Anti-TNF Alpha Fibronectin Type III Domain Based Scaffold Compositions, Methods and Uses

• US 20100144601A1
• Filed: 10/27/2009
• Published: 06/10/2010
• 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 8,415,291 B2
• Time in Patent Office: Days
• US Class Current: 514/2.400
• CPC Class Codes: A61K 38/39 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

A protein scaffold based on a consensus sequence of the tenth fibronectin type III (FN3) repeat from human fibronectin, preferably human Tenascin, that binds to human TNFα 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.

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 the Tenascin FN3 domains, wherein protein scaffold is capable of binding to human TNFα

.

2. The protein scaffold of claim 1, comprising 7 strands and 6 loops between the strands.

18. A composition comprising a protein scaffold according to any of claims 1-5 and at least one pharmaceutically acceptable carrier or diluent.

19. A composition according to claim 18, further comprising at least one compound or polypeptide selected from a detectable label or reporter, 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.

20. A medical device, comprising a protein scaffold according to any of claims 1-5, 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.

3. 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 having the amino acid sequence of any one of the B:

C or F;

G loops of SEQ ID NOS;

22-168, wherein the protein scaffold forms loops at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 and is capable of binding to human TNFα

.

4. 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;

22-77; 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;

78-168, wherein the protein scaffold is capable of binding to human TNFα

.

5. The protein scaffold of claim 4, 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;

22-77; and

an F;

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

16 comprising any one of SEQ ID NOS;

78-168, wherein the protein scaffold is capable of binding to human TNFα

.

6. The protein scaffold of claim 5, 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.

7. The protein scaffold of claim 5, wherein the loops form sites for binding to targets.

8. A method of constructing a library of a protein scaffold based on a fibronectin type III (FN3) domain capable of binding to human TNFα

, comprising the steps of;

providing the protein scaffold of claim 5; and

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

9. The method of claim 8, wherein the introducing diversity step comprises mutating at least one loop region selected from the group consisting of the residues at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of the protein scaffold amino acid sequence.

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

11. A method of generating a protein scaffold capable of binding to human TNFα

with a predefined binding affinity, comprising contacting the library of claim 10 with at least a portion of human TNFα

protein and isolating a protein scaffold binding to human TNFα

with the predefined affinity.

12. The method of claim 11, wherein the isolating step comprising isolating scaffold molecules binding to human TNFα

and testing the isolated scaffold molecules for binding affinity to human TNFα

.

13. The method of claim 12, wherein the affinity is a K_D less than or equal to about 10⁻

7M.

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

15. An isolated nucleic acid vector comprising the isolated nucleic acid molecule according to claim 14.

16. A prokaryotic or eukaryotic host cell comprising the isolated nucleic acid molecule according to claim 14.

17. The host cell according to claim 16, 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.

21. 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.

22. The article of manufacture of claim 21, 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.

23. Any invention described herein.