeutics, Inc.
• Inventors: Kuimelis, Robert G., Lipovsek, Dasa, Wagner, Richard W.
• Primary Examiner(s): Campell, Bruce R.
• Assistant Examiner(s): AUDET, MAURY A
• Application Number:
  US10/728,078
• Publication Number:
  US 20050038229A1
• Time in Patent Office: 1,035 Days
• Field of Search: 435/69.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/525 Tumour necrosis factor [TNF]
• 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 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• 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 2319/00 Fusion polypeptide
• 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: 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 an immunoglobulin fold and that can be used as scaffolds. 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 non-antibody protein comprising a domain having an immunoglobulin-like fold, said non-antibody protein deriving from a reference protein having an immunoglobulin-like fold by having a mutated amino acid sequence, wherein said domain having an immunoglobulin-like fold has a sequence that is at least 80% identical to the seciuence of SEQ ID NO:

81 and, wherein said non-antibody protein binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein.

2. The derivative protein of claim 1, said derivative protein binding with a Kd at least as tight as 500 nM.

3. The derivative protein of claim 2, said derivative protein binding with a Kd at least as tight as 100 nM.

4. The derivative protein of claim 3, said derivative protein binding with a Kd at least as tight as 10 nM.

5. The derivative protein of claim 4, said derivative protein binding with Kd at least as tight as 1 nM.

6. The derivative protein of claim 5, said derivative protein binding with a Kd at least as tight as 500 pM.

7. The derivative protein of claim 6, said derivative protein binding with a Kd at least as tight as 100 pM.

8. The derivative protein of claim 7, said derivative protein binding with a Kd at least as tight as 20 pM.

9. The derivative protein of claim 1, wherein said domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO:

81 contains one, two, or three mutated loops and wherein at least one of said loops contributes to the binding of said derivative protein to said compound.

10. The derivative protein of claim 9, wherein at least two of said loops contribute to said binding of said derivative protein to said compound.

11. The derivative protein of claim 9, wherein three of said loops contribute to said binding of said derivative protein to said compound.

12. The derivative protein of claim 1, wherein said reference protein lacks disulfide bonds.

13. The derivative protein of claim 12, wherein said derivative protein has at least one disulfide bond.

14. The derivative protein of claim 1, wherein said domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO:

81 has a molecular mass less than 10 kD.

15. The derivative protein of claim 1, wherein said domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO:

81 has a molecular mass greater than 7.5 kD.

16. The derivative protein of claim 1, wherein said domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO:

81 has a molecular mass between 7.5–

10 kD.

17. The derivative protein of claim 1, wherein said derivative protein is a monomer under physiological conditions.

18. The derivative protein of claim 1, wherein said derivative protein is a dimer under physiological conditions.

19. The derivative protein of claim 1, wherein said reference protein is a naturally-occurring mammalian protein.

20. The derivative protein of claim 1, wherein said domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO:

81 is mutated and comprises up to 34% mutated amino acids as compared to the immunoglobulin-like fold of said reference protein.

21. The derivative protein of claim 1, said derivative protein being immobilized on a solid support.

22. The derivative protein of claim 21, said derivative protein being part of an array immobilized on a solid support.

23. The derivative protein of claim 21, said solid support being a chip or bead.

24. The derivative protein of claim 1, wherein said derivative protein is derived from a fibronectin or fibronectin dimer.

25. A method of obtaining a derivative non-antibody protein which binds to a compound, said method comprising:

(a) providing a non-antibody scaffold protein comprising a domain having an immunoglobulin-like fold, wherein said scaffold protein does not bind to said compound with a Kd as tight as 1 μ

M;

(b) generating mutated derivatives of said non-antibody scaffold protein wherein said derivatives comprise a domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO;

81, thereby producing a library of mutated proteins;

(c) contacting said library with said compound;

(d) selecting from said library at least one derivative protein which binds to said compound with a Kd at least as tight as 1 μ

M; and

(e) optionally repeating steps (b)–

(d) substituting for the non-antibody scaffold protein in repeated step (b) the product from the previous step (d).

26. A method for obtaining a non-antibody protein which binds to a compound, said method comprising:

(a) contacting said compound with a candidate protein, said candidate protein being a derivative non-antibody protein comprising a domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO;

81, said non-antibody protein deriving from a reference protein by having a mutated amino acid sequence wherein said non-antibody protein binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein, wherein said contacting is carried out under conditions that allow compound-protein complex formation; and

(b) obtaining, from said complex, said derivative protein which binds to said compound.

27. A method for obtaining a compound which binds to a non-antibody protein, said non-antibody protein comprising a domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the sequence of SEQ ID NO:

81 and deriving from a reference protein by having a mutated amino acid sequence, wherein said non-antibody protein binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein, said method comprising;

(a) contacting said derivative protein with a candidate compound, wherein said contacting is carried out under conditions that allow compound-protein complex formation; and

(b) obtaining, from said complex, said compound which binds to said derivative proteins.

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

(a) contacting said sample with a non-antibody protein comprising a domain having an immunoglobulin-like fold and a sequence that is at least 80% identical to the SEQ ID NO;

81, said non-antibody protein deriving from a reference protein by having a mutated amino acid sequence wherein said non-antibody protein binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein, wherein said contacting is carried out under conditions that allow compound-protein complex formation; and

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

29. A non-antibody protein that binds tumor necrosis factor-α

(TNF-α

) with a Kd at least as tight as 1 μ

M, said protein having a sequence that is less than 20% identical to TNF-α

receptor and is at least 80% identical to the seciuence of SEQ ID NO;

81.

30. The non-antibody protein of claim 29, wherein said non-antibody protein comprises a mutated fibronectin type III domain and wherein said protein is mutated in the DE, BC, and FG loops.

31. The non-antibody protein of claim 30, wherein said FG loop is the same length as the wild-type FG loop.

32. The non-antibody protein of claim 29, wherein said protein comprises a sequence shown in FIG. 25.

33. A loop structure on a protein, said loop comprising an amino acid sequences that is at least 80% identical to the sequence of SEQ ID NO:

81, wherein said loop binds with a Kd at least as tight as 1 μ

M to a compound that is not bound as tightly by said reference protein.