Protein Scaffolds

• US 20100298541A1
• Filed: 10/31/2008
• Published: 11/25/2010
• Est. Priority Date: 10/31/2007
• Status: Active Grant

• Current Assignee: Medimmune LLC (Astrazeneca PLC)
• Original Assignee: Medimmune LLC (Astrazeneca PLC)
• Inventors: Swers, Jeffrey, Chacko, Benoy, Wu, Herren, Baca, Manuel
• Granted Patent: US 8,633,297 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/14 for baldness or alopecia
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• 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 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 16/2878 : against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• 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: C12N 15/1037 : Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1044 : Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: G01N 33/566 : using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention provides protein scaffolds and methods of preparing, screening, engineering and using such protein scaffolds.

Claims

1. A recombinant polypeptide scaffold comprising:

I. seven beta strand domains designated A, B, C, D, E, F, and G;

II. linked to six loop regions, wherein a loop region connects each beta strand and is designated AB, BC, CD, DE, EF, and FG loops;

III. wherein at least one loop regions is a non-naturally occurring variant of the cognate loop region in any of SEQ ID NOs;

1-32 or 68-88; and

IV. wherein at least one beta strand domain has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 95%, or at least 99% homology to the cognate beta strand domain in any of SEQ ID NOs;

1-32 or 68-88.

2. The scaffold of claim 1, wherein said beta strand domains comprises the polypeptide sequence encoded by any of SEQ ID NOs:

1-32 or 68-88.

3. The scaffold of claim 1, wherein said beta strand domains comprise:

a. amino acid sequences for the A beta strand (SEQ ID NO;

228), B beta strand (SEQ ID NO;

229), C beta strand (SEQ ID NO;

230), D beta strand (SEQ ID NO;

231), E beta strand (SEQ ID NO;

232), F beta strand (SEQ ID NO;

233) and G beta strand (SEQ ID NO;

234);

or

b. amino acid sequences for the A beta strand (SEQ ID NO;

235), B beta strand (SEQ ID NO;

229), C beta strand (SEQ ID NO;

230), D beta strand (SEQ ID NO;

236), E beta strand (SEQ ID NO;

232), F beta strand (SEQ ID NO;

237) and G beta strand (SEQ ID NO;

234).

4. The scaffold of claim 3, wherein said BC loop comprises 9 amino acids having a sequence of S-X-a-X-b-X-X-X-G, wherein X represents any amino acid, wherein (a) represents proline or alanine and wherein (b) represents alanine or glycine.

5. The scaffold of claim 3, wherein said BC loop comprises 11 amino acids having a sequence of S-P-c-X-X-X-X-X-X T G, wherein X represents any amino acid and wherein (c) represents proline, serine or glycine.

6. The scaffold of claim 3, wherein said BC loop comprises 12 amino acids having a sequence of A-d-P X-X-X-e-f-X-I-X-G, wherein X represents any amino acid, wherein (d) represents proline, glutamate or lysine, wherein (e) represents asparagine or glycine, and wherein (f) represents serine or glycine.

7. The scaffold of claim 3, wherein said FG loop comprises 9 amino acids having a sequence of X-a-X-X-G-X-X-X-S, wherein X represents any amino acid and wherein (a) represents asparagine, threonine or lysine.

8. The scaffold of claim 3, wherein said FG loop comprises 10 amino acids having a sequence of X-a-X-X-X-X-b-N-P-A, wherein X represents any amino acid, wherein (a) represents asparagine, threonine or lysine and wherein (b) represents serine or glycine.

9. The scaffold of claim 3, wherein said FG loop comprises 11 amino acids having a sequence of X-a-X-X-G-X-X-S-N-P-A, wherein X represents any amino acid, and wherein (a) represents asparagine, threonine or lysine.

10. The scaffold of claim 3, wherein said DE loop comprises 6 amino acids having a sequence of X-X-X-X-X-X, wherein X represents any amino acid.

11. The scaffold of claim 3, wherein said AB loop comprises 7 residues having the sequence:

K-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine.

12. The scaffold of claim 3, wherein said AB loop comprises 9 residues having the sequence:

K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine.

13. The scaffold of claim 3, wherein said CD loop comprises 7, 8, or 9 residues wherein each residue in the CD loop is randomized and wherein each residue may be asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine.

14. The scaffold of claim 3, wherein said EF loop comprises 8 residues having the sequence X-b-L-X-P-X-c-X, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, wherein (b) represents asparagine, lysine, arginine, aspartic acid, glutamic acid, or glycine, and wherein (c) represents isoleucine, threonine, serine, valine, alanine, or glycine.

15. The scaffold of claim 1, wherein said scaffold further comprises at least one disulfide bond to link any two of said seven beta strand domains.

16. The scaffold of claim 15, wherein said disulfide bond forms a link between:

a. the A beta strand and B beta strand;

b. the D beta strand and E beta strand;

c. the F beta strand and G beta strand;

or

d. the C beta strand and F beta strand.

17. The scaffold of claim 15, wherein said scaffold further comprises at least two disulfide bonds to link any of said seven beta strand domains.

18. The scaffold of claim 17, wherein said disulfide bonds form a first link between the F beta strand and the G beta strand, and a second link between the C beta strand and F beta strand.

19. The scaffold of claim 1, wherein said scaffold further comprises at least one disulfide bond in a loop region.

20. The scaffold of claim 15, wherein said scaffold comprises the sequence corresponding to Tn3^SS1(SEQ ID NO:

64), Tn3^SS2(SEQ ID NO;

210), Tn3^SS3(SEQ ID NO;

65), or Tn3^SS4(SEQ ID NO;

66).

21-96. -96. (canceled)