Pharmaceutically acceptable FN3 polypeptides for human treatments

• US 20060246059A1
• Filed: 07/07/2006
• Published: 11/02/2006
• Est. Priority Date: 12/10/1998
• Status: Abandoned Application

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Compound Therapeutics, Inc. (Bristol-Myers Squibb Company)
• Inventors: Kuimelis, Robert G., Lipovsek, Dasa, Wagner, Richard W.
• Application Number:
  US11/483,918
• Publication Number:
  US 20060246059A1
• Time in Patent Office: Days
• US Class Current: 424/133.100
• 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 pharmaceutically acceptable polypeptide comprising a tenth fibronectin type III (¹⁰Fn3) domain, wherein:

(a) the ¹⁰Fn3 domain has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of the human ¹⁰Fn3 domain;

(b) the ¹⁰Fn3 domain binds to a target compound that is not bound by the human ¹⁰Fn3 domain; and

(c) the integrin binding motif, RGD, of the ¹⁰Fn3 domain is replaced by an amino acid sequence as follows;

basic amino acid-neutral amino acid-acidic amino acid.

2. The polypeptide of claim 1, wherein the pharmaceutically acceptable Fn3 domain binds with a K_D of 500 nM or less to the target compound.

3. The polypeptide of claim 2, wherein the K_D is 1 nM or less.

4. The polypeptide of claim 2, wherein binding of the ¹⁰Fn3 domain to the target compound is mediated by one loop of the Fn3 domain.

5. The polypeptide of claim 2, wherein the loop is selected from the group consisting of the BC loop, the DE loop and the FG loop.

7. The polypeptide of claim 2, wherein binding of the ¹⁰Fn3 domain to the binds to the target compound is mediated by two loops of the Fn3 domain.

8. The polypeptide of claim 2, wherein binding of the ¹⁰Fn3 domain to the binds to the target compound is mediated by three loops of the ¹⁰Fn3 domain.

9. The polypeptide of claim 7, wherein the BC, DE and FG loops bind to the target compound.

10. The polypeptide of claim 1, wherein the FG loop binds to the target compound.

11. The polypeptide of claim 1, wherein the FG loop is longer than the FG loop of human ¹⁰Fn3 domain.

12. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain has an amino acid sequence that is at least 70% identical to the sequence of a human ¹⁰Fn3 domain.

13. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain contains no free sulfhydryl moieties and no disulfide bonds.

14. The polypeptide of claim 1, wherein the therapeutic protein is produced by expressing the polypeptide in a prokaryote.

15. The polypeptide of claim 1, wherein the ¹⁰Fn3 domain was identified by a screening method comprising:

(i) contacting the target compound with a candidate polypeptide including an ¹⁰Fn3 domain having at least one modified loop under conditions that allow target compound-polypeptide complex formation; and

(ii) obtaining the polypeptide which binds to the compound.

16. The polypeptide of claim 15, wherein the screening method further comprises:

(iii) repeating (i) and (ii) using a further randomized ¹⁰Fn3 domain.

17. The polypeptide of claim 1, further comprising a Fc region of an antibody.

18. The polypeptide of claim 1, further comprising a complement protein.

19. The polypeptide of claim 1, further comprising an albumin protein.

20. The polypeptide of claim 1, further comprising a toxin protein.

21. The polypeptide of claim 1, further comprising a human Fn3 domain.

22. The polypeptide of claim 21, wherein the human Fn3 domain:

(a) has at least one loop with a modified amino acid sequence relative to the sequence of the corresponding loop of a naturally occurring Fn3 domain; and

(b) binds to a second target compound that is not bound by the corresponding naturally-occurring Fn3 domain with a K_D of 500 nM or less.

23. A pharmaceutical composition comprising the polypeptide of claim 1.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is substantially free of endotoxin.

25. The pharmaceutical composition of claim 23, further comprising a physiologically acceptable carrier.