Fibronectin Type III Domain Based Scaffold Compositions, Methods and Uses

• US 20100216708A1
• Filed: 10/27/2009
• Published: 08/26/2010
• Est. Priority Date: 10/31/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: O'Neil, Karyn, Jacobs, Steven
• Granted Patent: US 8,278,419 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 39/02 Antidotes
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• 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

Abstract

A protein scaffold based on a consensus sequence of fibronectin type III (FN3) proteins, such as the tenth FN3 repeat from human fibronectin (human Tenascin), including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of making and using thereof have applications in diagnostic and/or therapeutic compositions, methods and devices. In particular, protein scaffold molecules binding to IgG have been identified as useful for diagnostic and/or therapeutic applications.

Claims

1. An isolated protein scaffold based on a fibronectin type III (FN3) domain comprising an amino acid sequence derived from a consensus sequence of an FN3 domain, wherein the amino acid sequence has at least 75% identity to the consensus FN3 domain sequence.

2. An isolated protein scaffold based on a fibronectin type III (FN3) domain comprising an amino acid sequence derived from a consensus sequence of the Tenascin FN3 domains, wherein the amino acid sequence has at least 75% identity to SEQ ID NO:

16.

3. An isolated protein scaffold based on a fibronectin type III (FN3) domain comprising an amino acid sequence derived from a consensus sequence of a FN3 domain, comprising 7 strands and 6 loops between the strands.

4. The protein scaffold of claim 3, wherein one or more of the loops is capable of binding to cellular proteins and/or nucleic acid molecules

5. The protein scaffold of claim 4, comprising the amino acid sequence of SEQ ID NO:

16 and forming loops at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO;

16, the loops capable of binding a target comprising cellular proteins and/or nucleic acid molecules.

6. The protein scaffold of claim 5, wherein one or more of the loops is altered in order to bind to the target.

7. The protein scaffold of claim 5, wherein said scaffold binds a target with at least one affinity selected from a K_D of at least 10⁻

9M, at least 10<sup>−

10</sup>M, at least 10<sup>−

11</sup>M, and at least 10<sup>−

12</sup>M, at least 10<sup>−

13</sup>M, at least 10<sup>−

14</sup>M, and at least 10<sup>−

15</sup>M, as determined by surface plasmon resonance or the Kinexa method.

21. An isolated nucleic acid molecule encoding the protein scaffold of claim 5.

22. The isolated nucleic acid molecule of claim 21, comprising the nucleotide sequence set forth in SEQ ID NO:

17.

23. An isolated nucleic acid vector comprising the isolated nucleic acid molecule according to claim 21.

24. A prokaryotic or eukaryotic host cell comprising the isolated nucleic acid molecule according to claim 21.

25. The host cell according to claim 24, wherein said host cell is at least one selected from E. coli BL21Star(DE3), other E. coli cell, yeast, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cell thereof.

26. A composition comprising the protein scaffold of claim 5 and at least one pharmaceutically acceptable carrier or diluent.

27. A composition according to claim 26, further comprising at least one compound or polypeptide selected from a detectable label or reporter, a TNF antagonist, an anti-infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an antineoplastic, an immunomodulation drug, an opthalmic, otic or nasal drug, a topical drug, a nutritional drug, a cytokine, and a cytokine antagonist.

28. A medical device, comprising the protein scaffold of claim 5, wherein said device is suitable for contacting or administering said protein scaffold by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

29. An article of manufacture for human pharmaceutical or diagnostic use, comprising packaging material and a container comprising a solution or a lyophilized form of the protein scaffold of claim 5.

30. The article of manufacture of claim 29, wherein said container is a component of a parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery device or system.

8. A method of constructing a library of a protein scaffold based on a fibronectin type III (FN3) domain derived from a consensus sequence of an FN3 domain, comprising the steps of:

providing a polypeptide derived from a consensus sequence of an FN3 domain; and

introducing diversity into copies of the polypeptide to form the protein scaffold library.

9. A library produced by the method of claim 8.

10. A method of generating a protein scaffold binding to a specific target with a predefined binding affinity, comprising contacting the library of claim 9 with the specific target and isolating a protein scaffold binding to the specific target with the predefined affinity.

11. The method of claim 10, wherein the isolating step comprising isolating scaffold molecules binding to the specific target and testing the isolated scaffold molecules for binding affinity to the specific target.

12. The method of claim 11, wherein the isolating step comprises panning the library with the specific target, identifying scaffold molecules binding to the specific target, and isolating the binding scaffold molecules.

13. The method of claim 11, wherein the affinity is less than or equal to about 10⁻

7M.

14. A method of constructing a library of a protein scaffold based on a fibronectin type III (FN3) domain, comprising the steps of:

providing a polypeptide having the amino acid sequence of SEQ ID NO;

16; and

introducing diversity into copies of the polypeptide having the amino acid sequence of SEQ ID NO;

16 to form the protein scaffold library.

15. The method of claim 14, wherein the introducing diversity step comprises mutating at least one loop region selected from the group consisting of residues at or about positions 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO:

16

16. A library produced by the method of claim 15.

17. A method of generating a protein scaffold binding to a specific target with a predefined binding affinity, comprising contacting the library of claim 16 with the specific target and isolating a protein scaffold binding to the specific target with the predefined affinity.

18. The method of claim 17, wherein the isolating step comprising isolating scaffold molecules binding to the specific target and testing the isolated scaffold molecules for binding affinity to the specific target.

19. The method of claim 18, wherein the isolating step comprises panning the library with the specific target, identifying scaffold molecules binding to the specific target, and isolating the binding scaffold molecules.

20. The method of claim 18, wherein the affinity is less than or equal to about 10⁻

7M.

31. An isolated protein scaffold based on a fibronectin type III (FN3) domain comprising an amino acid sequence derived from a consensus sequence of the Tenascin FN3 domains, wherein the amino acid sequence has at least 75% identity to any one of a B:

C or F;

G loop of SEQ ID NOS;

21-140 and the protein scaffold binds to human IgG.

32. The protein scaffold of claim 31, wherein the scaffold comprises 7 strands and 6 loops between the strands.

33. The protein scaffold of claim 31, comprising the amino acid sequence of any one of a B:

C or F;

G loop of SEQ ID NOS;

21-140, wherein the protein scaffold forms loops at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81.

34. An isolated protein scaffold based on a fibronectin type III (FN3) domain comprising:

a backbone portion having an amino acid sequence identical to SEQ ID NO;

16 at residues 1-21, 29-74, and 82-89;

a B;

C loop portion between residues 21 and 29 of SEQ ID NO;

16 having at least 75% identity to any one of SEQ ID NOS;

21-45; and

an F;

G loop portion between residues 74 and 82 of SEQ ID NO;

16 having at least 75% identity to any one of SEQ ID NOS;

46-140, wherein the protein scaffold is capable of binding to human IgG.

35. The protein scaffold of claim 34, comprising:

a backbone portion having an amino acid sequence identical to SEQ ID NO;

16 at residues 1-21, 29-74, and 82-89;

a B;

C loop portion between residues 21 and 29 of SEQ ID NO;

16 comprising any one of SEQ ID NOS;

21-45; and

an F;

G loop portion between residues 74 and 82 of SEQ ID NO;

16 comprising any one of SEQ ID NOS;

46-140, wherein the protein scaffold is capable of binding to human IgG.

36. The protein scaffold of claim 34, wherein said scaffold binds a target with at least one affinity selected from a K_D of less than or equal to 10⁻

9M, less than or equal to 10<sup>−

10</sup>M, less than or equal to 10<sup>−

11</sup>M, less than or equal to 10<sup>−

12</sup>M, less than or equal to 10<sup>−

13</sup>M, less than or equal to 10<sup>−

14</sup>M, and less than or equal to 10<sup>−

15</sup>M, as determined by surface plasmon resonance or the Kinexa method.

37. The protein scaffold of claim 35, wherein the loops form sites for binding to IgG.

38. An isolated protein scaffold based on a fibronectin type III (FN3) domain comprising an amino acid sequence derived from a consensus sequence of the Tenascin FN3 domains, wherein protein scaffold is capable of binding to human IgG.

39. Any invention described herein.